Archives of Toxicology

, Volume 78, Issue 4, pp 218–225 | Cite as

Low glutathione S-transferase dogs

  • Toshiyuki WatanabeEmail author
  • Tomomi Sugiura
  • Sunao Manabe
  • Wataru Takasaki
  • Yoshihiko Ohashi
Toxicokinetics and Metabolism


Liver and kidney glutathione S-transferase (GST) activities to 1,2-dichloro-4-nitrobenzene (DCNB) as a substrate (GST-D activities) were measured in 280 dogs from five different breeders, and significant individual differences in this activity were observed in both organs. Interestingly, 34 out of the 280 dogs (i.e. 12.1%) were those in which liver GST-D activities were less than 10 nmol/min per mg cytosolic protein, “low GST dogs”, and the other dogs were classified as “middle” and “high” GST dogs for which the liver GST-D activities were 10–80 and >80 nmol/min per mg protein, respectively, and occurred at similar percentages (41.4% for the middle GST dog and 46.4% for the high GST dog). Furthermore, the existence of the low GST dogs was not limited to one particular breeder. There was a good correlation (r=0.910) between the liver and kidney GST-D activities, showing low activity in not only the liver but also the kidney in the low GST dogs. Although liver GST activity to 1-chloro-2,4-dinitrobenzene as a substrate (GST-C activity), catalyzed by various GST isozymes in dogs, was significantly correlated with liver GST-D activity, GST-C activity showed more than 450 nmol/min per mg protein even in the low GST dogs. There was no significant difference in cytochrome P450 content, 7-ethoxycoumarin O-deethylase activity or UDP-glucuronosyltransferase activity to p-nitrophenol as a substrate between low GST dogs and the other dogs. Finally, remarkably high plasma concentrations of DCNB were observed in the low GST dogs after single doses of DCNB at 5 or 100 mg/kg. The individual differences in GST-D activity are probably attributable to the content and/or activity of the theta class GST isozyme YdfYdf since it has been reported that glutathione conjugation of DCNB is specifically catalyzed by GSTYdfYdf in dogs. In conclusion, we identified a number of low GST dogs in which the GST-D activities were not observed either in vivo or in vitro. The feasibility of using a single low dose of DCNB to phenotype dogs based on GST-D activity was confirmed. It was also suggested that low GST dogs have high susceptibility, including unexpected toxicity or abnormal exposure, to chemicals metabolized by GSTYdfYdf.


Glutathione S-transferase Low GST dog GSTYdfYdf Deficiency High susceptibility 



We are particularly grateful to Dr. Koichi Hirano, Mr. Yasumitsu Nakatsugawa and Mr. Hitoshi Onishi for their advice and technical assistance. We also wish to express our thanks to Ms Caroline Bertorelli for proof-reading of this manuscript. We declare that this experiment complied with the current laws of Japan.


  1. Ali-Osman F, Akande O, Antoun G, Mao JX, Buolamwini J (1997) Molecular cloning, characterization and expression in Escherichia coli of full-length cDNAs of three human glutathione S-transferase Pi gene variants. Evidence for differential catalytic activity of the encoded proteins. J Biol Chem 272:10004–10012PubMedGoogle Scholar
  2. Blackburn AC, Tzeng HF, Anders MW, Board PG (2000) Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis. Pharmacogenetics 10:49–57CrossRefPubMedGoogle Scholar
  3. Board PG, Baker RT, Chelvanayagam G, Jermiin LS (1997) Zeta, a novel class of glutathione transferases in a range of species from plants to humans. Biochem J 328:929–935PubMedGoogle Scholar
  4. Bock KW, Fröhling W, Remmer H, Rexer B (1973) Effects of phenobarbital and 3-methylcholanthrene on substrate specificity of rat liver microsomal UDP-glucuronyltransferase. Biochim Biophys Acta 327:46–56CrossRefPubMedGoogle Scholar
  5. Coles BF, Morel F, Rauch C, Huber WW, Yang M, Teitel CH, Green B, Lang NP, Kadlubar FF (2001) Effect of polymorphism in the human glutathione S-transferase A1 promoter on hepatic GSTA1 and A2 expression. Pharmacogenetics 11:663–669CrossRefPubMedGoogle Scholar
  6. Dirr H, Reinemer P, Huber R (1994) X-ray crystal structures of cytosolic glutathione S-transferases. Implications for protein architecture, substrate recognition and catalytic function. Eur J Biochem 220:645–661PubMedGoogle Scholar
  7. Eaton DL, Bammler TK (1999) Concise review of the glutathione S-transferases and their significance to toxicology. Toxicol Sci 49:156–164PubMedGoogle Scholar
  8. Habig WH, Pabst MJ, Jakoby WB (1974) Glutathione S-transferases. The first enzymatic step in mercapuric acid formation. J Biol Chem 249:7130–7139PubMedGoogle Scholar
  9. Hayes JD, Pulford DJ (1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol 30:445–600PubMedGoogle Scholar
  10. Hong SK, Anestis DK, Ball JG, Valentovic MA, Rankin GO (2002) In vitro nephrotoxicity induced by chloronitrobenzenes in renal cortical slices from Fischer 344 rats. Toxicol Lett 129:133–141CrossRefPubMedGoogle Scholar
  11. Igarashi T, Nanba E, Sagami F, Tsukidate K, Fukuda T, Horie T, Satoh T, Kitagawa H (1988) Dog liver glutathione S-transferase and its strong immunoreactivity with rat transferase-P(7-7). Biochem Pharmacol 37:4713–4718CrossRefPubMedGoogle Scholar
  12. Igarashi T, Kohara A, Shikata Y, Sagami F, Sonoda J, Horie T, Satoh T (1991) The unique feature of dog liver cytosolic glutathione S-transferases. An isozyme not retained on the affinity column has the highest activity toward 1,2-dichloro-4-nitrobenzene. J Biol Chem 266:21709–21717PubMedGoogle Scholar
  13. Inskip A, Elexperu-Camiruaga J, Buxton N, Dias PS, MacIntosh J, Campbell D, Jones PW, Yengi L, Talbot JA, Strange RC, Fryer AA (1995) Identification of polymorphism at the glutathione S-transferase, GSTM3 locus: evidence for linkage with GSTM1*A. Biochem J 312:713–716PubMedGoogle Scholar
  14. Ji X, von Rosenvinge EC, Johnson WW, Tomarev SI, Piatigorsky J, Armstrong RN, Gilliland GL (1995) Three-dimensional structure, catalytic properties, and evolution of a sigma class glutathione transferase from squid, a progenitor of the lens S-crystallins of cephalopods. Biochemistry 34:5317–5328PubMedGoogle Scholar
  15. Knowles SR, Uetrecht J, Shear NH (2000) Idiosyncratic drug reactions: the reactive metabolite syndromes. Lancet 356:1587–1591CrossRefPubMedGoogle Scholar
  16. Landi S (2000) Mammalian class theta GST and differential susceptibility to carcinogens: a review. Mutat Res 463:247–283PubMedGoogle Scholar
  17. Li Q, Minami M, Inagaki H (1998) Acute and subchronic immunotoxicity of p-chloronitrobenzene in mice. I. Effect of natural killer, cytotoxic T-lymphocyte activities and mitogen-stimulated lymphocyte proliferation. Toxicology 127:223–232CrossRefPubMedGoogle Scholar
  18. Li Q, Minami M, Hanaoka T, Yamamura Y (1999) Acute immunotoxicity of p-chloronitrobenzene in mice: II. Effect of p-chloronitrobenzene on the immunophenotype of murine splenocytes determined by flow cytometry. Toxicology 137:35–45CrossRefPubMedGoogle Scholar
  19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275Google Scholar
  20. Matsubara T, Otsubo S, Yoshihara E, Touchi A (1983) Biotransformation of coumarin derivatives (2). Oxidative metabolism of 7-alkoxycoumarin by microsomal enzymes and a simple assay procedure for 7-alkoxycoumarin O-dealkylase. Jpn J Pharmacol 33:41–56PubMedGoogle Scholar
  21. Meyer DJ, Coles B, Pemble SE, Gilmore KS, Fraser GM, Ketterer B (1991) Theta, a new class of glutathione transferases purified from rat and man. Biochem J 274:409–414PubMedGoogle Scholar
  22. Morrow CS, Cowan KH (1990) Glutathione S-transferases and drug resistance. Cancer Cells 2:15–22PubMedGoogle Scholar
  23. Nair RS, Johannsen FR, Levinskas GJ, Terrill JB (1986a) Subchronic inhalation toxicity of p-nitroaniline and p-nitrochlorobenzene in rats. Fundam Appl Toxicol 6:618–627PubMedGoogle Scholar
  24. Nair RS, Johannsen FR, Levinskas GJ, Terrill JB (1986b) Assessment of toxicity of o-nitrochlorobenzene in rats following a 4-week inhalation exposure. Fundam Appl Toxicol 7:609–614PubMedGoogle Scholar
  25. Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378Google Scholar
  26. Pemble S, Schroeder KR, Spencer SR, Meyer DJ, Hallier E, Bolt HM, Ketterer B, Taylor JB (1994) Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism. Biochem J 300:271–276PubMedGoogle Scholar
  27. Pemble SE, Wardle AF, Taylor JB (1996) Glutathione S-transferase class Kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homologue. Biochem J 319:749–754PubMedGoogle Scholar
  28. Petersen KU (2002) From toxic precursors to safe drugs. Mechanisms and relevance of idiosyncratic drug reactions. Arzneimittelforschung 52:423–429PubMedGoogle Scholar
  29. Pirmohamed M, Madden S, Park BK (1996) Idiosyncratic drug reactions. Metabolic bioactivation as a pathogenic mechanism. Clin Pharmacokinet 31:215–230PubMedGoogle Scholar
  30. Prohaska JR, Ganther HE (1976) Glutathione peroxidase activity of glutathione S-transferases purified from rat liver. Biochem Biophys Res Commun 76:437–445PubMedGoogle Scholar
  31. Pumford NR, Halmes NC (1997) Protein targets of xenobiotic reactive intermediates. Annu Rev Pharmacol Toxicol 37:91–117CrossRefPubMedGoogle Scholar
  32. Seidegard J, Vorachek WR, Pero RW, Pearson WR (1988) Hereditary differences in the expression of human glutathione transferase active on trans-stilbene oxide are due to a gene deletion. Proc Natl Acad Sci USA. 85:7293–7297Google Scholar
  33. Shreve MR, Morrissey PG, O’Brien PJ (1979) Lipid and steroid hydroperoxides as substrates for the non-selenium-dependent glutathione peroxidase. Biochem J 177:761–763PubMedGoogle Scholar
  34. Stücker I, Hirvonen A, de Waziers I, Cabelguenne A, Mitrunen K, Cenee S, Koum-Besson E, Hemon D, Beaune P, Loriot MA (2002) Genetic polymorphism of glutathione S-transferases as modulators of lung cancer susceptibility. Carcinogenesis 23:1475–1481CrossRefPubMedGoogle Scholar
  35. Toda G (1999) Liver injury induced by troglitazone. Endcrinol Diabetol 9:393–400Google Scholar
  36. Travlos GS, Mahler J, Ragan, HA, Chou BJ, Bucher JR (1996) Thirteen-week inhalation toxicity of 2- and 4-chloronitrobenzene in F344/N rats and B6C3F1 mice. Fundam Appl Toxicol 30:75–92CrossRefPubMedGoogle Scholar
  37. van Poppel G, de Vogel N, van Balderen, PJ, Kok FJ (1992) Increased cytogenetic damage in smokers deficient in glutathione S-transferase isozyme mu. Carcinogenesis 13:303–305PubMedGoogle Scholar
  38. Vlachodimitropoulos D, Norppa H, Autio K, Catalan J, Hirvonen A, Tasa G, Uuskula M, Demopoulos NA, Sorsa M (1997) GSTT1-dependent induction of centromere-negative and -positive micronuclei by 1,2:3,4-diepoxybutane in cultured human lymphocytes. Mutagenesis 12:397–403PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Toshiyuki Watanabe
    • 1
    Email author
  • Tomomi Sugiura
    • 1
  • Sunao Manabe
    • 1
  • Wataru Takasaki
    • 1
  • Yoshihiko Ohashi
    • 1
  1. 1.Medicinal Safety Research LaboratoriesSankyo Co. Ltd.ShizuokaJapan

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