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Effect of decreased glutathione levels in hereditary glutathione synthetase deficiency on dibromoethane-induced genotoxicity in human fibroblasts
Mutation Research 389 Ž1997. 291–297
Effect of decreased glutathione levels in hereditary glutathione synthetase deficiency on dibromoethane-induced genotoxicity in human fibroblasts Laurie D. DeLeve
1
Department of Pharmacology, DiÕision of Clinical Pharmacology, Research Institute, Hospital for Sick Children, and Department of Pharmacology, UniÕersity of Toronto, Toronto, Ontario, Canada, M5G 1X8 Received 30 July 1996; revised 23 September 1996; accepted 31 October 1996
Abstract The genotoxic effect of dibromoethane is thought to be due to glutathione S-transferase mediated metabolism. The purpose of this study was to determine whether variations in endogenous glutathione in human cells could modify the genotoxicity of dibromoethane. Genotoxicity of dibromoethane, assessed by sister chromatid exchange, was examined in normal human skin fibroblasts and fibroblasts obtained from individuals with hereditary generalized glutathione synthetase deficiency. Cell proliferation was examined as a measure of dibromoethane toxicity. The number of sister chromatid exchanges induced by dibromoethane was significantly lower in the fibroblasts with glutathione synthetase deficiency compared to control cells. Inhibition of cell proliferation was similar in the glutathione-deficient and normal fibroblasts. In conclusion, low endogenous glutathione levels are protective against dibromoethane-induced genotoxicity in human fibroblasts. Keywords: Glutathione; Sister chromatid exchange; Dibromoethane; Cells cultured; Methylnitronitrosoguanidine
1. Introduction Dibromoethane ŽDBE. is a widely used industrial chemical and fumigant. It is toxic w1–3x, possibly teratogenic w4,5x, mutagenic in a variety of test sys-
1
Present address: USC Health Science Campus, 1333 San Pablo St-MMR 401, Los Angeles CA 90033, USA. Tel.: Ž213. 342-3248; Fax: Ž213. 342-3243; E-mail: [email protected]
tems including the sister chromatid exchange assay used in this paper w6–9x and carcinogenic in rodents w6,10,11x. According to the currently accepted scheme, dibromoethane metabolism occurs by two pathways w2,12,13x. DBE is conjugated by glutathione S-transferase ŽGST. to glutathione ŽGSH., which yields a S-Ž2-bromoethyl.glutathione conjugate. This conjugate reacts intramolecularly to form an episulfonium ion. The episulfonium ion forms a ‘hard’ electrophile that preferentially will bind cova-
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L.D. DeLeÕer Mutation Research 389 (1997) 291–297
lently to nucleophiles such as DNA. This pathway is deemed responsible for the mutagenic effect of dibromoethane w6,7,14x. Thus, this is one of the unusual compounds for which conjugation to GSH functions as an activating rather than a detoxifying pathway. Oxidation of DBE by P450 yields 2bromoacetaldehyde, which can be conjugated to GSH to form S-Ž2-oxoethyl.glutathione or metabolized by aldehyde dehydrogenase to form bromoacetic acid w12x ŽFig. 1.. Bromoaldehydic metabolites generated by the P450-catalyzed reaction are thought to be responsible for much of the toxicity of DBE w2,3x, which may be mediated by lipid peroxidation and binding to cellular macromolecules w2,15x. Since DBE genotoxicity requires GST catalyzed conjugation to GSH, hereditary variations in either GST isozymes or GSH levels would be predicted to affect the risk of DBE-induced carcinogenicity. Studies have indeed shown that polymorphism of human class-theta GST correlates with differences in GSH conjugation to DBE w16x and alters mutagenicity of DBE w17x. However, no studies have ever looked at whether hereditary differences in GSH levels in eukaryotic cells might alter genotoxicity of compounds. Sister chromatid exchange ŽSCE. is widely used as a useful and sensitive test of genotoxicity w18x. It is considered a good predictor of mutagenicity and carcinogenicity w19x although the exact mechanism of formation of SCEs is unknown. In the current study we have examined the incidence of SCE in normal fibroblasts and in fibroblasts obtained from two individuals with greatly reduced intracellular GSH levels due to hereditary generalized GSH synthetase deficiency, an inborn error of GSH metabolism w20x
2. Materials and methods All chemicals were obtained from the Sigma Chemical Corporation ŽSt. Louis, MO.. Normal human skin fibroblasts grown from biopsies from the forearm were a kind gift from the Department of Genetics at the Hospital for Sick Children ŽToronto, Canada.. Two skin fibroblast strains obtained from individuals with hereditary GSH synthetase deficiency were used: one strain was previously described w21x and the other was obtained from the brother of the patient described in that report. Fibroblasts were plated at a density of 3000 cellsrcm2 , on slides for cells processed for SCE and in tissue culture dishes for measurements of GSH and toxicity. The day after plating cells were exposed to DBE for 2 h in a 378C, CO 2 incubator. At the end of the incubation period the dishes were washed once with phosphate buffered saline and fresh medium with 10 mM bromodeoxyuridine was added. For the next 46 h, the dishes were kept in the dark in a 378C, CO 2 incubator. Cells were arrested in metaphase by a 2-h incubation with 0.1 mM colchicine and were then lysed with 0.075 M potassium chloride. Slides were fixed with methanolrglacial acetic acid Ž3 : 1., incubated with 5 mgrml bisbenzamide for 15 min and exposed to ultraviolet light Žpeak wavelength 335 nm. for 2 h. After a 35-min incubation in 2 = standard saline citrate solution Žstandard saline citrate: 0.3 M sodium chlorider0.03 M trisodium citrate. at 658C, slides were stained with a Gurr stain Ž2 ml of Giemsa stain in 50 ml phosphate buffer, pH 6.8.. All experiments were done in duplicate. Only
Fig. 1. Postulated metabolism of dibromoethane.
L.D. DeLeÕer Mutation Research 389 (1997) 291–297
metaphases in which all 46 chromosomes were present were used to count exchanges. For each determination, SCEs were counted in 20 metaphases Ži.e., 40 cells for duplicate experiments.. Since the distribution of SCE was skew to the right, geometric means were used and this restored a normal distribution to the values of both the treated and control populations. Baseline cell number was determined in a control plate at the beginning of the incubation and cell growth for each cell strain was determined over 48 h. The effect of DBE on cell growth was assessed by expressing cell growth in the presence of DBE as a percentage of the cell number present in the solvent control. Cell number was determined by Coulter counter. Glutathione was measured by the method of Tietze w22x. In brief, cells were plated at the same density used in the actual SCE experiments, 3000 cellsrcm2 . The following day, i.e., the same time point at which cells assessed for SCE would be exposed to DBE, cells were scraped into 20 mM HCl and sonicated. 100 ml cell suspension, 0.6 mmol 5,5X-dithiobisŽ2-nitrobenzoic acid., 5 mM EDTA, and 0.24 mmol NADPH were added to a cuvet containing 0.1 M sodium phosphate buffer at pH 7.5 for a final volume of 1 ml; the reaction was initiated by the addition of 10 mg glutathione reductase. The production of 2-nitro-5-thiobenzoic acid was measured spectrophotometrically at 412 nm over 6 min. An aliquot was taken for protein determination by the method of Lowry w23x. Increase in SCEs were compared with two-factor repeated measures analysis of variance ŽANOVA. using the Microsoft Excel Analysis ToolPak. When dose-response curves were statistically significant by ANOVA, a` posteriori comparison of individual doses between dose-response curves was done by least significant difference ŽLSD.. A p value of F 0.05 was considered significant.
3. Results To document the magnitude of the difference in intracellular GSH in control and GSH synthetase-deficient cell strains, GSH levels were determined. Intracellular GSH concentrations were substantially
293
Table 1 Glutathione levels in fibroblast cell strains Cell strain
mg GSHrmg protein )
GSH synthetase deficient 1 GSH synthetase deficient 2 Control 1 Control 2 Control 3
1.0"0.7 0.9 † 14.4"5.5 10.4"0.3 11.8"1.5
) Glutathione ŽGSH. determined in subconfluent cells. Mean"SEM, ns 3. † ns1.
lower in the two GSH synthetase-deficient cell strains compared to the three cell strains from control patients ŽTable 1.. The GSH synthetase-deficient cell strains display significantly fewer exchanges with increasing concentrations of DBE than the control cells ŽFig. 2, Table 2.. The GSH synthetase-deficient cells were significantly different from one or two of the controls at 2.5 and 5 mM, but at 10 mM DBE the number of SCEs in both deficient cell strains was significantly different compared to the number of exchanges in each of the three control strains by least significant difference ŽFig. 2.. The baseline
Fig. 2. Sister chromatid exchanges induced by dibromoethane. Normal human skin fibroblasts and fibroblasts from patients with GSH synthetase deficiency were exposed to dibromoethane for 2 h and then assessed for sister chromatid exchange after 46 h. Two factor ANOVA with replication comparing the curves was significant at p- 0.0001. Means were compared a` posteriori by least significant difference Ž ns 4.: ) at 10 mM, GSHy 1 vs. control 3: p- 0.005, GSHy 1 vs. controls 1 and 2: p- 0.001: †at 10 mM, GSHy 2 vs. controls 1 and 3: p- 0.01, GSHy 2 vs. control 2: p- 0.001.
L.D. DeLeÕer Mutation Research 389 (1997) 291–297
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frequency of SCEs in GSHy 1 was at the upper range of those examined and in GSHy 2 at the lower range. Statistical comparison of the baseline frequency of SCEs of these two GSH synthetase-deficient cell strains demonstrated no statistical differences by least significant difference of these strains compared to any of the other cell strains. A possible confounder would be if the GSH synthetase-deficient cell strains respond differently in the SCE assay than other cells. However, in studies with N-methyl-N Xnitro-N-nitrosoguanidine one of the two GSH synthetase-deficient cell strains ŽGSHy 2. demonstrated an increase in SCE similar to control human fibrob-
lasts and the other cell strain ŽGSHy 1. demonstrated an increase in SCEs that was substantially less than control levels ŽFig. 3.. To further confirm the ability of GSHy 1 to respond normally in the SCE assay, this cell strain was studied in the presence of dinitrochlorobenzene ŽFig. 4.. At 2.5 and 5 mM dinitrochlorobenzene GSHy 1 demonstrated an increase in SCEs similar to the two control cell strains. Thus the decreased number of SCEs was not an artifact of the inborn error of GSH synthetase. Since severe toxicity has been shown to artificially increase SCEs w24x, the effect of DBE on cell proliferation was examined ŽTable 3.. Glutathione
Table 2 SCE frequencies in solvent or dibromoethane treated fibroblasts Exp. No.
0 mM
2.5 mM
5 mM
10 mM
1
1 2 3 4 mean " SE
11.22 " 0.33 11.83 " 0.70 12.03 " 0.23 9.37 " 0.02 11.11 " 0.61
11.51 " 0.91 12.85 " 0.29 11.38 " 0.21 10.63 " 0.36 11.59 " 0.46
12.47 " 1.77 11.17 " 1.68 13.19 " 0.44 10.41 " 0.15 11.81 " 0.63
13.27 " 2.46 11.78 " 0.78 16.47 " 0.98 10.72 a 13.06 " 1.25
GSHy 2
1 2 3 4 mean " SE
7.24 " 0.28 8.78 " 0.09 7.62 " 0.83 6.56 " 0.88 7.55 " 0.46
8.96 " 0.67 10.41 " 0.65 8.86 " 0.40 7.28 " 0.95 8.88 " 0.64
10.18 " 1.87 9.65 a 9.84 a 9.10 " 1.15 9.69 " 0.23
10.08 " 1.37 11.33 " 1.03 11.11 " 1.57 9.05 " 1.22 10.39 " 0.52
1 2 3 4 mean " SE
5.93 " 1.11 11.32 " 0.06 9.20 " 0.30 11.09 " 0.28 9.38 " 1.25
8.59 " 0.67 17.39 " 1.60 13.09 " 0.59 30.9 " 6.22 17.49 " 4.82
11.66 " 1.87 21.41 " 1.07 16.37 " 0.16 27.02 " 2.62 19.11 " 3.30
14.10 " 0.15 23.17 " 6.57 20.58 " 0.04 24.29 " 0.62 20.53 " 2.28
1 2 3 4 mean " SE
9.58 " 0.16 9.64 " 0.61 5.62 " 0.48 7.50 " 0.26 8.08 " 0.96
12.34 " 2.31 12.67 " 1.42 11.56 " 0.53 12.41 " 0.61 12.24 " 0.24
13.66 " 0.03 13.12 " 0.46 12.29 a 13.06 " 1.99 13.03 " 0.28
18.26 " 0.37 18.76 " 2.09 21.39 " 1.63 17.33 " 3.06 18.93 " 0.87
1 2 3 4 mean " SE
10.64 " 0.71 5.83 " 0.19 7.63 " 0.03 6.80 " 0.67 7.72 " 1.04
13.57 " 0.88 7.53 " 0.81 10.32 " 0.59 9.53 " 0.1 10.24 " 1.26
14.27 " 0.04 9.62 " 0.90 10.73 " 1.62 10.52 " 1.02 11.28 " 1.02
21.77 " 5.42 11.30 " 0.64 17.47 " 1.33 16.64 " 1.55 16.79 " 2.15
Cell strain y
GSH
Control 1
Control 2
Control 3
)
)
))
))
SCE frequencies from 4 separate experiments Žmean " SD. in 2 glutathione synthetase deficient ŽGSH y . cell strains and 3 control cell strains. Statistical comparison of SCE at 0 and 10 mM by paired t-test: ) p - 0.01; ) ) p - 0.005. a Single value derived from 20 metaphases.
L.D. DeLeÕer Mutation Research 389 (1997) 291–297
295
synthetase-deficient cells grow significantly slower than normal human fibroblasts w25x. In the two GSH synthetase-deficient cell strains used in this study, baseline cell growth rate was approximately half of that seen in the normal human fibroblasts Žsee values in footnote Table 3.. The degree of DBE-induced inhibition of proliferation in the GSH synthetase cell strains is not markedly different from two of the controls.
X
Fig. 3. Sister chromatid exchanges induced by N-methyl-N -nitroN-nitrosoguandine. Normal human skin fibroblasts and fibroblasts from patients with GSH synthetase deficiency were exposed to X N-methyl-N -nitro-N-nitrosoguandine for 2 h and then assessed for sister chromatid exchange after 46 h Ždata from 3–5 experiments..
Fig. 4. Sister chromatid exchange induced by dinitrochlorobenzene. Normal human skin fibroblasts and fibroblasts from a patient with GSH synthetase deficiency were exposed to dinitrochlorobenzene for 2 h and then assessed for sister chromatid exchange after 46 h Ž ns 2..
Table 3 Effect of dibromoethane on cell proliferation Cell strain y
GSH 1 GSHy 2 Control 1 Control 2 Control 3
2.5 mM
5 mM
10 mM
86"7 83"2 64"10 77"10 80"3
80"3 67"8 49"11 63"12 76"10
61"9 48"11 20"5 43 ) 51"10
Cell growth in solvent control Žincrease at time of harvest as % of cell number present at t s 0.: GSHy 1: 129%; GSHy 2: 140%; Controle 1: 264%; Controle 2: 242%; Controle 3: 212%. Values in the table reflect cell growth in the presence of DBE as a percentage of growth in solvent control Ž ns 4.. ) ns 2. GSHy, glutathione synthetase deficient.
4. Discussion This study examines whether hereditary differences in GSH levels in eukaryotic cells can modify the genotoxic response in vitro. The findings are consistent with the literature in this area. Thus low endogenous GSH levels in Salmonella typhimurium exposed to DBE in the Ames test are protective w8x and GSH depletion in rats with diethylmaleate or buthionine sulfoximine decrease DNA adduct formation in vivo w13x. The ability of polymorphisms of P450 and GST to modify the risk of carcinogenic compounds has been examined in the past, but this is the first demonstration that hereditary differences in GSH levels might have this effect in eukaryotic cells. The cell strains used are from individuals with an inborn error of GSH synthetase that leads to very low intracellular levels of GSH. Although such large variations in GSH levels may be needed to show differences in susceptibility in acute exposure experiments, much smaller variations might alter responses in chronic exposure to environmental toxins. Heterozygotes for GSH synthetase deficiency seem phenotypically normal and have normal GSH levels, but have an impaired ability to regenerate GSH and therefore have decreased GSH detoxification capacity w26x. Thus, heterozygosity for GSH synthetase deficiency might protect against genotoxicity from DBE, but might increase the risk for carcinogens detoxified by GSH. Furthermore, it is possible that significant variations in intracellular GSH in the ‘normal population’ would affect the response to chemical carcinogens. Unfortunately, little is known about the range of normal intracellular GSH in the population, on normal variability in GSH synthetic capacity or on the frequency of heterozygosity for
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inborn errors of the g-glutamyl cycle that lead to impaired GSH detoxification. The ability of DBE to induce genotoxicity in human cells assessed by SCE was previously reported in a study of the effect of DBE vapor in peripheral lymphocytes w9x. It is difficult to compare the response to a liquid dissolved in medium with the response to a gas, but the current study and the study in lymphocytes both show a similar increase in the frequency of exchanges. Dibromoethane toxicity is thought to be mainly due to P450-mediated metabolism w2,3x and mutagenicity to metabolites formed following conjugation to GSH w6,7,14x. Studies that used bromoheptane to deplete GSH in hepatocytes showed protection against dibromoethane toxicity and concluded that the glutathione conjugate also contributes to dibromoethane cytotoxicity w2x. In the current study GSH deficiency protected against genotoxicity, but toxicity as manifested by inhibition of cell proliferation was not markedly different from control cells. However, the GSH synthetase-deficient cells have a slow baseline proliferation rate and this complicates the interpretation of cell proliferation data as a measure of toxicity. Severe toxicity has been shown to cause a small artifactual increase in SCEs, perhaps by increased incorporation of bromodeoxyuridine due to reduced cell density w24x. Since dibromoethane was not much more toxic to the control cell strains than to the glutathione synthetase-deficient cell strains, the greater increase in SCEs in the control strains is unlikely to be due to the in vitro artifact. This in vitro artifact could also complicate the use of the SCE assay in slow growing cells, such as the GSH synthetase-deficient cells. However, no significant increase in baseline SCE frequency in the GSH synthetase-deficient cell strains was observed. Furthermore, if this artifact were significant in the setting of a toxin-induced reduction in cell growth in a slower growing cell line, this should artifactually increase SCE frequency. Thus, if this artifact were occurring in the current study, it would decrease the observed differences in SCE between control cells and the GSH synthetase-deficient cells, not enhance the differences. However, if GSH synthetase-deficient cell strains are used in future studies to evaluate compounds that are more genotoxic in GSH
deficiency, this artifact may complicate interpretation. Future studies will need to determine how large the normal variation is in endogenous GSH levels and whether such variations can alter susceptibility towards environmental or occupational exposure to chemical carcinogens.
Acknowledgements The author would like to thank the late Marilyn Cannon for her technical assistance and Dr. Stephen Spielberg for his support and advice.
References w1x Storer, R.D. and R.B. Conolly Ž1983. Comparative in vivo genotoxicity and acute hepatoxicity of three 1,2-dihaloalkanes, Carcinogenesis, 4, 1491–1494. w2x Khan, S., C. Sood and P.J. O’Brien Ž1993. Molecular mechanisms of dibromoalkane cytotoxicity in isolated rat hepatocytes, Biochem. Pharmacol., 45, 439–447. w3x Nichols, W.K., M.O. Covington, C.D. Seiders, S. Safiullah and G.S. Yost Ž1992. Bioactivation of halogenated hydrocarbons by rabbit pulmonary cells, Pharmacol. Toxicol., 71, 335–339. w4x Mitra, A., D.R. Hilbelink, J.J. Dwornik and A. Kulkarni Ž1992. Rat hepatic glutathione S-transferase-mediated embryotoxic bioactivation of ethylene dibromide, Teratology, 46, 439–446. w5x Mitra, A., D.R. Hilbelink, J.J. Dwornik and A. Kulkarni Ž1992. A novel model to assess developmental toxicity of dihaloalkanes in humans: bioactivation of 1,2-dibromoethane by the isozymes of human fetal liver glutathione S-transferase, Teratogrn. Carcinogen. Mutagen., 12, 113–127. w6x Rannug, U. Ž1980. Genotoxic effects of 1,2-dibromoethane and 1,2-dichloroethane, Mutation Res., 76, 269–295. w7x Buijs, W., A. van der Gen, G.R. Mohn and D.D. Breimer Ž1984. The direct mutagenic activity of a ,v-dihalogen alkanes in Salmonella typhimurium, Mutation Res., 141, 11–14. w8x Kerklaan, P., S. Bouter and G. Mohn Ž1983. Isolation of a mutant of Salmonella typhimurium strain TA 1535 with decreased levels of glutathione ŽGSHy .: primary charaterization and chemical mutagenesis studies, Mutation Res., 122, 257–266. w9x Tucker, J.D., J. Xu, J. Stewart and T.-M. Ong Ž1984. Detection of sister chromatid exchanges in human peripheral lymphocytes induced by ethylen dibromide vapor, Mutation Res., 138, 93–98. w10x Wong, L.C., J.M. Winston, C.B. Hong and H. Plotnick Ž1982. Carcinogenicity and toxicity of 1,2-dibromoethane in the rat, Toxicol. Appl. Pharmacol., 63, 155–165.
L.D. DeLeÕer Mutation Research 389 (1997) 291–297 w11x Huff, J.E. Ž1983. 1,2-Dibromoethane Žethylene dibromide., Env. Health Perspect., 47, 359–363. w12x Shih, T.W. and D.L. Hill Ž1981. Metabolic activation of 1,2-dibromoethane by glutathione transferase and microsomal mixed function oxidase: further evidence for formation of two reactive metabolites, Res. Commun. Chem. Pathol. Pharmacol., 33, 449–461. w13x Kim, D.H. and F.P. Guengerich Ž1990. Formation of the DNA adduct S-w2-Ž N 7-guanyl.ethylxglutathione from ethylene dibromide: effects of modulation of glutathione and glutathione S-transferase levels and lack of a role for sulfation, Carcinogenesis, 11, 419–424. w14x Ozawa, N. and F.P. Guengerich Ž1983. Evidence of formation of an S-w2-Ž N 7-guanyl.ethylxgutathione adduct in glutathione-mediated binding of 1,2-dibromoethane to DNA, Proc. Natl. Acad. Sci. USA, 80, 5266–5277. w15x Albano, E., G. Poli, A. Tomasi, A. Bini, V. Vannini and M.U. Dianzani Ž1984. Toxicity of 1,2-dibromoethane in isolated hepatocytes: role of lipid peroxidation, Chem.-Biol. Interact., 50, 255–265. w16x Ploemen, J.H.T.M., L.W. Wormhoudt, B. van Ommen, J.N.M. Commandeur, N.P.E. Vermeulen and P.J. van Bladeren Ž1995. Polymorphism in the glutathione conjugation activity of human erythrocytes towards ethylene dibromide and 1,2-epoxy-3-Ž p-nitrophenoxy.propane, Biochim. Biophys. Acta, 1243, 469–476. w17x Thier, R., S.E. Pemble, H. Kramer, J.B. Taylor, F.P. Guengerich and B. Ketterer Ž1996. Human glutathione S-transferase T1-1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4-diepoxybutane in Salmonella typhimurium, Carcinogenesis, 17, 163–166. w18x Tucker, J.D., A. Auletta, M.C. Cimino, K.L. Dearfield, D. Jacobson-Kram, R.R. Tice and A.V. Carrano Ž1993. Sister-
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chromatid exchange: second report of the Gene-Tox program, Mutation Res., 297, 101–180. Abe, S. and M. Sasaki Ž1982. Sister chromatid exchange as an index of mutagenesis andror carcinogenesis, in: A.A. Sandberg ŽEd.., Sister chromatid exchange, Alan R. Liss, New York, pp. 461–514. Larsson, A. and L. Hagenfeldt Ž1983. Hereditary glutathione synthetase deficiency in man, in: A. Larsson, S. Orrenius, A. Holmgren and B. Mannervik ŽEds.., Functions of glutathione –Biochemical, physiological, toxicological and clinical aspects, Raven Press, New York, pp. 317–324. Spielberg, S.P., L.I. Kramer, S.I. Goodman, J. Butler, F. Tietze, P. Quinn and J.D. Schulman Ž1977. 5-Oxoprolinuria: biochemical observations and case report, J. Pediatr., 91, 237–241. Tietze, F. Ž1969. Enzymic method for quantitative determination of nanogram amounts of total and oxidised glutathione: applications to mammalian blood and other tissues, Anal. Biochem., 27, 502–522. Lowry, O., N.J. Rosebrough, A.L. Farr and R.J. Randall Ž1951. Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193, 265–275. Galloway, S.M., D.A. Deasy, C.L. Bean, A.R. Kraynak, M.J. Armstrong and M.O. Bradley Ž1987. Effects of high osmotic strength on chromosome aberrations, sister-chromatid exchanges and DNA strand breaks, and the relation to toxicity, Mutation. Res., 189, 15-25. Larsson, A., B. Mattson, L. Hagenfeldt and P. Moldeus ´ Ž1983. Glutathione synthetase deficient human fibroblasts in culture, Clin. Chim. Acta, 135, 57–64. Spielberg, S.P. Ž1985. Acetaminophen toxicity in lymphocytes heterozygous for glutathione synthetase deficiency, Can. J. Physiol. Pharmacol., 63, 468–471.
Effect of decreased glutathione levels in hereditary glutathione synthetase deficiency on dibromoethane-induced genotoxicity in human fibroblasts Laurie D. DeLeve
1
Department of Pharmacology, DiÕision of Clinical Pharmacology, Research Institute, Hospital for Sick Children, and Department of Pharmacology, UniÕersity of Toronto, Toronto, Ontario, Canada, M5G 1X8 Received 30 July 1996; revised 23 September 1996; accepted 31 October 1996
Abstract The genotoxic effect of dibromoethane is thought to be due to glutathione S-transferase mediated metabolism. The purpose of this study was to determine whether variations in endogenous glutathione in human cells could modify the genotoxicity of dibromoethane. Genotoxicity of dibromoethane, assessed by sister chromatid exchange, was examined in normal human skin fibroblasts and fibroblasts obtained from individuals with hereditary generalized glutathione synthetase deficiency. Cell proliferation was examined as a measure of dibromoethane toxicity. The number of sister chromatid exchanges induced by dibromoethane was significantly lower in the fibroblasts with glutathione synthetase deficiency compared to control cells. Inhibition of cell proliferation was similar in the glutathione-deficient and normal fibroblasts. In conclusion, low endogenous glutathione levels are protective against dibromoethane-induced genotoxicity in human fibroblasts. Keywords: Glutathione; Sister chromatid exchange; Dibromoethane; Cells cultured; Methylnitronitrosoguanidine
1. Introduction Dibromoethane ŽDBE. is a widely used industrial chemical and fumigant. It is toxic w1–3x, possibly teratogenic w4,5x, mutagenic in a variety of test sys-
1
Present address: USC Health Science Campus, 1333 San Pablo St-MMR 401, Los Angeles CA 90033, USA. Tel.: Ž213. 342-3248; Fax: Ž213. 342-3243; E-mail: [email protected]
tems including the sister chromatid exchange assay used in this paper w6–9x and carcinogenic in rodents w6,10,11x. According to the currently accepted scheme, dibromoethane metabolism occurs by two pathways w2,12,13x. DBE is conjugated by glutathione S-transferase ŽGST. to glutathione ŽGSH., which yields a S-Ž2-bromoethyl.glutathione conjugate. This conjugate reacts intramolecularly to form an episulfonium ion. The episulfonium ion forms a ‘hard’ electrophile that preferentially will bind cova-
1383-5718r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 6 . 0 0 1 5 9 - 0
292
L.D. DeLeÕer Mutation Research 389 (1997) 291–297
lently to nucleophiles such as DNA. This pathway is deemed responsible for the mutagenic effect of dibromoethane w6,7,14x. Thus, this is one of the unusual compounds for which conjugation to GSH functions as an activating rather than a detoxifying pathway. Oxidation of DBE by P450 yields 2bromoacetaldehyde, which can be conjugated to GSH to form S-Ž2-oxoethyl.glutathione or metabolized by aldehyde dehydrogenase to form bromoacetic acid w12x ŽFig. 1.. Bromoaldehydic metabolites generated by the P450-catalyzed reaction are thought to be responsible for much of the toxicity of DBE w2,3x, which may be mediated by lipid peroxidation and binding to cellular macromolecules w2,15x. Since DBE genotoxicity requires GST catalyzed conjugation to GSH, hereditary variations in either GST isozymes or GSH levels would be predicted to affect the risk of DBE-induced carcinogenicity. Studies have indeed shown that polymorphism of human class-theta GST correlates with differences in GSH conjugation to DBE w16x and alters mutagenicity of DBE w17x. However, no studies have ever looked at whether hereditary differences in GSH levels in eukaryotic cells might alter genotoxicity of compounds. Sister chromatid exchange ŽSCE. is widely used as a useful and sensitive test of genotoxicity w18x. It is considered a good predictor of mutagenicity and carcinogenicity w19x although the exact mechanism of formation of SCEs is unknown. In the current study we have examined the incidence of SCE in normal fibroblasts and in fibroblasts obtained from two individuals with greatly reduced intracellular GSH levels due to hereditary generalized GSH synthetase deficiency, an inborn error of GSH metabolism w20x
2. Materials and methods All chemicals were obtained from the Sigma Chemical Corporation ŽSt. Louis, MO.. Normal human skin fibroblasts grown from biopsies from the forearm were a kind gift from the Department of Genetics at the Hospital for Sick Children ŽToronto, Canada.. Two skin fibroblast strains obtained from individuals with hereditary GSH synthetase deficiency were used: one strain was previously described w21x and the other was obtained from the brother of the patient described in that report. Fibroblasts were plated at a density of 3000 cellsrcm2 , on slides for cells processed for SCE and in tissue culture dishes for measurements of GSH and toxicity. The day after plating cells were exposed to DBE for 2 h in a 378C, CO 2 incubator. At the end of the incubation period the dishes were washed once with phosphate buffered saline and fresh medium with 10 mM bromodeoxyuridine was added. For the next 46 h, the dishes were kept in the dark in a 378C, CO 2 incubator. Cells were arrested in metaphase by a 2-h incubation with 0.1 mM colchicine and were then lysed with 0.075 M potassium chloride. Slides were fixed with methanolrglacial acetic acid Ž3 : 1., incubated with 5 mgrml bisbenzamide for 15 min and exposed to ultraviolet light Žpeak wavelength 335 nm. for 2 h. After a 35-min incubation in 2 = standard saline citrate solution Žstandard saline citrate: 0.3 M sodium chlorider0.03 M trisodium citrate. at 658C, slides were stained with a Gurr stain Ž2 ml of Giemsa stain in 50 ml phosphate buffer, pH 6.8.. All experiments were done in duplicate. Only
Fig. 1. Postulated metabolism of dibromoethane.
L.D. DeLeÕer Mutation Research 389 (1997) 291–297
metaphases in which all 46 chromosomes were present were used to count exchanges. For each determination, SCEs were counted in 20 metaphases Ži.e., 40 cells for duplicate experiments.. Since the distribution of SCE was skew to the right, geometric means were used and this restored a normal distribution to the values of both the treated and control populations. Baseline cell number was determined in a control plate at the beginning of the incubation and cell growth for each cell strain was determined over 48 h. The effect of DBE on cell growth was assessed by expressing cell growth in the presence of DBE as a percentage of the cell number present in the solvent control. Cell number was determined by Coulter counter. Glutathione was measured by the method of Tietze w22x. In brief, cells were plated at the same density used in the actual SCE experiments, 3000 cellsrcm2 . The following day, i.e., the same time point at which cells assessed for SCE would be exposed to DBE, cells were scraped into 20 mM HCl and sonicated. 100 ml cell suspension, 0.6 mmol 5,5X-dithiobisŽ2-nitrobenzoic acid., 5 mM EDTA, and 0.24 mmol NADPH were added to a cuvet containing 0.1 M sodium phosphate buffer at pH 7.5 for a final volume of 1 ml; the reaction was initiated by the addition of 10 mg glutathione reductase. The production of 2-nitro-5-thiobenzoic acid was measured spectrophotometrically at 412 nm over 6 min. An aliquot was taken for protein determination by the method of Lowry w23x. Increase in SCEs were compared with two-factor repeated measures analysis of variance ŽANOVA. using the Microsoft Excel Analysis ToolPak. When dose-response curves were statistically significant by ANOVA, a` posteriori comparison of individual doses between dose-response curves was done by least significant difference ŽLSD.. A p value of F 0.05 was considered significant.
3. Results To document the magnitude of the difference in intracellular GSH in control and GSH synthetase-deficient cell strains, GSH levels were determined. Intracellular GSH concentrations were substantially
293
Table 1 Glutathione levels in fibroblast cell strains Cell strain
mg GSHrmg protein )
GSH synthetase deficient 1 GSH synthetase deficient 2 Control 1 Control 2 Control 3
1.0"0.7 0.9 † 14.4"5.5 10.4"0.3 11.8"1.5
) Glutathione ŽGSH. determined in subconfluent cells. Mean"SEM, ns 3. † ns1.
lower in the two GSH synthetase-deficient cell strains compared to the three cell strains from control patients ŽTable 1.. The GSH synthetase-deficient cell strains display significantly fewer exchanges with increasing concentrations of DBE than the control cells ŽFig. 2, Table 2.. The GSH synthetase-deficient cells were significantly different from one or two of the controls at 2.5 and 5 mM, but at 10 mM DBE the number of SCEs in both deficient cell strains was significantly different compared to the number of exchanges in each of the three control strains by least significant difference ŽFig. 2.. The baseline
Fig. 2. Sister chromatid exchanges induced by dibromoethane. Normal human skin fibroblasts and fibroblasts from patients with GSH synthetase deficiency were exposed to dibromoethane for 2 h and then assessed for sister chromatid exchange after 46 h. Two factor ANOVA with replication comparing the curves was significant at p- 0.0001. Means were compared a` posteriori by least significant difference Ž ns 4.: ) at 10 mM, GSHy 1 vs. control 3: p- 0.005, GSHy 1 vs. controls 1 and 2: p- 0.001: †at 10 mM, GSHy 2 vs. controls 1 and 3: p- 0.01, GSHy 2 vs. control 2: p- 0.001.
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frequency of SCEs in GSHy 1 was at the upper range of those examined and in GSHy 2 at the lower range. Statistical comparison of the baseline frequency of SCEs of these two GSH synthetase-deficient cell strains demonstrated no statistical differences by least significant difference of these strains compared to any of the other cell strains. A possible confounder would be if the GSH synthetase-deficient cell strains respond differently in the SCE assay than other cells. However, in studies with N-methyl-N Xnitro-N-nitrosoguanidine one of the two GSH synthetase-deficient cell strains ŽGSHy 2. demonstrated an increase in SCE similar to control human fibrob-
lasts and the other cell strain ŽGSHy 1. demonstrated an increase in SCEs that was substantially less than control levels ŽFig. 3.. To further confirm the ability of GSHy 1 to respond normally in the SCE assay, this cell strain was studied in the presence of dinitrochlorobenzene ŽFig. 4.. At 2.5 and 5 mM dinitrochlorobenzene GSHy 1 demonstrated an increase in SCEs similar to the two control cell strains. Thus the decreased number of SCEs was not an artifact of the inborn error of GSH synthetase. Since severe toxicity has been shown to artificially increase SCEs w24x, the effect of DBE on cell proliferation was examined ŽTable 3.. Glutathione
Table 2 SCE frequencies in solvent or dibromoethane treated fibroblasts Exp. No.
0 mM
2.5 mM
5 mM
10 mM
1
1 2 3 4 mean " SE
11.22 " 0.33 11.83 " 0.70 12.03 " 0.23 9.37 " 0.02 11.11 " 0.61
11.51 " 0.91 12.85 " 0.29 11.38 " 0.21 10.63 " 0.36 11.59 " 0.46
12.47 " 1.77 11.17 " 1.68 13.19 " 0.44 10.41 " 0.15 11.81 " 0.63
13.27 " 2.46 11.78 " 0.78 16.47 " 0.98 10.72 a 13.06 " 1.25
GSHy 2
1 2 3 4 mean " SE
7.24 " 0.28 8.78 " 0.09 7.62 " 0.83 6.56 " 0.88 7.55 " 0.46
8.96 " 0.67 10.41 " 0.65 8.86 " 0.40 7.28 " 0.95 8.88 " 0.64
10.18 " 1.87 9.65 a 9.84 a 9.10 " 1.15 9.69 " 0.23
10.08 " 1.37 11.33 " 1.03 11.11 " 1.57 9.05 " 1.22 10.39 " 0.52
1 2 3 4 mean " SE
5.93 " 1.11 11.32 " 0.06 9.20 " 0.30 11.09 " 0.28 9.38 " 1.25
8.59 " 0.67 17.39 " 1.60 13.09 " 0.59 30.9 " 6.22 17.49 " 4.82
11.66 " 1.87 21.41 " 1.07 16.37 " 0.16 27.02 " 2.62 19.11 " 3.30
14.10 " 0.15 23.17 " 6.57 20.58 " 0.04 24.29 " 0.62 20.53 " 2.28
1 2 3 4 mean " SE
9.58 " 0.16 9.64 " 0.61 5.62 " 0.48 7.50 " 0.26 8.08 " 0.96
12.34 " 2.31 12.67 " 1.42 11.56 " 0.53 12.41 " 0.61 12.24 " 0.24
13.66 " 0.03 13.12 " 0.46 12.29 a 13.06 " 1.99 13.03 " 0.28
18.26 " 0.37 18.76 " 2.09 21.39 " 1.63 17.33 " 3.06 18.93 " 0.87
1 2 3 4 mean " SE
10.64 " 0.71 5.83 " 0.19 7.63 " 0.03 6.80 " 0.67 7.72 " 1.04
13.57 " 0.88 7.53 " 0.81 10.32 " 0.59 9.53 " 0.1 10.24 " 1.26
14.27 " 0.04 9.62 " 0.90 10.73 " 1.62 10.52 " 1.02 11.28 " 1.02
21.77 " 5.42 11.30 " 0.64 17.47 " 1.33 16.64 " 1.55 16.79 " 2.15
Cell strain y
GSH
Control 1
Control 2
Control 3
)
)
))
))
SCE frequencies from 4 separate experiments Žmean " SD. in 2 glutathione synthetase deficient ŽGSH y . cell strains and 3 control cell strains. Statistical comparison of SCE at 0 and 10 mM by paired t-test: ) p - 0.01; ) ) p - 0.005. a Single value derived from 20 metaphases.
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295
synthetase-deficient cells grow significantly slower than normal human fibroblasts w25x. In the two GSH synthetase-deficient cell strains used in this study, baseline cell growth rate was approximately half of that seen in the normal human fibroblasts Žsee values in footnote Table 3.. The degree of DBE-induced inhibition of proliferation in the GSH synthetase cell strains is not markedly different from two of the controls.
X
Fig. 3. Sister chromatid exchanges induced by N-methyl-N -nitroN-nitrosoguandine. Normal human skin fibroblasts and fibroblasts from patients with GSH synthetase deficiency were exposed to X N-methyl-N -nitro-N-nitrosoguandine for 2 h and then assessed for sister chromatid exchange after 46 h Ždata from 3–5 experiments..
Fig. 4. Sister chromatid exchange induced by dinitrochlorobenzene. Normal human skin fibroblasts and fibroblasts from a patient with GSH synthetase deficiency were exposed to dinitrochlorobenzene for 2 h and then assessed for sister chromatid exchange after 46 h Ž ns 2..
Table 3 Effect of dibromoethane on cell proliferation Cell strain y
GSH 1 GSHy 2 Control 1 Control 2 Control 3
2.5 mM
5 mM
10 mM
86"7 83"2 64"10 77"10 80"3
80"3 67"8 49"11 63"12 76"10
61"9 48"11 20"5 43 ) 51"10
Cell growth in solvent control Žincrease at time of harvest as % of cell number present at t s 0.: GSHy 1: 129%; GSHy 2: 140%; Controle 1: 264%; Controle 2: 242%; Controle 3: 212%. Values in the table reflect cell growth in the presence of DBE as a percentage of growth in solvent control Ž ns 4.. ) ns 2. GSHy, glutathione synthetase deficient.
4. Discussion This study examines whether hereditary differences in GSH levels in eukaryotic cells can modify the genotoxic response in vitro. The findings are consistent with the literature in this area. Thus low endogenous GSH levels in Salmonella typhimurium exposed to DBE in the Ames test are protective w8x and GSH depletion in rats with diethylmaleate or buthionine sulfoximine decrease DNA adduct formation in vivo w13x. The ability of polymorphisms of P450 and GST to modify the risk of carcinogenic compounds has been examined in the past, but this is the first demonstration that hereditary differences in GSH levels might have this effect in eukaryotic cells. The cell strains used are from individuals with an inborn error of GSH synthetase that leads to very low intracellular levels of GSH. Although such large variations in GSH levels may be needed to show differences in susceptibility in acute exposure experiments, much smaller variations might alter responses in chronic exposure to environmental toxins. Heterozygotes for GSH synthetase deficiency seem phenotypically normal and have normal GSH levels, but have an impaired ability to regenerate GSH and therefore have decreased GSH detoxification capacity w26x. Thus, heterozygosity for GSH synthetase deficiency might protect against genotoxicity from DBE, but might increase the risk for carcinogens detoxified by GSH. Furthermore, it is possible that significant variations in intracellular GSH in the ‘normal population’ would affect the response to chemical carcinogens. Unfortunately, little is known about the range of normal intracellular GSH in the population, on normal variability in GSH synthetic capacity or on the frequency of heterozygosity for
296
L.D. DeLeÕer Mutation Research 389 (1997) 291–297
inborn errors of the g-glutamyl cycle that lead to impaired GSH detoxification. The ability of DBE to induce genotoxicity in human cells assessed by SCE was previously reported in a study of the effect of DBE vapor in peripheral lymphocytes w9x. It is difficult to compare the response to a liquid dissolved in medium with the response to a gas, but the current study and the study in lymphocytes both show a similar increase in the frequency of exchanges. Dibromoethane toxicity is thought to be mainly due to P450-mediated metabolism w2,3x and mutagenicity to metabolites formed following conjugation to GSH w6,7,14x. Studies that used bromoheptane to deplete GSH in hepatocytes showed protection against dibromoethane toxicity and concluded that the glutathione conjugate also contributes to dibromoethane cytotoxicity w2x. In the current study GSH deficiency protected against genotoxicity, but toxicity as manifested by inhibition of cell proliferation was not markedly different from control cells. However, the GSH synthetase-deficient cells have a slow baseline proliferation rate and this complicates the interpretation of cell proliferation data as a measure of toxicity. Severe toxicity has been shown to cause a small artifactual increase in SCEs, perhaps by increased incorporation of bromodeoxyuridine due to reduced cell density w24x. Since dibromoethane was not much more toxic to the control cell strains than to the glutathione synthetase-deficient cell strains, the greater increase in SCEs in the control strains is unlikely to be due to the in vitro artifact. This in vitro artifact could also complicate the use of the SCE assay in slow growing cells, such as the GSH synthetase-deficient cells. However, no significant increase in baseline SCE frequency in the GSH synthetase-deficient cell strains was observed. Furthermore, if this artifact were significant in the setting of a toxin-induced reduction in cell growth in a slower growing cell line, this should artifactually increase SCE frequency. Thus, if this artifact were occurring in the current study, it would decrease the observed differences in SCE between control cells and the GSH synthetase-deficient cells, not enhance the differences. However, if GSH synthetase-deficient cell strains are used in future studies to evaluate compounds that are more genotoxic in GSH
deficiency, this artifact may complicate interpretation. Future studies will need to determine how large the normal variation is in endogenous GSH levels and whether such variations can alter susceptibility towards environmental or occupational exposure to chemical carcinogens.
Acknowledgements The author would like to thank the late Marilyn Cannon for her technical assistance and Dr. Stephen Spielberg for his support and advice.
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