IDENTIFICATION OF INTERSPECIFIC DIFFERENCES IN PHASE II REACTIONS: DETERMINATION OF METABOLITES IN THE URINE OF 16 MAMMALIAN SPECIES EXPOSED TO ENVIRONMENTAL PYRENE AKSORN SAENGTIENCHAI,yz YOSHINORI IKENAKA,*y SHOUTA M.M. NAKAYAMA,y HAZUKI MIZUKAWA,y MAYU KAKEHI,y NESTA BORTEY-SAM,y WAGEH SOBHY DARWISH,yx TOSHIO TSUBOTA,k MASANORI TERASAKI,# AMNART POAPOLATHEP,z and MAYUMI ISHIZUKAy yLaboratory of Toxicology, Department of Environmental Veterinary Science, Graduate, School of Veterinary Medicine, Hokkaido University, Sapporo, Japan zDepartment of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand xFood Control Department, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt kLaboratory of Wildlife Biology and Medicine, Department of Environmental Veterinary Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan #Institute for Environmental Sciences, University of Shizuoka, Shizuoka, Japan (Submitted 17 January 2014; Returned for Revision 28 February 2014; Accepted 29 May 2014) Abstract: 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 0 -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. Environ Toxicol Chem 2014;33:2062–2069. # 2014 SETAC Keywords: Pyrene Urine metabolite Mammal Interspecific difference INTRODUCTION Interspecific differences in the biotransformation and elimination of drugs and other xenobiotics are typically complex, making it difficult to predict the adverse consequences of environmental pollutants [1]. Studies on interspecific differ- ences in xenobiotic metabolism of various animals, such as domesticated livestock, companion animals, and captive and free-ranging wildlife, are becoming of increasing interest to ecotoxicologists [2]. The best-known species difference in phase II metabolism of xenobiotics is the biotransformation of drugs and structurally related phenolic compounds by glucuronidation in domestic cats. Slow glucuronidation of acetaminophen and acetylsalicylic acid (aspirin) accounts for the slow clearance and extreme sensitivity of the Felidae family to the adverse effects of these drugs in comparison with dogs and most other mammalian species [3,4]. In addition, other enzymes reduce susceptibility to toxins (e.g., glutathione-S-transferase in the mouse affords tolerance to the food-borne hepatocarcinogen aflatoxin B1 [5]). Studies on experimental exposure in vivo represent the best approach to characterizing interspecific differences in kinetics, metabolic pathways, and specific toxicological effects; yet, they are usually difficult to characterize in domesticated livestock, companion animals, and captive and free-ranging wildlife. However, genomic investigation and in vitro experiments (e.g., enzyme kinetics analysis, cell culture experiments) are recog- nized as powerful tools for predicting these differences. A previous report on genomics-based phylogenetic analysis of uridine 5 0 -diphospho-glucuronosyltransferase 1A6 (UGT1A6; i.e., the principal gene regulating the biotransformation of phenolic compounds) indicates that this gene occurs as a pseudogene throughout Felidae [6] and in other species such as the brown hyena (Hyaena brunnea) and the northern elephant seal (Mirounga angustirostris) [7]. This analysis predicts that the chemical sensitivity of these species, which do not express UGT1A6, is high. On the other hand, gene-based phylogeny is not always correlated with in vivo results. For example, ferret UGT1A6 is not a pseudogene, yet glucuronidation of acetaminophen in ferrets is relatively slow in comparison with other species [8]. Hence, both in vitro (genetic and enzymatic analysis) and in vivo studies are necessary to predict interspecific difference in xenobiotic metabolism. Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that can evoke carcinogenic and endocrine effects in humans and other animals. They are formed and emitted as a result of the incomplete combustion of organic material. They may also be released into the environment through the disposal of coal tars and other coal-processing wastes, petroleum sledges, and other wood preservative wastes. The biomarkers of PAH exposure have been widely reported. The induction of mRNA * Address correspondence to y_ikenaka@vetmed.hokudai.ac.jp. Published online 4 June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2656 Environmental Toxicology and Chemistry, Vol. 33, No. 9, pp. 2062–2069, 2014 # 2014 SETAC Printed in the USA 2062