Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Sep 15;326(Pt 3):837–846. doi: 10.1042/bj3260837

Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse. Comparison of the tissue distribution of GST T1-1 with that of classes Alpha, Mu and Pi GST in human.

P J Sherratt 1, D J Pulford 1, D J Harrison 1, T Green 1, J D Hayes 1
PMCID: PMC1218740  PMID: 9307035

Abstract

The cDNA encoding human glutathione S-transferase (GST) T1 has been expressed as two recombinant forms in Escherichia coli that could be purified by affinity chromatography on either IgG-Sepharose or nickel-agarose; one form of the transferase was synthesized from the pALP 1 expression vector as a Staphylococcus aureus protein A fusion, whereas the other form was synthesized from the pET-20b expression vector as a C-terminal polyhistidine-tagged recombinant. The yields of the two purified recombinant proteins from E. coli cultures were approx. 15 mg/l for the protein A fusion and 25 mg/l for the C-terminal polyhistidine-tagged GST T1-1. The purified recombinant proteins were catalytically active, although the protein A fusion was typically only 5-30% as active as the histidine-tagged GST. Both recombinant forms could catalyse the conjugation of glutathione with the model substrates 1,2-epoxy-3-(4'-nitrophenoxy)propane,4-nitrobenzyl chloride and 4-nitrophenethyl bromide but were inactive towards 1-chloro-2,4-dinitrobenzene, ethacrynic acid and 1-menaphthyl sulphate. Recombinant human GST T1-1 was found to exhibit glutathione peroxidase activity and could catalyse the reduction of cumene hydroperoxide. In addition, recombinant human GST T1-1 was found to conjugate glutathione with dichloromethane, a pulmonary and hepatic carcinogen in the mouse. Immunoblotting with antibodies raised against different transferase isoenzymes showed that GST T1-1 is expressed in a large number of human organs in a tissue-specific fashion that differs from the pattern of expression of classes Alpha, Mu and Pi GST. Most significantly, GST T1-1 was found in only low levels in human pulmonary soluble extract of cells, suggesting that in man the lung has little capacity to activate the volatile dichloromethane.

Full Text

The Full Text of this article is available as a PDF (431.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andersen M. E., Clewell H. J., 3rd, Gargas M. L., Smith F. A., Reitz R. H. Physiologically based pharmacokinetics and the risk assessment process for methylene chloride. Toxicol Appl Pharmacol. 1987 Feb;87(2):185–205. doi: 10.1016/0041-008x(87)90281-x. [DOI] [PubMed] [Google Scholar]
  2. Andersson C., Mosialou E., Weinander R., Morgenstern R. Enzymology of microsomal glutathione S-transferase. Adv Pharmacol. 1994;27:19–35. doi: 10.1016/s1054-3589(08)61028-5. [DOI] [PubMed] [Google Scholar]
  3. Berhane K., Widersten M., Engström A., Kozarich J. W., Mannervik B. Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1480–1484. doi: 10.1073/pnas.91.4.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Burek J. D., Nitschke K. D., Bell T. J., Wackerle D. L., Childs R. C., Beyer J. E., Dittenber D. A., Rampy L. W., McKenna M. J. Methylene chloride: a two-year inhalation toxicity and oncogenicity study in rats and hamsters. Fundam Appl Toxicol. 1984 Feb;4(1):30–47. doi: 10.1016/0272-0590(84)90217-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burgess J. R., Chow N. W., Reddy C. C., Tu C. P. Amino acid substitutions in the human glutathione S-transferases confer different specificities in the prostaglandin endoperoxide conversion pathway. Biochem Biophys Res Commun. 1989 Jan 31;158(2):497–502. doi: 10.1016/s0006-291x(89)80076-2. [DOI] [PubMed] [Google Scholar]
  7. Chang M., Hong Y., Burgess J. R., Tu C. P., Reddy C. C. Isozyme specificity of rat liver glutathione S-transferases in the formation of PGF2 alpha and PGE2 from PGH2. Arch Biochem Biophys. 1987 Dec;259(2):548–557. doi: 10.1016/0003-9861(87)90521-2. [DOI] [PubMed] [Google Scholar]
  8. Ellis E. M., Judah D. J., Neal G. E., O'Connor T., Hayes J. D. Regulation of carbonyl-reducing enzymes in rat liver by chemoprotectors. Cancer Res. 1996 Jun 15;56(12):2758–2766. [PubMed] [Google Scholar]
  9. Fjellstedt T. A., Allen R. H., Duncan B. K., Jakoby W. B. Enzymatic conjugation of epoxides with glutathione. J Biol Chem. 1973 May 25;248(10):3702–3707. [PubMed] [Google Scholar]
  10. Graves R. J., Coutts C., Eyton-Jones H., Green T. Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice. Carcinogenesis. 1994 May;15(5):991–996. doi: 10.1093/carcin/15.5.991. [DOI] [PubMed] [Google Scholar]
  11. Graves R. J., Coutts C., Green T. Methylene chloride-induced DNA damage: an interspecies comparison. Carcinogenesis. 1995 Aug;16(8):1919–1926. doi: 10.1093/carcin/16.8.1919. [DOI] [PubMed] [Google Scholar]
  12. Habig W. H., Jakoby W. B. Assays for differentiation of glutathione S-transferases. Methods Enzymol. 1981;77:398–405. doi: 10.1016/s0076-6879(81)77053-8. [DOI] [PubMed] [Google Scholar]
  13. Hayes J. D., Clarkson G. H. Purification and characterization of three forms of glutathione S-transferase A. A comparative study of the major YaYa-, YbYb- and YcYc-containing glutathione S-transferases. Biochem J. 1982 Dec 1;207(3):459–470. doi: 10.1042/bj2070459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hayes J. D., Gilligan D., Chapman B. J., Beckett G. J. Purification of human hepatic glutathione S-transferases and the development of a radioimmunoassay for their measurement in plasma. Clin Chim Acta. 1983 Oct 31;134(1-2):107–121. doi: 10.1016/0009-8981(83)90189-4. [DOI] [PubMed] [Google Scholar]
  15. Hayes J. D., Mantle T. J. Use of immuno-blot techniques to discriminate between the glutathione S-transferase Yf, Yk, Ya, Yn/Yb and Yc subunits and to study their distribution in extrahepatic tissues. Evidence for three immunochemically distinct groups of transferase in the rat. Biochem J. 1986 Feb 1;233(3):779–788. doi: 10.1042/bj2330779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hayes J. D., Pulford D. J. 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. 1995;30(6):445–600. doi: 10.3109/10409239509083491. [DOI] [PubMed] [Google Scholar]
  17. Hayes J. D. Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver. Biochem J. 1986 Feb 1;233(3):789–798. doi: 10.1042/bj2330789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hiratsuka A., Sebata N., Kawashima K., Okuda H., Ogura K., Watabe T., Satoh K., Hatayama I., Tsuchida S., Ishikawa T. A new class of rat glutathione S-transferase Yrs-Yrs inactivating reactive sulfate esters as metabolites of carcinogenic arylmethanols. J Biol Chem. 1990 Jul 15;265(20):11973–11981. [PubMed] [Google Scholar]
  19. Howie A. F., Forrester L. M., Glancey M. J., Schlager J. J., Powis G., Beckett G. J., Hayes J. D., Wolf C. R. Glutathione S-transferase and glutathione peroxidase expression in normal and tumour human tissues. Carcinogenesis. 1990 Mar;11(3):451–458. doi: 10.1093/carcin/11.3.451. [DOI] [PubMed] [Google Scholar]
  20. Hussey A. J., Hayes J. D. Characterization of a human class-Theta glutathione S-transferase with activity towards 1-menaphthyl sulphate. Biochem J. 1992 Sep 15;286(Pt 3):929–935. doi: 10.1042/bj2860929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hussey A. J., Hayes J. D. Human Mu-class glutathione S-transferases present in liver, skeletal muscle and testicular tissue. Biochim Biophys Acta. 1993 Nov 10;1203(1):131–141. doi: 10.1016/0167-4838(93)90047-u. [DOI] [PubMed] [Google Scholar]
  22. Hussey A. J., Kerr L. A., Cronshaw A. D., Harrison D. J., Hayes J. D. Variation in the expression of Mu-class glutathione S-transferase isoenzymes from human skeletal muscle. Evidence for the existence of heterodimers. Biochem J. 1991 Jan 15;273(Pt 2):323–332. doi: 10.1042/bj2730323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jakobsson P. J., Mancini J. A., Ford-Hutchinson A. W. Identification and characterization of a novel human microsomal glutathione S-transferase with leukotriene C4 synthase activity and significant sequence identity to 5-lipoxygenase-activating protein and leukotriene C4 synthase. J Biol Chem. 1996 Sep 6;271(36):22203–22210. doi: 10.1074/jbc.271.36.22203. [DOI] [PubMed] [Google Scholar]
  24. Jemth P., Stenberg G., Chaga G., Mannervik B. Heterologous expression, purification and characterization of rat class theta glutathione transferase T2-2. Biochem J. 1996 May 15;316(Pt 1):131–136. doi: 10.1042/bj3160131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Juronen E., Tasa G., Uusküla M., Pooga M., Mikelsaar A. V. Production and characterization of monoclonal antibodies against class theta glutathione S-transferase T1-1. Hybridoma. 1996 Feb;15(1):77–82. doi: 10.1089/hyb.1996.15.77. [DOI] [PubMed] [Google Scholar]
  26. Juronen E., Tasa G., Uusküla M., Pooga M., Mikelsaar A. V. Purification, characterization and tissue distribution of human class theta glutathione S-transferase T1-1. Biochem Mol Biol Int. 1996 May;39(1):21–29. doi: 10.1080/15216549600201021. [DOI] [PubMed] [Google Scholar]
  27. Katoh T., Nagata N., Kuroda Y., Itoh H., Kawahara A., Kuroki N., Ookuma R., Bell D. A. Glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) genetic polymorphism and susceptibility to gastric and colorectal adenocarcinoma. Carcinogenesis. 1996 Sep;17(9):1855–1859. doi: 10.1093/carcin/17.9.1855. [DOI] [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Lam B. K., Penrose J. F., Freeman G. J., Austen K. F. Expression cloning of a cDNA for human leukotriene C4 synthase, an integral membrane protein conjugating reduced glutathione to leukotriene A4. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7663–7667. doi: 10.1073/pnas.91.16.7663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lear J. T., Heagerty A. H., Smith A., Bowers B., Payne C. R., Smith C. A., Jones P. W., Gilford J., Yengi L., Alldersea J. Multiple cutaneous basal cell carcinomas: glutathione S-transferase (GSTM1, GSTT1) and cytochrome P450 (CYP2D6, CYP1A1) polymorphisms influence tumour numbers and accrual. Carcinogenesis. 1996 Sep;17(9):1891–1896. doi: 10.1093/carcin/17.9.1891. [DOI] [PubMed] [Google Scholar]
  31. Mainwaring G. W., Nash J., Davidson M., Green T. Isolation of a mouse theta glutathione S-transferase active with methylene chloride. Biochem J. 1996 Mar 1;314(Pt 2):445–448. doi: 10.1042/bj3140445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mainwaring G. W., Williams S. M., Foster J. R., Tugwood J., Green T. The distribution of theta-class glutathione S-transferases in the liver and lung of mouse, rat and human. Biochem J. 1996 Aug 15;318(Pt 1):297–303. doi: 10.1042/bj3180297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meyer D. J., Coles B., Pemble S. E., Gilmore K. S., Fraser G. M., Ketterer B. Theta, a new class of glutathione transferases purified from rat and man. Biochem J. 1991 Mar 1;274(Pt 2):409–414. doi: 10.1042/bj2740409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meyer D. J., Lalor E., Coles B., Kispert A., Alin P., Mannervik B., Ketterer B. Single-step purification and h.p.l.c. analysis of glutathione transferase 8-8 in rat tissues. Biochem J. 1989 Jun 15;260(3):785–788. doi: 10.1042/bj2600785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Meyer D. J. Significance of an unusually low Km for glutathione in glutathione transferases of the alpha, mu and pi classes. Xenobiotica. 1993 Aug;23(8):823–834. doi: 10.3109/00498259309059411. [DOI] [PubMed] [Google Scholar]
  36. Meyer D. J., Thomas M. Characterization of rat spleen prostaglandin H D-isomerase as a sigma-class GSH transferase. Biochem J. 1995 Nov 1;311(Pt 3):739–742. doi: 10.1042/bj3110739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Müller M., Meijer C., Zaman G. J., Borst P., Scheper R. J., Mulder N. H., de Vries E. G., Jansen P. L. Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):13033–13037. doi: 10.1073/pnas.91.26.13033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. NASH T. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem J. 1953 Oct;55(3):416–421. doi: 10.1042/bj0550416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pemble S. E., Wardle A. F., Taylor J. B. Glutathione S-transferase class Kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homologue. Biochem J. 1996 Nov 1;319(Pt 3):749–754. doi: 10.1042/bj3190749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pemble S., Schroeder K. R., Spencer S. R., Meyer D. J., Hallier E., Bolt H. M., Ketterer B., Taylor J. B. Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism. Biochem J. 1994 May 15;300(Pt 1):271–276. doi: 10.1042/bj3000271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rowe J. D., Nieves E., Listowsky I. Subunit diversity and tissue distribution of human glutathione S-transferases: interpretations based on electrospray ionization-MS and peptide sequence-specific antisera. Biochem J. 1997 Jul 15;325(Pt 2):481–486. doi: 10.1042/bj3250481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schröder K. R., Hallier E., Meyer D. J., Wiebel F. A., Müller A. M., Bolt H. M. Purification and characterization of a new glutathione S-transferase, class theta, from human erythrocytes. Arch Toxicol. 1996;70(9):559–566. doi: 10.1007/BF03035371. [DOI] [PubMed] [Google Scholar]
  43. Schröder K. R., Hallier E., Peter H., Bolt H. M. Dissociation of a new glutathione S-transferase activity in human erythrocytes. Biochem Pharmacol. 1992 Apr 15;43(8):1671–1674. doi: 10.1016/0006-2952(92)90695-f. [DOI] [PubMed] [Google Scholar]
  44. Seaton M. J., Follansbee M. H., Bond J. A. Oxidation of 1,2-epoxy-3-butene to 1,2:3,4-diepoxybutane by cDNA-expressed human cytochromes P450 2E1 and 3A4 and human, mouse and rat liver microsomes. Carcinogenesis. 1995 Oct;16(10):2287–2293. doi: 10.1093/carcin/16.10.2287. [DOI] [PubMed] [Google Scholar]
  45. Singhal S. S., Zimniak P., Sharma R., Srivastava S. K., Awasthi S., Awasthi Y. C. A novel glutathione S-transferase isozyme similar to GST 8-8 of rat and mGSTA4-4 (GST 5.7) of mouse is selectively expressed in human tissues. Biochim Biophys Acta. 1994 Feb 16;1204(2):279–286. doi: 10.1016/0167-4838(94)90019-1. [DOI] [PubMed] [Google Scholar]
  46. Stirling D. A., Petrie A., Pulford D. J., Paterson D. T., Stark M. J. Protein A-calmodulin fusions: a novel approach for investigating calmodulin function in yeast. Mol Microbiol. 1992 Mar;6(6):703–713. doi: 10.1111/j.1365-2958.1992.tb01519.x. [DOI] [PubMed] [Google Scholar]
  47. Tamai K., Shen H. X., Tsuchida S., Hatayama I., Satoh K., Yasui A., Oikawa A., Sato K. Role of cysteine residues in the activity of rat glutathione transferase P (7-7): elucidation by oligonucleotide site-directed mutagenesis. Biochem Biophys Res Commun. 1991 Sep 16;179(2):790–797. doi: 10.1016/0006-291x(91)91886-h. [DOI] [PubMed] [Google Scholar]
  48. Tan K. L., Chelvanayagam G., Parker M. W., Board P. G. Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues. Biochem J. 1996 Oct 1;319(Pt 1):315–321. doi: 10.1042/bj3190315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tew K. D. Glutathione-associated enzymes in anticancer drug resistance. Cancer Res. 1994 Aug 15;54(16):4313–4320. [PubMed] [Google Scholar]
  50. Thier R., Müller M., Taylor J. B., Pemble S. E., Ketterer B., Guengerich F. P. Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione S-transferase. Chem Res Toxicol. 1995 Apr-May;8(3):465–472. doi: 10.1021/tx00045a019. [DOI] [PubMed] [Google Scholar]
  51. Thier R., Pemble S. E., Kramer H., Taylor J. B., Guengerich F. P., Ketterer B. Human glutathione S-transferase T1-1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4-diepoxybutane in Salmonella typhimurium. Carcinogenesis. 1996 Jan;17(1):163–166. doi: 10.1093/carcin/17.1.163. [DOI] [PubMed] [Google Scholar]
  52. Thier R., Taylor J. B., Pemble S. E., Humphreys W. G., Persmark M., Ketterer B., Guengerich F. P. Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8576–8580. doi: 10.1073/pnas.90.18.8576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Welsch D. J., Creely D. P., Hauser S. D., Mathis K. J., Krivi G. G., Isakson P. C. Molecular cloning and expression of human leukotriene-C4 synthase. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9745–9749. doi: 10.1073/pnas.91.21.9745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wiencke J. K., Pemble S., Ketterer B., Kelsey K. T. Gene deletion of glutathione S-transferase theta: correlation with induced genetic damage and potential role in endogenous mutagenesis. Cancer Epidemiol Biomarkers Prev. 1995 Apr-May;4(3):253–259. [PubMed] [Google Scholar]
  55. Wilce M. C., Board P. G., Feil S. C., Parker M. W. Crystal structure of a theta-class glutathione transferase. EMBO J. 1995 May 15;14(10):2133–2143. doi: 10.1002/j.1460-2075.1995.tb07207.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wong L. C., Winston J. M., Hong C. B., Plotnick H. Carcinogenicity and toxicity of 1,2-dibromoethane in the rat. Toxicol Appl Pharmacol. 1982 Apr;63(2):155–165. doi: 10.1016/0041-008x(82)90036-9. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES