Researcher Database

Mayumi Ishizuka
Faculty of Veterinary Medicine Veterinary Medicine Environmental Veterinary Sciences

Researcher Profile and Settings


  • Faculty of Veterinary Medicine Veterinary Medicine Environmental Veterinary Sciences

Job Title

  • Professor

J-Global ID

Research Interests

  • シトクロムP450   環境化学物質   エストロゲン   野生動物   マイクロアレイ   アフリカ   ルシフェラーゼ   neonatal imprinting   レポーターアッセイ   アロマターゼ   レポーター   トランスジェニック   トキシコロジー   in vivoイメージング   ERE   インプリンティング   視床下部   環境汚染   ステロイドホルモン   環境ホルモン   ビスフェノールA   ダイオキシン受容体   PPAR   フタル酸エステル   バイオマーカー   環境汚染物質   ダイオキシン類   CYP1A   海棲哺乳類   ドブネズミ   

Research Areas

  • Environmental science/Agricultural science / Landscape science
  • Environmental science/Agricultural science / Environmental agriculture
  • Environmental science/Agricultural science / Environmental effects of chemicals
  • Environmental science/Agricultural science / Environmental effects of radiation
  • Life sciences / Applied molecular and cellular biology
  • Life sciences / Healthcare management, medical sociology
  • Life sciences / Hygiene and public health (non-laboratory)
  • Life sciences / Hygiene and public health (laboratory)
  • Life sciences / Hygiene and public health (non-laboratory)
  • Life sciences / Hygiene and public health (laboratory)
  • Life sciences / Healthcare management, medical sociology
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine

Academic & Professional Experience

  • 2011 - 2012 北海道大学 (連合)獣医学研究科 教授

Association Memberships


Research Activities

Published Papers

  • Shouta M M Nakayama, Ayuko Morita, Yoshinori Ikenaka, Yusuke K Kawai, Kensuke P Watanabe, Chihiro Ishii, Hazuki Mizukawa, Yared B Yohannes, Keisuke Saito, Yukiko Watanabe, Masaki Ito, Natsuo Ohsawa, Mayumi Ishizuka
    Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 228 108635 - 108635 1532-0456 2020/02 [Refereed][Not invited]
    Worldwide use of anticoagulant rodenticides (ARs) for rodents control has frequently led to secondary poisoning of non-target animals, especially raptors. In order to suggest some factors that may help considering the mechanism of the incidents, this study focused on the avian vitamin K 2, 3-epoxide reductase (VKOR) that is the target protein of ARs. We addressed the interspecific differences in VKOR activity and inhibition related to amino acid sequence and mRNA expression of VKORC1 and VKORC1-like1 (VKORC1L1). Poultry have been considered to be more tolerant to ARs than mammals. However, VKOR activity of owls, hawks, falcon and surprisingly, canaries, was lower and inhibited by warfarin more easily than that of chickens and turkeys. The amino acid sequence of VKORC1 and VKORC1L1 implied that the value of Ki for VKOR activity to ARs could depend on the amino acid at position 140 in the TYX warfarin-binding motif in VKORC1, and other amino acid mutations in VKORC1L1. The mRNA expression ratio of VKORC1:VKORC1L1 differed between turkey (8:1) and chicken (2:3) liver. VKORC1L1 has been reported to be resistant to warfarin compared to VKORC1. Hence, both the Ki of specific VKORC1 and VKORC1L1, and the mRNA expression ratio would cause avian interspecific difference of the VKOR inhibition. Our study also suggested the high inhibition of VKOR activities in raptors and surprisingly that in canaries as well. These factors are the most likely to contribute to the high sensitivity to ARs found in raptors.
  • Masao Togao, Shouta M M Nakayama, Yoshinori Ikenaka, Hazuki Mizukawa, Yoshiki Makino, Ayano Kubota, Takehisa Matsukawa, Kazuhito Yokoyama, Takafumi Hirata, Mayumi Ishizuka
    Chemosphere 238 124581 - 124581 0045-6535 2020/01 [Refereed][Not invited]
    Lead (Pb) pollution is one of the most serious environmental problems and has attracted worldwide attention. Pb causes hematological, central nervous system, as well as renal toxicity, and so on. Although many investigations about Pb in blood to evaluate pollution status and toxic effects have been reported, there are open question about biological behavior of Pb. In order to reveal any toxicological mechanisms or influences, we focused on the local distribution of Pb in mice organs. Lead acetate (100 mg/L and 1000 mg/L) in drinking water were given to the BALB/c mice (male, seven weeks of age, N = 24) for three weeks. Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) analysis revealed a homogenous distribution of Pb in the liver and inhomogeneous distribution in the kidney and brain. The hippocampus, thalamus, and hypothalamus had higher concentrations than other areas such as the white matter. Surprisingly, in the kidney, Pb tended to accumulate in the medulla rather than the cortex, strongly suggesting that high sensitivity areas and high accumulation areas differ. Moreover, distribution of stromal interacting protein 1 (STIM1) which is candidate gene of Pb pathway to the cells was homogenous in the liver and kidney whereas inhomogeneous in the brain. In contrast to our hypothesis, interestingly, Pb exposure under the current condition did not induce mRNA expressions for any candidate channel or transporter genes. Thus, further study should be conducted to elucidate the local distribution of Pb and other toxic metals, and pathway that Pb takes to the cells.
  • Kosuke Tanaka, Hideshige Takada, Yoshinori Ikenaka, Shouta M M Nakayama, Mayumi Ishizuka
    Marine pollution bulletin 150 110732 - 110732 0025-326X 2020/01 [Refereed][Not invited]
    In the ocean, plastic debris containing chemical additives is fragmented into smaller pieces that can be ingested by a wide range of organisms, potentially exposing them to additives. However, the levels of additives retained in marine plastic fragments have rarely been assessed. In this study, 141 plastic fragments from a beach in Kauai were analyzed piece-by-piece for 12 compounds, including UV stabilizers and brominated flame retardants. UV stabilizers (UV-326, UV-328, UV-327, and BP-12) were found in 13% of "small" fragments (4-7 mm) with levels of up to 315 μg/g and in 33% of "large" fragments (15-80 mm) with levels of up to 1130 μg/g. This observation suggests that exhaustive leaching of additives does not occur during fragmentation and that significant levels of additives, comparable to those of the original products, can be retained in fragments of marine plastic, indicating their importance as a vector of chemical additive exposure.
  • Kraisiri Khidkhan, Hazuki Mizukawa, Yoshinori Ikenaka, Shouta M M Nakayama, Kei Nomiyama, Nozomu Yokoyama, Osamu Ichii, Wageh Sobhy Darwish, Mitsuyoshi Takiguchi, Shinsuke Tanabe, Mayumi Ishizuka
    Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 226 108613 - 108613 1532-0456 2019/12 [Refereed][Not invited]
    Cats have been known to be extremely sensitive to chemical exposures. To understand these model species' sensitivity to chemicals and their toxicities, the expression profiles of xenobiotic-metabolizing enzymes should be studied. Unfortunately, the characterization of cytochrome P450 (CYP), the dominant enzyme in phase I metabolism, in cats has not extensively been studied. Polychlorinated biphenyls (PCBs) are known as CYP inducers in animals, but the information regarding the PCB-induced CYP expression in cats is limited. Therefore, in the present study, we aimed to elucidate the mRNA expression of the CYP1-CYP3 families in the cat tissues and to investigate the CYP mRNA expression related to PCB exposure. In cats, the greatest abundance of CYP1-CYP3 (CYP1A2, CYP2A13, CYP2C41, CYP2D6, CYP2E1, CYP2E2, CYP2F2, CYP2F5, CYP2J2, CYP2U1, and CYP3A132) was expressed in the liver, but some extrahepatic isozymes were found in the kidney (CYP1A1), heart (CYP1B1), lung (CYP2B11 and CYP2S1) and small intestine (CYP3A131). In cats, CYP1A1, CYP1A2 and CYP1B1 were significantly upregulated in the liver as well as in several tissues exposed to PCBs, indicating that these CYPs were distinctly induced by PCBs. The strong correlations between 3,3',4,4'-tetrachlorobiphenyl (CB77) and CYP1A1 and CYP1B1 mRNA expressions were noted, demonstrating that CB77 could be a potent CYP1 inducer. In addition, these CYP isoforms could play an essential role in the PCBs biotransformation, particularly 3-4 Cl-PCBs, because a high hydroxylated metabolite level of 3-4 Cl-OH-PCBs was observed in the liver.
  • Kodai Motohira, Yoshinori Ikenaka, Yared Beyene Yohannes, Shouta M M Nakayama, Victor Wepener, Nico J Smit, Johan H J VAN Vuren, Ana Catarina Sousa, Alex Ajeh Enuneku, Emmanuel Temiotan Ogbomida, Mayumi Ishizuka
    The Journal of veterinary medical science 81 (11) 1575 - 1579 0916-7250 2019/11/14 [Refereed][Not invited]
    Dichlorodiphenyltrichloroethane (DDT) is an organochlorine insecticide that has been used for indoor residual spraying for the control of mosquito-borne diseases including malaria. However, due to its toxicity and environmental persistence, there are concerns about its potential deleterious effects in humans and wildlife. Therefore, the current study aimed to monitor and estimate the level of DDTs in human communities. The accumulation of DDT and its metabolites was evaluated in house rat (as sentinel) livers collected in an area where DDT was sprayed. DDTs were measured using a gas chromatography / Electron Capture Detector. The results revealed high concentrations of DDTs in the rat livers and the levels of DDTs were similar to findings reported from the same area in 2014.
  • Lesa A Thompson, Yoshinori Ikenaka, Wageh Sobhy Darwish, Shouta M M Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    Environmental toxicology and pharmacology 72 103249 - 103249 1382-6689 2019/11 [Refereed][Not invited]
    The organochlorine pesticide dichloro-diphenyl-trichloroethane (DDT) is persistent in the environment and leads to adverse human health effects. High levels in breast milk pose a threat to both breast tissue and nursing infants. The objectives of this study were to investigate DDT-induced transcriptomic alterations in enzymes and transporters involved in xenobiotic metabolism, immune responses, oxidative stress markers, and cell growth in a human breast cancer cell line. MCF-7 cells were exposed to both environmentally-relevant and previously-tested concentrations of p,p'-DDT in a short-term experiment. Significant up-regulation of metabolizing enzymes and transporters (ACHE, GSTO1, NQO1 and ABCC2) and oxidative stress markers (CXCL8, HMOX-1, NFE2L2 and TNF) was clearly observed. Conversely, UGT1A6, AHR and cell growth genes (FGF2 and VEGFA) were severely down-regulated. Identification of these genes helps to identify mechanisms of p,p'-DDT action within cells and may be considered as useful biomarkers for exposure to DDT contamination.
  • Kohki Takaguchi, Hiroyuki Nishikawa, Hazuki Mizukawa, Rumi Tanoue, Nozomu Yokoyama, Osamu Ichii, Mitsuyoshi Takiguchi, Shouta M M Nakayama, Yoshinori Ikenaka, Tatsuya Kunisue, Mayumi Ishizuka, Shinsuke Tanabe, Hisato Iwata, Kei Nomiyama
    The Science of the total environment 688 1172 - 1183 0048-9697 2019/10/20 [Refereed][Not invited]
    Polychlorinated biphenyls (PCBs) and their hydroxylated metabolites (OH-PCBs) might disrupt thyroid function. However, there is no clear evidence of PCB exposure disrupting thyroid hormone (TH) homeostasis in dogs and cats. The present study conducted in vivo experiments to evaluate the effects of a mixture of 12 PCB congeners (CB18, 28, 70, 77, 99, 101, 118, 138, 153, 180, 187 and 202, each congener 0.5 mg/kg BW, i.p. administration) on serum TH levels in male dogs (Canis lupus familiaris) and male cats (Felis silvestris catus). In PCB-exposed dogs, the time courses of higher-chlorinated PCBs and L-thyroxine (T4)-like OH-PCBs (4-OH-CB107 and 4-OH-CB202) concentrations were unchanged or tended to increase, whereas those of lower-chlorinated PCBs and OH-PCBs tended to decrease after 24 h. In PCB-exposed cats, concentrations of PCBs increased until 6 h and then remained unchanged. The levels of lower-chlorinated OH-PCBs including 4'-OH-CB18 increased until 96 h and then decreased. In PCB-exposed dogs, free T4 concentrations were higher than those in the control group at 48 and 96 h after PCB administration and positively correlated with the levels of T4-like OH-PCBs, suggesting competitive binding of T4 and T4-like OH-PCBs to a TH transporter, transthyretin. Serum levels of total T4 and total 3,3',5-triiodo-L-thyronine (T3) in PCB-exposed dogs were lower than in the control group at 24 and 48 h and negatively correlated with PCB concentrations, implying that PCB exposure enhanced TH excretion by increasing TH uptake and TH conjugation enzyme activities in the dog liver. In contrast, no obvious changes in TH levels were observed in PCB-exposed cats. This could be explained by the lower levels of T4-like OH-PCBs and lower hepatic conjugation enzyme activities in cats compared with dogs. Different effects on serum TH levels in PCB-exposed dogs and cats are likely to be attributable to species-specific PCB and TH metabolism.
  • Tarryn L Botha, Sarel J Brand, Yoshinori Ikenaka, Shouta M M Nakayama, Mayumi Ishizuka, Victor Wepener
    Aquatic toxicology (Amsterdam, Netherlands) 215 105287 - 105287 0166-445X 2019/10 [Refereed][Not invited]
    Gold nanoparticles are used as drug delivery vectors based on the assumption that they have low toxicity. Literature has, however, produced conflicting results over the last few years. As such, this study aimed to investigate the toxicological effects of nanogold (nAu) on several indicators that range from subcellular to whole-organism level. Gene regulation, changes in oxidative stress biomarkers and swimming performance were assessed in Danio rerio (zebrafish) following exposures to nAu. Adult zebrafish were exposed in vivo to nAu for 96 h and swimming performance measured post-exposure. Liver tissue was collected for DNA microarray and Real-Time Polymerase Chain Reactions (RT-PCR) analyses to determine changes in gene expression (catalase, superoxide dismutase and metallothioneins) and protein biomarker analyses (catalase, superoxide dismutase, acetylcholine esterase, malondialdehyde, cellular energy allocation and metallothionein) were performed on whole-body samples. Swimming behaviour was assessed in 1.1 L Tecniplast™ tanks for a period of six hours and videos were analysed using Noldus EthoVision software. Critical swimming speed was measured in a Loligo® swimming tunnel. The DNA microarray revealed that fish exposed to 20 mg/L differed most from the control group. At 20 mg/L there was a significant increase in gene expression for all genes analysed but this didn't translate to significant responses in protein biomarker levels except for an increase in protein carbonyl formation. The behaviour results demonstrated significant changes in distance moved, swimming speed, acceleration bouts, zone alterations and time spent within the top zone - responses that are usually observed in fish responding to toxicological stress. Furthermore, the critical swimming speed of exposed fish was decreased significantly compared to the control. Since swimming performance and social interaction among zebrafish is essential to their survival, whole-organism behaviour that suggests a toxicological response after exposure to nAu is in agreement with the genetic responses measured in this study.
  • Shouta M M Nakayama, Hokuto Nakata, Yoshinori Ikenaka, John Yabe, Balazs Oroszlany, Yared B Yohannes, Nesta Bortey-Sam, Kaampwe Muzandu, Kennedy Choongo, Takeshi Kuritani, Mitsuhiro Nakagawa, Mayumi Ishizuka
    Environmental pollution (Barking, Essex : 1987) 252 (Pt B) 1267 - 1276 0269-7491 2019/09 [Refereed][Not invited]
    Metal pollution has been associated with anthropogenic activities, such as effluents and emissions from mines. Soil could be exposure route of wild rats to metals, especially in mining areas. The aim of this study was to verify whether soil exposure under environmentally relevant circumstances results in metal accumulation and epigenetic modifications. Wistar rats were divided to three groups: 1) control without soil exposure, 2) low-metal exposure group exposed to soil containing low metal levels (Pb: 75 mg/kg; Cd: 0.4), and 3) high-metal exposure group exposed to soil (Pb: 3750; Cd: 6). After 1 year of exposure, the metal levels, Pb isotopic values, and molecular indicators were measured. Rats in the high-group showed significantly greater concentrations of Pb and Cd in tissues. Higher accumulation factors (tissue/soil) of Cd than Pb were observed in the liver, kidney, brain, and lung, while the factor of Pb was higher in the tibia. The obtained results of metal accumulation ratios (lung/liver) and stable Pb isotope ratios in the tissues indicated that the respiratory exposure would account for an important share of metal absorption into the body. Genome-wide methylation status and DNA methyltransferase (Dnmt 3a/3b) mRNA expressions in testis were higher in the high-group, suggesting that exposure to soil caused metal accumulation and epigenetic alterations in rats.
  • Ryu Miura, Atsuko Araki, Machiko Minatoya, Kunio Miyake, Mei-Lien Chen, Sumitaka Kobayashi, Chihiro Miyashita, Jun Yamamoto, Toru Matsumura, Mayumi Ishizuka, Takeo Kubota, Reiko Kishi
    Scientific reports 9 (1) 12369 - 12369 2019/08/26 [Refereed][Not invited]
    Exposure to bisphenol A (BPA) in utero is associated with adverse health outcome of the offspring. Differential DNA methylation at specific CpG sites may link BPA exposure to health impacts. We examined the association of prenatal BPA exposure with genome-wide DNA methylation changes in cord blood in 277 mother-child pairs in the Hokkaido Study on Environment and Children's Health, using the Illumina HumanMethylation 450 BeadChip. We observed that a large portion of BPA-associated differentially methylated CpGs with p-value < 0.0001 was hypomethylated among all newborns (91%) and female infants (98%), as opposed to being hypermethylated (88%) among males. We found 27 and 16 CpGs with a false discovery rate (FDR) < 0.05 in the analyses for males and females, respectively. Genes annotated to FDR-corrected CpGs clustered into an interconnected genetic network among males, while they rarely exhibited any interactions in females. In contrast, none of the enrichment for gene ontology (GO) terms with FDR < 0.05 was observed for genes annotated to the male-specific CpGs with p < 0.0001, whereas the female-specific genes were significantly enriched for GO terms related to cell adhesion. Our epigenome-wide analysis of cord blood DNA methylation implies potential sex-specific epigenome responses to BPA exposure.
  • Wilson Mwandira, Kazunori Nakashima, Satoru Kawasaki, Mayumi Ito, Tsutomu Sato, Toshifumi Igarashi, Meki Chirwa, Kawawa Banda, Imasiku Nyambe, Shouta Nakayama, Hokuto Nakata, Mayumi Ishizuka
    Chemosphere 228 17 - 25 0045-6535 2019/08 [Refereed][Not invited]
    Environmental impacts resulting from historic lead and zinc mining in Kabwe, Zambia affect human health due to the dust generated from the mine waste that contains lead, a known hazardous pollutant. We employed microbially induced calcium carbonate precipitation (MICP), an alternative capping method, to prevent dust generation and reduce the mobility of contaminants. Pb-resistant Oceanobacillus profundus KBZ 1-3 and O. profundus KBZ 2-5 isolated from Kabwe were used to biocement the sand that would act as a cover to prevent dust and water infiltration. Sand biocemented by KBZ 1-3 and KBZ 2-5 had maximum unconfined compressive strength values of 3.2 MPa and 5.5 MPa, respectively. Additionally, biocemented sand exhibited reduced water permeability values of 9.6 × 10-8 m/s and 8.9 × 10-8 m/s for O. profundus KBZ 1-3 and KBZ 2-5, respectively, which could potentially limit the entrance of water and oxygen into the dump, hence reducing the leaching of heavy metals. We propose that these isolates represent an option for bioremediating contaminated waste by preventing both metallic dust from becoming airborne and rainwater from infiltrating into the waste. O. profundus KBZ 1-3 and O. profundus KBZ 2-5 isolated form Kabwe represent a novel species that has, for the first time, been applied in a bioremediation study.
  • Yasuhiro Uno, Rika Takahira, Norie Murayama, Shunsuke Onozeki, Shu Kawamura, Shotaro Uehara, Yoshinori Ikenaka, Mayumi Ishizuka, Shinichi Ikushiro, Hiroshi Yamazaki
    Biochemical pharmacology 163 335 - 344 0006-2952 2019/05 [Refereed][Not invited]
    UDP-glucuronosyltransferases (UGTs) are essential enzymes metabolizing endogenous and exogenous chemicals. However, characteristics of UGTs have not been fully investigated in molecular levels of cynomolgus macaques, one of non-human primates widely used in preclinical drug metabolism studies. In this study, three UGT2A cDNAs (UGT2A1, 2A2, and 2A3) were isolated and characterized along with seven UGT2Bs previously identified in cynomolgus macaques. Several transcript variants were found in cynomolgus UGT2A1 and UGT2A2, like human orthologs. Cynomolgus UGT2A and UGT2B amino acid sequences were highly identical (87-96%) to their human counterparts. By phylogenetic analysis, all these cynomolgus UGT2s were more closely clustered with their human homologs than with dog, rat, or mouse UGT2s. Especially, UGT2As showed orthologous relationships between humans and cynomolgus macaques. All the cynomolgus UGT2 mRNAs were expressed in livers, jejunum, and/or kidneys abundantly, except that UGT2A1 and UGT2A2 mRNAs were predominantly expressed in nasal mucosa, like human UGT2s. UGT2A and UGT2B genes together form a gene cluster in the cynomolgus and human genome. Among the seven cynomolgus UGT2Bs heterologously expressed in yeast, UGT2B9 and UGT2B30 showed activities in estradiol 17-O-glucuronidation and morphine 3-O-glucuronidation but did not show activities in estradiol 3-O-glucuronidation, similar to human UGT2Bs. In liver microsomes, cynomolgus macaques showed higher estradiol 17-O-glucuronidase and morphine 3-O-glucuronidase activities than humans, suggesting functional activities of the responsible UGT2B enzymes in cynomolgus macaques. Therefore, cynomolgus UGT2s had overall molecular similarities to human UGT2s, but also showed some differences in UGT2B enzyme properties.
  • Wilson Mwandira, Kazunori Nakashima, Satoru Kawasaki, Mayumi Ito, Tsutomu Sato, Toshifumi Igarashi, Kawawa Banda, Meki Chirwa, Imasiku Nyambe, Shouta Nakayama, Mayumi Ishizuka
    Environmental science and pollution research international 26 (15) 15653 - 15664 0944-1344 2019/05 [Refereed][Not invited]
    Biocementation of hazardous waste is used in reducing the mobility of contaminants, but studies on evaluating its efficacy have not been well documented. Therefore, to evaluate the efficacy of this method, physicochemical factors affecting stabilized hazardous products of in situ microbially induced calcium carbonate precipitation (MICP) were determined. The strength and leach resistance were investigated using the bacterium Pararhodobacter sp. Pb-contaminated kiln slag (KS) and leach plant residue (LPR) collected from Kabwe, Zambia, were investigated. Biocemented KS and KS/LPR had leachate Pb concentrations below the detection limit of < 0.001 mg/L, resisted slaking, and had maximum unconfined compressive strengths of 8 MPa for KS and 4 MPa for KS/LPR. Furthermore, biocemented KS and KS/LPR exhibited lower water absorption coefficient values, which could potentially reduce the water transportation of Pb2+. The results of this study show that MICP can reduce Pb2+ mobility in mine wastes. The improved physicochemical properties of the biocemented materials, therefore, indicates that this technique is an effective tool in stabilizing hazardous mine wastes and, consequently, preventing water and soil contamination.
  • Yusuke K Kawai, So Shinya, Yoshinori Ikenaka, Aksorn Saengtienchai, Takamitsu Kondo, Wageh Sobhy Darwish, Shota M M Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 217 5 - 14 1532-0456 2019/03 [Refereed][Not invited]
    Birds are exposed to many xenobiotics during their lifetime. For accurate prediction of xenobiotic-induced toxic effects on avian species, it is necessary to understand metabolic capacities in a comprehensive range of bird species. However, there is a lack of information about avian xenobiotic metabolizing enzymes (XMEs), particularly in wild birds. Uridine diphosphate glucuronosyltransferase (UGT) is an XME that plays an important role in phase II metabolism in the livers of mammals and birds. This study was performed to determine the characteristics of UGT1E isoform in avian species, those are related to mammals UGT 1A. To understand the characteristics of avian UGT1E isoforms, in vitro metabolic activity and genetic characteristics were investigated. Furthermore, mRNA expression levels of all chicken UGT1E isoforms were measured. On in vitro enzymatic analysis, the white-tailed eagle, great horned owl, and Humboldt penguin showed lower UGT-dependent activity than domestic birds. In synteny analysis, carnivorous birds were shown to have fewer UGT1E isoforms than herbivorous and omnivorous birds, which may explain why they have lower in vitro UGT activity. These observations suggested that raptors and seabirds, in which UGT activity is low, may be at high risk if exposed to elevated levels of xenobiotics in the environment. Phylogenetic analysis suggested that avian UGT1Es have evolved independently from mammalian UGT1As. We identified the important UGT isoforms, such as UGT1E13, and suspected their substrate specificities in avian xenobiotic metabolism by phylogenetic and quantitative real-time PCR analysis. This is the first report regarding the genetic characteristics and interspecies differences of UGT1Es in avian species.
  • Shouta M M Nakayama, Ayuko Morita, Yoshinori Ikenaka, Hazuki Mizukawa, Mayumi Ishizuka
    The Journal of veterinary medical science 81 (2) 298 - 313 0916-7250 2019/02/28 [Refereed][Not invited]
    Worldwide use of anticoagulant rodenticides (ARs) for rodents control has frequently led to secondary poisoning of non-target animals, especially raptors. In spite of the occurrence of many incidents of primary or secondary AR-exposure and poisoning of non-target animals, these incidents have been reported only for individual countries, and there has been no comprehensive worldwide study or review. Furthermore, the AR exposure pathway in raptors has not yet been clearly identified. The aim of this review is therefore to comprehensively analyze the global incidence of primary and secondary AR-exposure in non-target animals, and to explore the exposure pathways. We reviewed the published literature, which reported AR residues in the non-target animals between 1998 and 2015, indicated that various raptor species had over 60% AR- detection rate and have a risk of AR poisoning. According to several papers studied on diets of raptor species, although rodents are the most common diets of raptors, some raptor species prey mainly on non-rodents. Therefore, preying on targeted rodents does not necessarily explain all causes of secondary AR-exposure of raptors. Since AR residue-detection was also reported in non-target mammals, birds, reptiles and invertebrates, which are the dominant prey of some raptors, AR residues in these animals, as well as in target rodents, could be the exposure source of ARs to raptors.
  • Yoshinori Ikenaka, Yuichi Miyabara, Takahiro Ichise, Shouta Nakayama, Collins Nimako, Mayumi Ishizuka, Chiharu Tohyama
    Environmental toxicology and chemistry 38 (1) 71 - 79 0730-7268 2019/01 [Refereed][Not invited]
    Neonicotinoid insecticides that have been on the market since 1992 have been used globally including in Japan. Because they are sprayed over forests and agricultural areas, inadvertent toxicity in nontarget insects (especially honey bees) and humans is a matter of public concern. However, information on exposure levels and potential health impacts of neonicotinoids in children living around sprayed areas is scarce. Thus, we determined neonicotinoid exposure levels in children living in communities where thiacloprid was used to control pine wilt disease. A total of 46 children (23 males and 23 females) were recruited for the present study, and informed written consent was obtained from their guardians. Urine specimens were collected before, during, and after insecticide spraying events; and atmospheric particulate matter was also collected. Concentrations of thiacloprid and 6 other neonicotinoid compounds were determined in urine samples and in atmospheric particulate matter specimens using liquid chromatography-electrospray ionization-tandem mass spectrometry. In urine specimens, thiacloprid concentrations were <0.13 μg/L and were detectable in approximately 30% of all samples. Concentrations of the other neonicotinoids, N-dm-acetamiprid, thiamethoxam, dinotefuran, and clothianidin, were 18.7, 1.92, 72.3, and 6.02 µg/L, respectively. Estimated daily intakes of these neonicotinoids were then calculated from urinary levels; although the estimated daily intakes of the neonicotinoids were lower than current acceptable daily intake values, the children were found to be exposed to multiple neonicotinoids on a daily basis. Environ Toxicol Chem 2019;38:71-79. © 2018 SETAC.
  • Go Ichikawa, Ryota Kuribayashi, Yoshinori Ikenaka, Takahiro Ichise, Shouta M M Nakayama, Mayumi Ishizuka, Kumiko Taira, Kazutoshi Fujioka, Toshimi Sairenchi, Gen Kobashi, Jean-Marc Bonmatin, Shigemi Yoshihara
    PloS one 14 (7) e0219208  2019 [Refereed][Not invited]
    OBJECTIVES: Neonicotinoid insecticides are widely used systemic pesticides with nicotinic acetylcholine receptor agonist activity that are a concern as environmental pollutants. Neonicotinoids in humans and the environment have been widely reported, but few studies have examined their presence in fetuses and newborns. The objective of this study is to determine exposure to neonicotinoids and metabolites in very low birth weight (VLBW) infants. METHODS: An analytical method for seven neonicotinoids and one neonicotinoid metabolite, N-desmethylacetamiprid (DMAP), in human urine using LC-ESI/MS/MS was developed. This method was used for analysis of 57 urine samples collected within 48 hours after birth from VLBW infants of gestational age 23-34 weeks (male/female = 36/21, small for gestational age (SGA)/appropriate gestational age (AGA) = 6/51) who were admitted to the neonatal intensive care unit of Dokkyo Hospital from January 2009 to December 2010. Sixty-five samples collected on postnatal day 14 (M/F = 37/22, SGA/AGA = 7/52) were also analyzed. RESULTS: DMAP, a metabolite of acetamiprid, was detected in 14 urine samples collected at birth (24.6%, median level 0.048 ppb) and in 7 samples collected on postnatal day 14 (11.9%, median level 0.09 ppb). The urinary DMAP detection rate and level were higher in SGA than in AGA infants (both p<0.05). There were no correlations between the DMAP level and infant physique indexes (length, height, and head circumference SD scores). CONCLUSION: These results provide the first evidence worldwide of neonicotinoid exposure in newborn babies in the early phase after birth. The findings suggest a need to examine potential neurodevelopmental toxicity of neonicotinoids and metabolites in human fetuses.
  • Aksorn Saengtienchai, Yoshinori Ikenaka, Minami Kawata, Yusuke Kawai, Kazuki Takeda, Takamitsu Kondo, Nesta Bortey-Sam, Shouta M M Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    Comparative biochemistry and physiology. Toxicology & pharmacology : CBP 214 28 - 35 1532-0456 2018/12 [Refereed][Not invited]
    There have been many reports regarding toxic chemicals in birds. Chemicals are mainly metabolized in the liver through phase I oxidation by cytochrome P450 (CYP) and phase II conjugation by conjugated enzymes, such as UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), glutathione-S-transferase (GST), etc. Xenobiotic metabolism differs among bird species, but little detailed information is available. In the present study, the four-ring polycyclic aromatic hydrocarbon (PAH), pyrene, was used as a model xenobiotic to clarify the characteristics of xenobiotic metabolism in birds compared with laboratory animals by in vivo and in vitro studies. Plasma, bile, and excreta (urine and feces) were collected after oral administration of pyrene and analyzed to clarify xenobiotic metabolism ability in chickens and quails. Interestingly, pyrenediol-glucuronide sulfate (PYDOGS) and pyrenediol-diglucuronide (PYDOGG) were present in chickens and quails but not in rats. In addition, the area under the curve (AUC), maximum plasma concentration (Cmax), and time to maximum plasma concentration (Tmax) of pyrene-1-sulfate (PYOS) were higher than those of the parent molecule, pyrene, while the elimination half-life (t1/2) and mean residence time (MRT) were faster than those of the parent pyrene. With regard to sulfation of 1-hydroxypyrene (PYOH), the maximum velocity (Vmax) and Michaelis constant (Km) of rat liver cytosol were greater than those of chicken and quail liver cytosol. Furthermore, Vmax/Km of UGT activity in rat liver microsomes was also greater than those of chicken and quail liver microsomes. Characterization of xenobiotic metabolism revealed species differences between birds and mammals, raising concerns about exposure to various xenobiotics in the environment.
  • Chihiro Ishii, Shouta M M Nakayama, Andrew Kataba, Yoshinori Ikenaka, Keisuke Saito, Yukiko Watanabe, Yoshiki Makino, Takehisa Matsukawa, Ayano Kubota, Kazuhito Yokoyama, Hazuki Mizukawa, Takafumi Hirata, Mayumi Ishizuka
    Chemosphere 212 994 - 1001 0045-6535 2018/12 [Refereed][Not invited]
    Lead (Pb) poisoning in raptors and water birds is a serious problem in many countries. However, only a small fraction of Pb poisoning has been detected in birds. Bone specimens may be useful indices of Pb exposure because bones contain ∼90% of the total Pb body burden. The original purpose of this study was to comprehensively analyze Pb accumulation in various bone types using inductively coupled plasma-mass spectrometry (ICP-MS). Since our results showed that Pb accumulation differed greatly depending on bone type, a secondary objective was defined, aiming to investigate the fine Pb distribution and its relation to bone structure and bone marrow by using laser ablation (LA)-ICP-MS. Our findings suggested that bone samples (1) consisting of trabecular tissue and (2) those that contain bone marrow could accumulate high levels of Pb following acute exposure. The shorter turnover time of trabecular bone can cause a rapid accumulation of Pb, and bone marrow may have an important role for internal exposure of Pb to bone tissue. Pb is accumulated in bones via blood flow, and bone marrow receives blood from outside the bones. In conclusion, bone samples provide valuable information on Pb exposure and could be useful to investigate and understand mortalities related to suspected Pb poisoning.
  • Nico J Wolmarans, Louis H Du Preez, Yared Beyene Yohannes, Yoshinori Ikenaka, Mayumi Ishizuka, Nico J Smit, Victor Wepener
    Ecotoxicology (London, England) 27 (9) 1203 - 1216 0963-9292 2018/11 [Refereed][Not invited]
    Organochlorine pesticides are highly persistent in aquatic ecosystems. Amphibians, specifically anurans, play an intricate part in the aquatic food web, and have very permeable skin which makes them prone to bioaccumulation of persistent pollutants. In this study the bioaccumulation of various legacy organochlorine pesticides (OCPs)-including dichlorodiphenyltrichloroethane (DDT), currently used for malaria vector control (MVC)-was assessed along with a set of biomarker responses in Müller's clawed frog Xenopus muelleri collected from the lower Phongolo River floodplain in South Africa. Possible relationships between bioaccumulation and biomarkers (of exposure, oxidative stress biomarkers, and cellular energy allocation) alongside their temporal changes were investigated. The OCP concentrations showed a significant increase over time for the duration of the study. The increase correlated negatively with rainfall from the region. DDT levels were well below expected effects levels with p,p-DDE being the main contributing metabolite. The results of this study indicate OCPs actively accumulate at sub-lethal levels in aquatic frogs from the study area, while showing possible relations towards some of the biochemical stress responses measured. Most notable were negative relationships indicated between p,p-DDE and acetylcholinesterase, malondialdehyde, and carbohydrates and protein energy availability. Levels of DDT were not found to be significantly higher than other legacy pesticides in the frog tissue, although evidence of newly introduced DDT in the frog tissue was found. Further investigation about sub-lethal effects of these pesticides on anurans is required to gain better insight into their full impact on animal livelihood.
  • Yasuhiro Uno, Rika Takahira, Norie Murayama, Yu Ishii, Yoshinori Ikenaka, Mayumi Ishizuka, Hiroshi Yamazaki, Shinichi Ikushiro
    Biochemical pharmacology 155 172 - 181 0006-2952 2018/09 [Refereed][Not invited]
    UDP-glucuronosyltransferases (UGTs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics. Molecular characteristics of UGTs have been extensively investigated in humans, but in cynomolgus macaques, a non-human primate species widely used in drug metabolism studies, remain to be investigated. In this study, 12 UGT1A cDNAs (UGT1A1, 1A2, 1A4A, 1A4B, 1A5A, 1A5B, 1A5C, 1A6, 1A7, 1A8, 1A9, and 1A10) were isolated and characterized in cynomolgus macaques. UGT1A5C cDNA did not contain a complete coding region due to nonsense mutations, and was excluded from further analysis. Amino acid sequences of all 11 cynomolgus UGT1As had high sequence identities (92-95%) with human UGT1As and were phylogenetically close to human UGT1As. These cynomolgus UGT1A genes shared exons 2-5, and contained a variable exon 1 unique to each gene, similar to human UGT1A genes. Moreover, cynomolgus and human UGT1A gene clusters were located in corresponding regions in the genome. Among the 10 tissue types analyzed, cynomolgus UGT1A mRNAs were most abundantly expressed in the liver, jejunum, and/or kidney, the drug-metabolizing organs, similar to human UGT1As. Among these 11 cynomolgus UGT1A mRNAs, cynomolgus UGT1A2, UGT1A9, and UGT1A10 mRNAs were most abundantly expressed in the liver, kidney, and jejunum, respectively. Cynomolgus liver microsomes and UGT1A proteins catalyzed glucuronidation of the substrates human UGT1As catalyze, including 4-methylumbelliferone, 4-nitrophenol, estradiol, trifluoperazine, serotonin, and propofol, although trifluoperazine glucuronidation was not catalyzed by any cynomolgus UGT1A proteins. These results suggest that cynomolgus UGT1As are functional enzymes with molecular similarities to human UGT1As.
  • Osei Akoto, Nesta Bortey-Sam, Shouta M M Nakayama, Yoshinori Ikenaka, Elvis Baidoo, Joseph Apau, Jemima Tiwaa Marfo, Mayumi Ishizuka
    Journal of health & pollution 8 (19) 180902 - 180902 2018/09 [Refereed][Not invited]
    Background: Soil contamination with heavy metals and metalloids has become an increasingly important issue in recent years. Objectives: The present study examines possible contamination of the environment with metals from gold mining activities in Obuasi, Ghana. Methods: Soil samples were collected from commercial and residential areas and tailing dams in Obuasi in order to investigate the extent of lead (Pb), cadmium (Cd), mercury (Hg), copper (Cu), zinc (Zn), cobalt (Co), chromium (Cr), nickel (Ni) and arsenic (As) pollution, create thematic maps showing the extent of heavy metals pollution, identify the sources of pollution, and to assess risks to humans and the surrounding ecological system. Results: Mean concentrations of metals from the study were found in the order of As > Cu > Zn > Cr > Ni > Pb > Co > Hg > Cd. The results showed that all communities were severely polluted with As, and distribution maps highlighted two hot spots at the sulfite treatment plant and Pompura treatment plant tailings dams. Additionally, the levels of Pb, Cu and Zn were elevated around the city center where vehicular traffic is very dense. Principal component analysis indicated that mining activities may have significantly contributed to metal levels in Obuasi soils. The potential ecological risk (RI) indicated that soils in 41% of the communities pose very high risks to the surrounding ecological system, 50% pose considerable risk, and 9% pose a moderate risk. Arsenic and Hg contributed 73 and 15% of the RI, respectively. The average hazard quotient due to soil As exposure was 2.51 ± 1.23 and ingestion of soils in 95% of the communities in the study area could pose non-carcinogenic health risks to children. Moreover, the average cancer risk for children from the communities was 1.13 × 10-3. Based on the United States Environmental Protection Agency (USEPA) recommendation for cancer risk of 10-6 to 10-4, the cancer risk for children (> 10-3) was higher in 45% of the studied communities. Conclusions: The central part of the study area is polluted with Pb, Zn and Cu, and As pollution is severe in all of the studied communities. The RI from all study sites revealed very high risk to the ecological system, including mammals. There could be non-cancer and cancer risks to Obuasi residents due to ingestion of As-contaminated soils, and children are particularly vulnerable. Competing Interests: The author declares no competing financial interests.
  • Wihan Pheiffer, Nico J Wolmarans, Ruan Gerber, Yared B Yohannes, Yoshinori Ikenaka, Mayumi Ishizuka, Nico J Smit, Victor Wepener, Rialet Pieters
    The Science of the total environment 628-629 517 - 527 0048-9697 2018/07/01 [Refereed][Not invited]
    Organochlorine pesticides (OCPs) in South Africa have for the most part been banned, except dichlorodiphenyltrichloroethane (DDT) which is still used as malaria vector control. The aim of this study was to determine OCP residues in the aquatic fauna of one of South Africa's most populated areas, Soweto. Risk to human health through OCP exposure via fish consumption was investigated. Clarias gariepinus was chosen as bioindicator because it is an apex predator that is in abundance, but is also a valued food source. Dichlorodiphenyltrichloroethanes (DDTs), hexachlorocyclohexanes (HCHs), and chlordanes (CHLs) were detected in the fish tissue with the DDTs being the most prevalent at all sites. Of the three locations, Fleurhof, Orlando, and Lenasia, the latter location's fish had the highest ΣOCP load, ranging between 81 and 1190ng/gwm. The DDTs were determined to be from historic use, whereas the CHL levels indicated more recent inputs. Although the possibility of illegal use cannot be excluded completely, the presence of OCPs outside of their allowed areas of use indicate that these compounds not only stay in the aquatic systems long term, but may be of concern in areas previously not considered high risk areas. The OCP residues in C. gariepinus from the study area pose an extremely high risk to human health when consumed, and has a cancer risk as high as 1 in 10. This potential problem should be kept in consideration when developing national health and conservation strategies.
  • John Yabe, Shouta M M Nakayama, Yoshinori Ikenaka, Yared B Yohannes, Nesta Bortey-Sam, Abel Nketani Kabalo, John Ntapisha, Hazuki Mizukawa, Takashi Umemura, Mayumi Ishizuka
    Chemosphere 202 48 - 55 0045-6535 2018/07 [Refereed][Not invited]
    Lead (Pb) and cadmium (Cd) are toxic metals that exist ubiquitously in the environment. Children in polluted areas are particularly vulnerable to metal exposure, where clinical signs and symptoms could be nonspecific. Absorbed metals are excreted primarily in urine and reflect exposure from all sources. We analyzed Pb and Cd concentrations in blood, feces and urine of children from polluted townships near a lead-zinc mine in Kabwe, Zambia, to determine concurrent childhood exposure to the metals. Moreover, the study determined the Pb and Cd relationships among urine, feces and blood as well as accessed the potential of urine and fecal analysis for biomonitoring of Pb and Cd exposure in children. Fecal Pb (up to 2252 mg/kg, dry weight) and urine Pb (up to 2914 μg/L) were extremely high. Concentrations of Cd in blood (Cd-B) of up to 7.7 μg/L, fecal (up to 4.49 mg/kg, dry weight) and urine (up to 18.1 μg/L) samples were elevated. metal levels were higher in younger children (0-3 years old) than older children (4-7). Positive correlations were recorded for Pb and Cd among blood, urine and fecal samples whereas negative correlations were recorded with age. These findings indicate children are exposed to both metals at their current home environment. Moreover, urine and feces could be useful for biomonitoring of metals due to their strong relationships with blood levels. There is need to conduct a clinical evaluation of the affected children to fully appreciate the health impact of these metal exposure.
  • Miura R, Araki A, Miyashita C, Kobayashi S, Kobayashi S, Wang SL, Chen CH, Miyake K, Ishizuka M, Iwasaki Y, Ito YM, Kubota T, Kishi R
    Environment international 115 21 - 28 0160-4120 2018/06 [Refereed][Not invited]
  • Kumiko Yamamoto, Yoshinori Ikenaka, Takahiro Ichise, Tomoki Bo, Mayumi Ishizuka, Hironobu Yasui, Wakako Hiraoka, Tohru Yamamori, Osamu Inanami
    Free radical research 52 (6) 648 - 660 1071-5762 2018/06 [Refereed][Not invited]
    To evaluate the metabolic responses in tumour cells exposed to ionizing radiation, oxygen consumption rate (OCR), cellular lipid peroxidation, cellular energy status (intracellular nucleotide pool and ATP production), and mitochondrial reactive oxygen species (ROS), semiquinone (SQ), and iron-sulphur (Fe-S) cluster levels were evaluated in human cervical carcinoma HeLa cells at 12 and 24 h after X-irradiation. LC/MS/MS analysis showed that levels of 8-iso PGF2α and 5-iPF2α-VI, lipid peroxidation products of membrane arachidonic acids, were not altered significantly in X-irradiated cells, although mitochondrial ROS levels and OCR significantly increased in the cells at 24 h after irradiation. LC/UV analysis revealed that intracellular AMP, ADP, and ATP levels increased significantly after X-irradiation, but adenylate energy charge (adenylate energy charge (AEC) = [ATP + 0.5 × ADP]/[ATP + ADP + AMP]) remained unchanged after X-irradiation. In low-temperature electron spin resonance (ESR) spectra of HeLa cells, the presence of mitochondrial SQ at g = 2.004 and Fe-S cluster at g = 1.941 was observed and X-irradiation enhanced the signal intensity of SQ but not of the Fe-S cluster. Furthermore, this radiation-induced increase in SQ signal intensity disappeared on treatment with rotenone, which inhibits electron transfer from Fe-S cluster to SQ in complex I. From these results, it was suggested that an increase in OCR and imbalance in SQ and Fe-S cluster levels, which play a critical role in the mitochondrial electron transport chain (ETC), occur after X-irradiation, resulting in an increase in ATP production and ROS leakage from the activated mitochondrial ETC.
  • Kazuki Takeda, Yoshinori Ikenaka, Kazuyuki D Tanaka, Shouta M M Nakayama, Tsutomu Tanikawa, Hazuki Mizukawa, Mayumi Ishizuka
    Pesticide biochemistry and physiology 148 42 - 49 0048-3575 2018/06 [Refereed][Not invited]
    Anti-blood coagulation rodenticides, such as warfarin, have been used all over the world. They inhibit vitamin K epoxide reductase (VKOR), which is necessary for producing several blood clotting factors. This inhibition by rodenticides results in lethal hemorrhage in rodents. However, heavy usage of these agents has led to the appearance of rodenticide-resistant rats. There are two major mechanisms underlying this resistance, i.e., mutation of the target enzyme of warfarin, VKOR, and enhanced metabolism of warfarin. However, there have been few studies regarding the hepatic metabolism of warfarin, which should be related to resistance. To investigate warfarin metabolism in resistant rats, in situ liver perfusion of warfarin was performed with resistant black rats (Rattus rattus) from Tokyo, Japan. Liver perfusion is an in situ methodology that can reveal hepatic function specifically with natural composition of the liver. The results indicated enhanced hepatic warfarin hydroxylation activity compared with sensitive black rats. On the other hand, in an in vitro microsomal warfarin metabolism assay to investigate kinetic parameters of cytochrome P450, which plays a major role in warfarin hydroxylation, the Vmax of resistant rats was slightly but significantly higher compared to the results obtained in the in situ study. These results indicated that another factor like electron donators may also contribute to the enhanced metabolism in addition to high expression of cytochrome P450.
  • Emmanuel Temiotan Ogbomida, Shouta M M Nakayama, Nesta Bortey-Sam, Balazs Oroszlany, Isioma Tongo, Alex Ajeh Enuneku, Ogbeide Ozekeke, Martins Oshioriamhe Ainerua, Iriagbonse Priscillia Fasipe, Lawrence Ikechukwu Ezemonye, Hazuki Mizukawa, Yoshinori Ikenaka, Mayumi Ishizuka
    Ecotoxicology and environmental safety 151 98 - 108 0147-6513 2018/04/30 [Refereed][Not invited]
    The use of free range animals for monitoring environmental health offers opportunities to detect exposure and assess the toxicological effects of pollutants in terrestrial ecosystems. Potential human health risk of dietary intake of metals and metalloid via consumption of offal and muscle of free range chicken, cattle and goats by the urban population in Benin City was evaluated. Muscle, gizzard, liver and kidney samples were analyzed for Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Cd, and Pb concentrations using inductively coupled plasma mass spectrometer (ICP-MS) while Hg was determined using Hg analyzer. Mean concentrations of metals (mg/kg ww) varied significantly depending upon the tissues and animal species. Human health risk estimations for children and adults showed estimated daily intake (EDI) values of tissues below oral reference dose (RfD) threshold for non essential metals Cd, As, Pb and Hg thus strongly indicating no possible health risk via consumption of animal based food. Calculated Hazard quotient (THQ) was less than 1 (< 1) for all the metals analyzed for both adult and children. However, Cd and As had the highest value of THQ suggestive of possible health risk associated with continuous consumption of Cd and As contaminated animal based foods. Hazard Index (HI) for additive effect of metals was higher in chicken liver and gizzard for children and chicken liver for adults. Thus, HI indicated that chicken liver and gizzard may contribute significantly to adult and children dietary exposure to heavy metals. Principal component analysis (PCA) showed a clear species difference in metal accumulation between chickens and the ruminants. This study provides baseline data for future studies and also valuable evidence of anthropogenic impacts necessary to initiate national and international policies for control of heavy metal and metalloid content in food items.
  • Nesta Bortey-Sam, Yoshinori Ikenaka, Osei Akoto, Shouta M M Nakayama, Kwadwo A Asante, Elvis Baidoo, Christian Obirikorang, Hazuki Mizukawa, Mayumi Ishizuka
    Environmental pollution (Barking, Essex : 1987) 235 163 - 170 0269-7491 2018/04 [Refereed][Not invited]
    Heavy metals and metalloids contamination in soils, water, food and livers of wild rats have been studied in Kumasi, Ghana and despite the estimated risks to residents, there is no epidemiological study to ascertain these projections. In addition, the World Health Organization and International Agency for Research on Cancer have reported an increase in respiratory diseases and cancers, in Ghana. The study's purpose was therefore to explore the potential associations between metal exposure and occurrences of respiratory diseases, lipid peroxidation and/or DNA damage to different age groups and sexes in Kumasi. Human urine was collected from the general population in urban and control sites in Kumasi and nine metals were measured in each sample. Results showed that although Zn was the most abundant total urinary As concentration was higher in 83% of samples compared to reference values. Urinary concentrations of metals, malondialdehyde (MDA) and 8-hydroxy-2-deoxy-guanosine (8-OHdG) were higher in urban sites compared to the control site. Based on the results obtained, there was no significant correlation between urinary metals and age. However, urinary Cd and MDA were highest in age groups 61-85 and 3-20 years, respectively. Significantly higher levels of urinary Co, As and Cd were detected in female participants. The study revealed that exposure to As was significantly associated with increased odds of asthma (odds ratio (OR) = 2.76; CI: 1.11-6.83) and tachycardia (OR = 3.93; CI: 1.01-15.4). Significant association was observed between urinary metals and MDA and 8-OHdG indicating possibility of lipid peroxidation and/or DNA damage in Kumasi residents.
  • Nesta Bortey-Sam, Yoshinori Ikenaka, Osei Akoto, Shouta M M Nakayama, Jemima T Marfo, Aksorn Saengtienchai, Hazuki Mizukawa, Mayumi Ishizuka
    The Journal of veterinary medical science 80 (2) 375 - 381 0916-7250 2018/03/02 [Refereed][Not invited]
    Industrialization, economic and population growth rates in Ghana have increased the release of contaminants including polycyclic aromatic hydrocarbons (PAHs) into the environment through which humans and animals are exposed. Cattle is reported to be exposed to high levels of PAHs through feed and inhalation. Once exposed, PAHs are metabolized and excreted in urine, feces or bile. In a previous study, cattle in Ghana was reported to excrete high levels of 1-hydroxypyrene (1-OHPyr) due to high exposure to the parent compound, pyrene. 1-OHPyr is further metabolized to glucuronide and sulfate conjugates. Thus, the aim of this study was to investigate the sex and site differences in urinary excretion of conjugated pyrene metabolites using cattle urine collected from rural and urban sites of the Ashanti region, Ghana. From the results, geometric mean concentration adjusted by specific gravity indicated that 1-OHPyreneGlucuronide (PyG) was the most abundant conjugate followed by PyrenediolSulfate (M3). The sum of conjugated pyrene metabolites and sum of both conjugated and deconjugated pyrene metabolites correlated significantly with PyG, PydiolSulfate (M2) and PydiolSulfate (M3). The study revealed no significant difference in urinary excretion of conjugated pyrene metabolites between rural and urban sites. This indicated that similar to urban sites, cattle in rural sites were exposed to high levels of pyrene. There was no significant difference in urinary concentrations of conjugated pyrene metabolites between sexes.
  • Wageh Sobhy Darwish, Yoshinori Ikenaka, Shouta Nakayama, Hazuki Mizukawa, Lesa A Thompson, Mayumi Ishizuka
    Environmental science and pollution research international 25 (7) 6320 - 6328 0944-1344 2018/03 [Refereed][Not invited]
    Benzo[a]pyrene (B[a]P) is one of the polycyclic aromatic hydrocarbons which is formed due to smoking of foods, incomplete combustion of woods, vehicle exhausts, and cigarettes smokes. B[a]P gets entry into human and animal bodies mainly through their diets. Metabolic activation of B[a]P is required to induce mutagenesis and carcinogenesis in animal and human studies. Carotenoids and retinoids are phytochemicals that if ingested have multiple physiological interferences in the human and animal bodies. In this study, we firstly investigated the protective effects of β-carotene, β-apo-8-carotenal, retinol, and retinoic acid against B[a]P-induced mutagenicity and oxidative stress in human HepG2 cells. Secondly, we tested the hypothesis of modulating xenobiotic metabolizing enzymes (XMEs) by carotenoids and retinoids as a possible mechanism of protection by these micronutrients against B[a]P adverse effects. The obtained results declared that β-carotene and retinol significantly reduced B[a]P-induced mutagenicity and oxidative stress. Tested carotenoids and retinoids reduced B[a]P-induced phase I XMEs and induced B[a]P reduced phase II and III XMEs. Thus, the protective effects of these micronutrients are probably due to their ability of induction of phase II and III enzymes and interference with the induction of phase I enzymes by the promutagen, B[a]P. It is highly recommended to consume foods rich in these micronutrients in the areas of high PAH pollution.
  • Yoshinori Ikenaka, Kazutoshi Fujioka, Tomonori Kawakami, Takahiro Ichise, Nesta Bortey-Sam, Shouta M M Nakayama, Hazuki Mizukawa, Kumiko Taira, Keisuke Takahashi, Keisuke Kato, Koji Arizono, Mayumi Ishizuka
    Toxicology reports 5 744 - 749 2018 [Refereed][Not invited]
    Tea is one of the world's most popular beverages due to health promoting effects. Despite these, there have been concerns about the adverse effects of tea contamination by neonicotinoid insecticides. Only a handful of studies on neonicotinoid insecticides in tea have been carried out and this study was therefore performed to determine the concentrations of seven neonicotinoid insecticides and 20 metabolites in Japanese green tea leaves, and black tea leaves from Sri Lanka; and assess the Maximum Daily Intake (MDI) of neonicotinoid insecticides. From the results, the seven parent compounds were detected in Japanese tea leaves and beverages. Dinotefuran (3004 ng/g) was found at the highest level in green tea leaves. Ten of the 20 metabolites were detected in Japanese tea products. Dinotefuran-urea (92%) and thiacloprid-amide (89%) were most frequently detected in Japanese tea leaves. Clothianidin-urea (100 ng/g) was found at the highest level in green tea leaves. Neonicotinoid insecticides and metabolites were not detected in Sri Lankan black tea leaves. The concentrations and MDI of neonicotinoid insecticides in tea leaves were below the Maximum Residual Levels (MRLs) and Acceptable Daily Intakes (ADIs), respectively.
  • Lesa A Thompson, Yoshinori Ikenaka, Wageh S Darwish, Yared B Yohannes, Johan J van Vuren, Victor Wepener, Nico J Smit, Atnafu G Assefa, Ahmed Tharwat, Walaa Fathy Saad Eldin, Shouta M M Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    PloS one 13 (10) e0204400  2018 [Refereed][Not invited]
    The objective of this study was to identify potential mRNA expression changes in chicken livers associated with environmental exposure to dichloro-diphenyl-trichloroethane (DDT) and its metabolites (DDTs). In particular, we focused on genes relating to the immune system and metabolism. We analyzed liver samples from free-ranging chickens in KwaZulu-Natal, South Africa, for contamination by DDTs. This area predominantly uses DDT in its malaria control program, and homes are sprayed annually with the pesticide. Genes relating to the immune system and metabolism were selected as potential genetic biomarkers that could be linked to higher contamination with DDTs. RT-qPCR analysis on 39 samples showed strong correlations between DDTs contamination and mRNA expression for the following genes: AvBD1, AvBD2, AvBD6 and AvBD7 (down-regulated), and CYP17, ELOVL2 and SQLE (up-regulated). This study shows for the first time interesting and significant correlations between genetic material collected from environmentally-exposed chickens and mRNA expression of several genes involved in immunity and metabolism. These findings show the usefulness of analysis on field samples from a region with high levels of environmental contamination in detecting potential biomarkers of exposure. In particular, we observed clear effects from DDT contamination on mRNA expression of genes involved in immune suppression, endocrine-disrupting effects, and lipid dysregulation. These results are of interest in guiding future studies to further elucidate the pathways involved in and clinical importance of toxicity associated with DDT exposure from contaminated environments, to ascertain the health risk to livestock and any subsequent risks to food security for people.
  • Yusuke K Kawai, Yoshinori Ikenaka, Mayumi Ishizuka, Akira Kubota
    PloS one 13 (10) e0205266  2018 [Refereed][Not invited]
    UDP-glycosyltransferase (UGT) catalyzes the transfer of glycosyl groups (e.g., glucuronic acid) to exogenous or endogenous chemicals and plays an important role in conjugation reactions. In vertebrates, UGT genes are divided into 5 families: UGT1, UGT2, UGT3, UGT5, and UGT8. Among these UGT enzymes, UGT1 and UGT2 enzymes are known to be important xenobiotic metabolizing enzymes in mammals. However, little is known about UGT1 and UGT2 genes in avian species. In this study, we therefore aimed to classify avian UGT1 and UGT2 genes based on their evolutionary relationships. We also investigated the association between UGT molecular evolution and ecological factors, specifically feeding habits, habitat, and migration. By examining the genomes of 43 avian species with differing ecology, we showed that avian UGT1E genes are divided into 6 groups and UGT2 genes into 3 groups. Correlations between UGT gene count and ecological factors suggested that the number of UGT1E genes is decreasing in carnivorous species. Estimates of selection pressure also support the hypothesis that diet influenced avian UGT1E gene evolution, similar to mammalian UGT1A and UGT2B genes.
  • Chihiro Ishii, Shouta M. M. Nakayama, Yoshinori Ikenaka, Hokuto Nakata, Keisuke Saito, Yukiko Watanabe, Hazuki Mizukawa, Shinsuke Tanabe, Kei Nomiyama, Terutake Hayashi, Mayumi Ishizuka
    CHEMOSPHERE 186 367 - 373 0045-6535 2017/11 [Refereed][Not invited]
    Lead (Pb) poisoning is widespread among raptors and water birds. In Japan, fragments of Pb ammunition are still found in endangered eagles although more than 10 years have passed since legislation regarding use of Pb ammunition was introduced. This study was performed to investigate Pb exposure in raptors from various locations in Japan. We measured hepatic and renal Pb concentrations and hepatic Pb isotope ratios of Steller's sea eagles (Haliaeetus pelagicus), white-tailed sea eagles (Haliaeetus albicilla), golden eagles (Aquila chrysaetos), and 13 other species (total 177 individuals) that were found dead, as well as blood samples from three eagles found in a weakened state during 1993-2015 from Hokkaido (northern part), Honshu (the main island), and Shikoku (a southern island) of japan. In the present study in Hokkaido, one quarter of the sea eagles showed a high Pb concentration, suggesting exposure to abnormally high Pb levels and Pb poisoning. Pb isotope ratios indicated that endangered Steller's sea eagle and white-tailed sea eagle were poisoned by Pb ammunition that was used illegally in Hokkaido. In other areas of Japan, both surveillance and regulations were less extensive than in Hokkaido, but Pb poisoning in raptors was also noted. Therefore, Pb poisoning is still a serious problem in raptors in various areas of Japan due to accidental ingestion of materials containing Pb, especially Pb ammunition. (C) 2017 Published by Elsevier Ltd.
  • Noboru Sasaki, Kazuhiro Ishi, Nobuki Kudo, Shouta M. M. Nakayama, Kensuke Nakamura, Keitaro Morishita, Hiroshi Ohta, Mayumi Ishizuka, Mitsuyoshi Takiguchi
    PLOS ONE 12 (11) e0188093  1932-6203 2017/11 [Refereed][Not invited]
    Non-muscle invasive bladder cancer is one of the most common tumors of the urinary tract. Despite the current multimodal therapy, recurrence and progression of disease have been challenging problems. We hereby introduced a new approach, ultrasound-assisted intravesical chemotherapy, intravesical instillation of chemotherapeutic agents and microbubbles followed by ultrasound exposure. We investigated the feasibility of the treatment for nonmuscle invasive bladder cancer. In order to evaluate intracellular delivery and cytotoxic effect as a function to the thickness, we performed all experiments using a bladder cancer mimicking 3D culture model. Ultrasound-triggered microbubble cavitation increased both the intracellular platinum concentration and the cytotoxic effect of cisplatin at the thickness of 70 and 122 mu m of the culture model. The duration of enhanced cytotoxic effect of cisplatin by ultrasound-triggered microbubble cavitation was approximately 1 hr. Based on the distance and duration of delivery, we further tested the feasibility of repetition of the treatment. Triple treatment increased the effective distance by 1.6-fold. Our results clearly showed spatial and temporal profile of delivery by ultrasound-triggered microbubble cavitation in a tumor-mimicking structure. Furthermore, we demonstrated that the increase in intracellular concentration results in the enhancement of the cytotoxic effect in a structure with the certain thickness. Repetition of ultrasound exposure would be treatment of choice in future clinical application. Our results suggest ultrasound-triggered microbubble cavitation can be repeatable and is promising for the local control of non-muscle invasive bladder cancer.
  • Hazuki Mizukawa, Kei Nomiyama, Susumu Nakatsu, Miyuki Yamamoto, Mayumi Ishizuka, Yoshinori Ikenaka, Shouta M. M. Nakayama, Shinsuke Tanabe
    ENVIRONMENTAL SCIENCE & TECHNOLOGY 51 (19) 11354 - 11362 0013-936X 2017/10 [Refereed][Not invited]
    Present study determined concentrations and residue patterns of bromophenols (BPhs) in whole blood samples of pet cats and pet dogs collected from veterinary hospitals in Japan. BPhs concentrations were higher in cat blood than in dog blood, with statistically insignificant differences (p = 0.07). Among the congeners, 2,4,6-tribromophenol (TBPh) constituted the majority of BPhs (>90%) detected in both species. Analysis of commercial pet food to estimate exposure routes showed that the most abundant congener in all pet food samples was 2,4,6-TBPh, accounting for >99% of total BPhs. This profile is quite similar to the blood samples of the pets, suggesting that diet might be an important exposure route for BPhs in pets. After incubation in polybrominated diphenyl ether (PBDE) mixtures (BDE-47, BDE-99 and BDE-209), 2,4,5-TBPh was found in dog liver microsomes but not in cat liver microsomes, implying species-specific metabolic capacities for PBDEs. Formation of 2,4,5-TBPh occurred by hydroxylation at the 1' carbon atom of the ether bond of BDE-99 is similar to human study reported previously. Hydroxylated PBDEs were not detected in cats or dogs; therefore, diphenyl ether bond cleavage of PBDEs can also be an important metabolic pathway for BPhs formation in cats and dogs.
  • Yared Beyene Yohannes, Yoshinori Ikenaka, Gengo Ito, Shouta M. M. Nakayama, Hazuki Mizukawa, Victor Wepener, Nico J. Smit, Johan H. J. Van Vuren, Mayumi Ishizuka
    ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH 24 (30) 23763 - 23770 0944-1344 2017/10 [Refereed][Not invited]
    Ethiopia and South Africa are among the few countries to still implement indoor residual spraying with dichloro-diphenyl-trichloroethane (DDT) for malaria vector control. In this study, we investigated the levels and ecological risks of DDT and its metabolites in liver tissues of house rat, as a sentinel animal, for providing an early warning system for public health and wildlife intervention from Ethiopia and South Africa. The results showed that Sigma DDT concentration ranged from 127 to 9155 mu g/kg wet weight, and the distribution order of DDT and its metabolites in the analyzed liver samples was p,p'-DDD > p,p'-DDE >> p,p'-DDT, o,p'-DDT, and o,p'-DDD. The risk assessment indicated a potential adverse impact on humans, especially for pregnant women and children, because they spend majority of their time in a DDT-sprayed house. The ecological assessment also showed a concern for birds of prey and amphibians like frogs. This study is the first report on DDT contamination in liver tissues of house rats from Ethiopia and South Africa, and henceforth, the data will serve as a reference data for future studies.
  • Nesta Bortey-Sam, Yoshinori Ikenaka, Osei Akoto, Shouta M. M. Nakayama, Kwadwo A. Asante, Elvis Baidoo, Christian Obirikorang, Aksorn Saengtienchai, Norikazu Isoda, Collins Nimako, Hazuki Mizukawa, Mayumi Ishizuka
    ENVIRONMENTAL POLLUTION 228 311 - 320 0269-7491 2017/09 [Refereed][Not invited]
    Studies of polycyclic aromatic hydrocarbons (PAHs) and its metabolites in PM10, soils, rat livers and cattle urine in Kumasi, Ghana, revealed high concentrations and cancer potency. In addition, WHO and IARC have reported an increase in cancer incidence and respiratory diseases in Ghana. Human urine were therefore collected from urban and control sites to: assess the health effects associated with PAHs exposure using malondialdehyde (MDA) and 8-hydroxy-2-deoxyguanosine (8-OHdG); identify any association between OH-PAHs, MDA, 8-OHdG with age and sex; and determine the relationship between PAHs exposure and occurrence of respiratory diseases. From the results, urinary concentrations of the sum of OH-PAHs (Sigma OHPAHs) were significantly higher from urban sites compared to the control site. Geometric mean concentrations adjusted by specific gravity, GM(SG), indicated 2-OHNaphthalene (2-OHNap) (6.01 +/- 4.21 ng/mL) as the most abundant OH-PAH, and exposure could be through the use of naphthalene-containing-mothballs in drinking water purification, insect repellent, freshener in clothes and/or "treatment of various ailments". The study revealed that exposure to naphthalene significantly increases the occurrence of persistent cough (OR = 2.68, CI: 1.43-5.05), persistent headache (OR = 1.82, CI: 1.02-3.26), tachycardia (OR = 3.36, CI: 1.39-8.10) and dyspnea (OR = 3.07, CI: 1.27-7.43) in Kumasi residents. Highest level of urinary 2-OHNap (224 ng/mL) was detected in a female, who reported symptoms of persistent cough, headache, tachycardia, nasal congestion and inflammation, all of which are symptoms of naphthalene exposure according to USEPA. The Sigma OHPAHs, 2-OHNap, 2-3-OHFluorenes, and -OHPhenanthrenes showed a significantly positive correlation with MDA and 4-OHPhenanthrene with 8-OHdG, indicating possible lipid peroxidation/cell damage or degenerative disease in some participants. MDA and 8-OHdG were highest in age group 21-60. The present study showed a significant sex difference with higher levels of urinary OH-PAHs in females than males. (C) 2017 Published by Elsevier Ltd.
  • Sumitaka Kobayashi, Fumihiro Sata, Chihiro Miyashita, Ryu Miura, Kaoru Azumi, Sachiko Kobayashi, Houman Goudarzi, Atsuko Araki, Mayumi Ishizuka, Takashi Todaka, Jumboku Kajiwara, Tsuguhide Hori, Reiko Kishi
    TOXICOLOGY 390 135 - 145 0300-483X 2017/09 [Refereed][Not invited]
    Background: Associations between prenatal exposure to polychlorinated biphenyls (PCBs) and reduced birth size, and between DNA methylation of insulin-like growth factor-2 (IGF-2), HI 9 locus, and long interspersed nuclear element-1 (LINE-1) and reduced birth-size are well established. To date, however, studies on the associations between prenatal exposure to PCBs and alterations in methylation of IGF-2, H19, and LINE-1 are lacking. Thus, in this study, we examined these associations with infant-gender stratification. Methods: We performed a prospective birth cohort study using the Sapporo cohort from the previously described Hokkaido Birth Cohort Study on Environment and Children's Health conducted between 2002 and 2005 in Japan. In the final 169 study participants included in this study, we measured the concentrations of various non dioxin-like PCBs in maternal blood during pregnancy using high-resolution gas chromatography/high-resolution mass spectrometry. IGF-2, H19 and LINE-1 methylation levels in cord blood were measured using the bisulfite pyrosequencing methods Finally, we assessed the associations between prenatal exposure to various PCBs and the gene methylation levels using multiple regression models stratified by infant gender. Results: We observed a 0.017 (95% confidence interval [CI]: 0.003-0.031) increase in the log(10)-transformed H19 methylation levels (%) in cord blood for each ten-fold increase in the levels of decachlorinated biphenyls (decaCBs) in maternal blood among all infants. Similarly, a 0.005 (95% CI: 0.000-0.010) increase in the log(10)-transformed LINE-1 methylation levels (%) in cord blood was associated with each ten-fold increase in heptachlorinated biphenyls (heptaCBs) in maternal blood among all infants. In particular, we observed a dose-dependent association of the decaCB levels in maternal blood with the H19 methylation levels among female infants (P value for trend = 0.040); likewise a dose-dependent association of heptaCB levels was observed with LINE-I methylation levels among female infants (P value for trend = 0.015). Moreover, these associations were only observed among infants of primiparous women. Conclusion: Our results suggest that the dose-dependent association between prenatal exposure to specific non-dioxin-like PCBs and increases in the H19 and LINE-1 methylation levels in cord blood might be more predominant in females than in males.
  • Osei Akoto, Nesta Bortey-Sam, Yoshinori Ikenaka, Shouta M M Nakayama, Elvis Baidoo, Yared Beyene Yohannes, Mayumi Ishizuka
    Journal of health & pollution 7 (15) 28 - 39 2017/09 [Refereed][Not invited]
    Background: Environmental contamination with heavy metals and metalloids due to industrial, smelting and mining activities have become common in large and growing cities. Kumasi is one of the most industrialized cities in Ghana and experiences metal pollution due to recent and past activities. Although metals are naturally abundant in the area, their accumulation in soils could potentially lead to adverse effects on local ecosystems. Objectives: The aims of this study were to determine the distribution, enrichment, geoaccumulation and sources of metals in Kumasi soils and to estimate the contamination factor (CF) and pollution load index (PLI) of these metals in soils. Methods: Concentrations of eight heavy metals and a metalloid were determined in 112 soil samples randomly collected from 31 sampling sites in the area. In addition, 5 soil samples were collected from a pristine site (Kwame Nkrumah University of Science and Technology Botanical Gardens) for data comparison, to determine the local background values for metal concentrations and to evaluate the extent of metal pollution in the study area. Results: Heavy metals such as zinc (Zn), lead (Pb), cadmium (Cd) and chromium (Cr) were enriched in 65, 32, 58 and 93% of the sampling sites, respectively, and geo-accumulation indexes for Cr, Zn, Cd, mercury (Hg) and Pb showed moderate to extreme contamination in 100, 97, 77, 65 and 45% of the sampling sites, respectively. Principal component and cluster analyses revealed that industrial activities including mining were the major sources of metals in Kumasi soils with high metal input in the community of Suame. Distribution maps revealed hotspots of Cd, nickel (Ni), arsenic (As), cobalt (Co), copper (Cu) and Pb in Suame. The highest CFs for Cu, Cd, Ni, As, Co and Pb highlighted anthropogenic inputs in Suame, while Hg was highest in Mbrom, Zn in Suntreso, and Cr in Aboabo. Conclusions: The PLI of metals revealed Suame as the most polluted study site, while Anomangye and Bomso were the least polluted.
  • Takamitsu Kondo, Yoshinori Ikenaka, Shouta M. M. Nakayama, Yusuke K. Kawai, Hazuki Mizukawa, Yoko Mitani, Kei Nomiyama, Shinsuke Tanabe, Mayumi Ishizuka
    TOXICOLOGICAL SCIENCES 158 (1) 90 - 100 1096-6080 2017/07 [Refereed][Not invited]
    UDP-glucuronosyltransferases (UGTs) are among the most important xenobiotic metabolizing enzymes that conjugate a wide range of chemicals. Previous studies showed that Felidae and Pinnipedia species have very low UGT activities toward some phenolic compounds because of the UGT1A6 pseudogene and small numbers of UGT1A isozymes. In addition to the UGT1As, UGT2Bs isozymes also conjugate various endogenous (eg, estrogens, androgens, and bile acids) and exogenous compounds (opioids, non-steroidal anti-inflammatory drugs, and environmental pollutants). However UGT2B activity and genetic background are unknown in carnivore species. Therefore, this study was performed to elucidate the species differences of UGT2Bs. Using typical substrates for UGT2Bs, UGT activity was measured in vitro. In addition, UGT2B genetic features are analyzed in silico. Results of UGT activity measurement indicate marked species differences between dogs and other carnivores (cats, Northern fur seals, Steller sea lions, Harbor seals, and Caspian seals). Dogs have very high V-max/K-m toward estradiol (17-glucuronide), estrone, lorazepam, oxazepam, and temazepam. Conversely, cats and pinniped species (especially Caspian seals and Harbor seals) have very low activities toward these substrates. The results of genetic synteny analysis indicate that Felidae and pinniped species have very small numbers of UGT2B isozymes (one or none) compared with dogs, rodents, and humans. Furthermore, Felidae species have the same nonsense mutation in UGT2B, which suggests that Felidae UGT2B31-like is also a pseudogene in addition to UGT1A6. These findings of lower activity of UGT2B suggest that Felidae and some pinniped species have very low UGT activity toward a wide range of chemicals. These results are important for Felidae and Pinnipedia species that are frequently exposed to drugs and environmental pollutants.
  • Sachiko Kobayashi, Kaoru Azumi, Houman Goudarzi, Atsuko Araki, Chihiro Miyashita, Sumitaka Kobayashi, Sachiko Itoh, Seiko Sasaki, Mayumi Ishizuka, Hiroyuki Nakazawa, Tamiko Ikeno, Reiko Kishi
    JOURNAL OF EXPOSURE SCIENCE AND ENVIRONMENTAL EPIDEMIOLOGY 27 (3) 251 - 259 1559-0631 2017/05 [Refereed][Not invited]
    Prenatal exposure to perfluoroalkyl acids (PFAAs) influences fetal growth and long-term health. However, whether PFAAs affect offspring DNA methylation patterns to influence health outcomes is yet to be evaluated. Here, we assessed effect of prenatal PFAA exposure on cord blood insulin-like growth factor 2 (IGF2), H19, and long interspersed element 1 (LINE1) methylation and its associations with birth size. Mother-child pairs (N = 177) from the Hokkaido Study on Environment and Children's Health were included in the study. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) levels in maternal serum were measured by liquid chromatography-tandem mass spectrometry. IGF2, H19, and LINE1 methylation in cord blood DNA was determined by pyrosequencing. After full adjustment in multiple linear regression models, IGF2 methylation showed a significant negative association with log-unit increase in PFOA (partial regression coefficient = -0.73; 95% confidence interval: -1.44 to -0.02). Mediation analysis suggested that reduced IGF2 methylation explained similar to 21% of the observed association between PFOA exposure and reduced ponderal index of the infant at birth. These results indicated that the effects of prenatal PFOA exposure could be mediated through DNA methylation. Further study will be required to determine the potential for long-term adverse health effects of reduced IGF2 methylation induced by PFOA exposure.
  • Lesa A. Thompson, Wageh Sobhy Darwish, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (4) 751 - 764 0916-7250 2017/04 [Refereed][Not invited]
    Organochlorine pesticides (OCPs) have been used worldwide, particularly in Africa, for several decades. Although many are banned, several African countries still use OCPs especially for the prevention and control of malaria. OCPs are characterized by their bio-accumulation in the environment, especially in the food chain, where they find their way into the human body. Despite no clear epidemiological studies confirming hazardous effects of these chemicals on human health, many studies have reported positive associations between the use of OCPs and neurological and reproductive disorders, and cancer risk. There is a clear gap in published reports on OCPs in Africa and their potential health hazards. Thus, the aim of this review is to summarize the incidence of OCP contamination in various foods in Africa, to demonstrate the potential transmission of these chemicals to people and to discuss their possible health hazards.
  • Chihiro Ishii, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Yared Beyene Yohannes, Yutaka Watanuki, Masaaki Fukuwaka, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 79 (4) 807 - 814 0916-7250 2017/04 [Refereed][Not invited]
    Seabirds are marine top predators and accumulate high levels of metals and metalloids in their tissues. Contamination by metals in the highly productive offshore region has become a matter of public concern. It is home to 80% of the seabird population in the U.S.A., 95% of northern fur seals (Callorhinus ursinus), and major populations of Steller sea lions (Eumetopias jubatus), walruses (Odobenus rosmarus) and whales. Here, the concentrations of eight heavy metals (Hg, Cd, Cr, Co, Ni, Cu, Zn and Pb) and a metalloid (As) in the liver and kidneys of the northern fulmar (Fulmarus glacialis), thick-billed murre (Uria lomvia), short-tailed shearwater (Puffinus tenuirostris), tufted puffin (Fratercula cirrhata) and horned puffin (Fratercula corniculata) collected in the Bering Sea were measured. As proxies of trophic level and habitat, nitrogen (delta N-15) and carbon (delta C-13) stable isotope ratios of breast muscles were also measured. Hepatic Hg concentration was high in northern fulmar, whereas Cd level was high in tufted puffin and northern fulmar. The Hg concentration and d15N value were positively correlated across individual birds, suggesting that Hg uptake was linked to the trophic status of consumed prey. Furthermore, Hg concentration in our study was higher than those of the same species of seabirds collected in 1990.
  • Hazuki Mizukawa, Yoshinori Ikenaka, Mayu Kakehi, Shouta Nakayama, Mayumi Ishizuka
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 137 (3) 257 - 263 0031-6903 2017/03 [Refereed][Not invited]
    The ability to metabolize xenobiotics in organisms has a wide degree of variation among organisms. This is caused by differences in the pattern of xenobiotic bioaccumulation among organisms, which affects their tolerance. It has been reported in the veterinary field that glucuronidation (UGT) activity in cats, acetylation activity in dogs and sulfation (SULT) activity in pigs are sub-vital in these species, respectively, and require close attention when prescribing the medicine. On the other hand, information about species differences in xenobiotics metabolism remains insufficient, especially in non-experimental animals. In the present study, we tried to elucidate xenobiotic metabolism ability, especially in phase II UGT conjugation of various non-experimental animals, by using newly constructed in vivo, in vitro and genomic techniques. The results indicated that marine mammals (Steller sea lion, northern fur seal, and Caspian seal) showed UGT activity as low as that in cats, which was significantly lower than in rats and dogs. Furthermore, UGT1A6 pseudogenes were found in the Steller sea lion and Northern fur seal; all Otariidae species are thought to have the UGT1A6 pseudogene as well. Environmental pollutants and drugs conjugated by UGT are increasing dramatically in the modern world, and their dispersal into the environment can be of great consequence to Carnivora species, whose low xenobiotic glucuronidation capacity makes them highly sensitive to these compounds.
  • Yared Beyene Yohannes, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    BULLETIN OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY 98 (2) 172 - 177 0007-4861 2017/02 [Refereed][Not invited]
    Concentrations of ten trace elements (Hg, As, Cd, Pb, Co, Cr, Cu, Ni, Se and Zn) were determined in different tissues (liver, kidney, muscle, heart and brain) of African sacred ibis (Threskiornis aethiopicus), Hamerkop (Scopus umbretta), marabou stork (Leptoptilos crumeniferus) and great white pelican (Pelecanus onocrotalus) inhabiting the Ethiopian Rift Valley region. There were differences in trace element patterns among the bird species. Significantly (p < 0.05) higher concentrations of Cd (5.53 A mu g/g dw +/- 2.94) in kidney and Hg (0.75 A mu g/g ww +/- 0.30) in liver were observed in the great white pelican compared to the other species, and liver concentrations of these two elements showed positive correlations with trophic level. Concentrations of toxic elements (As, Cd, Pb and Hg) in liver were below their respective toxicological thresholds, indicating that the data may provide baseline information for future studies.
  • Hokuto Nakata, Shouta M M Nakayama, Balazs Oroszlany, Yoshinori Ikenaka, Hazuki Mizukawa, Kazuyuki Tanaka, Tsunehito Harunari, Tsutomu Tanikawa, Wageh Sobhy Darwish, Yared B Yohannes, Aksorn Saengtienchai, Mayumi Ishizuka
    International journal of environmental research and public health 14 (1) 1661-7827 2017/01/10 [Refereed][Not invited]
    Although Japan has been considered to have little lead (Pb) pollution in modern times, the actual pollution situation is unclear. The present study aims to investigate the extent of Pb pollution and to identify the pollution sources in Japan using stable Pb isotope analysis with kidneys of wild rats. Wild brown (Rattus norvegicus, n = 43) and black (R. rattus, n = 98) rats were trapped from various sites in Japan. Mean Pb concentrations in the kidneys of rats from Okinawa (15.58 mg/kg, dry weight), Aichi (10.83), Niigata (10.62), Fukuoka (8.09), Ibaraki (5.06), Kyoto (4.58), Osaka (4.57), Kanagawa (3.42), and Tokyo (3.40) were above the threshold (2.50) for histological kidney changes. Similarly, compared with the previous report, it was regarded that even structural and functional kidney damage as well as neurotoxicity have spread among rats in Japan. Additionally, the possibility of human exposure to a high level of Pb was assumed. In regard to stable Pb isotope analysis, distinctive values of stable Pb isotope ratios (Pb-IRs) were detected in some kidney samples with Pb levels above 5.0 mg/kg. This result indicated that composite factors are involved in Pb pollution. However, the identification of a concrete pollution source has not been accomplished due to limited differences among previously reported values of Pb isotope composition in circulating Pb products. Namely, the current study established the limit of Pb isotope analysis for source identification. Further detailed research about monitoring Pb pollution in Japan and the demonstration of a novel method to identify Pb sources are needed.
  • Yared Beyene Yohannes, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    SCIENCE OF THE TOTAL ENVIRONMENT 574 1389 - 1395 0048-9697 2017/01 [Refereed][Not invited]
    Despite the presence of a wide variety and number of birds, there is exceedingly little data on organochlorine pesticide (OCP) residues in birds inhabiting in Africa. In the present study, concentrations of dichlorodiphenyl-trichloroethanes (DDTs), hexachlorocyclohexanes, drins, cyclodienes, and hexachlorobenzene were measured in liver, kidney, heart and brain of 4 bird species from the Rift Valley region, Ethiopia. Indoor residual spraying of DDT for malaria vector control, and indiscriminate and illegal use of pesticides underline the relevance of this study. Levels of Sigma OCPs ranged from 1.87 to 4586 ng/g wet weight, and the scavenger bird species Leptoptilos crumeniferus had the highest level in liver. In all tissues, contamination profiles of OCPs within the species were similar, with DDTs >> other OCPs. Among the DDTs, p,p'-DDE was the most abundant compound and had significantly a higher burden in all tissues. The risk characterization demonstrated potential risks to the studied birds associated with DDE exposure. Maximum hepatic levels of p,p'-DDE exceeded the levels reported to trigger adverse effects. The detection of p,p'-DDT in all bird tissues suggests the release of fresh DDT to the environment. This is the first study to assay OCPs in different tissues of birds from the Ethiopian Rift Valley region, and henceforth the data will serve as a reference data for future studies. (C) 2016 Elsevier B.V. All rights reserved.
  • Aksorn Saengtienchai, Yoshinori Ikenaka, Nesta Bortey-Sam, Usuma Jermnark, Hazuki Mizukawa, Yusuke K. Kawai, Shouta M. M. Nakayama, Mayumi Ishizuka
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY 190 38 - 47 1532-0456 2016/12 [Refereed][Not invited]
    The African hedgehog, Atelerix albiventris, is a spiny mammal that has become popular as an exotic pet in many countries. To elucidate the ability of hedgehogs to metabolize xenobiotics, the animals were exposed to polycyclic aromatic hydrocarbon, pyrene. The in vivo exposure study indicated that pyrene was biotransformed to glucuronide and sulfate conjugates, such as pyrene-l-glucuronide, pyrene-1-sulfate, and pyrenediol-sulfate, and excreted in the urine. Pyrene-l-glucuronide was the main metabolite, and limited sulfate conjugate excretion was observed. The main products excreted in feces were 1-hydroxypyrene and pyrene. Based on the results of the in vivo exposure study, in vitro enzymatic kinetic experiments were performed using various substrates and compared to rats and pigs. The enzyme efficiencies of cytochrome P450 (CYP)-mediated ethoxyresorufin 0-deethylase activity and warfarin 4 '-, 6-, and 8-hydroxylation activity in hedgehogs were lower than those of rats. Furthermore, UDP-glucuronosyltransferase activity in hedgehogs also had a lower Km value than that in pigs. Interestingly, the enzyme efficiencies of sulfation activity toward 1-hydroxypyrene and beta-estradiol in hedgehogs were significantly lower than those in pigs. These observations suggested that phenol and estrogen sulfotransferases may have limited roles in xenobiotic metabolism in hedgehogs. (C) 2016 Elsevier Inc. All rights reserved.
  • Nesta Bortey-Sam, Yoshinori Ikenaka, Osei Akoto, Shouta M. M. Nakayama, Jemima Marfo, Aksorn Saengtienchai, Hazuki Mizukawa, Mayumi Ishizuka
    ENVIRONMENTAL POLLUTION 218 331 - 337 0269-7491 2016/11 [Refereed][Not invited]
    Previous studies of polycyclic aromatic hydrocarbons (PAHs) in particulate matter, soils and livers of wild rats indicated that the city centre of Kumasi, Ghana has been severely polluted with high cancer potency. Cattle urine were therefore collected from Kumasi (urban) and Offinso (rural), Ghana: to determine concentrations of urinary PAH metabolites (OH-PAHs); and find their association with sex; and to estimate exposure of cattle to PAHs from the different sites. From the results, geometric mean concentrations (adjusted by specific gravity), GM(SG), showed that 2-OHNaphthalene (2-OHNap) was the most abundant OH-PAH in cattle urine from all study sites, and naphthalene-containing-mothballs might have contributed significantly to the levels. There was no significant difference between urinary OH-PAHs concentrations in cattle from urban and rural sites except for 2-OHPhe and 4-OHPhe, and similar to urban areas, rural sites could also be polluted with PAHs. GMSG of 2-OHNap in cattle urine in Kokote (21.9 +/- 6.51 ng/mL; a rural area), was significantly higher compared to the other sites followed by Oforikrom (4.15 +/- 4.37 ng/mL; urban). The GMSG concentration (ng/mL) of the sum of OH-PAHs decreased in the order, Kokote (44.7) > Oforikrom (7.87) > Saboa (6.98) > Santasi (6.68) > and Twumasen Estate (5.23). The high concentrations of urinary 2-OHNap, 2-OHPhe, 3-OHPhe and 4-OHPhe in Kokote indicated high PAHs exposure to cattle in this area or different/specific source of PAHs exposure. GMSG of 2-OHNap was significantly higher in male cattle compared to females while 1-9-OHPhe was significantly higher in females. (C) 2016 Published by Elsevier Ltd.
  • Kazuki Takeda, Yoshinori Ikenaka, Tsutomu Tanikawa, Kazuyuki D. Tanaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 134 1 - 7 0048-3575 2016/11 [Refereed][Not invited]
    Roof rats (Rattus rattus) live mainly in human habitats. Heavy use of rodenticides, such as warfarin, has led to the development of drug resistance, making pest control difficult. There have been many reports regarding mutations of vitamin K epoxide reductase (VKOR), the target enzyme of warfarin, in resistant rats. However, it has been suggested there are other mechanisms of warfarin resistance. To confirm these possibilities, closed colonies of warfarin-susceptible roof rats (S) and resistant rats from Tokyo (R) were established, and the pharmacokinetics/ pharmacodynamics of warfarin in rats from both colonies was investigated. R rats had low levels of warfarin in serum and high clearance activity. These rats can rapidly metabolize warfarin by hydroxylation. The levels of accumulation in the organs were lower than those of S rats. R rats administered warfarin showed high expression levels of CYP2B, 2C, and 3A, which play roles in warfarin hydroxylation, and may explain the high clearance ability of R rats. The mechanism of warfarin resistance in roof rats from Tokyo involved not only mutation of VKOR but also high clearance ability due to high levels of CYP2B, 2C and 3A expression possibly induced by warfarin. (C) 2016 Elsevier Inc. All rights reserved.
  • Yoshihiro Nishiyama, Shouta M. M. Nakayama, Kensuke P. Watanabe, Yusuke K. Kawai, Marumi Ohno, Yoshinori Ikenaka, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (4) 675 - 680 0916-7250 2016/04 [Refereed][Not invited]
    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.
  • Ruan Gerber, Nico J. Smit, Johan H. J. Van Vuren, Shouta M. M. Nakayama, Yared B. Yohannes, Yoshinori Ikenaka, Mayumi Ishizuka, Victor Wepener
    SCIENCE OF THE TOTAL ENVIRONMENT 550 522 - 533 0048-9697 2016/04 [Refereed][Not invited]
    With the second highest gross domestic product in Africa, South Africa is known to have a high pesticide usage, including the highly persistent and banned group of organochlorine pesticides (OCPs). South Africa is also one of few countries to still actively spray DDT as malaria vector control. The aim of the study was to determine the degree to which aquatic biota in selected rivers of the world renowned Kruger National Park (KNP) arc exposed to by use of OCPs in the catchments outside the KNP and how this exposure relates to human health. Tigerfish (Hydrocynus vittatus) arc economically important apex predators and was selected as bioindicator for this study. Fish were sampled from the KNP sections of the Luvuvhu, Letaba and Olifants rivers during the high and low flow periods from 2010 to 2011 within the KNP and 19 OCPs were determined in muscle tissue using GC-ECD techniques. Significant flow related and spatial OCP bioaccumulation was observed. Tigerfish from the Luvuvhu River displayed the highest OCP bioaccumulation. Concentrations of the majority of the OCPs including the DDTs were the highest levels ever recorded from South African freshwater systems and in many cases the concentrations were higher than most contaminated areas from around the world. The concentrations found in H. vittatus muscle also exceeded maximum residue levels in edible fat as set by the European Union. The health risk assessment also demonstrated that the levels of OCPs pose very high cancer risks to the local populations consuming tigerfish, as high as 2 in 10 increased risk factor. This is of concern not only when managing the water resources of the conservation area but also for surrounding communities consuming freshwater fish. Contaminants enter the park from outside the borders and pose potential risks to the mandated conservation of aquatic biota within the KNP. (C) 2016 Elsevier B.V. All rights reserved.
  • Abdallah Fikry A. Mahmoud, Yoshinori Ikenaka, Yared B. Yohannes, Wageh S. Darwish, Elsaid A. Eldaly, Alaa Eldin M. A. Morshdy, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    CHEMOSPHERE 144 1365 - 1371 0045-6535 2016/02 [Refereed][Not invited]
    Food consumption is an important route of human exposure to organochlorine pesticides (OCPs). In order to assess the potential human health risks associated with OCPs, edible cattle tissues (liver, kidney and tongue) were collected from three slaughter houses in Mansoura, Zagazig and Ismailia cities, Egypt. Levels of 22 OCPs such as hexachlorocyclohexanes (HCHs), dichlorodiphenyltrichloroethanes (DDTs), aldrin, dieldrin and endrin (Drins), chlordanes (CHLs), heptachlors (HPTs) and hexachlorobenzene (HCB) residues were investigated. Among the investigated OCPs, HCHs represented the most dominant group with high proportions of gamma-HCH isomer (53-91% of total HCHs). Mansoura city had the highest OCPs contamination load ranged from 0.1 to 2827 ng g(-1) lw (lipid weight). Surprisingly, tongue samples collected from Mansoura showed the highest concentration of HCHs (448 ng g(-1) lw) in comparison to liver (152 ng g(-1) lw) and kidney (266 ng g(-1) lw). Generally, contamination pattern of OCPs was in the order of HCHs > Drins > CHLs > DDTs congruent to HCB and HPTs. Estimated daily intakes (EDIs) through dietary consumption of cattle tissues were lower than the recommended acceptable daily intakes (ADIs) established by FAO/WHO. However, the hazard ratios (HRs) based on cancer risk were greater than 1.0 for HCHs based on the average and 95th centile concentrations, indicating carcinogenic effects to consumers through cattle tissues consumption. (C) 2015 Elsevier Ltd. All rights reserved.
  • Wageh Sobhy Darwish, Yoshinori Ikenaka, Alaa Eldin Morshdy, Kamal Ibrahim Eldesoky, Shouta Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 78 (2) 351 - 354 0916-7250 2016/02 [Refereed][Not invited]
    The aim of this study was to estimate total carotenoids, beta-carotene and retinol concentrations in the livers and muscles of some ungulates (cattle, buffalo, sheep, goats and horses) in comparison with the Wistar rats as a control. Cattle and horses had the highest contents of total carotenoids and beta-carotene. Unexpectedly, sheep was the highest accumulator of retinol with a mean concentration of 203 +/- 23.34 mu g/g, while the least accumulator was buffalo, having a mean value of 58.28 +/- 13.77 mu g/g. Livers had higher contents of the examined phytochemicals than muscles. Consumption of these tissues may provide human with some needs from these important phytochemicals, though ingestion of livers, especially that of the sheep, is not advisable for the pregnant women.
  • Hokuto Nakata, Shouta M. M. Nakayama, John Yabe, Allan Liazambi, Hazuki Mizukawa, Wageh Sobhy Darwish, Yoshinori Ikenaka, Mayumi Ishizuka
    ENVIRONMENTAL POLLUTION 208 (Pt B) 395 - 403 0269-7491 2016/01 [Refereed][Not invited]
    Stable Pb isotope ratios (Pb-IRs) have been recognized as an efficient tool for identifying sources. This study carried out at Kabwe mining area, Zambia, to elucidate the presence or absence of Pb isotope fractionation in goat and chicken, to evaluate the reliability of identifying Pb pollution sources via analysis of Pb-IRs, and to assess whether a threshold for blood Pb levels (Pb-B) for biological fractionation was present The variation of Pb-IRs in goat decreased with an increase in Pb-B and were fixed at certain values close to those of the dominant source of Pb exposure at Pb-B > 5 mu g/dL. However, chickens did not show a clear relationship for Pb-IRs against Pb-B, or a fractionation threshold. Given these, the biological fractionation of Pb isotopes should not occur in chickens but in goats, and the threshold for triggering biological fractionation is at around 5 mu g/dL of Pb-B in goats. (C) 2015 Elsevier Ltd. All rights reserved.
  • Wageh S. Darwish, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    JOURNAL OF FOOD SCIENCE 81 (1) T275 - T281 0022-1147 2016/01 [Refereed][Not invited]
    Lead (Pb) is an environmental pollutant that can get entry into human body through contaminated foods, drinks, and inhaled air leading to severe biological consequences, and has been responsible for many deaths worldwide. The objectives of this study were 1st to investigate the modulatory effects of environmentally relevant concentrations of Pb on AhR gene battery, which is controlling xenobiotics metabolism. 2nd, trials to reduce Pb-induced adverse effects were done using some phytochemicals like beta-carotene or ascorbic acid. Human hepatoma (HepG2) cell lines were exposed to a wide range of Pb concentrations varying from physiological to toxic levels (0 to 10 mg/L) for 24 h. High Pb concentrations (1 to 10 mg/L) significantly reduced phase I (CYP1A1 and 1A2) and phase II (UGT1A6 and NQO1) xenobiotic metabolizing enzyme mRNA expression in a mechanistic manner through the AhR regulation pathway. Additionally, these Pb concentrations induced oxidative stress in HepG2 cells in terms of production of reactive oxygen species (ROS) and induced heme oxygenase-1 mRNA expression in a concentration-dependent phenomenon. Coexposure of HepG2 cells to physiological concentrations of some micronutrients, like beta-carotene (10 mu M) or ascorbic acid (0.1 mM), along with Pb (1 mg/L) for 24 h significantly reduced the levels of ROS production and recovered AhR mRNA expression into the normal levels. Thus, consumption of foods rich in these micronutrients may help to reduce the adverse effects of lead in areas with high levels of pollution.
  • Hazuki Mizukawa, Kei Nomiyama, Susumu Nakatsu, Hisato Iwata, Jean Yoo, Akira Kubota, Miyuki Yamamoto, Mayumi Ishizuka, Yoshinori Ikenaka, Shouta M. M. Nakayama, Tatsuya Kunisue, Shinsuke Tanabe
    ENVIRONMENTAL SCIENCE & TECHNOLOGY 50 (1) 444 - 452 0013-936X 2016/01 [Refereed][Not invited]
    There are growing concerns about the increase in hyperthyroidism in pet cats due to exposure to organo-halogen contaminants and their hydroxylated metabolites. This study investigated the blood contaminants polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) and their hydroxylated and methoxylated derivatives (OH-PCBs, OH-PBDEs, and MeO-PBDEs), in pet dogs and cats. We also measured the residue levels of these compounds in commercially available pet foods. Chemical analyses of PCBs and OH-PCBs showed that the OH-PCB levels were 1 to 2 orders of magnitude lower in cat and dog food products than in their blood, suggesting that the origin of OH-PCBs in pet dogs and cats is PCBs ingested with their food. The major congeners of OH-/MeO-PBDEs identified in both pet food products and blood were natural products (60H-/MeO-BDE47 and 2'OH-/MeO-BDE68) from marine organisms. In particular, higher concentrations of 60H-BDE47 than 2'OH-BDE68 and two MeO-PBDE congeners were observed in the cat blood, although MeO-BDEs were dominant in cat foods, suggesting the efficient biotransformation of 60H-BDE47 from 6MeO-BDE47 in cats. We performed in vitro demethylation experiments to confirm the biotransformation of MeO-PBDEs to OH-PBDEs using liver microsomes. The results showed that 6MeO-BDE47 and 2'MeO-BDE68 were demethylated to 60H-BDE47 and 2'OH-BDE68 in both animals, whereas no hydroxylated metabolite from BDE47 was detected. The present study suggests that pet cats are exposed to MeO-PBDEs through cat food products containing fish flavors and that the OH-PBDEs in cat blood are derived from the CYP-dependent demethylation of naturally occurring MeO-PBDE congeners, not from the hydroxylation of PBDEs.
  • Ruan Gerber, Nico J. Smit, Johan H. J. van Vuren, Shouta M. M. Nakayama, Yared B. Yohannes, Yoshinori Ikenaka, Mayumi Ishizuka, Victor Wepener
    ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH 22 (24) 19971 - 19989 0944-1344 2015/12 [Refereed][Not invited]
    The physical and chemical characteristics of surface sediments from a leading conservation area, namely the Kruger National Park, were determined in order to identify potential stressors in the systems that may contribute to overall deterioration in sediment quality within the reserve, leading to potential threats to the aquatic biota conserved within these stretches of river. Sediment samples were collected during four surveys (two low flow and two high flow) from 2009 to 2011. Samples were analysed for organic content, grain size determination, metals and various organochlorine pesticides. Results indicated that the Olifants River sediments did not show any great improvement over the years and point towards the continued input of pollutants into this system. Sediment quality in the Luvuvhu and Letaba Rivers is better than that of sediments from the Olifants River in terms of metals, but metal concentrations are still comparable and point towards anthropogenic inputs of metals into these rivers. Even though the data indicate that these systems are being contaminated with both metals and organochlorine pesticides (OCPs), levels were still below contaminated sediments from around the globe. Sediment Quality Index scores showed that the sediment quality of these rivers is in a relatively good state. High metal concentrations were the drivers behind lowered sediment quality, and in some cases certain OCPs played a role. Both metals and OCP concentrations were highly correlated with finer grain sizes. Sediment assessments are not routinely applied in South Africa resulting in very little reference or background data available for the area. The metal concentrations for the study area were generally lower than those for other studies in more polluted regions. The study also contributes to the available knowledge on surrounding metal pollution in riverine sediments in South Africa.
  • Hiroki Teraoka, Erika Okamoto, Moe Kudo, Shouta M. M. Nakayama, Yoshinori Ikenaka, Mayumi Ishizuka, Tetsuya Endo, Takio Kitazawa, Takeo Hiraga
    ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 122 557 - 564 0147-6513 2015/12 [Refereed][Not invited]
    The red-crowned (Japanese) crane Grus japonensis is native to east Hokkaido, Japan, in contrast to the East Asia mainland. Previously, we reported that red-crowned cranes in Hokkaido were highly contaminated with mercury in the 1990s and that the contamination rapidly decreased to a moderate level in the 2000s. In the present study, we determined levels of organic mercury (O-Hg) in the liver and kidney of cranes in east Hokkaido in comparison with levels of total mercury (T-Hg). T-Hg levels in the kidneys were higher than those in the livers in adults but not in subadults and juveniles; however, the reverse was the case for O-Hg even for adults. The ratio of O-Hg to T-Hg in both the liver and kidney decreased as T-Hg increased in the three developmental stages. While the ratios of O-Hg to T-Hg in the liver and kidney of adults were significantly lower than those of juveniles, the ratios were similar for adults and juveniles in a lower range of T-Hg. The ratio of selenium (Se) to T-Hg decreased as T-Hg increased in both the liver and kidney, irrespective of stages. Mercury burdens in feathers were about 59% and 67% of the total body burdens for juveniles and adults, respectively. Furthermore, ratios of carbon and nitrogen stable isotopes to T-Hg varied greatly, with no relation to mercury level in the liver. The results suggest slow accumulation of inorganic mercury in the kidney of red-crowned cranes in east Hokkaido, Japan. (C) 2015 Elsevier Inc. All rights reserved.
  • Jemima Tiwaa Mario, Kazutoshi Fujioka, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Yoshiko Aoyama, Mayumi Ishizuka, Kumiko Taira
    PLOS ONE 10 (11) e0142172  1932-6203 2015/11 [Refereed][Not invited]
    Neonicotinoid insecticides are nicotinic acetylcholine receptor agonists used worldwide. Their environmental health effects including neurotoxicity are of concern. We previously determined a metabolite of acetamiprid, N-desmethyl-acetamiprid in the urine of a patient, who exhibited some typical symptoms including neurological findings. We sought to investigate the association between urinary N-desmethyl-acetamiprid and the symptoms by a prevalence case-control study. Spot urine samples were collected from 35 symptomatic patients of unknown origin and 50 non-symptomatic volunteers (non-symptomatic group, NSG, 4-87 year-old). Patients with recent memory loss, finger tremor, and more than five of six symptoms (headache, general fatigue, palpitation/chest pain, abdominal pain, muscle pain/weakness/spasm, and cough) were in the typical symptomatic group (TSG, n = 19, 5-69 year-old); the rest were in the atypical symptomatic group (ASG, n = 16, 5-78 year-old). N-desmethyl-acetamiprid and six neonicotinoids in the urine were quantified by liquid chromatography-tandem mass spectrometry. The detection of N-desmethyl-acetamiprid was the most frequent and highest in TSG (47.4%, 6.0 ppb (frequency, maximum)), followed by in ASG (12.5%, 4.4 ppb) and in NSG (6.0%, 2.2 ppb), however acetamiprid was not detected. Thiamethoxam was detected in TSG (31.6%, 1.4 ppb), in ASG (6.3%, 1.9 ppb), but not in NSG. Nitenpyram was detected in TSG (10.5%, 1.2 ppb), in ASG (6.3%, not quantified) and in NSG (2.0%, not quantified). Clothianidin was only detected in ASG (6.3%, not quantified), and in NSG (2.0%, 1.6 ppb). Thiacloprid was detected in ASG (6.3%, 0.1 ppb). The cases in TSG with detection of N-desmethyl-acetamiprid and thiamethoxam were aged 5 to 62 years and 13 to 62 years, respectively. Detection of N-desmethyl-acetamiprid was associated with increased prevalence of the symptoms (odds ratio: 14, 95% confidence interval: 3.5-57). Urinary N-desmethyl-acetamiprid can be used as a biomarker for environmental exposure to acetamiprid. Further multi-centered clinical research in larger patients groups with more metabolites analysis is needed.
  • Wageh Sobhy Darwish, Yoshinori Ikenaka, Shouta Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    JAPANESE JOURNAL OF VETERINARY RESEARCH 63 (4) 173 - 182 0047-1917 2015/11 [Refereed][Not invited]
    Cooking of meat usually aims in producing microbiologically safe food suitable for human consumption. However, doing so at such high temperatures may produce some cooking toxicants or mutagens. The objectives of this study were to investigate the mutagenicity of modelled-heat-treated meat after different cooking methods (boiling, pan-frying and charcoal grilling) using Ames Salmonella typhimurium mutagenicity assay. In addition, the content of benzo[a]pyrene (B[a]P) in the meat extracts prepared under different cooking methods were measured using HPLC. In a trial to investigate the causes behind the mutagenicity of different meat extracts, HepG2 cell line was exposed to different modelled-heat-treated meat extracts. mRNA expression levels of various phase I and II xenobiotic metabolizing enzymes (XMEs) were examined using real time PCR. The results obtained declared that pan-fried and charcoal grilled-meat extracts significantly induced production of histidine+ revertants in the Ames mutagenicity assay. Grilled-meat extracts had the highest residual concentrations of B[a]P followed by pan-fried-meat, boiled meat and raw meat extracts, respectively. Induction of XMEs especially CYP1A1, CYP1A2 and NQO1 may contribute to the mutagenic ability of these extracts. It is highly advisable to control cooking temperature, time and method in order to reduce cooked-meat mutagens.
  • Kensuke P. Watanabe, Minami Kawata, Yoshinori Ikenaka, Shouta M. M. Nakayama, Chihiro Ishii, Wageh Sobhi Darwish, Aksorn Saengtienchai, Hazuki Mizukawa, Mayumi Ishizuka
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 34 (10) 2328 - 2334 0730-7268 2015/10 [Refereed][Not invited]
    Coumarin-derivative anticoagulant rodenticides used for rodent control are posing a serious risk to wild bird populations. For warfarin, a classic coumarin derivative, chickens have a high median lethal dose (LD50), whereas mammalian species generally have much lower LD50. Large interspecies differences in sensitivity to warfarin are to be expected. The authors previously reported substantial differences in warfarin metabolism among avian species; however, the actual in vivo pharmacokinetics have yet to be elucidated, even in the chicken. In the present study, the authors sought to provide an in-depth characterization of warfarin metabolism in birds using in vivo and in vitro approaches. A kinetic analysis of warfarin metabolism was performed using liver microsomes of 4 avian species, and the metabolic abilities of the chicken and crow were much higher in comparison with those of the mallard and ostrich. Analysis of in vivo metabolites from chickens showed that excretions predominantly consisted of 4-hydroxywarfarin, which was consistent with the in vitro results. Pharmacokinetic analysis suggested that chickens have an unexpectedly long half-life despite showing high metabolic ability in vitro. The results suggest that the half-life of warfarin in other bird species could be longer than that in the chicken and that warfarin metabolism may not be a critical determinant of species differences with respect to warfarin sensitivity. Environ Toxicol Chem 2015;34:2328-2334. (c) 2015 SETAC
  • Hokuto Nakata, Shouta M. M. Nakayama, Yoshinori Ikenaka, Hazuki Mizukawa, Chihiro Ishii, Yared B. Yohannes, Satoru Konnai, Wageh Sobhy Darwish, Mayumi Ishizuka
    ENVIRONMENTAL POLLUTION 205 8 - 15 0269-7491 2015/10 [Refereed][Not invited]
    Nairobi city in Kenya produces 2000 tons/day of garbage, and most of it is dumped onto the Dandora dumping site, home to a quarter-million residents. This study was conducted (1) to assess the contamination levels of nine metals and a metalloid (arsenic) in the blood of pigs, goats, sheep and cattle from Dandora, and (2) to identify a possible source of lead (Pb) pollution. Cadmium (Cd, 0.17-435 mu g/kg, dry-wt) and Pb (90-2710 mu g/kg) levels in blood were generally high, suggesting human exposure to Cd through livestock consumption and Pb poisoning among pigs (2600 mu g/kg) and cattle (354 mu g/kg). Results of Pb isotope ratios indicated that the major exposure route might differ among species. Our results also suggested a possibility that the residents in Dandora have been exposed to the metals through livestock consumption. (C) 2015 Elsevier Ltd. All rights reserved.
  • Aksorn Saengtienchai, Yoshinori Ikenaka, Wageh Sobhy Darwish, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 77 (10) 1261 - 1267 0916-7250 2015/10 [Refereed][Not invited]
    Pyrene (PY) is a polycyclic aromatic hydrocarbon (PAH) that is often used as a biomarker for human and wildlife exposure to PAHs. As the metabolites of PAHs, similar to their parent compounds, pose public health risks, it is necessary to study their characteristics and tissue-specific distribution. The present study was performed to experimentally characterize PY metabolites and analyze the tissue-specific distribution of the conjugated metabolites after oral administration of PY to rats. PY metabolites, such as pyrenediol-disulfate (PYdiol-diS), pyrenediol-sulfate (PYdiol-S), pyrene-1-sufate (PYOS), pyrene-l-glucuronide (PYOG) and 1-hydroxypyrene (PYOH), were detected in rat urine. Although glucuronide conjugate was the predominant metabolite, the metabolite composition varied among tissues. Interestingly, the proportion of PYOH was high in the large intestine. Furthermore, PYOH was the only PY metabolite detected in feces.
  • Mayu Kakehi, Yoshinori Ikenaka, Shouta M. M. Nakayama, Yusuke K. Kawai, Kensuke P. Watanabe, Hazuki Mizukawa, Kei Nomiyama, Shinsuke Tanabe, Mayumi Ishizuka
    TOXICOLOGICAL SCIENCES 147 (2) 360 - 369 1096-6080 2015/10 [Refereed][Not invited]
    There are various interspecies differences in xenobiotic-metabolizing enzymes. It is known that cats show slow glucuronidation of drugs such as acetaminophen and strong side effects due to the UGT1A6 pseudogene. Recently, the UGT1A6 pseudogene was found in the Northern elephant seal and Otariidae was suggested to be UGT1A6-deficient. From the results of measurements of uridine diphosphate-glucuronosyltransferase (UGT) activity using liver microsomes, the Steller sea lion, Northern fur seal, and Caspian seal showed UGT activity toward 1-hydroxypyrene and acetaminophen as low as in cats, which was significantly lower than in rat and dog. Furthermore, UGT1A6 pseudogenes were found in Steller sea lion and Northern fur seal, and all Otariidae species were suggested to have the UGT1A6 pseudogene. The UGT1 family genes appear to have undergone birth-and-death evolution based on a phylogenetic and synteny analysis of the UGT1 family in mammals including Carnivora. UGT1A2-1A5 and UGT1A7-1A10 are paralogous genes to UGT1A1 and UGTA6, respectively, and their numbers were lower in cat, ferret and Pacific walrus than in human, rat, and dog. Felidae and Pinnipedia, which are less exposed to natural xenobiotics such as plant-derived toxins due to their carnivorous diet, have experienced fewer gene duplications of xenobiotic-metabolizing UGT genes, and even possess UGT1A6 pseudogenes. Artificial environmental pollutants and drugs conjugated by UGT are increasing dramatically, and their elimination to the environment can be of great consequence to cat and Pinnipedia species, whose low xenobiotic glucuronidation capacity makes them highly sensitive to these compounds.
  • Nesta Bortey-Sam, Shouta M M Nakayama, Osei Akoto, Yoshinori Ikenaka, Elvis Baidoo, Hazuki Mizukawa, Mayumi Ishizuka
    International journal of environmental research and public health 12 (9) 11448 - 65 1661-7827 2015/09/11 [Refereed][Not invited]
    Heavy metals and a metalloid in agricultural soils in 19 communities in Tarkwa were analyzed to assess the potential ecological risk. A total of 147 soil samples were collected in June, 2012 and analyzed for As, Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn. Mean concentrations (mg/kg dw) of heavy metals in the communities decreased in order of Zn (39) ˃ Cr (21) ˃ Pb (7.2) ˃ Cu (6.2) ˃ As (4.4) ˃ Ni (3.7) ˃ Co (1.8) ˃ Hg (0.32) ˃ Cd (0.050). Correlations among heavy metals and soil properties indicated that soil organic matter could have substantial influence on the total contents of these metals in soil. From the results, integrated pollution (C(deg)) in some communities such as, Wangarakrom (11), Badukrom (13) and T-Tamso (17) indicated high pollution with toxic metals, especially from As and Hg. Potential ecological risk (RI) indices indicated low (Mile 7) to high risks (Wangarakrom; Badukrom) of metals. Based on pollution coefficient (C(i)(f)), C(deg), monomial ecological risk (E(i)(r)) and RI, the investigated soils fall within low to high contamination and risk of heavy metals to the ecological system especially plants, soil invertebrates and/or mammalian wildlife. This represented moderate potential ecological risk in the study area, and mining activities have played a significant role.
  • Nesta Bortey-Sam, Shouta M M Nakayama, Osei Akoto, Yoshinori Ikenaka, Julius N Fobil, Elvis Baidoo, Hazuki Mizukawa, Mayumi Ishizuka
    International journal of environmental research and public health 12 (8) 8811 - 27 1661-7827 2015/07/28 [Refereed][Not invited]
    This study was carried out to assess the extent of heavy metals and metalloid accumulation from agricultural soils to foodstuffs (viz, M. esculenta (cassava) and Musa paradisiaca (plantain)) around thirteen neighboring communities within Tarkwa, Ghana; and to estimate the human health risk associated with consumption of these foodstuffs. Concentrations of As, Cd, Co, Cr, Cu, Ni, Pb, and Zn were measured with an inductively coupled plasma-mass spectrometer and mercury analysis was done using a mercury analyzer. From the results, 30% of cassava samples collected, contained higher concentrations of Pb when compared to Codex Alimentarius Commission standard values. Bioconcentration factor indicated that Ni had higher capacity of absorption into food crops from soil than the other heavy metals. For both children and adults, the target hazard quotient (THQ) of Pb in cassava in communities such as Techiman, Wangarakrom, Samahu, and Tebe (only children) were greater than 1, which is defined as an acceptable risk value. This indicated that residents could be exposed to significant health risks associated with cassava consumption.
  • Nesta Bortey-Sam, Yoshinori Ikenaka, Osei Akoto, Shouta M. M. Nakayama, Yared Beyene Yohannes, Elvis Baidoo, Hazuki Mizukawa, Mayumi Ishizuka
    ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH 22 (13) 9658 - 9667 0944-1344 2015/07 [Refereed][Not invited]
    Airborne particulate samples were collected on quartz filters to determine the concentrations, sources and health risks of polycyclic aromatic hydrocarbons (PAHs) in air in Kumasi, Ghana. A total of 32 air samples were collected in Kwame Nkrumah University of Science and Technology (KNUST) campus (pristine site) and city centre (CC). Samples were extracted with 1:2v/v acetone/hexane mixture prior to GC-MS analyses. The sum of concentrations of 17 PAHs in air ranged from 0.51 to 16 (KNUST) and 19-38 ng/m(3) (CC). The concentration of benzo[a]pyrene, BaP, ranged from below detection limit to 0.08 ng/m(3) (KNUST) and 1.6 to 5.6 ng/m(3) (CC). Chemical mass balance model showed that PAHs in air in Kumasi were mainly from fuel combustion. The total BaP equivalent concentration (BaPeq) in CC was 18 times higher compared to KNUST; based on the European Legislation and Swedish and UK Standards for BaP in air, CC could be classified as highly polluted. Estimated carcinogenicity of PAHs in terms of BaPeq indicated that BaP was the principal PAH contributor in CC (70 %). Health risk to adults and children associated with PAH inhalation was assessed by taking into account the lifetime average daily dose and corresponding incremental lifetime cancer risk (ILCR). The ILCR was within the acceptable range (10(-6) to 10(-4)), indicating low health risk to residents.
  • Nesta Bortey-Sam, Shouta M. M. Nakayama, Yoshinori Ikenaka, Osei Akoto, Elvis Baidoo, Hazuki Mizukawa, Mayumi Ishizuka
    ENVIRONMENTAL MONITORING AND ASSESSMENT 187 (7) 397  0167-6369 2015/07 [Refereed][Not invited]
    Concentrations of heavy metals and metalloid in borehole drinking water from 18 communities in Tarkwa, Ghana, were measured to assess the health risk associated with its consumption. Mean concentrations of heavy metals (mu g/L) exceeded recommended values in some communities. If we take into consideration the additive effect of heavy metals and metalloid, then oral hazard index (HI) results raise concerns about the noncarcinogenic adverse health effects of drinking groundwater in Huniso. According to the US Environmental Protection Agency's (USEPA) guidelines, HI values indicating noncarcinogenic health risk for adults and children in Huniso were 0.781 (low risk) and 1.08 (medium risk), respectively. The cancer risk due to cadmium (Cd) exposure in adults and children in the sampled communities was very low. However, the average risk values of arsenic (As) for adults and children through drinking borehole water in the communities indicated medium cancer risk, but high cancer risk in some communities such as Samahu and Mile 7. Based on the USEPA assessment, the average cancer risk values of As for adults (3.65E-05) and children (5.08E-05) indicated three (adults) and five (children) cases of neoplasm in a hundred thousand inhabitants. The results of this study showed that residents in Tarkwa who use and drink water from boreholes could be at serious risk from exposure to these heavy metals and metalloid.
  • Wageh S. Darwish, Shouta M. M. Nakayama, Yuumi Itotani, Marumi Ohno, Yoshinori Ikenaka, Mayumi Ishizuka
    JOURNAL OF FOOD SCIENCE 80 (7) T1627 - T1632 0022-1147 2015/07 [Refereed][Not invited]
    Heterocyclic amines get entry into human body mainly through ingestion of pan-fried meats cooked at high temperatures. Exposure of the gastrointestinal tract (GIT) to ingested xenobiotics prior to delivery to the liver may lead to metabolic activation, which may explain the high incidence of GIT carcinogenesis. Therefore, this study investigated the mutagenic activation of 2 heterocyclic amines, 2-aminoanthracene (2-AA) and 3-amino-1-methyl-5H-prydo[4,3-b]indole (Trp-P-2), in the GIT of rats. In addition, the constitutive mRNA expression profiles of xenobiotic-metabolizing enzymes (XMEs) in the GIT of rats were examined. Metabolic activation of 2-AA was detected in all GIT tissues except the duodenum and rectum, and it was detected at high levels in the ileum and cecum. Furthermore, we revealed high metabolic activation of 2-AA and Trp-P-2 in the jejunum. The mRNA expression of phase I and II enzymes in rat GIT corresponded with their mutagenic activation ability. In conclusion, our results suggest that different expression levels of XME among GIT tissues may contribute to the tissue-specific differences in metabolic activation of xenobiotics such as heterocyclic amines in rats. Practical Application This study declares mutagenic activation of 2 heterocyclic amines namely 2-aminoanthracene (2-AA) and 3-amino-1-methyl-5H-prydo[4,3-b]indole (Trp-P-2), in the gastrointestinal tract (GIT) of rats. In addition, results obtained in this study suggest that GIT tissue-specific expression of xenobiotic metabolizing enzymes may contribute to the tissue-specific mutagenesis/carcinogenesis.
  • Yoshinori Ikenaka, Shouta M. M. Nakayama, Mami Oguri, Aksorn Saengtienchai, Hazuki Mizukawa, Jun Kobayashi, Wageh Sobhy Darwish, Mayumi Ishizuka
    ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 39 (3) 1148 - 1153 1382-6689 2015/05 [Refereed][Not invited]
    Red gourami (Colisa labiosa) have previously been shown to have low levels of pyrene-metabolizing activity. In this study, other pharmacokinetic factors of pyrene in C. labiosa were compared to those in Japanese medaka (Oryzias latipes). Results indicated that the two species labiosa absorbed pyrene in similar amounts. However, excretion of pyrene metabolites from C. labiosa over an 8-day period was lower than those from O. latipes. These findings show that C. labiosa has low ability to metabolize pyrene and to excrete pyrene and its metabolites from the body, and is therefore considered an accumulator of these chemicals. C. labiosa has unique characteristics with regard to pyrene pharmacokinetics. Knowledge about interspecies differences in pharmacokinetics is crucial in determining the endangered species to xenobiotic exposure. (C) 2015 Elsevier B.V. All rights reserved.
  • Hazuki Mizukawa, Kei Nomiyama, Tatsuya Kunisue, Michio X. Watanabe, Annamalai Subramanian, Hisato Iwata, Mayumi Ishizuka, Shinsuke Tanabe
    ENVIRONMENTAL RESEARCH 138 255 - 263 0013-9351 2015/04 [Refereed][Not invited]
    The concentrations of polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and their hydroxylated metabolites (OH-PCBs and OH-PBDEs) were measured in the blood of Eurasian wild pigs (Sus scrofa) from a municipal waste open dumping site (DS) and a reference site (RS) in South India. We showed that contamination with OH-PCBs was higher in female pigs from the DS than in all other adult pigs. The highest OH-PCB concentrations were found in piglets from the DS. Moreover, the hepatic expression levels of CYP1A and CYP2B were higher in piglets than in their dam, implying metabolism of PCBs by cytochrome P450 (CYP) enzymes. The OH-PCB congener profiles differed according to sex and collection sites, possibly because of variations in the expression levels of phase I and phase II enzymes among individual pigs, differences in the exposure sources, and maternal transfer of parent PCBs. The hepatic CYP1A expression levels were positively correlated with the blood concentrations of 4OH-CB107, 4OH-CB162, and 4OH-CB187, implying CYP1A-dependent formation of these OH-PCBs in the pig liver. We found no significant correlations between the blood concentrations of OH-PCBs and thyroid hormones (THs); however, the thyroxin (T-4) levels were lower in pigs from the DS than in pigs from the RS. Our limited dataset suggest that induced CYP enzymes accelerate the metabolism of xenobiotics and endogenous molecules in pigs. Thus, besides parental compounds, the risk of hydroxylated metabolites entering wildlife and humans living in and around municipal open waste dumping sites should be considered. (C) 2015 Elsevier Inc. All rights reserved.
  • John Yabe, Shouta M. M. Nakayama, Yoshinori Ikenaka, Yared B. Yohannes, Nesta Bortey-Sam, Balazs Oroszlany, Kaampwe Muzandu, Kennedy Choongo, Abel Nketani Kabalo, John Ntapisha, Aaron Mweene, Takashi Umemura, Mayumi Ishizuka
    CHEMOSPHERE 119 941 - 947 0045-6535 2015/01 [Refereed][Not invited]
    Childhood lead poisoning is a serious public health concern worldwide. Blood lead levels exceeding 5 mu g dL(-1) are considered elevated. In Kabwe, the capital of Zambia's Central Province, extensive Pb contamination of township soils in the vicinity of a Pb-Zn mine and posing serious health risk to children has been reported. We investigated BLLs in children under the age of 7 years in townships around the mine; where blood samples were collected and analyzed using an ICP-MS. All of the sampled children had BLLs exceeding 5 mu g dL(-1). Children in these areas could be at serious risk of Pb toxicity as 18% of the sampled children in Chowa, 57% (Kasanda) and 25% (Makululu) had BLLs exceeding 65 mu g dL(-1). Eight children had BLLs exceeding 150 mu g dL(-1) with the maximum being 427.8 mu g dL(-1). We recommend that medical intervention be commenced in the children with BLL exceeding 45 mu g dL(-1). (C) 2014 Elsevier Ltd. All rights reserved.
  • Nesta Bortey-Sam, Shouta M. M. Nakayama, Yoshinori Ikenaka, Osei Akoto, Elvis Baidoo, Yared Beyene Yohannes, Hazuki Mizukawa, Mayumi Ishizuka
    ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 111 160 - 167 0147-6513 2015/01 [Refereed][Not invited]
    Heavy metal and metalloid contamination in food resulting from mining is of major concern due to the potential risk involved. Food consumption is the most likely route of human exposure to metals. This study was therefore to assess metals in different organs and different animal species near gold mines used for human consumption (free-range chicken, goat and sheep) in Tarkwa, Ghana, and to estimate the daily intake and health risk. The concentrations of Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Cd, and Pb were measured with an inductively coupled plasma-mass spectrometer and Hg analysis was done using the mercury analyzer. Principal component analysis of the results showed a clear separation between chicken, grouped on one side, and the ruminants clustered on another side in both offal and muscle. Interestingly, As, Cd, Hg, Mn and Pb made one cluster in the offal of chicken. Chicken muscle also showed similar distribution with As, Hg and Pb clustered together. The daily intake of metals (mu g/kg body weight/day) were in the following ranges; As [0.002 (kidneys of goat and sheep)-0.19 (chicken gizzard)], Cd [0.003 (chicken muscle)-0.55 (chicken liver)], Hg [0.002 (goat muscle)-0.29 (chicken liver)], Pb [0.01 (muscles and kidneys of goat and sheep)-0.96 (chicken gizzard)] and Mn [0.13 (goat kidney)-8.92 (sheep liver)]. From the results, daily intakes of As, Cd, Hg, Pb and Mn in these food animals were low compared to the provisional tolerable daily intake guidelines. The THQs although less than one, indicated that contributions of chicken gizzard and liver to toxic metal exposure in adults and especially children could be significant. (C) 2014 Published by Elsevier Inc.
  • Nesta Bortey-Sam, Yoshinori Ikenaka, Shouta M. M. Nakayama, Osei Akoto, Yared Beyene Yohannes, Elvis Baidoo, Hazuki Mizukawa, Mayumi Ishizuka
    SCIENCE OF THE TOTAL ENVIRONMENT 496 471 - 478 0048-9697 2014/10 [Refereed][Not invited]
    As a developing country, the economic and population growth rates in Ghana over the past few years have seen a tremendous increase. The growing rate of industrialization is gradually leading to contamination and deterioration of the environment and pollution is likely to reach disturbing levels. Surface soil samples were collected randomly from 36 communities in the Kumasi Metropolis, Ghana, to determine the concentrations, distribution, sources and toxic potential of emission of polycyclic aromatic hydrocarbons (PAHs) since no such comprehensive study had been conducted. The mean concentration of total PAHs in the surface soils in each community ranged from 14.78 at Ahinsan to 2084 ng/g dry weight at Adum with an average of 442.5 +/- 527.2 ng/g dry weight. Diagnostic ratios and chemical mass balance models of the results showed that PAHs in surface soil samples from the study area were mainly from fuel combustion. Carcinogenic potency of PAH load from the city centre was approximately 150 times higher as compared to a pristine site, Kwame Nkrumah University of Science and Technology's Botanical Garden. BaP, a human carcinogen, contributed 70% of the total PAHs toxicity level from the city centre of Kumasi. (C) 2014 Elsevier B.V. All rights reserved.
  • Nozomi Fujisawa, Shouta M. M. Nakayama, Yoshinori Ikenaka, Mayumi Ishizuka
    ARCHIVES OF TOXICOLOGY 88 (9) 1739 - 1748 0340-5761 2014/09 [Refereed][Not invited]
    Halogenated aromatic hydrocarbons, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are known to cause severe heart defects in avian species. However, the mechanism of TCDD-induced chick cardiovascular toxicity is unclear. In this study, we investigated cyclooxygenase-2 (COX-2) as a possible mechanism of TCDD-induced cardiotoxicity. Fertile chicken eggs were injected with TCDD and a COX-2 selective inhibitor, NS398, and we investigated chick heart failure on day 10. We found that the chick heart to body weight ratio and atrial natriuretic factor mRNA expression were increased, but this increase was abolished with treatment of NS398. In addition, the morphological abnormality of an enlarged ventricle resulting from TCDD exposure was also abolished with co-treatment of TCDD and NS398. Our results suggested that TCDD-induced chick heart defects are mediated via the nongenomic pathway and that they do not require the genomic pathway.
  • Aksorn Saengtienchai, Yoshinori Ikenaka, Shouta M. M. Nakayama, Hazuki Mizukawa, Mayu Kakehi, Nesta Bortey-Sam, Wageh Sobhy Darwish, Toshio Tsubota, Masanori Terasaki, Amnart Poapolathep, Mayumi Ishizuka
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 33 (9) 2062 - 2069 0730-7268 2014/09 [Refereed][Not invited]
    Interspecific differences in xenobiotic metabolism are a key to determining relative sensitivities of animals to xenobiotics. However, information on domesticated livestock, companion animals, and captive and free-ranging wildlife is incomplete. The present study evaluated interspecific differences in phase II conjugation using pyrene as a nondestructive biomarker of polycyclic aromatic hydrocarbon (PAH) exposure. Polycyclic aromatic hydrocarbons and their metabolites have carcinogenic and endocrine-disrupting effects in humans and wildlife and can have serious consequences. The authors collected urine from 16 mammalian species and analyzed pyrene metabolites. Interspecific differences in urinary pyrene metabolites, especially in the concentration and composition of phase II conjugated metabolites, were apparent. Glucuronide conjugates are dominant metabolites in the urine of many species, including deer, cattle, pigs, horses, and humans. However, they could not be detected in ferret urine even though the gene for ferret Uridine 5'-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) 1A6 is not a pseudogene. Sulfate conjugates were detected mainly in the urine of cats, ferrets, and rabbits. Interestingly, sulfate conjugates were detected in pig urine. Although pigs are known to have limited aryl sulfotransferase activity, the present study demonstrated that pig liver was active in 1-hydroxypyrene sulfation. The findings have some application for biomonitoring environmental pollution. (c) 2014 SETAC
  • Yared Beyene Yohannes, Yoshinori Ikenaka, Shouta M. M. Nakayama, Mayumi Ishizuka
    ENVIRONMENTAL POLLUTION 192 121 - 128 0269-7491 2014/09 [Refereed][Not invited]
    Organochlorine pesticides (OCPs) and stable isotopes were measured in muscle from 4 bird and 5 fish species from the Ethiopian Rift Valley region where DDT is used for malaria control and vast agricultural activities are carried out. We investigated the bioaccumulation of OCPs such as DDTs, HCHs, chlordanes, and heptachlors between the species, and examined the potential risk posed by these compounds for bird species. Significant differences in contaminant profiles and levels were observed within the species. Levels of total OCPs ranged from 3.7 to 148.7 mu g/g lipid in bird and 0.04 to 10.9 mu g/g lipid in fish species. DDTs were the predominant contaminant, and a positive relationship between delta N-15 and Sigma DDT concentrations was found. The main DDT metabolite, p,p'-DDE was the most abundant and significantly greater concentrations in bird species (up to 138.5 mu g/g lipid), which could have deleterious effects on survival and/or reproduction of birds. (C) 2014 Elsevier Ltd. All rights reserved.
  • Zein Shaban Ibrahim, Mohamed Mohamed Ahmed, Samir Ahmed El-Shazly, Mayumi Ishizuka, Shoichi Fujita
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 (9) 1550 - 1559 0916-8451 2014/09 [Refereed][Not invited]
    Peroxisome proliferator activated receptor alpha (PPAR alpha) ligands, fibrates used to control hyperlipidemia. We demonstrated CYP2B induction by clofibric acid (CFA) however, the mechanism was not clear. In this study, HepG2 cells transfected with expression plasmid of mouse constitutive androstane receptor (CAR) or PPAR alpha were treated with CFA, phenobarbital (PB) or TCPOBOP. Luciferase assays showed that CFA increased CYP2B1 transcription to the same level as PB, or TCPOBOP in HepG2 transfected with mouse CAR But failed to induce it in PPAR alpha transfected cells. CYP2B expressions were increased with PB or CFA in Wistar female rats (having normal levels of CAR) but not in Wistar Kyoto female rats (having low levels of CAR). The induction of CYP2B by PB or CFA was comparable to nuclear CAR levels. CAR nuclear translocation was induced by CFA in both rat strains. This indicates that fibrates can activate CAR and that fibrates-insulin sensitization effect may occur through CAR, while hypolipidemic effect may operate through PPAR alpha.
  • Yoshinori Shimamoto, Kensuke Watanabe, Hiromi Ikadai, Masashi Okamura, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (8) 1147 - 1151 0916-7250 2014/08 [Refereed][Not invited]
    To examine the effect of Babesia infection on the level of the drug-metabolizing enzyme hepatic cytochrome P450 (CYP) 2D, we intraperitoneally inoculated Babesia microti into male ICR mice. CYP2D protein and CYP2D9 mRNA were significantly decreased at 12 days after infection with B. microti. The activity of bunitrolol 4-hydroxylase, which is catalyzed by CYP2D, was also significantly decreased. The mRNA levels of transcriptional regulators of CYP2D9, hepatocyte nuclear factor 4 alpha and signal transducer and activator of transcription 5b, were markedly suppressed. These results suggest that Babesia infection represses CYP2D expression in the mouse liver. The decline in CYP2D-dependent drug metabolism might be involved in the incidence of adverse drug reactions in patients with babesiosis.
  • Yared B. Yohannes, Yoshinori Ikenaka, Aksorn Saengtienchai, Kensuke P. Watanabe, Shouta M. M. Nakayam, Mayumi Ishizuka
    ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 106 95 - 101 0147-6513 2014/08 [Refereed][Not invited]
    Fish consumption is known to have several health benefits for humans. However, the accumulation of organic pollutants, like organochlorine pesticides (OCPs) could pose health hazards. Thus, OCPs in edible fish species (Oreochromis niloticus, Tilapia zillii, Carassius spp., and Clarias gariepinus) from Lake Ziway, an Ethiopian Rift Valley Lake were investigated to assess the potential human health hazards of these contaminants. Dichlorodiphenyltrichloroethanes (DDTs), hexachlorocyclohexanes (HCHs), chlordanes, and heptachlors were observed with Sigma OCPs concentration ranging from 1.41 to 63.8 ng g(-1) ww. DDTs were the predominant contaminants (0.9 to 61.9 ng g(-1) ww), followed by HCHs. The predominance of DDTs may be attributed to their current use in vector control and contamination from past usage. The estimated daily intakes (EDIs) of OCPs from all fish species were much lower than the acceptable daily intakes (ADIs), indicating that consumption of fish is at little risk to human health at present. However, the cancer risk estimates in the area of concern and the hazard ratios (HRs) of HCHs, DDTs, and heptachlors exceeded the threshold value of one, indicating daily exposure to these compounds is a potential concern. This may result in a lifetime cancer risk greater than of I in 10(6). (C) 2014 Elsevier Inc. All rights reserved.
  • Samak Sutjarit, Shota M. M. Nakayama, Yoshinori Ikenaka, Mayumi Ishizuka, Wijit Banlunara, Worawut Rerkamnuaychoke, Susumu Kumagai, Amnart Poapolathep
    TOXICOLOGY LETTERS 229 (1) 292 - 302 0378-4274 2014/08 [Refereed][Not invited]
    Fusarenon-X (FX), a type B trichothecene mycotoxin, is mainly produced by Fusarium crookwellense, which occurs naturally in agricultural commodities, such as wheat and barley. FX has been shown to exert a variety of toxic effects on multiple targets in vitro. However, the embryonic toxicity of FX in vivo remains unclear. In the present study, we investigated FX-induced apoptosis and the relationship between the genetic regulatory mechanisms and FX-induced apoptosis in the developing mouse brain of FX-treated pregnant mice. Pregnant mice were orally administered FX (3.5 mg/kg b.w.) and were assessed at 0,12, 24 and 48 h after treatment (HAT). Apoptosis in the fetal brain was determined using hematoxylin and eosin staining, the TUNEL method, immunohistochemistry for PCNA and electron microscopy. Gene expressions were evaluated using microarray and real time-reverse transcription polymerase chain reaction (qRT-PCR). Histopathological changes showed that the number of apoptotic cells in the telencephalon of the mouse fetus peaked at 12 HAT and decreased at 24 and 48 HAT. FX induced the up-regulation of Bax, Trp53 and Casp9 and down-regulated Bcl2 but the expression levels of Fas and Casp8 mRNA remained unchanged. These data suggested that FX induces apoptosis in the developing mouse brain in FX-treated dams. Moreover, the genetic regulatory mechanisms of FX-induced apoptosis are regulated by Bax, Bcl2, Trp53 and Casp9 or can be defined via an intrinsic apoptotic pathway. (C) 2014 Elsevier Ireland Ltd. All rights reserved.
  • Hazuki Mizukawa, Yoshinori Ikenaka, Shouta M. M. Nakayama, Kentaro Q. Sakamoto, Shoichi Fujita, Mayumi Ishizuka
    JAPANESE JOURNAL OF VETERINARY RESEARCH 62 (3) 107 - 115 0047-1917 2014/08 [Refereed][Not invited]
    Contamination levels of coplanar polychlorinated biphenyls (Co-PCBs), polycyclic aromatic hydrocarbons (PAHs), and dichlorodiphenyltrichloroethane (DDTs) were measured in the entire body of the large Japanese field mouse (Apodemus speciosus) collected from Hokkaido (Ishikari and Rankoshi) and Aomori prefecture (Takko) in Japan. Higher concentrations of PCBs including Co-PCBs, were observed in the mice collected from Ishikari than those from Rankoshi. The concentration of PAHs in the soil from Ishikari was also higher than that in the other sampling sites. The findings suggest that Ishikari is the most polluted area, probably because of human activities, depending on the population distribution. However, the observed contaminant levels were extremely lower compared to those in previous studies. The ratio of testis weight to body weight (TW/BW) was the lowest in the mice collected from Ishikari, which is the area contaminated with PAHs and p,p'-dichlorodiphenyldichloroethylene (DDE). However, the serum testosterone levels of mice from the Ishikari area were higher than those from the non-contaminated other areas although no significant differences. Previous studies have shown that a low-level exposure to dioxin related compounds (DRCs) disturbances in sexual function, resulting in the production of testosterone. This study showed that POPs exposure is one of the possibility of the high testosterone concentration in the mice of the Ishikari area in addition to a cause of biological and environmental factors such as habitat density, age, temperatures and/or food riches.
  • Chihiro Ishii, Yoshinori Ikenaka, Shouta M. M. Nakayama, Yuya Suzuki, Yutaka Watanuki, Yuji Watanabe, Yared Beyene Yohannes, Hazuki Mizukawa, Mayumi Ishizuka
    JAPANESE JOURNAL OF VETERINARY RESEARCH 62 (3) 143 - 149 0047-1917 2014/08 [Refereed][Not invited]
    Teuri Island, Hokkaido in Japan is an important place for seabirds breeding. We measured the concentrations of heavy metals (Hg, Cd, Cr, Co, Ni, Cu, Zn, and Pb) and a metalloid (As) in rhinoceros auklet (Cerorhinca monocerata) (n = 7), thick-billed murre (Uria lomvia) (n = 2), spectacled guillemot (Cepphus carbo) (n = 6), slaty-backed gull (Larus schistisagus) (n = 15), jungle crow (Corvus macrorhynchos) (n = 3), Japanese anchovy (Engraulis japonica) (n = 6) and Atka mackerel (Pleurogrammus azonus) (n = 2). Spectacled guillemot had high As concentrations, with its source being their feeding habitat. Concentration of Hg in kidney of jungle crow was higher than other seabird species at Teuri.
  • Wageh Sobhy Darwish, Yoshinori Ikenaka, Shouta M. M. Nakayama, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 76 (6) 789 - 797 0916-7250 2014/06 [Refereed][Not invited]
    Mycotoxins are fungal secondary metabolites that contaminate various feedstuffs and agricultural crops. The contamination of food by mycotoxins can occur before production, during storage, processing, transportation or marketing of the food products. High temperature, moisture content and water activity are among the predisposing factors that facilitate the production of mycotoxins in food. Aflatoxins, ochratoxins, fumonisins, deoxynivalenol and zearalenone are all considered the major mycotoxins produced in food and feedstuffs. In Africa, mycotoxin contamination is considered to be a major problem with implications that affect human and animal health and economy. Aflatoxin-related hepatic diseases are reported in many African countries. Ochratoxin and fumonisin toxicity in humans and animals is widespread in Africa. The available, updated information on the incidence of mycotoxin contamination, decontamination and its public health importance in Africa is lacking. The aim of this review is to highlight, update and discuss the available information on the incidence of mycotoxins in African countries. The public health implications and the recommended strategies for control of mycotoxins in food and agricultural crops are also discussed.
  • Wageh Sobhy Darwish, Yoshinori Ikenaka, Shouta Nakayama, Mayumi Ishizuka
    BIOLOGICAL TRACE ELEMENT RESEARCH 158 (2) 243 - 248 0163-4984 2014/05 [Refereed][Not invited]
    Copper (Cu2+) is an essential element that plays important roles in physiological functions of the body. However, high Cu2+ levels can have toxic implications. This study aims to investigate the constitutive response to Cu2+ exposure of xenobiotic-metabolizing enzymes in cultured rat liver (H4-II-E) cell lines. Rat cells were exposed to copper sulfate (0-500 mu M) for 24 h. The effects of Cu2+ on the messenger RNA (mRNA) expressions of phase I and II enzymes and regulatory elements were examined using real-time PCR. Metallothionein mRNA expression was induced in a dose-dependent manner after treatment with Cu2+. mRNA expressions of phase I enzymes such as cytochrome P450 1A1 and 1A2 (CYP1A1 and CYP1A2) were slightly induced after exposure to low concentrations of Cu2+; however, CYP1A1 and CYP1A2 mRNA expressions were significantly downregulated at higher Cu2+ concentrations. These effects corresponded with expression of aryl hydrocarbon receptor mRNA. The mRNA expressions of phase II enzymes were reduced upon exposure to Cu2+. In conclusion, phase I and II enzyme expressions were significantly modulated upon Cu2+ exposure. These results indicated that Cu2+ exposure had toxicological implications for cultured H4-II-E cells.
  • Thanit Saengtienchai, Saranya Poapolathep, Supaporn Isariyodom, Yoshinori Ikenaka, Mayumi Ishizuka, Amnart Poapolathep
    FOOD AND CHEMICAL TOXICOLOGY 66 307 - 312 0278-6915 2014/04 [Refereed][Not invited]
    Fusarenon-X (FX) is one of the trichothecene mycotoxins mainly produced by Fusarium crookwellense, which naturally occurs in agricultural commodities such as wheat and barley. To investigate the toxicokinetics of FX and its metabolite nivalenol (NIV), FX was then administered intravenously or orally to piglets at a dosage of 1 mg/1<g body weight. The concentrations of FX and NIV in the plasma and various tissues were measured using LC-MS/MS. The plasma concentrations of FX in the piglets were determined up to 24 h and 48 h after iv and po administration, respectively, and the concentration of NIV was detected up to 12 h after both types of administration. The C-p(0) of FX was 580.28 +/- 140.81 ng/ml after iv administration. The values of t(1/2 beta), V-ss and F-oral were 1.71 +/- 0.74 h, 0.009 +/- 0.002 ml and 74.40 +/- 18.96%, respectively. FX and NIV were detectable in the vital organs up to 24 h after po administration. The peak level of FX in the liver, the kidney, and the spleen, respectively, were 165.95 +/- 9.68 ng/g, 66.29 +/- 8.48 and 7.35 +/- 0.69 ng/g at 3 h following po administration. In vitro of liver postmitochondrial fractions with FX demonstrated that the liver and kidney are capable of FX-to-NIV metabolism. (C) 2014 Elsevier Ltd. All rights reserved.
  • Yared Beyene Yohannes, Yoshinori Ikenaka, Aksorn Saengtienchai, Kensuke P. Watanabe, Shouta M. M. Nakayama, Mayumi Ishizuka
    ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH 20 (12) 8663 - 8671 0944-1344 2013/12 [Refereed][Not invited]
    Dichlorodiphenyltrichloroethanes (DDTs) and heavy metals are ubiquitous contaminants with high bioaccumulation and persistence in the environment, which can have adverse effects on humans and animals. Although applications of DDTs have been banned in many countries, developing countries like Ethiopia are still using these for agricultural and medicinal purposes. In addition, heavy metals are naturally present in the aquatic environment and distributed globally. In this study, the occurrence, distribution, and ecological risk of DDTs and heavy metals in surface sediments from one of the Ethiopian rift valley lakes were studied. Twenty-five surface sediment samples from Lake Awassa, Ethiopia were collected and analyzed for DDTs and heavy metals. Results showed that concentrations of total DDTs ranged from 3.64 to 40.2 ng/g dry weight. High levels of DDTs were observed in the vicinity of inflow river side and coastal areas with agricultural activities. The heavy metals content were followed the order Zn > Ni > Pb > Cu > Cr > Co > As > Cd > Hg. Correlation analysis and principal components analysis demonstrated that heavy metals were originated from both natural and anthropogenic inputs. The levels of DDE and DDD in surface sediments exceeded the sediment quality guideline values, indicating that adverse effects may occur to the lake. A method based on toxic-response factor for heavy metals revealed that the calculated potential ecological risk indices showed low ecological risk for the water body.
  • Nozomi Fujisawa, Yusuke K. Kawai, Shouta M. M. Nakayama, Yoshinori Ikenaka, Hideaki Yamamoto, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (12) 1577 - 1583 0916-7250 2013/12 [Refereed][Not invited]
    There are two arylhydrocarbon receptor (AhR) isoforms in birds, AhR1 and AhR2. The varying sensitivity of AhR is reported to be related to two critical amino acids at positions 325 and 381 in the AhR1 ligand-binding domain. In this study, seven avian species whose in vivo dioxin sensitivity was known, and 13 species with no data regarding their in vivo dioxin sensitivity were examined. The two critical amino acids in the ligand-binding domain were investigated in avian species, and the results were compared with the taxonomy or phylogenetic trees for the bird AhR proteins. We found that the two critical amino acids did not correlate with the taxonomy or phylogeny of these proteins, suggesting that dioxin sensitivity was independent of taxonomy.
  • Hiroshi Kitamura, Shunsuke Kimura, Yoshinori Shimamoto, Jun Okabe, Masatoshi Ito, Tomomi Miyamoto, Yoshinori Naoe, Chisato Kikuguchi, Bob Meek, Chitoku Toda, Shiki Okamoto, Katsushi Kanehira, Koji Hase, Hiroshi Watarai, Mayumi Ishizuka, Assam El-Osta, Osamu Ohara, Ichiro Miyoshi
    FASEB JOURNAL 27 (12) 4940 - 4953 0892-6638 2013/12 [Refereed][Not invited]
    Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2-69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/FABP4 and PAI-1/SERPINE1 genes were increased by 4- and 1.8-fold, respectively, after short hairpin RNA-mediated knockdown (KD) of the USP2 gene, and such expression was alleviated by overexpression of USP2-69 in human myeloid cell lines. Supernatants derived from USP2-KD cells induced IL6 (similar to 6-fold) and SAA3 (similar to 15-fold) in 3T3-L1 adipocytes to suggest the anti-inflammatory properties of USP2. In addition, we observed a 30% decrease in the number of macrophages in mesenteric adipose tissue derived from USP2-69 transgenic mice fed a high-fat diet for 14 wk compared with that in their C57BL/6 littermates (P<0.01), which was consistent with a similar to 40% decrease in transcription of aP2 and PAI-1. The aP2 locus exhibited elevated chromatin accessibility (>2.1-fold), methylation of histone H3 lysine 4 (>4.5-fold), and acetylation of histone H4 (>2.5-fold) in USP2-KD cells. Transfection of isopeptidase-mutated USP2-69 did not alter chromatin conformation on the aP2 locus in USP2-KD cells. Our results suggest that USP2-69 suppresses meta-inflammatory molecules involved in the development of type-2 diabetes.
  • Yoshinori Shimamoto, Hiroshi Kitamura, Kimie Niimi, Yasunaga Yoshikawa, Fumio Hoshi, Mayumi Ishizuka, Eiki Takahashi
    MOLECULAR BIOLOGY REPORTS 40 (12) 6747 - 6755 0301-4851 2013/12 [Refereed][Not invited]
    The common marmoset (Callithrix jacchus) is increasingly being used as a non-human primate animal model in biomedical research. To perform accurate quantitative analysis of gene expression by quantitative reverse transcription polymerase chain reaction, reliable reference genes should be selected. In this study, we evaluated the expressions of 11 widely used reference genes: ACTB, ATP5F1, B2M, GAPDH, HPRT1, PGK1, PPIA, RN18S1, RPLP0, TBP and UBC in 12 tissues and five brain areas of healthy common marmosets. NormFinder and geNorm indicated that the most suitable reference genes for cross-sectional studies of the 17 tissues were RN18S1 and RPLP0. Conversely, ACTB and PPIA were the most suitable for analyzing brain samples; however, the expression of PGK1 fluctuated among brain areas. These results indicate that suitable reference genes differ between the tissues examined. This study provides fundamental information for gene expression studies of the common marmoset and highlights the importance of validating reference genes before quantification of target mRNAs.
  • Nobuo Sasaki, Tomoyuki Matumoto, Yoshinori Ikenaka, Shouta M. M. Nakayama, Mayumi Ishizuka, Akio Kazusaka, Shoichi Fujita
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (11) 1497 - 1502 0916-7250 2013/11 [Refereed][Not invited]
    The nitrofuran antimicrobial agent, furazolidone (FZ), is still used in veterinary medicine in some countries in the Middle and Far Eastern countries. The present study aimed to investigate the effects of successive bolus doses of FZ and its metabolite 3-amino-2-oxazolidinone (AOZ) on cytochrome P450 (CYP)-related activities in the livers of rats and chickens. Female Wistar rats and white Leghorn chickens were orally administered FZ once a day for 4 consecutive days. FZ-treated chickens showed an increase in multiple CYP-related activities, however, rats treated with FZ did not show these changes. In chickens, treatment with FZ also induced production of microsomal CYP2C6-like apoprotein. The present study demonstrated that FZ caused a multiple-type induction of CYP-related activities in chickens, but not in rats.
  • Yusuke K. Kawai, Kensuke P. Watanabe, Akihiro Ishii, Aiko Ohnuma, Hirofumi Sawa, Yoshinori Ikenaka, Mayumi Ishizuka
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY D-GENOMICS & PROTEOMICS 8 (3) 201 - 208 1744-117X 2013/09 [Refereed][Not invited]
    The cytochrome P450 (CYP) 1-3 families are involved in xenobiotic metabolism, and are expressed primarily in the liver. Ostriches (Struthio camelus) are members of Palaeognathae with the earliest divergence from other bird lineages. An understanding of genes coding for ostrich xenobiotic metabolizing enzyme contributes to knowledge regarding the xenobiotic metabolisms of other Palaeognathae birds. We investigated CYP1-3 genes expressed in female ostrich liver using a next-generation sequencer. We detected 10 CYP genes: CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2W2, CYP2AC1, CYP2AC2, CYP2AF1, and CYP3A37. We compared the gene expression levels of CYP1A5, CYP2C23, CYP2C45, CYP2D49, CYP2G19, CYP2AF1, and CYP3A37 in ostrich liver and determined that CYP2G19 exhibited the highest expression level. The mRNA expression level of CYP2G19 was approximately 2-10 times higher than those of other CYP genes. The other CYP genes displayed similar expression levels. Our results suggest that CYP2G19, which has not been a focus of previous bird studies, has an important role in ostrich xenobiotic metabolism. (C) 2013 Elsevier Inc. All rights reserved.
  • Yoshinori Ikenaka, Mami Oguri, Aksorn Saengtienchai, Shouta M M Nakayama, Shigeho Ijiri, Mayumi Ishizuka
    Environmental toxicology and pharmacology 36 (2) 567 - 578 1382-6689 2013/09 [Refereed][Not invited]
    Metabolic activity, particularly conjugation, was examined in fish by analyzing pyrene (a four-ring, polycyclic aromatic hydrocarbon) metabolites using high-performance liquid chromatography (HPLC) with fluorescence detector (FD), a mass spectrometry (MS) system, and kinetic analysis of conjugation enzymes. Fourteen fresh water fish species, including Danio rerio and Orizias latipes, were exposed to aqueous pyrene, and the resulting metabolites were collected. Identification of pyrene metabolites by HPLC/FD and ion-trap MS indicated that the major metabolites were pyrene glucuronide and pyrene sulfate in all 14 species. Differences were observed in pyrene glucuronide:pyrene sulfate ratio and in the total amount of pyrene conjugates excreted between fish species. Furthermore, a correlation was found between the amount of pyrene glucuronide present and the total amount of the pyrene metabolite eliminated. Kinetic analysis of conjugation by hepatic microsomes in vitro indicated that the differences in excreted metabolites reflected the differences in enzymatic activities.
  • Kensuke P. Watanabe, Yusuke K. Kawai, Yoshinori Ikenaka, Minami Kawata, Shin-Ichi Ikushiro, Toshiyuki Sakaki, Mayumi Ishizuka
    PLOS ONE 8 (9) e75689  1932-6203 2013/09 [Refereed][Not invited]
    Cytochrome P450 (CYP) of chicken and other avian species have been studied primarily with microsomes or characterized by cloning and protein expression. However, the overall existing isoforms in avian CYP1-3 families or dominant isoforms in avian xenobiotic metabolism have not yet been elucidated. In this study, we aimed to clarify and classify all of the existing isoforms of CYP1-3 in avian species using available genome assemblies for chicken, zebra finch, and turkey. Furthermore, we performed qRT-PCR assay to identify dominant CYP genes in chicken liver. Our results suggested that avian xenobiotic-metabolizing CYP genes have undergone unique evolution such as CYP2C and CYP3A genes, which have undergone avian-specific gene duplications. qRT-PCR experiments showed that CYP2C45 was the most highly expressed isoform in chicken liver, while CYP2C23b was the most highly induced gene by phenobarbital. Considering together with the result of further enzymatic characterization, CYP2C45 may have a dominant role in chicken xenobiotic metabolism due to the constitutive high expression levels, while CYP2C23a and CYP2C23b can be greatly induced by chicken xenobiotic receptor (CXR) activators. These findings will provide not only novel insights into avian xenobiotic metabolism, but also a basis for the further characterization of each CYP gene.
  • Shouta M. M. Nakayama, Yoshinori Ikenaka, Kyohei Hamada, Kaampwe Muzandu, Kennedy Choongo, John Yabe, Takashi Umemura, Mayumi Ishizuka
    ENVIRONMENTAL MONITORING AND ASSESSMENT 185 (6) 4907 - 4918 0167-6369 2013/06 [Refereed][Not invited]
    The lead-zinc (Pb-Zn) mine in Kabwe City and the copper-cobalt (Cu-Co) mine in the Copperbelt Province are major mining areas in Zambia. To examine the effects of metal pollution on wildlife, wild black rats (Rattus rattus and Rattus tanezumi) were captured in Kabwe and Chingola (in the Copperbelt Province), and in Lusaka (a noncontaminated site). Wild black rats in Kabwe accumulated significantly higher concentrations of Pb and Cd in various organs than rats from Lusaka. In Chingola, significantly higher concentrations of Cu, Co, Pb, and Cd were accumulated in wild black rats than in rats from Lusaka. These results were in accordance with metal accumulation patterns in soil. From toxicological aspects, concentrations of Pb and Cd in rats were generally low. However, metallothionein-1 (MT-1) and metallothionein-2 (MT-2) mRNA expression levels in wild black rats from Kabwe were significantly higher than those in rats from Lusaka. A generalized linear model (GLM) showed that concentrations of Zn and Cu had positive effects on the MT-1 and MT-2 mRNA expression. These results suggest that wild black rats in Zambian mining sites were exposed to metals that accumulated in their organs, causing biological responses such as MT mRNA induction. GLM indicated that heme oxygenase-1 (HO-1) mRNA expression could be a marker for Cr exposure.
  • Yared Beyene Yohannes, Yoshinori Ikenaka, Shouta M. M. Nakayama, Aksorn Saengtienchai, Kensuke Watanabe, Mayumi Ishizuka
    CHEMOSPHERE 91 (6) 857 - 863 0045-6535 2013/05 [Refereed][Not invited]
    The levels and bioaccumulation of organochlorine pesticides (OCPs) and heavy metals were studied in muscle and liver of three fish species, with two trophic levels, from Lake Awassa, Ethiopia. DDTs were the predominant organic pollutant in all species with a maximum level of 73.28 ng g(-1) wet weight (ww). p,p'-DDE was the predominate congener and showed a significant (p <0.001) relationship with delta N-15, which indicates that DDTs could biomagnified in the food web of the lake. Generally, high levels of heavy metals (Cd, Co, Cr, Cu, Ni, Pb, Zn and Hg) were found in liver samples as compared to muscles. The levels of Cd, Co, Cu, Ni, and Pb in liver samples showed negative correlation with delta N-15. They were found markedly higher in the lower trophic level fish species (p < 0.05) that indicates biodilution whereas; Zn level showed positive correlation with delta N-15. (C) 2013 Elsevier Ltd. All rights reserved.
  • Kazuyuki D. Tanaka, Yusuke K. Kawai, Yoshinori Ikenaka, Tsunehito Harunari, Tsutomu Tanikawa, Shoichi Fujita, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 75 (2) 135 - 139 0916-7250 2013/02 [Refereed][Not invited]
    Warfarin is a rodenticide commonly used worldwide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. Recently, warfarin-resistant brown rats (Rattus norvegicus) were found around the Aomori area of Japan. There is no significant difference in the metabolic activity of warfarin in sensitive and resistant brown rats. To clarify the mechanism underlying warfarin resistance, we cloned the VKORC1 gene from rats and identified a novel substitution of arginine to proline at position 33 of the VKORC1 amino acid sequence. Then, we determined the differences in kinetics of VKOR activity between warfarin-resistant and sensitive rats. Hepatic microsomal VKOR-dependent activity was measured over a range of vitamin K epoxide concentrations from 6.25 to 150 mu M. The V-max, values of resistant rats (0.0029 +/- 0.020 nmol/min/mg) were about one tenth of those of sensitive rats (0.29 +/- 0.12 nmol/min/mg). The K-m values of resistant rats (47 +/- 32 mu M) were similar to those of sensitive rats (59 +/- 18 mu M). Warfarin-sensitive rats exhibited enzyme efficiencies (V-max/K-m) which were ten-fold greater than those observed in resistant rats. It may mean that VKOR activity of warfarin-resistant Aomori rats is almost lost, because their enzymatic efficiencies are very low even without warfarin. Further studies are needed to clarify how these rats can survive with a markedly reduced VKOR activity and how they simultaneously exhibit warfarin resistance.
  • Yoshinori Ikenaka, Masaki Sakamoto, Takamaru Nagata, Hirokazu Takahashi, Yuichi Miyabara, Takayuki Hanazato, Mayumi Ishizuka, Tomohiko Isobe, Jun-Woo Kim, Kwang-Hyeon Chang
    JOURNAL OF TOXICOLOGICAL SCIENCES 38 (1) 131 - 136 0388-1350 2013/02 [Refereed][Not invited]
    We estimated acute toxicity of benzo[a]pyrene (B[a]P) using two cladoceran species, Ceriodaphnia reticulata and Daphnia magna, and also analyzed its impact on zooplankton community throughout an exposure experiment using small-scale mesocosms. LC50 of B[a]P for C. reticulata and D. magna was 4.3 and 4.7 mu g/l, respectively. However, individuals fed with Chlorella showed higher LC50, 6.1 mu g/l for C. reticulata and 8.0 mu g/l for D. magna. In the exposure experiment, we examined the impact of B[a]P on zooplankton community using conceivable concentrations in the environment (5 and 10 mu g/l) using typical zooplankton community in eutrophicated systems. Despite the residence time of B[a]P in the water column was short as < 4 days, application of B[a]P induced decrease of zooplankton abundance. However, the recovery pattern was different among cladocerans and rotifers. Consequently, B[a]P showed insecticide-like impacts, suppressing cladoceran populations and inducing the dominance of rotifers particularly under high concentration (10 mu g/l). Results have suggested that, even such short duration of B[a] P in the water body can have impact on zooplankton abundance and community structure. Since B[a]P easily precipitate to the bottom and rapidly disappears from the water body, careful monitoring and further assessment of the potential toxicity of polycyclic aromatic hydrocarbons are necessary.
  • Darwish WS, Eldaly EA, El-Abbasy MT, Ikenaka Y, Nakayama S, Ishizuka M
    The Japanese journal of veterinary research 61 Suppl S13 - 22 0047-1917 2013/02 [Refereed][Not invited]
  • Biological responses of xenobiotic metabolizing enzymes to lead exposure in cultured H4IIE rat cells.
    Wageh Sobhy Darwish, Yoshinori Ikenaka, Mayumi Ishizuka
    The Japanese journal of veterinary research 61 Suppl S48-53 - 53 0047-1917 2013/02 [Refereed][Not invited]
    This study was undertaken to investigate the constitutive response of xenobiotic metabolizing enzymes (XMEs) to lead (Pb2+) exposure in cultured rat liver (H4IIE) cell lines. Phase I enzymes such as CYP1A1 and CYP1A2 had mRNA expressions that were slightly induced after exposure to low concentrations of Pb2+; however, under higher concentrations of Pb2+, the mRNA expressions of CYP1A1 and CYP1A2 were significantly down-regulated. These effects were in correspondence with AhR mRNA expression. Phase II enzymes had mRNA expressions that were reduced upon exposure to Pb2+. Metallothionein mRNA expression was induced after treatment with Pb2+ in a dose-dependent trend. In conclusion, Phase I and II enzymes were significantly modulated upon lead exposure indicating some toxicological implications for lead exposure in cultured H4IIE cells.
  • Determination of organochlorine pesticides (OCPs) in the edible offal of Egyptian buffalo.
    Abdallah Fikry A Mahmoud, Wageh Sobhy Darwish, Alaa Eldin M A Morshdy, Elsaid A Eldaly, Yoshinori Ikenaka, Mayumi Ishizuka
    The Japanese journal of veterinary research 61 Suppl S58-63 - 63 0047-1917 2013/02 [Refereed][Not invited]
    Environmental contamination by OCPs has a great concern, since most of these pesticide compounds are very toxic and harmful to human and ecosystems. This study was conducted to determine the concentrations of OCPs residues in the edible offal (livers, kidneys and tongues) of Egyptian buffalo collected from three locations (Zagazig, Ismailia and Mansoura) in Egypt. Examined samples from Mansura city had the highest OCPs contamination load. Tongues had the highest concentration of these toxic residues in a comparison to livers and kidneys in the examined samples. The overall results showed that OCPs residues did not exceed the Egyptian maximum permissible limits in all of the samples analyzed from the three different locations.
  • Distribution of metals in organs of Clarias gariepinus, Heterobranchus bidorsalis, and Chrysichthys nigrodigitatus from the Offin River at Dunkwa-on-Offin, Ghana.
    Jemima T Marfo, Osei Akoto, Shouta M M Nakayama, Elvis Baidoo, Yoshinori Ikenaka, Mayumi Ishizuka
    The Japanese journal of veterinary research 61 Suppl S69-71 - 71 0047-1917 2013/02 [Refereed][Not invited]
    All heavy metals are potentially harmful to most organisms at some level of exposure and absorption. Concentrations of Co, Cr, Cu, Ni, Zn, Hg, Cd, As, and Pb were determined by atomic absorption spectrophotometry (AAS) in three fish species (C. gariepinus, C. nigrodigitatus, and H. bidorsalis) from the Offin River in Dunkwa township, Ghana. In the fish species, gills, livers, and muscles were analyzed. The metal that recorded the highest concentration was Zn, which was highly accumulated in the liver of C. gariepinus, but had the lowest concentration in the muscles of C. nigrodigitatus.
  • Determination of benzo[a]pyrene levels in ambient air and the source of polycyclic aromatic hydrocarbons using a diagnostic ratio method in Ghana.
    Bortey-Sam N, Akoto O, Ikenaka Y, Nakayama SM, Ishizuka M
    The Japanese journal of veterinary research 61 Suppl S72 - 4 0047-1917 2013/02 [Refereed][Not invited]
  • Heavy metal pollution in Japanese seabirds.
    Ishii C, Ikenaka Y, Nakayama SM, Suzuki Y, Watanuki Y, Watanabe Y, Fukuwaka MA, Yohannes YB, Kawai YK, Ishizuka M
    The Japanese journal of veterinary research 61 Suppl S75 - 6 0047-1917 2013/02 [Refereed][Not invited]
  • Metabolism of pyrene, a polycyclic aromatic hydrocarbon in freshwater turtles.
    Oroszlany B, Ikenaka Y, Saengtiengchan A, Oguri M, Nakayama SM, Ishizuka M
    The Japanese journal of veterinary research 61 Suppl S77 - 8 0047-1917 2013/02 [Refereed][Not invited]
  • Metal contaminated soil from mining area caused metal accumulation and biological responses in rats.
    Nakayama SM, Ikenaka Y, Muzandu K, Choongo K, Yabe J, Umemura T, Ishizuka M
    The Japanese journal of veterinary research 61 Suppl S79 - 81 0047-1917 2013/02 [Refereed][Not invited]
  • In vitro diazepam metabolism in horses.
    Hayami A, Darwish WS, Ikenaka Y, Nakayama SM, Ishizuka M
    The Japanese journal of veterinary research 61 Suppl S82 - 4 0047-1917 2013/02 [Refereed][Not invited]
  • Shouta M M Nakayama, Yoshinori Ikenaka, Kaampwe Muzandu, Kennedy Choongo, John Yabe, Taro Muroya, Shigeho Ijiri, Masao Minagawa, Takashi Umemura, Mayumi Ishizuka
    Archives of environmental contamination and toxicology 64 (1) 119 - 29 0090-4341 2013/01 [Refereed][Not invited]
    The Copperbelt region, upstream of the Kafue River, including Lake Itezhi-tezhi (ITT), in Zambia has extensive copper (Cu) mines. In our field study, geographic information system analysis in lake sediment indicated that the northern part of the lake, i.e., the Copperbelt region, could be the source of Cu pollution. Concentrations of Cu in stomach contents between fish species were not significantly different. However, Oreochromis spp. liver showed significantly greater Cu concentrations than those in other fish species. Log liver [Cu], standard length, and nitrogen stable isotope ratio were positively correlated only in Oreochromis spp. In the laboratory study, O. niloticus and O. latipes were exposed to Cu for 4 days, and recovery phases ≤ 28 days were examined. O. niloticus showed significantly greater concentrations of Cu compared with O. latipes at all sampling points. Significantly greater concentrations of Hg in Schilbe intermedius liver than for other fish species were observed, whereas O. macrochir showed significantly greater concentrations of cadmium. In conclusion, the northern part of the lake could be the source of Cu pollution in Lake ITT. Diet may not be the reason for high Cu accumulation in Oreochromis spp. Results from both field and laboratory studies imply that Oreochromis spp. contain high concentrations of Cu under normal physiological conditions.
  • John Yabe, Shouta M. M. Nakayama, Yoshinori Ikenaka, Kaampwe Muzandu, Kennedy Choongo, Geoffrey Mainda, Matthew Kabeta, Mayumi Ishizuka, Takashi Umemura
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 32 (1) 189 - 192 0730-7268 2013/01 [Refereed][Not invited]
    Concentrations of Pb, Cd, and other metals in tissues of 17 free-range and 32 commercial broiler chickens from the Kabwe mining town in Zambia were determined. Mean concentrations of Pb and Cd exceeded maximum levels for human consumption in some organs including muscle (Pb only) in free-range chickens, in contrast to low levels in broiler chickens. Human consumers in Kabwe could be exposed to Pb and Cd in free-range chickens. Environ. Toxicol. Chem. 2013;32:189192. (c) 2012 SETAC
  • Dominic Mureithi, Wageh Sobhy Darwish, Yoshinori Ikenaka, Laetitia Kanja, Mayumi Ishizuka
    JAPANESE JOURNAL OF VETERINARY RESEARCH 60 (4) 205 - 210 0047-1917 2012/11 [Refereed][Not invited]
    The cytochrome P450 (CYP) 3A family is involved in the elimination processes of almost 50% of commonly used drugs. CYP3A mRNA expressions in goat and rat gastrointestinal tracts in comparison to the liver were investigated using real-time PCR. In goats, the expression of CYP3A-like mRNAs was comparatively higher in the liver than in the gastrointestinal tract. The intestinal expression of CYP3A-like mRNA showed a gradual decrease from the duodenum to the ileum. In rats, the highest CYP3A62 mRNA expression was found in the duodenum followed by the liver. This study provides insights into the contribution of CYP3A enzymes to xenobiotic metabolism, especially in small ruminants such as goats.
  • John Yabe, Shouta M. M. Nakayama, Yoshinori Ikenaka, Kaampwe Muzandu, Mayumi Ishizuka, Takashi Umemura
    JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (10) 1345 - 1347 0916-7250 2012/10 [Refereed][Not invited]
    Intensive agricultural practices are recognized as significant sources of metal pollution in soils and pasture. This study investigated metal contamination in cattle offal from an agricultural area in Zambia, where inorganic fertilizers, agricultural lime, and pesticides are routinely applied. The highest median values (mg/kg, wet weight) of Cu (40.9), Zn (35.2), Cr (1.35) and Ni (0.594) were recorded in the liver, whereas the highest median values of Pb (0.061) and Cd (0.049) were found in kidneys. Maximum levels of Hg, As and Co were under 0.2 mg/kg in both organs. Pb and Cd did not exceed the benchmark values in cattle offal for human consumption and did not pose immediate health risks. Concentrations of Ni and Cr could present a public health concern. Monitoring of metal accumulations in offal of cattle, not only from well-known polluted environments but also agricultural areas, should be done regularly for the health of human consumers.
  • Yoshinori Ikenaka, Shouta M. M. Nakayama, Taro Muroya, John Yabe, Satoru Konnai, Wageh Sobhy Darwish, Kaampwe Muzandu, Kennedy Choongo, Geoffrey Mainda, Hiroki Teraoka, Takashi Umemura, Mayumi Ishizuka
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 31 (10) 2300 - 2305 0730-7268 2012/10 [Refereed][Not invited]
    The Republic of Zambia is rich in mineral resources, such as zinc (Zn) and lead (Pb), and mining is a key industry in Zambia. A previous study of Pb pollution in Kabwe, one of the main mining areas, found that soil was contaminated with high levels of toxic metals over a substantial area. In the present study, the authors focus on toxic metal pollution in cattle, one of the most important domestic animals in Zambia. Blood samples from cattle in Kabwe and a control area (Lusaka) were tested for toxic metal content. They also measured mRNA expression of metal-responsive proteins and cytokines in white blood cells using real-time reverse transcriptase polymerase chain reaction. In the present in vitro study, The authors cultured peripheral blood mononuclear cells (PBMCs) from cattle, exposing them to Pb acetate for 24?h and analyzing mRNA expression of metal-responsive proteins and selected cytokines. Lead concentrations in cattle blood from Kabwe were significantly greater than those from Lusaka, as were the mRNA expressions of metallothionein-2 (MT-2), tumor necrosis factor-a (TNF-a), interferon-? (IFN-?), interleukin-1 beta (IL-1 beta), IL-6, and inducible nitric oxide synthase (iNOS). The present in vitro study demonstrated that Pb exposure led to an increase in the expressions of MT-2, TNF-a, IL-1 beta, and iNOS, similar to those found in vivo. These results indicate the possibility of immune system modulations in cattle from the Kabwe area. Environ. Toxicol. Chem. 2012; 31: 23002305. (c) 2012 SETAC
  • Ethel M'kandawire, Michelo Syakalima, Kaampwe Muzandu, Girja Pandey, Martin Simuunza, Shouta M. M. Nakayama, Yusuke K. Kawai, Yoshinori Ikenaka, Mayumi Ishizuka
    GENE 506 (2) 310 - 316 0378-1119 2012/09 [Refereed][Not invited]
    The study determined heavy metal concentrations and MT1 nucleotide sequence [phylogeny] in liver of the Kafue lechwe. Applicability of MT1 as a biomarker of pollution was assessed. cDNA-encoding sequences for lechwe MT1 were amplified by RT-PCR to characterize the sequence of MT1 which was subjected to BLAST searching at NCBI. Phylogenetic relationships were based on pairwise matrix of sequence divergences calculated by Clustal W. Phylogenetic tree was constructed by NJ method using PHILLIP program. Metals were extracted by acid digestion and concentrations of Cr, Co, Cu, Zn, Cd, Pb, and Ni were determined using an AAS. MT1 mRNA expression levels were measured by quantitative comparative real-time RT-PCR. Lechwe MT1 has a length of 183 bp, which encode for MT1 proteins of 61AA, which include 20 cysteines. Nucleotide sequence of lechwe MT1 showed identity with sheep MT (97%) and cattle MT1E (97%). Phylogenetic tree revealed that lechwe MT1 was clustered with sheep MT and cattle MT1E. Cu and Ni concentrations and MT1 mRNA expression levels of lechwe from Blue Lagoon were significantly higher than those from Lochinvar (p<0.05). Concentrations of Cd and Cu, Co and Cu, Co and Pb, Ni and Cu, and Ni and Cr were positively correlated. Spearman's rank correlations also showed positive correlations between Cu and Co concentrations and MT mRNA expression. PCA further suggested that MT mRNA expression was related to Zn and Cd concentrations. Hepatic MT1 mRNA expression in lechwe can be used as biomarker of heavy metal pollution. (C) 2012 Elsevier B.V. All rights reserved.
  • Tomohiro Okagawa, Satoru Konnai, Hirohisa Mekata, Naftaly Githaka, Saori Suzuki, Edward Kariuki, Francis Gakuya, Esther Kanduma, Tatsuya Shirai, Ryoyo Ikebuchi, Yoshinori Ikenaka, Mayumi Ishizuka, Shiro Murata, Kazuhiko Ohashi
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 148 (3-4) 373 - 379 0165-2427 2012/08 [Refereed][Not invited]
    Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus coffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo. (c) 2012 Elsevier B.V. All rights reserved.
  • Tomohide Uno, Mayumi Ishizuka, Takao Itakura
    ENVIRONMENTAL TOXICOLOGY AND PHARMACOLOGY 34 (1) 1 - 13 1382-6689 2012/07 [Refereed][Not invited]
    Cytochrome P450 (CYP) enzymes are members of the hemoprotein superfamily, and are involved in the mono-oxygenation reactions of a wide range of endogenous and exogenous compounds in mammals and plants. Characterization of CYP genes in fish has been carried out intensively over the last 20 years. In Japanese pufferfish (Takifugu rubripes), 54 genes encoding P450s have been identified. Across all species of fish, 137 genes encoding P450s have been identified. These genes are classified into 18 CYP families: namely, CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP17, CYP19, CYP20, CYP21, CYP24, CYP26, CYP27, CYP39, CYP46 and CYP51. We pinpointed eight CYP families: namely, CYP1, CYP2, CYP3, CYP4, CYP11, CYP17, CYP19 and CYP26 in this review because these CYP families are studied in detail. Studies of fish P450s have provided insights into the regulation of P450 genes by environmental stresses including water pollution. In this review, we present an overview of the CYP families in fish. (C) 2012 Elsevier B.V. All rights reserved.
  • Shouta M. M. Nakayama, Yoshinori Ikenaka, Kaampwe Muzandu, Kennedy Choongo, Ethel M'kandawire, Jun Yasuda, Mayumi Ishizuka
    ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 80 333 - 338 0147-6513 2012/06 [Refereed][Not invited]
    Hippopotami (Hippopotamus amphibius L) are large semi-aquatic mammals that can be exposed to metals and metalloid from both terrestrial and aquatic environments. Therefore, knowledge of metal and metalloid accumulation characteristics in hippopotami living in the national park is important from ecotoxicological point of view. Levels of toxic metals (Cd, Pb and Hg) and metalloid (As) in hippopotami liver from the South Luangwa National Park in Zambia were far lower compared to the established values of toxic levels in cattle. No temporal variations of metal levels in hippopotami were observed, probably because of good management condition and the lack of anthropogenic activities around the national park. However, hippopotami liver accumulated significantly higher concentrations of Hg compared to soil, sediment and their food (plants), most likely due to a process of biomagnification throughout a trophic chain. Moreover, hippopotami liver and land plants showed significantly higher Cd levels than those of soil. These results strongly suggest that hippopotami liver accumulate higher levels of these metals if surrounding environment is contaminated. Levels of Cr and Ni in hippopotami liver were higher compared to other toxic metals. Since this is the first report to show the Cr and Ni levels and bio-accumulation characteristics of Hg and Cd in hippopotami, we concluded that continuous monitoring and evaluation of toxic effects of these metals on hippopotami should be conducted. (C) 2012 Elsevier Inc. All rights reserved.
  • Yoshinori Shimamoto, Mizuki Sasaki, Hiromi Ikadai, Mayumi Ishizuka, Naoaki Yokoyama, Ikuo Igarashi, Fumio Hoshi, Hiroshi Kitamura
    JOURNAL OF VETERINARY MEDICAL SCIENCE 74 (2) 241 - 245 0916-7250 2012/02 [Refereed][Not invited]
    To investigate effects of Babesia infection on drug metabolism, we intraperitoneally inoculated B. microti into ICR mice and measured the expression and activity of hepatic cytochrome P450 (CYP) 3A, a major drug-metabolizing enzyme. Twelve days after infection, CYP3A11 mRNA, CYP3A protein and activity and mRNAs of nuclear receptors, which participate in CYP3A expression, were significantly reduced. These results suggest that B. microti infection suppresses CYP3A-dependent drug metabolism. Additionally, tumor necrosis factor (TNF)-alpha and nitric oxide synthase (NOS) 2 mRNAs were induced in the infected mouse liver. Since TNT-a is one of the potent mediators that induce NOS2 and repress CYP3A transcription, the possible involvement of TNF-alpha in this downregulation of CYP3A was discussed.
  • Marumi Ohno, Yoshinori Ikenaka, Mayumi Ishizuka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 417 (1) 484 - 489 0006-291X 2012/01 [Refereed][Not invited]
    Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are well-conserved transcription factors among species. However, there are a very limited number of reports on the physiological function of AHR, particularly on the regulation of AHR by endogenous compounds. We hence investigated the effects of all-trans retinoic acid (atRA) on cytochrome P450 (CYP)1A1 gene transcription as a model of AHR-regulated transcription mechanisms in HepG2 cells, a human hepatoma cell line. Treatment with atRA significantly reduced transactivation and expression of CYP1A1 mRNA to less than half of its control value, and this inhibitory effect was mediated by RAR alpha. The result of chromatin immunoprecipitation assay indicated that treatment with atRA at 1-100 nM drastically inhibited the recruitment of ARNT to DNA regions containing xenobiotic responsive elements. In conclusion, atRA at physiological concentrations could reduce AHR-mediated gene transcription via the inhibition of recruitment of ARNT to relevant DNA regions. (C) 2011 Elsevier Inc. All rights reserved.
  • Marumi Ohno, Yoshinori Ikenaka, Mayumi Ishizuka
    JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY 26 (1) 16 - 22 1095-6670 2012/01 [Refereed][Not invited]
    Sudan dyes possess a high affinity to the aryl hydrocarbon receptor (AHR) and potently induce its target genes, such as cytochrome P450 (CYP) 1A1, through unknown mechanisms. We investigated a detailed event occurring in cells after binding of Sudan dye to AHR in HepG2 cells. Treatment with 10 mu M Sudan III caused rapid translocation of AHR into the nucleus and increased expression levels of human CYP1A1 mRNA by approximately 20-fold after 16 and 24 h. The transactivation was due to the activation of a region located at -1137 to +59 bp from CYP1A1, in particular, four xenobiotic responsive elements (XREs) existing in the region. AHR and the Ah receptor nuclear translocator interacted with XRE sequences in a gel shift assay using nuclear extract from Sudan III-treated HepG2 cells. Moreover, we suggest that constitutive androstane receptor could modify CYP1A1 transactivation by Sudan III. (C) 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:16-22 2012; View this article online at DOI 10.1002/jbt.20408
  • Haruki Ueda, Yoshinori Ikenaka, Shouta M. M. Nakayama, Tomoko Tanaka-Ueno, Mayumi Ishizuka
    AQUATIC TOXICOLOGY 105 (3-4) 337 - 343 0166-445X 2011/10 [Refereed][Not invited]
    The present study examines amphibian metabolic activity - particularly conjugation - by analysis of pyrene (a four ring, polycyclic aromatic hydrocarbon) metabolites using high-performance liquid chromatography (HPLC) with fluorescence detector (FD), a mass spectrometry detector (MS) system and kinetic analysis of conjugation enzymes. Six amphibian species were exposed to pyrene (dissolved in water): African claw frog (Xenopus laevis); Tago's brown frog (Rana tagoi); Montane brown frog (Rana ornativentris); Wrinkled frog (Rana rugosa); Japanese newt (Cynops pyrrhogaster); and Clouded salamander (Hynobius nebulosus); plus one fish species, medaka (Oryzias latipes); and a fresh water snail (Clithon retropictus), and the resultant metabolites were collected. Identification of pyrene metabolites by HPLC and ion-trap MS system indicated that medaka mainly excreted pyrene-1-glucuronide (PYOG), while pyrene-1 -sulfate (PYOS) was the main metabolite in all amphibian species. Pyrene metabolites in amphibians were different from those in invertebrate fresh water snails. Inter-species differences were also observed in pyrene metabolism among amphibians. Metabolite analysis showed that frogs relied more strongly on sulfate conjugation than did Japanese newts and clouded salamanders. Furthermore, urodelan amphibians, newts and salamanders, excreted glucose conjugates of pyrene that were not detected in the anuran amphibians. Kinetic analysis of conjugation by hepatic microsomes and cytosols indicated that differences in excreted metabolites reflected differences in enzymatic activities. Furthermore, pyrenediol (PYDOH) glucoside sulfate was detected in the Japanese newt sample. This novel metabolite has not been reported previously to this report, in which we have identified unique characteristics of amphibians in phase II pyrene metabolism. (C) 2011 Elsevier B.V. All rights reserved.
  • John Yabe, Shouta M. M. Nakayama, Yoshinori Ikenaka, Kaampwe Muzandu, Mayumi Ishizuka, Takashi Umemura
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 30 (8) 1892 - 1897 0730-7268 2011/08 [Refereed][Not invited]
    Concentrations of lead, cadmium, and other metals in the liver and kidneys of cattle near a lead-zinc mine in Kabwe (Zambia), which is ranked among the 10 worst polluted places on earth, were compared with other Zambian towns. Metal concentrations were measured in the liver and kidneys of 51 cattle from Kabwe and other Zambian towns. The maximum metal concentrations, expressed in mg/kg and dry weight, in the liver or kidneys were 398.4 (Cu), 252.6 (Zn), 77.81 (Cr), 19.37 (Cd), 7.57 (Ni), 1.8 (Pb), 1.04 (Co), 0.112 (Hg), and 0.05 (As). Concentrations of Pb and Cd in Kabwe cattle were higher than levels in other Zambian towns. The mean concentration of Cd exceeded benchmark values in offal destined for human consumption. Levels of Ni and Cr may also pose public health concerns. Concentrations of Pb and Cr, Pb and Cu, Cd and Zn, Cd and Hg, Zn and Cu, Cu and Co, as well as Co and Ni were positively correlated. The present study also highlighted the dangers of exposure of animals and humans to a mixture of toxic metals. Environ. Toxicol. Chem. 2011;30:1892-1897. (C) 2011 SETAC
  • Marumi Ohno, Wageh S. Darwish, Yoshinori Ikenaka, Wataru Miki, Mayumi Ishizuka
    FOOD AND CHEMICAL TOXICOLOGY 49 (6) 1285 - 1291 0278-6915 2011/06 [Refereed][Not invited]
    Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase! metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax-containing oil (100 mg Ax/kg body weight/day for 3 days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-, 8.5-, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax-containing oil. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax-containing oil intake can alter CYP1A-dependent activities through two different mechanisms: (1) induction of CYP1A1 mRNA, protein expression, and activity; and (2) inhibition of the electron supply for the enzyme. (C) 2011 Elsevier Ltd. All rights reserved.
  • Shouta M. M. Nakayama, Yoshinori Ikenaka, Kyohei Hamada, Kaampwe Muzandu, Kennedy Choongo, Hiroki Teraoka, Naoharu Mizuno, Mayumi Ishizuka
    ENVIRONMENTAL POLLUTION 159 (1) 175 - 181 0269-7491 2011/01 [Refereed][Not invited]
    Metal (Cr, Co, Cu, Zn, Cd, Pb, Ni) and metalloid (As) accumulation was studied in roadside soil and wild rat (Rattus sp.) samples from near a Pb Zn mine (Kabwe, Zambia) and the capital city of Zambia (Lusaka). The concentrations of the seven metals and As in the soil samples and Pb in the rat tissue samples were quantified using atomic absorption spectroscopy. The concentrations of Pb, Zn, Cu, Cd, and As in Kabwe soil were much higher than benchmark values. Geographic Information System analysis indicated the source of metal pollution was mining and smelting activity. Interestingly, the area south of the mine was more highly contaminated even though the prevailing wind flow was westward. Wild rats from Kabwe had much higher tissue concentrations of Pb than those from Lusaka. Their body weight and renal Pb levels were negatively correlated, which suggests that mining activity might affect terrestrial animals in Kabwe. (C) 2010 Published by Elsevier Ltd.
  • Wageh S. Darwish, Yoshinori Ikenaka, Marumi Ohno, Elsaid A. Eldaly, Mayumi Ishizuka
    FOOD AND CHEMICAL TOXICOLOGY 48 (11) 3201 - 3208 0278-6915 2010/11 [Refereed][Not invited]
    Ungulates (deer, cattle and horses) are reported as animal species which show extreme-accelerated metabolism of CYP1A substrates, such as ethoxyresorufin compared to rats. This study was undertaken to investigate whether accumulation of carotenoids is a possible cause for inter-species difference in CYP1A-dependent activity in this group of animals. The relationship between inter-species differences in CYP1A-dependent activity and the accumulated carotenoids and retinoids as candidates of dietary CYP1A inducers in ungulate species was clarified. Interestingly, there were positive correlations between the accumulated carotenoids, such as beta-carotene, with both EROD activity and CYP1A protein expression. These correlations were negative with the accumulated retinoids, such as retinol. The beta-carotene was major component of carotenoids in ungulates, and known as an inducer of CYP1A. On the other hand, the retinol is reported as the inhibitor of CYP1A. Other factors which affect CYP1A1 expression, such as polycyclic aromatic hydrocarbons, were also analyzed. To cancel the effects of inter-species difference in CYP1A induction signal cascade among these animals, the rat cell line (H4-II-cells) was treated with the extracted carotenoids from the examined animals. In conclusion, carotenoids and retinoids may have direct effects on the inter-species differences in CYP1A-dependent activity and protein expression. (C) 2010 Elsevier Ltd. All rights reserved.
  • Darwish WS, Kawai Y, Ikenaka Y, Yamamoto H, Muroya T, Ishizuka M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 9 72 (9) 1237 - 1241 0916-7250 2010/09 [Refereed][Not invited]
  • Fumihiro Sata, Norie Kurahashi, Susumu Ban, Kimihiko Moriya, Kazuyuki D. Tanaka, Mayumi Ishizuka, Hiroyuki Nakao, Yuichiro Yahata, Hirohisa Imai, Hidehiro Kakizaki, Katsuya Nonomura, Reiko Kishi
    JOURNAL OF SEXUAL MEDICINE 8 7 (8) 2729 - 2738 1743-6095 2010/08 [Refereed][Not invited]
    Introduction. Hypospadias is a common congenital anomaly caused by incomplete fusion of urethral folds. Development of the urethra and external genital system in the male fetus is an androgen-dependent process. In this regard, enzymes 17 beta-hydroxvsteroid dehydrogenase type 3 (17 beta HSD3, encoded by HSD17B3) and steroid 5 alpha-reductase type 2 (encoded by SRD5A2) play crucial roles. Aim. To investigate the possible associations between common polymorphisms in HSD17B3 as well as well-known V89L polymorphism in SRD5A2 and risk of hypospadias. Methods. A case-control study was performed between 1999 and 2005. There were 89 Japanese boys with hypospadias and 291 newborn controls. We genotyped HSD17B3 -1999T>C, +10A>G, +20A>G, +139G>A (V31I), +913G>A (G289S), and SRD5A2 +336G>C (V89L) polvmorphisms by allelic discrimination assay. We measured mRNA expression of the wildtype G289 allele and the mutant S289 allele of the HSD17B3 gene in the transfected human fetal kidney HEK293 cells. Main Outcome Measures. Assessment of hypospadias including its severity and HSD17B3 and SRD5A2 genes using DNA blood samples: allele and genotype distribution of single nucleotide polymorphisms in these two genes in cases and controls. Results. In our study, the risk of hypospadias was significantly higher in subjects carrying homozygous HSD17133 +913A (289S) alleles (odds ratio [OR]: 3.06; 95% confidence interval [CI]: 1.38-6.76). The risk of severe hypospadias was much higher in these subjects (OR: 3.93; 95% CI: 1.34-11.49). The mRNA expression levels of HSD17B3 G289 were higher than those of IISD17B3 S289 mutant (P < 0.001). In addition, the risk of severe hypospadias increased in boys carrying the SRD5A2 +336C (89L) allele (OR: 3.19; 95% CI: 1.09-9.36). Conclusions. These results suggest that the HSD17B3 G289S polymorphism may be a potential risk modifier for hypospadias. Our findings provide evidence that a certain genotype related to androgen production may potentiate risk of hypospadias. Sata F, Kurahashi N, Ban S, Moriya K, Tanaka KD, Ishizuka M, Nakao H, Yahata Y, Imai H, Kakizaki H, Nonomura K, and Kishi R. Genetic polymorphisms of 17 beta-hydroxysteroid dehydrogenase 3 and the risk of hypospadias. J Sex Med 2010;7:2729-2738.
  • Shouta M. M. Nakayama, Yoshinori Ikenaka, Kaampwe Muzandu, Kennedy Choongo, Balazs Oroszlany, Hiroki Teraoka, Naoharu Mizuno, Mayumi Ishizuka
    ARCHIVES OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY 59 (2) 291 - 300 0090-4341 2010/08 [Refereed][Not invited]
    We measured the level of heavy metal accumulation in lake sediments, herbivorous (Oreochromis niloticus) and carnivorous (Serranochromis thumbergi) fish, and crayfish (Cherax quadricarinatus) from Lake Itezhi-tezhi (ITT) and Lake Kariba. We used atomic absorption spectrophotometry to quantify the levels of seven heavy metals (Cr, Co, Cu, Zn, Cd, Pb, and Ni). The sediment and the herbivorous fish O. niloticus accumulated a very high concentration of Cu in Lake ITT, most likely due to the discharge of Cu waste from a mining area 450 km upstream. The aquatic species we sampled in Lake Kariba had higher concentrations of Cr, Ni, and Pb relative to those in Lake ITT. This is most likely due to anthropogenic activities, such as the use of leaded petrol and antifouling agents in marine paints. Interestingly, we observed a negative correlation between the coefficient of condition (K) and Ni concentration in the crayfish hepatopancreas. Both O. niloticus and the crayfish had much higher biota-sediment accumulation factors (BSAF) for Cu, Zn, and Cd relative to Cr, Co, Pb, and Ni. The rank of BSAF values for O. niloticus (Cu > Cd > Zn) and C. quadricarinatus (Zn > Cd > Cu) differed from the expected ranks based on the general order of affinity of metals (Cd >> Zn > Cu).
  • Mami Takiguchi, Wageh S. Darwish, Yoshinori Ikenaka, Marumi Ohno, Mayumi Ishizuka
    TOXICOLOGICAL SCIENCES 116 (1) 79 - 91 1096-6080 2010/07 [Refereed][Not invited]
    Xenobiotic metabolism in oral tissues, especially in the tongue, has never been reported. In the present study, the metabolic activation/detoxification ability of promutagens in the tongue and the expression levels of related enzymes were investigated. Quantitative PCR analysis of rat tongue demonstrated constitutive messenger RNA (mRNA) expression of numerous drug-metabolizing enzymes. In particular, we detected mRNA, protein expression, and enzymatic activity of cytochrome P450 (CYP)1A1 in the tongue tissue. Metabolic activation of promutagens in the tongue was estimated using benzo[a]pyrene or heterocyclic amines (HCAs), found in cooked meat and tobacco products. Metabolic activation levels of HCAs in the tongue were comparable to those in the liver. In contrast, the expression levels of glutathione-S-transferase (GST) and uridine diphosphate-glucuronosyltransferase (UGT) in the tongue were considerably lower compared with those in the liver, and as a result, the mutagenic activity in the tongue was not decreased by GST- or UGT-dependent conjugation. Treatment of rats with sudan III, a typical inducer of CYP1A1, resulted in markedly increased CYP1A1 mRNA, protein expressions, and CYP1A-dependent enzymatic and mutagenic activities. In addition, CYP1A1 mRNA expression in carcinoma cells (SAS) was induced by sudan III exposure. In conclusion, mutagenic activation of xenobiotics and an increased risk of cancer in the tongue were observed in this study. Furthermore, ingestion of drug-metabolizing enzyme inducers has the potential to increase the metabolic activation in the tongue tissue and increase the risk of biomolecular attack by promutagens.
  • Kensuke P. Watanabe, Aksorn Saengtienchai, Kazuyuki D. Tanaka, Yoshinori Ikenaka, Mayumi Ishizuka
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY C-TOXICOLOGY & PHARMACOLOGY 152 (1) 114 - 119 1532-0456 2010/06 [Refereed][Not invited]
    Scattering coumarin derivative rodenticides in broad areas have caused primary- and secondary-poisoning incidents in non-target wild birds. In this study, we compared factors determining warfarin sensitivity between bird species and rats based on vitamin K 2,3-epoxide reductase (VKOR) kinetics, VKOR inhibition by warfarin and warfarin metabolism assays. In VKOR characterization, chickens and ostriches showed significantly lower enzymatic efficiencies than rats (one-sixth and one-third, respectively), suggesting bird species depend more on a non-VKOR vitamin K source. On the other hand, the inhibition constants (K-i) of VKOR for warfarin were significantly different between chickens and ostriches (113 +/- 2.5 mu M and 0.64 +/- 0.39 mu M, respectively). Interestingly, the ostrich K-i was similar to the values for rats (0.28 +/- 0.09 mu M). The K-i results reveal a surprising possibility that VKOR in some bird species are easily inhibited by warfarin. Warfarin metabolism assays also showed a large inter-species difference in bird species. Chickens and ostriches showed higher metabolic activity than that of rats, while mallards and owls showed only a slight ability to metabolize warfarin. In this study, we clarified the wide inter-species difference that exists among birds in xenobiotic metabolism and sensitivity to a rodenticide. (C) 2010 Elsevier Inc. All rights reserved.
  • Yusuke K. Kawai, Yoshinori Ikenaka, Shoichi Fujita, Mayumi Ishizuka
    MAMMALIAN GENOME 21 (5-6) 320 - 329 0938-8990 2010/06 [Refereed][Not invited]
    Members of the cytochrome P450 family 1 (CYP1s) are involved in the detoxification and bioactivation of numerous environmental pollutants and phyto-chemicals such as polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and flavonoids. The vertebrate CYP1 gene comprises four subfamilies: CYP1A, CYP1B, CYP1C, and CYP1D. Recently, the CYP1D gene was identified in fish, and subsequently in the platypus. These findings indicate the possibility that all vertebrates have a functional CYP1D subfamily. However, there is no information on the mammalian CYP1D gene. In this study we investigated the genomic location of CYP1D genes in mammals and other vertebrates in silico. We also performed phylogenetic analysis and calculated the identities and similarities of CYP1D sequences. The data from synteny and phylogenetic analyses of CYP1D genes demonstrated the evolutionary history of the CYP1 gene family. The results suggested that CYP1D became a nonfunctional pseudogene in human and bovine species; however, several other mammals possess functional CYP1D genes. The promoter regions of CYP1D genes were also examined. Unlike other CYP1 isoforms, few xenobiotic responsive element (XRE)-like sequences were found upstream of the CYP1D genes. Analysis of mammalian CYP1Ds also provided new insight into the relationship between CYP1 genes and the aryl hydrocarbon receptor.
  • Hye-Sook Chang, Miyan Ko, Mayumi Ishizuka, Shoichi Fujita, Akira Yabuki, Mohammad Alamgir Hossain, Osamu Yamato
    NUTRITION RESEARCH 30 (6) 435 - 440 0271-5317 2010/06 [Refereed][Not invited]
    There is evidence that onions and garlic protect against cancer in humans. It has been suggested that this effect is partly due to the organosulfur compounds in Allium vegetables and that these substances act through induction of phase II detoxification enzymes. Here, we hypothesized that alk (en)yl thiosulfates, sodium n-propyl thiosulfate (NPTS), and sodium 2-propenyl thiosulfate (2PTS), which were identified in onions and garlic, respectively, may induce phase II enzymes. Therefore, rat hepatoma cells (H4IIE) were cultured with 1 to 100 mu mol/L of NPTS or 2PTS for 48 hours at 37 degrees C; and the activities and messenger RNA (mRNA) expression levels of phase II enzymes in H4IIE cells were investigated. The effects of diallyl trisulfide and tert-butylhydroquinone, known as phase 11 inducers, were also examined as positive controls and compared with the responses of NPTS and 2PTS. Quinone reductase (QR) activity and mRNA expression levels of QR and epoxide hydrolase 1 were significantly increased by 2PTS (P < .05-.005). In particular, QR activity was increased at a relatively low concentration of 2PTS (10 mu mol/L). However, glutathione S-transferase activity and mRNA expression levels of glutathione S-transferase A5 and uridine diphosphate glucuronosyl transferase 1A1 were not changed by 2PTS. In contrast, NPTS did not affect the activities and mRNA expression levels of these phase II enzymes. These results show that 2PTS can induce phase II enzymes, and its inductive effect is comparable or superior to that of diallyl trisulfide and tert-butylhydroquinone. (C) 2010 Elsevier Inc. All rights reserved.
  • Wageh Sobhy Darwish, Yoshinori Ikenaka, Elsaid Abozeid Eldaly, Marumi Ohno, Kentaro Q. Sakamoto, Shoichi Fujita, Mayumi Ishizuka
    JOURNAL OF VETERINARY MEDICAL SCIENCE 72 (5) 561 - 566 0916-7250 2010/05 [Refereed][Not invited]
    The objective of this study was to investigate and characterize the metabolic activities of CYP1A in deer, cattle and horses in comparison to those of rats using ethoxyresorufin O-deethylation (EROD) and methoxyresorufin O-demethylation (MROD) assays. We performed an inhibition study for these activities using anti-rat CYP1A1 antibody and identified that these activities were due to the CYP1A subfamily. Interspecies differences in the CYP1A-dependent activities were highly observed in this study. In particular, we found that the horse had the highest EROD and MROD activities among the examined animal species. In the kinetic analysis, the horses showed the highest Vmax and catalytic efficiency (Vmax/Km), followed by the cattle, deer and rats.
  • Kentaro Q. Sakamoto, Katsufumi Sato, Akiko Kato, Daisuke Fukui, Gen Bando, Yasuhiko Naito, Yoshiaki Habara, Mayumi Ishizuka, Shoichi Fujita
    PHYSIOLOGICAL AND BIOCHEMICAL ZOOLOGY 2 83 (2) 232 - 238 1522-2152 2010/03 [Refereed][Not invited]
    Prolonged abnormal vomiting causes metabolic alkalosis. Many seabirds are known to feed their chicks by regurgitation. We hypothesized that metabolic alkalosis occurs in seabirds even under natural conditions during the breeding season. Adelie penguins Pygoscelis adeliae feed their chicks by regurgitating food for 50-60 d until the chicks fledge. In this study, the concentrations of Cl(-), HCO(3)(-), Na(+), K(+), pH, and Pco(2) in the blood of breeding Adelie penguins were measured throughout the chick-rearing season. The pH of penguin venous blood shifted from 7.54 in the guarding period to 7.47 in the creche period. Decreasing Cl(-) and increasing HCO(3)(-) blood concentrations in parents were associated with increasing mass of their brood in the guarding period, the early phase of the rearing season, indicating that regurgitating to feed chicks causes loss of gastric acid and results in relative metabolic alkalosis. The inverse trend was observed during the creche period, the latter phase of the rearing season, when parents spent more time at sea and have fewer opportunities for gastric acid loss. This was assumed to be the recovery phase. These results indicate that regurgitation might cause metabolic alkalosis in breeding Adelie penguins. To our knowledge, this is the first report to indicate that seabirds exhibit metabolic alkalosis due to regurgitation to feed chicks under natural conditions.
  • Darwish WS, Morshdy AE, Ikenaka Y, Ibrahim ZS, Fujita S, Ishizuka M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 2 72 (2) 221 - 224 0916-7250 2010/02 [Refereed][Not invited]
  • Kim HS, Sakai N, Saito K, Fujita S, Ishizuka M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 1 72 (1) 7 - 11 0916-7250 2010/01 [Refereed][Not invited]
  • Noriaki Sakai, Mayumi Ishizuka
    EXPERT OPINION ON DRUG METABOLISM & TOXICOLOGY 5 (11) 1421 - 1433 1742-5255 2009/11 [Refereed][Not invited]
    Rats are an important tool in pharmacology and toxicology. The authors focus on rat P450s in relation to diazepam metabolism. In particular, considerable attention is devoted to the CYP2D subfamily, which is a group of highly polymorphic enzymes. First, the metabolic profiles of diazepam of humans and other animals are compared. In this review, the authors describe a novel genetic polymorphism of diazepam observed in commonly used rat strains and compare it to human genetic polymorphisms. The genetic basis underlying diazepam polymorphism in rats is also discussed. The authors conclude that the metabolic capacities and major metabolic pathways of diazepam are quite different among rat strains and in the Wistar strain due to CYP2D3 genetic polymorphism, which is independent of the debrisoquine polymorphism catalyzed by CYP2D2. The situation, in which major metabolites differ depending on animal strain, will be highly problematic not only in pharmacokinetic studies of test compounds, but also in pharmacological or toxicological tests. This may provide researchers who use experimental animals insights into important aspects of the genetic background of experimental animals. Thus, great caution must be taken in the choice of rat strains for studies of drug metabolism.
  • Nakayama SM, Tanaka-Ueno T, Sakamoto KQ, Fujita S, Ishizuka M
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 10 71 (10) 1407 - 1411 0916-7250 2009/10 [Refereed][Not invited]
  • Marumi Ohno, Kentaro Q. Sakamoto, Mayumi Ishizuka, Shoichi Fujita
    PHYTOTHERAPY RESEARCH 23 (8) 1134 - 1139 0951-418X 2009/08 [Refereed][Not invited]
    Polyphenols have been shown to have potent antioxidant activity, and therefore, food containing polyphenols is expected to contribute to the prevention of cancer. However, food contains not only polyphenols but also various other constituents. We used the Ames test to investigate the effects of crude extracts of whole cacao products, which are known to be rich in polyphenols, on the mutagenicity of benzo[a]pyrene (B[a]P) in Salmonella typhimurium strain TA 98 and tert-butyl hydroperoxide (t-BuOOH) in S. typhimurium strain TA 102. B[a]P induces mutagenicity by metabolic activation and t-BuOOH induces it by generation of free radicals. While white chocolate did not modulate the numbers of revertant colonies produced by B[a]P treatment, milk chocolate and cacao powder extracts did. On the other hand, surprisingly, none of the cacao products tested affected the number of revertant colonies when t-BuOOH was used as the mutagen. At maximum concentration (13.25 mg cacao powder/ml), the crude cacao powder extract reduced ethoxyresorufin O-deethylase activity to 17.4% of the control, suggesting that whole cacao products inhibit cytochrome P450 (CYP) 1A activity. In conclusion, inhibition of CYP1A activity by cacao products may prevent DNA damage by reducing metabolic activation of carcinogens. Copyright (C) 2009 John Wiley & Sons, Ltd.
  • Noriaki Sakai, Kentaro Q. Sakamoto, Shoichi Fujita, Mayumi Ishizuka
    DRUG METABOLISM AND DISPOSITION 37 (8) 1703 - 1710 0090-9556 2009/08 [Refereed][Not invited]
    Cytochrome P450 (P450) 2D2 (CYP2D2) enzyme is known to metabolize the majority of typical substrates of the human CYP2D6 enzyme, which is the most extensively characterized polymorphic drug-metabolizing enzyme. Despite its impact on drug metabolism in rats, the transcriptional regulation of CYP2D2 remains to be elucidated. We clarified the molecular mechanism of CYP2D2 gene expression. The CYP2D2 gene was positively regulated by the poly(C)-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K) through a transcriptional regulatory element located in the 5'-flanking region from -94 to -113. To date, nothing is known about the potential role of hnRNP K in P450 gene regulation. Thus, this is the first report that hnRNP K protein is involved in CYP2D2 gene regulation. Furthermore, we elucidated the genetic basis of the extremely low expression of CYP2D2 mRNA in Dark Agouti (DA) rats. Because of its relatively low abundance, DA rats have been frequently used for the study of CYP2D substrate metabolism as the animal model of the poor metabolizer phenotype for CYP2D6 compared with Sprague-Dawley rats as an extensive metabolizer phenotype. We found a single substitution within the transcriptional regulatory element of the CYP2D2 gene in DA rats. The mutation was detected in the polypyrimidine sequence that is the preferred binding site for hnRNP K protein. The mutation within the transcriptional regulatory element attenuated the binding of hnRNP K protein. In conclusion, decreased recruitment of hnRNP K protein to the mutated sequence causes the low expression of CYP2D2 mRNA in DA rats.
  • Khlood M. El Bohi, Yoshiharu Hashimoto, Kaampwe Muzandu, Yoshinori Ikenaka, Zein Shaban Ibrahim, Akio Kazusaka, Shoichi Fujita, Mayumi Ishizuka
    JAPANESE JOURNAL OF VETERINARY RESEARCH 57 (2) 109 - 118 0047-1917 2009/08 [Refereed][Not invited]
    Pleurotus cornucopiae (PC) mushrooms are found in the field and commonly known in Japan as Tamogidake mushrooms. The present study investigated the protective effects of an aqueous extract of PC on carbon tetrachloride (CCl4)-induced hepatotoxicity and the possible mechanism involved in this protection including cytochrome P450 (CYP) 2E1. Wistar rats were pretreated with aqueous extracts of PC (0, 100, 200, and 400 mg/kg) orally for 8 days prior to the intraperitoneal. administration of a single dose of CCl4 (0.5 ml/kg) or corn oil. Pretreatment with PC mushroom extract significantly prevented the increased serum enzyme activities of alanine and aspartate aminotransferases in a dose-dependent manner, and suppressed the expression of CYP2E1. PC mushroom extract also protected hepatocytes from the damage effects of CCl4 as remarked by histological and electromicroscopical findings. It was concluded that repeated daily doses of aqueous extracts of PC mushroom reduced the toxic effects exerted by CCl4 on the liver.
  • Kenzi Nakano, Mayumi Ishizuka, Kentaro Q. Sakamoto, Shoichi Fujita
    BIOMETALS 22 (2) 345 - 351 0966-0844 2009/04 [Refereed][Not invited]
    Accumulating evidence, including experiments using cytochrome P450 1a2 (Cyp1a2) gene knock-out mice (Cyp1a2(-/-)), indicates that the development of chemically induced porphyria requires the expression of CYP1A2. It has also been demonstrated that iron enhances and expedites the development of experimental uroporphyria, but that iron alone without CYP1A2 expression, as in Cyp1a2(-/-) mice, does not cause uroporphyria. The role of iron in the development of porphyria has not been elucidated. We examined the in vivo effect of iron deficiency on hepatic URO accumulation in experimental porphyria. Mice were fed diets containing low (iron-deficient diet (IDD), 8.5 mg iron/kg) or normal (normal diet (ND), 213.7 mg iron/kg) levels of iron. They were treated with 3-methylcholanthrene (MC), an archetypal inducer of CYP1A, and 5-aminolevulinate (ALA), precursors of porphyrin and heme. We found that uroporphyrin (URO) levels and uroporphyrinogen oxidation (UROX) activity were markedly increased in ND mice treated with MC and ALA, while the levels were not raised in IDD mice with the same treatments. CYP1A2 levels and methoxyresorufin O-demethylase (MROD) activities, the CYP1A2-mediated reaction, were markedly induced in the livers of both ND and IDD mice treated with MC and ALA. UROX activity, supposedly a CYP1A2-dependent activity, was not enhanced in iron-deficient mice in spite of the fact of induction of CYP1A2. We showed that a sufficient level of iron is essential for the development of porphyria and UROX activity.
  • Kentaro Q. Sakamoto, Katsufumi Sato, Mayumi Ishizuka, Yutaka Watanuki, Akinori Takahashi, Francis Daunt, Sarah Wanless
    PLOS ONE 4 (4) e5379  1932-6203 2009/04 [Refereed][Not invited]
    An ethogram is a catalogue of discrete behaviors typically employed by a species. Traditionally animal behavior has been recorded by observing study individuals directly. However, this approach is difficult, often impossible, in the case of behaviors which occur in remote areas and/or at great depth or altitude. The recent development of increasingly sophisticated, animal-borne data loggers, has started to overcome this problem. Accelerometers are particularly useful in this respect because they can record the dynamic motion of a body in e. g. flight, walking, or swimming. However, classifying behavior using body acceleration characteristics typically requires prior knowledge of the behavior of free-ranging animals. Here, we demonstrate an automated procedure to categorize behavior from body acceleration, together with the release of a user-friendly computer application, "Ethographer''. We evaluated its performance using longitudinal acceleration data collected from a foot-propelled diving seabird, the European shag, Phalacrocorax aristotelis. The time series data were converted into a spectrum by continuous wavelet transformation. Then, each second of the spectrum was categorized into one of 20 behavior groups by unsupervised cluster analysis, using k-means methods. The typical behaviors extracted were characterized by the periodicities of body acceleration. Each categorized behavior was assumed to correspond to when the bird was on land, in flight, on the sea surface, diving and so on. The behaviors classified by the procedures accorded well with those independently defined from depth profiles. Because our approach is performed by unsupervised computation of the data, it has the potential to detect previously unknown types of behavior and unknown sequences of some behaviors.
  • Kentaro Q. Sakamoto, Katsufumi Sato, Yasuhiko Naito, Yoshiaki Habara, Mayumi Ishizuka, Shoichi Fujita
    JOURNAL OF VETERINARY MEDICAL SCIENCE 71 (3) 341 - 344 0916-7250 2009/03 [Refereed][Not invited]
    We investigated the blood status of 9 Weddell seal mothers and 9 pups during the breeding season ill a field study conducted from November to December 2004 at a breeding colony in Antarctica. The blood glucose and total cholesterol concentrations were higher ill the Pups than in the mothers. On the other hand, the blood urea nitrogen concentration was lower in the pups than in the mothers. Growth-associated depletion of blood triglyceride was observed in the pups and may have been due to the post-weaning fast. The results characterize the blood status of Weddell seals in relation to physiological adaptations for breeding.
  • Noriaki Sakai, Konomu Saito, Kentaro Q. Sakamoto, Mayumi Ishizuka, Shoichi Fujita
    DRUG METABOLISM AND DISPOSITION 37 (2) 268 - 271 0090-9556 2009/02 [Refereed][Not invited]
    Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) is widely used as a sedative, hypnotic, and anti-anxiety drug. At low diazepam concentrations, p-hydroxylation is the major metabolic pathway in rat liver microsomes. However, there are marked (similar to 300-fold) inter-and intrastrain differences in the activity among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. In our previous study, we determined that a deficiency of CYP2D3 protein, not CYP2D2, was responsible for the inter-and intrastrain differences in diazepam p-hydroxylation (Drug Metab Dispos 33:1657-1660, 2005). Quantitative real-time polymerase chain reaction (PCR) did not provide enough evidence to explain the inter-and intrastrain differences in the expression of CYP2D3 protein. Nucleotide sequence analysis revealed the insertion of a thymine in exon 8 of the CYP2D3 gene in the poor diazepam metabolizers. This single nucleotide mutation caused a shift in the reading frame and introduced a premature termination signal. It is noteworthy that the heme binding region, which is essential to maintain proper heme binding and active cytochrome P450 enzymes, was consequently deleted by the premature termination signal. In contrast, no mutation was detected in the CYP2D3 gene of extensive metabolizers. Thus, the truncated CYP2D3 must be a nonfunctional enzyme in poor metabolizers. In addition, we developed a convenient and specific genotyping assay using PCR-restriction, fragment-length polymorphism to distinguish homozygotes from heterozygotes. The genotyping gave results fully consistent with those of the inter-and intrastrain differences in diazepam p-hydroxylation.
  • Zein S. Ibrahim, Mayumi Ishizuka, Mohamed Soliman, Khlood ElBohi, Wageh Sobhy, Kaampwe Muzandu, Azza M. Elkattawy, Kentaro Q. Sakamoto, Shoichi Fujita
    JAPANESE JOURNAL OF VETERINARY RESEARCH 56 (3) 119 - 128 0047-1917 2008/11 [Refereed][Not invited]
    Nigella sativa (family Ranunculaceae) is an annual plant that has been traditionally used on the Indian subcontinent and in Middle Eastern countries. In this study, we investigated the effect of N. sativa oil on the drug-metabolizing cytochrome P450 (CYP) enzymes and whether it has a protective effect against the acute hepatotoxicity Of CCl4. Intraperitoneal injection of rats with CCl4 drastically decreased CYP2E1, CYP2B, CYP3A2, CYP2C11, and CYP1A2 mRNA and protein expressions. Oral administration of 1 ml/kg N. sativa oil every day for one week prior to CCl4 injection alleviated CCl4-induced suppression of CYP2B, CYP3A2, CYP2C11, and CYP1A2. Moreover, CCl4 increased iNOS and TNF alpha mRNA, while N. sativa oil administration for one week prior to CCl4 injection downregulated the CCl4-induced iNOS mRNA and up-regulated IL-10 mRNA. These results indicate that N. sativa oil administration has a protective effect against the CCl4-mediated suppression of hepatic CYPs and that this protective effect is partly due to the downregulation of NO production and up-regulation of the anti-inflammatory IL-10.
  • Mayumi Ishizuka, Kazutoshi Fujioka, Takayuki Shibamoto
    JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 56 (15) 6093 - 6096 0021-8561 2008/08 [Refereed][Not invited]
    A recently developed headspace/solid-phase microextraction/gas chromatograph (GC) equipped with a nitrogen-phosphorus detector (NPD) (HS/SPME/GC/NPD) method was used to analyze acrylamide formed in an aqueous polyacrylamide solution (25%) treated by heat or photo-irradiation. Original polyacrylamide contained 0.43 +/- 0.11 mu g/mL of acrylamide. When polyacrylamide solution was heated at 70 degrees C for 16 h with 0.5% potassium persulfate, the amount of acrylamide increased to 1.02 +/- 0.11 mu g/mL. When polyacrylamide solution was irradiated by UV (lambda = 300 nm) for 16 h with 0.05% 2-anthraquinone sulfate sodium salt, the amount of acrylamide increased to 1.14 +/- 0.54 mu g/mL. Polyacrylamide has been used in cosmetic formulations. The present study, therefore, suggests that there is another route of acrylamide exposure to humans in addition to foods and beverages.
  • Mayumi Ishizuka, Tsutomu Tanikawa, Kazuyuki D. Tanaka, Min Heewon, Fumie Okajima, Kentaro Q. Sakamoto, Shoichi Fujita
    JOURNAL OF TOXICOLOGICAL SCIENCES 33 (3) 283 - 291 0388-1350 2008/08 [Refereed][Not invited]
    Warfarin is commonly used worldwide as a rodenticide. It inhibits coagulation of blood by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity. An inadequate supply of vitamin K blocks the production of prothrombin and causes hemorrhage. It has been reported that repeated or long-term treatments with this drug cause resistance in wild rodents. However, the mechanism of warfarin resistance in rodents is still not known precisely. Recent studies reported and identified the function of the molecule, vitamin K epoxide reductase complex subunit 1 (VKORC1), which is the main unit of VKOR. An amino acid substitution in VKORC1 is one of the supposed mechanisms of warfarin resistance. An accelerated detoxification system involving cytochrome P450 (CYP) could also cause the rodenticide resistance. Administration of SKF-525A, a potent inhibitor for P450, increased the mortality due to reduction of warfarin metabolism in warfarin-resistant rats. Meanwhile, the appearance of warfarin-resistant rodents has led to the development of the more effective and toxic rodenticide superwarfarin, which is widely used in Europe and the USA. However, animals resistant to this second-generation rodenticide have already been reported in Europe. In this review, we focus on the mechanism and the pleiotropic effects of pesticide resistance in wild rodents.
  • Downregulation of male-specific cytochrome P450 by profenofos
    Gihan G. Moustafa, Zein S. Ibrahim, Mohamed M. Ahmed, Mervat H. Ghoneim, Kentaro Q. Sakamoto, Mayumi Ishizuka, Shoichi Fujita
    JAPANESE JOURNAL OF VETERINARY RESEARCH 56 (2) 109 - 118 0047-1917 2008/08 [Refereed][Not invited]
    The health hazards of individual organophosphorus insecticides have been characterized by their acute toxicity, mainly by investigating their cholinesterase inhibition. However, the chronic effects of most of these toxicants on the drug-metabolizing enzymes have not been investigated. Profenofos (O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate) is an organophosphorus pesticide widely used in cotton cultivation. In the present study, we investigated the effect of profenofos on male-specific cytochrome P450 (CYP) enzymes in adult Wistar rats. We orally administered 17.8 mg/kg body weight, twice weekly for 65 days. Profenofos downregulated levels of hepatic and testicular CYP2C11 and CYP3A2 mRNA and protein expression. Testicular aromatase (CYP19A) mRNA was decreased in the profenofos-treated rats compared to controls. Overall, the present study suggests that profenofos acts as an endocrine disruptor of male-specific CYP enzymes and affects testosterone concentration, which implicates its deleterious effects on animal or human males chronically exposed to organophosphorus pesticide.
  • Seiko Sasaki, Fumihiro Sata, Shizue Katoh, Yasuaki Saijo, Sonomi Nakajima, Noriaki Washino, Kanae Konishi, Susumu Ban, Mayumi Ishizuka, Reiko Kishi
    AMERICAN JOURNAL OF EPIDEMIOLOGY 167 (6) 719 - 726 0002-9262 2008/03 [Refereed][Not invited]
    Maternal smoking during pregnancy can result in both pregnancy complications and reduced size of the fetus and neonate. Among women who smoke, genetic susceptibility to tobacco smoke also is a likely causative factor in adverse pregnancy outcomes. A prospective cohort study was conducted among 460 pregnant women who delivered live singletons in Sapporo, Japan, from 2002 to 2005. Multiple linear regression models were used to estimate associations of maternal smoking and polymorphisms in two genes encoding N-nitrosamine-metabolizing enzymes-NAD(P)H: quinone oxidoreductase 1 (NQO1) and cytochrome P-450 2E1 (CYP2E1)-with birth size. Among infants born to smokers with the NQO1 homozygous wild-type allele, birth weight, birth length, and birth head circumference were significantly reduced (p < 0.01 for each factor). For the homozygous wild-type CYP2E1 allele, birth weight was lower by an estimated 195 g (standard error, 55; p < 0.001) among smokers. These genotypes did not confer adverse effects among women who had never smoked or who quit smoking during the first trimester. The adverse effects of maternal smoking on infant birth size may be modified by maternal genetic polymorphisms in N-nitrosamine-metabolizing enzymes among Japanese subjects. These results may help in directing smoking cessation interventions during pregnancy, especially among susceptible women.
  • Sasaki N, Sakai N, Ikenaka Y, Kamiya T, Heewon M, Sakamoto KQ, Ishizuka M, Fujita S
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 3 70 (3) 223 - 226 0916-7250 2008/03 [Refereed][Not invited]
  • Nahla A. G. Ahmed Refat, Zein Shaban Ibrahim, Gihan Gamal Moustafa, Kentaro Q. Sakamoto, Mayumi Ishizuka, Shoichi Fujita
    JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY 22 (2) 77 - 84 1095-6670 2008 [Refereed][Not invited]
    Azo dyes form a major class of chemically related compounds that are ubiquitous in foods, paints, printing inks, cosmetics, and also used as biological stains in histological and histopathological laboratories and clinics. Sudan I, sudan III, and sudan IV have been classified as category 3 carcinogens by International Agency for Research on Cancer. In this study, we investigated the difference between these three sudan dyes in induction of CYP1A1. We intraperitoneally treated Wistar rats with each of the three sudan dyes (I, III, and IV) for 3 days. Treatment of Wistar rats with sudan I produced the highest induction of CYP1A1 protein and mRNA whereas treatment of Wistar rats with sudan III produced about two third of CYP1A1 protein and mRNA than induced by sudan I. Furthermore, treatment of Wistar rats with sudan IV produced the lowest induction of CYP1A1 protein and mRNA which is about two third of that induced with sudan III treatment. We further investigated the effect of these sudan dyes on CYP1A1 transcription through investigating the xenobiotic response element (XRE) reporter activity in HepG2. The XRE reporter activity study showed the same trend of activity of sudan dyes comparable to the effects on CYP1A1 mRNA and protein. Immunohistochemical study revealed a differential pattern of distribution of CYP1A1 protein in rat liver among the three sudan dyes, apparent in the centrilobular and midzonal region with sudan III, progressing to panlobular with sudan I, whereas sudan IV showed a reversal of pattern of induction with the most intense staining in the periportal region. Our results suggest that there is an inverse relationship between the molecular size of the three sudan dyes and their ability to induce CYP1A. (c) 2008 Wiley Periodicals, Inc.
  • Gihan Gamal Moustafa, Zein Shaban Ibrahim, Yoshiharu Hashimoto, M. Alkelch Alkelch, Kentaro Q. Sakamoto, Mayumi Ishizuka, Shoichi Fujita
    ARCHIVES OF TOXICOLOGY 81 (12) 875 - 881 0340-5761 2007/12 [Refereed][Not invited]
    To investigate the effect of the phosphorothoate insecticide profenofos on male specific gene expression on rat testis, 16-week-old Wistar rats were orally administered at dose of 17.8 mg/kg twice weekly for 65 days. Gene expression in the testes was monitored by DNA microarray analysis and real-time RT-PCR, which revealed that genes related to steroidogenesis including cytochrome P450 17A1 (CYP17A1), steroidogenic acute regulatory protein (StAR) and CYP11A1 were significantly increased. Besides the testes were histopathologicaly examined, which revealed testicular destruction and degeneration represented by a layer of columnar epithelium, oedematous changes surrounding the seminiferous tubules besides vacuolated spermatogonial cells and more elongated Leydig cells. These data suggest that profenofos considered as one of the male reproductive toxicants. Furthermore, we propose that the above three steroidogenic-related genes and the gene of acrosomal reaction as potential biomarkers of testicular toxicity.
  • Ban S, Kondo T, Ishizuka M, Sasaki S, Konishi K, Washino N, Fujita S, Kishi R
    Fukuoka igaku zasshi = Hukuoka acta medica 5 98 208 - 214 0016-254X 2007/05 [Refereed][Not invited]
  • Mayumi Ishizuka, Fumie Okajima, Tsutomu Tanikawa, Heewon Min, Kazuyuki D. Tanaka, Kentaro Q. Sakamoto, Shoichi Fujita
    DRUG METABOLISM AND DISPOSITION 35 (1) 62 - 66 0090-9556 2007/01 [Refereed][Not invited]
    Wild roof rats (Rattus rattus) live in proximity to human habitats, and they may carry numerous pathogens of infectious diseases. Pest control is important for public health, and warfarin is a commonly used rodenticide worldwide. However, continual use of warfarin may cause drug resistance in rodents and lead to failure of their control, especially in urbanized areas. In warfarin-resistant rats, the warfarin level in plasma was significantly lower after oral administration than that in the control warfarin-sensitive rats. Warfarin is metabolized by cytochrome P450 (P450), and hydroxylation of warfarin by P450 isoforms was significantly higher in warfarin-resistant rats (2-fold). Western blot analysis indicated that the level of CYP3A2 expression in warfarin-resistant rats was significantly larger than in warfarin-sensitive rats. The NADPH-P450 reductase activities in resistant rats were 8-fold higher than those in sensitive rats. In vivo, the administration of the P450 potent inhibitor proadifen (SKF-525A) increased the mortality of warfarin in the warfarin-resistant roof rats. We concluded that the mechanism of warfarin resistance in Tokyo roof rats is caused by increased clearance of warfarin.
  • Kaampwe Muzandu, Mayumi Ishizuka, Kentaro Q. Sakamoto, Zein Shaban, Khlood El Bohi, Akio Kazusaka, Shoichi Fujita
    TOXICOLOGY AND APPLIED PHARMACOLOGY 215 (3) 330 - 340 0041-008X 2006/09 [Refereed][Not invited]
    Peroxynitrite formed by the reaction of superoxide and nitric oxide is a highly reactive species with a role in various pathological processes such as cancer, chronic inflammation, and cardiovascular and neurological diseases. In the present study, the effect of the carotenoids, lycopene and beta-carotene, on peroxynitrite-mediated modifications in plasmid DNA as well as cellular DNA and proteins were investigated. In pUC18 plasmid DNA, these carotenoids strongly inhibited DNA strand breaks caused by peroxynitrite generated from 3-morpholinosydnonimine (SIN-1). SIN-1 was also used to determine effects on DNA damage and protein tyrosine nitration in Chinese hamster lung fibroblasts. SIN-1 dose-dependently increased nitration of proteins in cells above basal levels as determined by Western blotting. This nitration was inhibited in the presence of the uric acid as well as lycopene. Physiological concentrations (0.31 - 10 mu M) of lycopene and beta-carotene also had protective effects on DNA damage, as measured by the comet assay. Lycopene significantly reduced DNA damage particularly, in the median range of concentrations (2.5 mu M). The protective effects of lycopene and beta-carotene could be due to their scavenging of reactive oxygen (ROS) and/or nitrogen species (RNS) as they reduce the amount of intracellular ROS/RNS produced following treatment with SIN-1 by as much as 47.5% and 42.4%, respectively. The results obtained in this study suggest that carotenoids may alleviate some of the deleterious effects of peroxynitrite and possibly other reactive nitrogen species as well in vivo. (c) 2006 Elsevier Inc. All rights reserved.
  • A Jinno, Y Maruyama, M Ishizuka, A Kazusaka, A Nakamura, S Fujita
    JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 98 (1) 48 - 55 0960-0760 2006/01 [Refereed][Not invited]
    Equilenin is one of 10 kinds of estrogens that are found in pregnant mares' urine. lt has been used extensively for estrogen replacement therapy in postmenopausal women. Typical inducers of the cytochrome P4501A1 (CYP1A1), such as TCDD, benzo(a)pyrene (B(a)P) and 3-methylcholanthrene, have a planar molecular structure in common and bind to the aryl hydrocarbon receptor (AhR). The structure of equilenin differs from classic estrogens by the presence of two additional double bonds in ring B of the steroid nucleus, and it is planar. This structural similarity of equilenin to the typical AhR agonist prompted us to investigate the capability of equilenin to induce CYP1A1 expression. Administration of equilenin to two mouse strains (C57BL and DBA) that exhibit different degrees of responsiveness to an Ahreceptor agonist and showed that equilenin was capable of dose-dependently increasing both the ethoxyresorufin O-deethylase activity and CYP1a proteins in both strains of mice. Equilenin also induced CYP1A1 mRNA in treated HepG2 cell lines and transcriptional activity in an XRE-directed luciferase reporter gene. Competitive binding studies using C57BL AhR indicated equilenin weakly displaced 3 H-B(a)P from AhR. Together, these data show that equilenin, an equine steroid hormone, served as an AhR ligand in the present study. (c) 2005 Elsevier Ltd. All rights reserved.
  • N Sakai, K Saito, HS Kim, A Kazusaka, M Ishizuka, Y Funae, S Fujita
    DRUG METABOLISM AND DISPOSITION 33 (11) 1657 - 1660 0090-9556 2005/11 [Refereed][Not invited]
    Diazepam was metabolized to three primary metabolites, 3-hydroxy-diazepam, N-desmethyl-diazepam, and p-hydroxy-diazepam. Our previous studies reported metabolic position-specific inter- or intrastrain differences in diazepam metabolism among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. Especially, there were marked (similar to 300 fold) inter- or intrastrain differences in diazepam p-hydroxylation activity at low concentration of substrate. In this study, we investigated the enzyme that catalyzes diazepam p-hydroxylation. The activity toward diazepam p-hydroxylation was inhibited by anti-cytochrome P450 2D (CYP2D) antibody, suggesting that this activity was catalyzed by CYP2D isoforms. Comparing the expression levels of the CYP2D subfamily in liver microsomes from various strains of rats using anti-CYP2D2 antibody, we found that there was a band of protein that was consistent with the phenotype of diazepam p-hydroxylation. N-terminal amino acid sequences of the specific protein exactly corresponded to those of CYP2D3, indicating that CYP2D3 might be involved in diazepam p-hydroxylation. Moreover, using rat CYP2D isoforms expressed in yeast, we tested CYP2Ds to catalyze diazepam p-hydroxylation. CYP2D1 and CYP2D2 practically did not participate in diazepam metabolism. On the other hand, diazepam p-hydroxylation was catalyzed by CYP2D3. CYP2D4 had high activity toward diazepam N-desmethylation, but not p-hydroxylation. In conclusion, the polymorphic expression of CYP2D3 caused the inter- or intrastrain differences in diazepam p-hydroxylation among rat strains or intrastrain differences in diazepam p-hydroxylation among rat strains or individuals.
  • K Muzandu, Z Shaban, M Ishizuka, A Kazusaka, S Fujita
    FREE RADICAL RESEARCH 39 (4) 389 - 398 1071-5762 2005/04 [Refereed][Not invited]
    Catechol estrogens (CEs), such as 4-hydroxyestradiol (4-OHE2), undergo redox cycling during which reactive oxygen species (ROS) such as superoxide (O-2(-)) and the chemically reactive estrogen semiquinone (CE-SQ) and quinone (CE-Q) intermediates are produced. The quinone's putative mutagenicity may be enhanced by ROS and/or reactive nitrogen species. High concentrations of nitric oxide (NO) present during inflammatory conditions may react with (O-2(-)) to form peroxynitrite (ONOO-), a potent oxidant implicated in many pathological conditions. In this study, the possible generation of peroxynitrite from the interaction of CEs and NO and its effect on plasmid DNA and intact cells were investigated. A combination of 4-OHE2 and NO increased the level of single strand breaks (SSB) in plasmid DNA by more than 60% compared to vehicle controls in a metal-free buffer system. 4-OHE2 alone or NO alone had no effect. Results obtained from use of different antioxidants and ROS scavengers suggested a role of peroxynitrite in oxidative stress. In cells, 4-OHE2 or NO alone induced dose-dependent DNA damage as assessed by single cell gel electrophoresis. Co-treatment with 4-OHE2 and NO had an additive effect at lower doses. Generation of intracellular ROS was measured by the oxidation of carboxy-2',7'-dichlorofluorescein diacetate to the fluorescent compound carboxy-2',7'-dichlorofluorescein. NO alone, in oxygenated media, generated little ROS whereas 4-OHE2 produced approximately 70% increase in fluorescence. When added together 4-OHE2 and NO, produced a 2-fold increase in ROS. The generation and involvement of peroxynitrite to this increase was implied since uric acid inhibited it. Generation of peroxynitrite was also observed by use of dihydrorhodamine 123. Therefore, we conclude that combined treatments with 4-OHE2 and NO generated peroxynitrite seen from increased fluorescence and its inhibition by uric acid or combined SOD and catalase treatments. Results reported here suggest a role of peroxynitrite in causing damage to biomolecules when CEs and NO are present simultaneously. This may have biological relevance as high concentrations of NO formed during inflammatory conditions may exacerbate cancers due to estrogens.
  • KM El Bohi, L Sabik, K Muzandu, Z Shaban, M Soliman, M Ishizuka, A Kazusaka, S Fujita
    JAPANESE JOURNAL OF VETERINARY RESEARCH 52 (4) 163 - 172 0047-1917 2005/02 [Refereed][Not invited]
    Pleurotus cornucopiae (PC) mushroom with a brilliant yellow pileus is found in the field and known in Japan as Tamogi dake mushroom. The purpose of this paper is to investigate the mechanism of the antimutagenic effect of PC mushroom using both the Ames test and Comet assay. We have found a strong inhibitory effect of both aqueous and organic PC extracts on the mutagenicity elicited by benzo [a] pyrene (B [a] P). This inhibition was dose-dependent in reaction mixtures containing cytosolic and microsomal fractions (S-9) from untreated rat liver as well as in those containing S-9 from aryl hydrocarbon receptor (Ah) ligand of Sudan Ill-treated rats. Sudan III was a potent inducer of cytochrome P450 1A (CYP1A) activity. We treated rats with Sudan III to enhance the metabolic activation of B [a] P by the S-9 fraction. To explain whether this antimutagenicity was due to the inhibition of CYP1A activity that metabolically activates B [a] P, we tested the effects of the extracts on activities of CYP1A1 and CYP1A2, represented by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD), respectively. Both aqueous and organic extracts inhibited EROD activity at all dose levels, while the inhibitory effect was only observed at high doses with regard to MROD activity. Furthermore, pre-treatment of Chinese hamster V79cells with PC extracts significantly reduced H2O2-induced-DNA damage, indicating that PC extracts provide a protective effect against oxidative DNA damage. These results indicate that whole-mushroom extracts contain compounds that may inhibit the metabolic activation of B [a] P by CYP1A1 as well as prevent oxidative DNA damage.
  • K Muzandu, K El Bohi, Z Shaban, M Ishizuka, A Kazusaka, S Fujita
    JAPANESE JOURNAL OF VETERINARY RESEARCH 52 (4) 173 - 184 0047-1917 2005/02 [Refereed][Not invited]
    The consumption of fruits and vegetables is associated with a reduced risk of various ailments, including cancer and cardiovascular diseases. Carotenoids, such as lycopene and beta-carotene, are natural constituents of edible plants and may protect against disease. In this study, the influence of lycopene and beta-carotene on DNA damage caused by catechol-estrogens in vitro is examined. One possible mechanism by which catechol estrogens such as 4-hydroxyestradiol (4-OHE2) and 2-hydroxyestradiol, which cause DNA damage in naked plasmid DNA as well as in cells, contributing to the process of carcinogenesis, is through the generation of reactive oxygen species. It was found that both carotenoids at concentrations ranging from 0.25 to 10 mu M significantly inhibit strand breakage induced by 4-OHE2/copper sulphate by up to 90 %in plasmid DNA with beta-carotene being slightly more effective. No pro-oxidant or cytotoxic effects were observed at the concentrations tested. These carotenoids had a similar, though reduced effect on DNA damage as measured by the comet assay, in Chinese hamster lung fibroblasts. The results obtained show that both lycopene and beta-carotene, most probably and mainly through their potent antioxidant properties, are able to inhibit catechol-estrogen-mediated DNA damage.
  • S Nikaidou, M Ishizuka, Y Maeda, Y Hara, A Kazusaka, S Fujita
    JAPANESE JOURNAL OF VETERINARY RESEARCH 52 (4) 185 - 192 0047-1917 2005/02 [Refereed][Not invited]
    Green tea contains catechins and caffeine as major constituents. Treatment of rats with green tea (2.5% w/v) significantly increased 7-ethoxycoumarin O-deethylase(7-ECOD), caffeine N-1 demethylase (CN1D) and UDP -glucuronyltransferase (UGT) activities. Treatment with caffeine similarly activated CYP1A2 and related monooxygenases as well as UGT, while treatment with catechins induced UGT activity but not 7-ECOD or CN1D activity. Numbers of benzo [a] pyrene (BP)-induced revertant colonies in an Ames test (mutation assay) with S. typhimurium TA98 as the test strain were markedly larger when BP was preincubated with the liver S-9 (9000 Xg supernatant of liver homogenate) from green tea-treated rats than when preincubated with that from control rats. In a modified Ames assay system in which UGT was activated by the addition of UDP-glucuronic acid to the preincubation mixture, the numbers of revertant colonies in the assay using liver S-9 from green tea-treated rats decreased to a similar level to that in the assay using S-9 from controls. The acceleration of two enzymatic reactions may contribute to the rapid elimination of BP; the first step, the formation of a metabolic intermediate (which is mutagenic) by CYP1A2 and the second, the conjugation of active metabolic intermediates by UGT. We speculated that green tea can reduce the amount of time carcinogens reside in the body and the chance that body tissues will be exposed to active metabolites of carcinogens thorough rapid elimination due to the simultaneous induction of CYP1A2 and UGT activities.
  • Nikaidou S, Ishizuka M, Maeda Y, Hara Y, Kazusaka A, Fujita S
    The Journal of veterinary medical science / the Japanese Society of Veterinary Science 1 67 (1) 137 - 138 0916-7250 2005/01 [Refereed][Not invited]
  • Z Shaban, S El-Shazly, S Abdelhady, Fattouh, I, K Muzandu, M Ishizuka, K Kimura, A Kazusaka, S Fujita
    JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (11) 1377 - 1386 0916-7250 2004/11 [Refereed][Not invited]
    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachloro dibenzo-p-dioxin (TCDD) and related compounds. Peroxisome proliferator activated receptor alpha (PPARalpha) is a nuclear receptor involved in the maintenance of lipid and glucose homeostasis. In this study we hypothesized that one of the possible mechanisms for the effect of TCDD and its related chemicals on fat metabolism could be through down regulation of PPARalpha functions. We treated Wistar rats with an AhR ligand, Sudan III (S.III), and/or PPARalpha ligand, Clofibric Acid (CA), for 3 days. We analysed the expression of one of the PPARalpha-target gene products, CYP4A protein and its mRNA. We also tested HepG2 cells with the afore-mentioned treatments and evaluated their effects on PPARalpha and RXRalpha protein. Treatment of Wistar rats with S.III was found to down regulates CYP4A protein expression and reduced its induction with CA. It also decreased mRNA expressions of CYP4A1, CYP4A2, CYP4A3 and PPARalpha. In HepG2 cells, PPARalpha and RXRalpha protein expression was decreased by S.III treatment in a dose dependent manner. Our results suggest that AhR has an inhibitory effect on PPARa function and a new pathway by which AhR ligands could disturb lipid metabolism.
  • Z Shaban, S El-Shazly, M Ishizuka, K Kimura, A Kazusaka, S Fujita
    ARCHIVES OF TOXICOLOGY 78 (9) 496 - 507 0340-5761 2004/09 [Refereed][Not invited]
    Fibrates, hypolipidemic drugs, have been reported to suppress the metabolic activities of cytochrome P450 1A1 and 1A2 in rats but the mechanism has not been elucidated. In the present study we tested the hypothesis that the inhibitory effect of fibrates on arylhydrocarbon receptor (AhR) function may be due to their stimulatory effects on PPARalpha. Sudan III (S.III) treatment induced CYP 1A1 and CYP 1A2 protein expression, mRNA and their metabolic activities, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD), in Wistar rats higher than those in the control. Co-treatment of rats with S.III and clofibric acid (CA) caused a 40-50% decrease in the induced levels of CYP1A1 and CYP1A2 protein, mRNA expression and their metabolic activities and reduced AhR protein expression. When we treated HepG2 cells with S.III and/or CA, no suppressive effect on S.III-induced CYP1A1 protein expression due to CA was found. HepG2 cells were transiently transfected with increasing concentrations of PPARalpha mammalian expression vector and exposed to the same treatment. CA co-treatment with S.III decreased AhR protein and S.III-induced CYP1A1 protein expression with increasing dose of PPARalpha transfected into HepG2 cells. Our results demonstrate that the suppressive effect of fibrates on CYP1A is PPARalpha-dependent and suggest that PPARalphahas an inhibitory effect on AhR function.
  • Strain differences in diazepam metabolism at its three metabolic sites in sprague-dawley, brown norway, dark agouti, and wistar strain rats.
    Saito K, Sakai N, Kim HS, Ishizuka M, Kazusaka A, Fujita S
    Drug metabolism and disposition: the biological fate of chemicals 9 32 (9) 959 - 965 0090-9556 2004/09 [Refereed][Not invited]
  • HS Kim, M Ishizuka, A Kazusaka, S Fujita
    JOURNAL OF VETERINARY MEDICAL SCIENCE 66 (9) 1119 - 1124 0916-7250 2004/09 [Refereed][Not invited]
    Di-(2-ethylhexyl) phthalate (DEHP), a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand, alters the lipid composition of rat testis, yet the mechanism is unclear. In this study, we investigated the effect of DEHP on the synthesis and metabolism of arachidonic acid (AA), a precursor of eicosanoids, in the testis of prepubertal rats. DEHP (100 and 1,000 mg/kg, 5 days) administration caused a significant reduction in activity of cytosolic phospholipase A(2) (cPLA(2)), the rate-limiting enzyme in the AA and eicosanoid synthesis pathways. DEHP increased the expression of 12-lipoxygenase (12-LOX) in rat testis, whereas cyclooxygenase-2 (COX-2) expression was not altered. Cytochrome P450 4A1 (CYP4A1), a product of a PPARalpha-regulated gene, was markedly increased in the testis by DEHP administration. Taken together, DEHP suppresses cPLA(2) activity and induces the AA metabolizing enzymes such as 12-LOX and CYP4A1, resulting in the reduction of AA level. These data suggest that altered AA metabolic cascades may be related to the decrease of testosterone concentration in DEHP-induced testicular atrophy.
  • KQ Sakamoto, M Ishizuka, A Kazusaka, S Fujita
    JAPANESE JOURNAL OF VETERINARY RESEARCH 52 (2) 85 - 94 0047-1917 2004/08 [Refereed][Not invited]
    In order to examine whether iodine supplements may have caused a global decline in sperm concentrations during the past several decades, the synchronicity of the decline in mean sperm counts and the introduction of iodine supplements was analyzed statistically. A positive correlation between the incidence of thyroid disease and sperm counts has been detected in Europe. In addition, it has been shown that sperm counts began falling around 1965 in the United States, 40years after iodine supplements were introduced. Mean sperm counts before and after 1965 were 111 x 10(6)/ml and 70 x 10(6)/ml, respectively, in calculations weighted by the number of subjects included in each individual publication. The timing of the declines coincided with the introduction of iodine supplements in the United States, France, and the United Kingdom. The implications are that the global decline in sperm concentrations may be caused by iodine intake. Geographical variation in the types of sperm count decline also appears to be present.
  • K Saito, HS Kim, N Sakai, M Ishizuka, A Kazusaka, S Fujita
    JOURNAL OF PHARMACEUTICAL SCIENCES 93 (5) 1271 - 1278 0022-3549 2004/05 [Refereed][Not invited]
    We observed variations in the metabolism of diazepam in Wistar rats. We studied these variations carefully, and found that the variations are dimorphic and about 17% of male rats of Wistar strain we examined showed two times higher diazepam metabolic activities in their liver microsomes than the rest of animals at the substrate concentrations less than 5 muM. We classified them as extensive metabolizer (EM) and poor metabolizer (PM) of diazepam. No sex difference was observed in the frequency of appearance of EM. Activities of the primary metabolic pathways of diazepam were examined to elucidate the cause of this polymorphism in male Wistar rats. No significant differences were observed in activities of neither diazepam 3-hydroxylation or N-desmethylation between EM and PM rats, while activity of diazepam p-hydroxylation was markedly (more than 200 times) higher in EM rats, indicating that this reaction is responsible for the polymorphism of diazepam metabolism in Wistar rats. We examined the expression levels of CYP2D1, which was reported to catalyze diazepam p-hydroxylation in Wistar rats to find no differences in the expression levels of CYP2D1 between EM and PM rats. The kinetic study on diazepam metabolism in male Wistar rats revealed that EM rats had markedly higher V-max and smaller K-m in diazepam p-hydroxylation than those of PM rats, indicating the presence of high affinity high capacity p-hydroxylase enzyme in EM rats. As a consequence, at low concentrations of diazepam, major pathways of diazepam metabolism were p-hydroxylation and 3-hydroxylation in male EM rats, while in male PM rats, 3-hydroxylation followed byN-desmethylation. Due to this kinetic nature of p-hydroxylase activity, EM rats had markedly higher total CLint of diazepam than that of PM rats. Polymorphism in diazepam metabolism in humans is well documented, but this is the first report, revealing the presence of the polymorphism in diazepam metabolism in rats. The current results infer polymorphic expression of new diazepam p-hydroxylating enzyme with lower K-m than CYP2D1 in EM Wistar rats. (C) 2004Wiley-Liss, Inc. and the American Pharmacists Association.
  • KQ Sakamoto, K Nakai, T Aoto, A Yokoyama, R Ushikoshi, H Hirose, M Ishizuka, A Kazusaka, S Fujita
    CHEMOSPHERE 51 (6) 491 - 500 0045-6535 2003/05 [Refereed][Not invited]
    Accumulations of polychlorinated dibenzo-p-dioxins, dibenzofurans and coplanar polychlorinated biphenyls were analyzed in common carp (Cyprinus carpio) collected in the Hikiji River, Kanagawa Prefecture, Japan in which dioxin contaminated effluent was released during the period starting from November 1992 to March 2000. Higher levels of 2,3,7,8-tetrachlorinated dibenzo-p-dioxin toxic equivalents were observed in carps collected downstream to the dioxin release site (contaminated site) than the reference site. Modulations of cytochrome P450 (CYP) enzyme in liver; serum estrogen concentration and gonadal somatic index (GSI) were also measured as biomarkers for the contaminants. Total CYP content in livers was markedly higher in male and female carps from the contaminated site relative to the reference site fish. The expression level of the cytochrome P450 1A and Ethoxyresorufin 0-deethylase activity were significantly higher in female carps from the contaminated site than from the reference site. A lower level of plasma estrogen was observed in carps from the contaminated site. The GSI in female carps from the contaminated site was smaller than that recorded at the reference site. The present study indicates that dioxins released to the Hikiji River might induce the CYP enzyme and inhibit the reproductive functions in common carps dwelling downstream from the release site. (C) 2003 Elsevier Science Ltd. All rights reserved.
  • HS Kim, K Saito, M Ishizuka, A Kazusaka, S Fujita
    ARCHIVES OF TOXICOLOGY 77 (8) 446 - 451 0340-5761 2003 [Refereed][Not invited]
    Exposure of pubertal rats to di-(2-ethylhexyl) phthalate (DEHP) for 14 days was reported to result in reduced testosterone (T) biosynthesis by altering androstenedione 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity. However, our study indicated that shorter period exposure of DEHP (100 or 1000 mg/kg for 5 days) to 4-week-old male rats did not affect the activity of 17beta-HSD, the rate-limiting enzyme of T biosynthesis in the testis. Testosterone 5alpha-reductase (T5alpha-R) activity in the testis was significantly enhanced, while aromatase mRNA was significantly reduced by increasing doses of DEHP. The expressions of cytochrome P450 (CYP) isoforms, CYP2C11 and CYP3A, in the testis increased along with their enzymatic activities, T16alpha- and T6beta-hydroxylation, respectively. Thus, the current study clearly indicates that the short period exposure to DEHP alters T metabolism through altering activities of T5alpha-R, aromatase and CYP2C11/3A2 in the testis of prepubertal rats, and that they are more sensitive marker enzymes to the DEHP exposure than those of biosynthetic enzymes of T from androstenedione.
  • M Ishizuka, J Yonemoto, H Zaha, C Tohyama, H Sone
    JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY 17 (5) 278 - 285 1095-6670 2003 [Refereed][Not invited]
    The cytochrome P450 (CYP) isoform CYP2C11 is specifically expressed in the liver of adult male rats, and 5alpha-reductase is specifically expressed in the liver of the adult female rats. The sexually dimorphic expressions of these hepatic enzymes are regulated by the sex-dependent profiles of the circulating growth hormone (GH). However, it is not well known whether hormonal imprinting or activation factors in the neonatal brain influence the sexually dimorphic expression patterns of hepatic enzymes. We therefore examined the effect of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on sex-dependent expressions of hepatic enzymes. Pregnant rats were treated with TCDD at a dose of 0, 200, or 800 ng/kg on gestation day 15, exposing the pups to the chemical. Although the expression of CYP2C11 protein in the livers of male pups on postnatal day (PND) 49 was significantly higher than that of the controls, but the 5alpha-reductase activities in the livers of female pups were not altered by exposure to TCDD. Focusing on perinatal periods, testosterone and estrogen levels significantly increased in the brain of male pups on PND 2. The results suggest that the alteration of testosterone and estrogen levels affect hormonal imprinting in the neonatal brain of male pups, and thus induces a change in the level of male-specific hepatic CYP2C11. We conclude that perinatal exposure to TCDD at low doses may change the sexual differentiation of the neonatal brain in male rats. (C) 2003 Wiley Periodicals, Inc.
  • Chiba, I, A Sakakibara, H Iwata, M Ishizuka, S Tanabe, F Akahori, A Kazusaka, S Fujita
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 21 (4) 794 - 806 0730-7268 2002/04 [Refereed][Not invited]
    From 16 largha seats (Phoca largha) and 15 ribbon seals (Phoca fasciata) in the coastal waters of Hokkaido, Japan, blubber chlorinated hydrocarbon (CHC) levels and hepatic cytochrome P450 (CYP) catalytic activities and their immunochemically detected protein content levels were measured. Concentrations of DDTs (2,2-bis(4-chlorophenyl)-1,1-dichloroethylene,p,p'-DDE; 2,2-bis(4-chlorophenyl)-1,1-dichloroethane,p,p'-DDD; dichlorodiphenyltrichloroethane, p,p'-DDT), polychlorinated biphenyl congeners (PCBs), and chlordane compounds (oxychlordane, chlordanes, and nonachlors) in both species were in the range of 290 to 5,300, 420 to 4,000, and 130 to 1,500 ng/g lipid weight, respectively. Aryl hydrocarbon receptor (AhR) agonists, non-ortho (IUPAC 77 and 126) and mono-ortho (IUPAC 105, 118, and 156) coplanar PCB congeners, were also detected, and the 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) toxic equivalents (TEQs) were 4.9 to 120 pg TEQ/g lipid weight. Cross-reactive proteins with polyclonal antibodies against rat CYP1A1 and CYP3A2 were notably detected in seal liver microsomes. Interestingly, a polyclonal antibody against rat CYP2B1 recognized proteins only at trace levels. In largha seals, both levels of alkoxyresorufin- (methoxy-, ethoxy-, pentoxy-, and benzyloxyresorufin) O-dealkylase (AROD) activities and proteins detected by polyclonal antibodies against rat CYP1A1 were significantly correlated with the concentrations of individual coplanar PCB congeners, total TEQs, and total PCBs. Threshold concentrations for TEQs in blubber of the largha seal to induce hepatic CYP1A protein and EROD activity were estimated to be 8.5 and 19 pg TEQ/g fat weight, respectively. In ribbon seals, similar correlations were not detected, although the TEQ levels were not significantly lower than those in largha seals. These results suggest that AROD activity and CYP1A1 protein in the liver of the largha seal could be a biomarker for the exposure to AhR agonists such as coplanar PCB congeners. This study also indicates differences in AhR-mediated responses to the CHC exposures between largha and ribbon seals.
  • KQ Sakamoto, T Kunisue, M Watanabe, Y Masuda, H Iwata, S Tanabe, F Akahori, M Ishizuka, A Kazusaka, S Fujita
    ENVIRONMENTAL TOXICOLOGY AND CHEMISTRY 21 (4) 842 - 847 0730-7268 2002/04 [Refereed][Not invited]
    Polychlorinated biphenyls (PCBs), including coplanar congeners, hexachlorocyclohexane isomers, chlordane-related compounds, and hexachlorobenzene, were found in the breast muscle of Steller's sea eagles (SSE) and white-tailed sea eagles (WSE) threatened species, collected in Hokkaido, Japan, during the two years from 1998 to 1999. Both PCBs and DDTs were the most notable compounds, with concentrations one to two orders of magnitude higher than the other compounds, that is, from 120 to 39,000 and from 68 to 15,000 ng/g wet weight, respectively. Non-ortho (International Union of Pure and Applied Chemistry [IUPAC] 77, 126, and 169) and mono-ortho (IUPAC 105, 118, and 156)-substituted coplanar PCB congeners amounted to 9.2 to 740 pg/g of 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents derived from the World Health Organization, Paris, France (WHO), toxic equivalent factors. The atmospheric PCBs and DDTs in eastern Siberian cities, such as Khabarovsk and Magadan, have been reported to be much higher than Hokkaido and the North Pacific. Thus, we speculated that the eagles might have been contaminated in these areas, where they spend most of the year except winter, which they spend in eastern Siberia. Adult eagles accumulated more PCBs and DDTs than younger ones. The patterns of PCB congeners were also found to change, depending on the age of the eagle examined; adult eagles showed relatively higher proportions of highly chlorinated PCBs than juvenile eagles did. This difference would be related to the efficiency of the excretion and the metabolism of each PCB congener in the eagles.

Books etc


Awards & Honors

  • 2015 ソロプチミスト日本財団 女性研究者賞
    受賞者: 石塚真由美
  • 2015 北海道大学 研究総長賞(優秀賞)
    受賞者: 石塚真由美
  • 2009 文部科学省 科学技術分野の文部科学大臣表彰・若手科学者賞
    受賞者: 石塚真由美
  • 2007 日本農学会 日本農学進歩賞
    受賞者: 石塚真由美
  • 2007 日本トキシコロジー学会 奨励賞
    受賞者: 石塚真由美

Educational Activities

Teaching Experience

  • 獣医科学・感染症学基礎科目 生物統計学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 環境汚染、環境修復、環境毒性学、資源開発、環境経済
  • 獣医科学・感染症学基礎科目 研究機器演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Basic Toxicology and Environmental Toxicology
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 公害、薬害、毒性発現、異物代謝、排泄、動態、用量-反応、毒性試験、環境汚染物質、リスクアセスメント
  • 獣医科学特別研究
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • Environmental Hygiene
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 環境破壊、環境汚染、化学汚染物質、生体防御、規制
  • ケミカルハザード対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • Practice in Toxicology
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
    キーワード : 化学物質 毒性発現目がニズム 毒性試験法 体内動態 環境動態
  • 獣医科学特論演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 生物統計学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 研究機器演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 環境毒性学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学特別研究
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学特論演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • インターンシップ
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学基礎科目 生物統計学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学基礎科目 研究機器演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 獣医科学基礎科目 環境獣医科学特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 先端獣医科学科目 環境毒性学演習
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • ケミカルハザード対策専門特論
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 毒性学演習
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • 研究・臨床セミナー
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部
  • アドバンスト演習
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 獣医学部

Campus Position History

  • 2013年4月1日 
  • 2015年4月1日 
  • 2017年10月26日 
  • 2017年4月1日 
  • 2019年4月1日 
  • 2019年4月1日 
  • 2019年4月1日 

Position History

  • 2013年4月1日 
  • 2015年4月1日 
  • 2017年10月26日 
  • 2017年4月1日 
  • 2019年4月1日 
  • 2019年4月1日 
  • 2019年4月1日 

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