Human Glutathione 5\"Transferase Deficiency as a Marker of Susceptibility to Epoxide-induced Cytogenetic Damage1

(CANCER RESEARCH 50, 1585-1590, March l, 1990]

Human Glutathione 5"-Transferase Deficiency as a Marker of Susceptibility to Epoxide-induced Cytogenetic Damage1 John K. Wiencke,2 Karl T. Kelsey, Rosito A. Lamela, and William A. Toscano, Jr. Department of Epidemiology and Biostatistics, Laboratory of Radiobiology and Environmental Health, School of Medicine, University of California, San Francisco, California 94143 [J. K. W., R. A. L.J; Occupational Health Program, Department of Environmental Science and Physiology and Department of Cancer Biology, Division of Biological Sciences, School of Public Health, Harvard University, Boston, Massachusetts 02135 [K. T. K]; and Division of Environmental and Occupational Health, University of Minnesota, Minneapolis, Minnesota 55455 [W. A. T.]

ABSTRACT The identification of genetic traits that predispose individuals to envi ronmentally induced cancers is one of the most important problems in cancer risk assessment. Genetic deficiency in the jj-isozyme of the glutathione (GSH) 5-transferases (EC 2.5.1.18) has recently been associated with increased lung cancer risk. To test whether this association could arise from a metabolically mediated sensitivity to mutagenic substrates, cytogenetic damage in lymphocytes from 21 isozyme-deficient and 24 nondeficient individuals was induced. Cells were treated with tranxstilbene oxide, an excellent substrate for GSH 5-transferase n, or cisstilbene oxide, a poor substrate for the isozyme. Sister eliminatili ex change induction was measured as an indicator of cytogenetic damage. A trimodal distribution of min.v-stilhcnc oxide-induced sister chromatid exchanges was observed in the population, including resistant, moderate, and highly sensitive groups. Glutathione 5-transferase Mdeficiency was associated with both moderate and high sensitivity to rrans-stilbene oxide-induced damage but had no effect on c/s-stilbene oxide-induced sister chromatid exchange. The results indicate that GSH 5-transferase n, a proposed marker of cancer susceptibility, is also a marker of susceptibility to the induction of cytogenetic damage by a certain class of mutagens. The differential effects of the t-i'.v-and rrans-isomers of stilbene oxide illustrate that the stereoselectivity of GSH 5-transferase Mtoward various alkene epoxide substrates can be an important factor affecting individual sensitivity to DNA-damaging epoxides.

INTRODUCTION GSH3 5-transferases

•¿

(EC 2.5.1.18) are a family of multifunc

tional enzymes that bind organic anions and detoxify reactive electrophiles capable of damaging DNA (1-4). In humans, the three main classes of glutathione 5-transferases, a, /¿,and ir, are the products of three separate gene families located on different chromosomes (5-11). Approximately 50% of Cauca sians are genetically deficient in one isozyme of this family, GSH 5-transferase /*. This isozyme is highly efficient in the detoxification of fraws-stilbene oxide (12-16), a potent epoxide mutagen. Recent evidence indicates that hereditary differences in the expression of glutathione transferase ß are due to a gene deletion (17). Persons deficient in the /u-isozyme have been reported to be at high risk for smoking-induced lung cancer (18). If the enzymatic deficiency in epoxide detoxification causes the observed increase in carcinogenic risk, then somatic cells having this defect could be more susceptible to chromoReceived 3/29/89; revised 8/21/89, 11/8/89; accepted 11/27/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in part by USPHS Grant P02 CA 43764 from the National Cancer Institute, Bethesda, MD (J. K. W.), by USPHS Grant GM/ES02866 (W. A. T.), by Center Grant ES-0002 from the National Institutes of Health. Bethesda, MD (W. A. T., K. T. K.), by a Faculty Development Award, Mellon Foundation (K. T. K.), and by the Office of Health and Environmental Research, United States Department of Energy Contract DE-AC03-76-SF01012 (J. K. W.). 2To whom requests for reprints should be addressed, c/o Editor, Department of Epidemiology and Biostatistics, School of Medicine, University of California, San Francisco. CA 94143-0560. 3The abbreviations used are: GSH, glutathione; SCE, sister chromatid ex change; TSO, frans-stilbene oxide; CSO, c/s-stilbene oxide.

somal damage induced by epoxide substrates of the enzyme. Hypersensitivity to the induction of cytogenetic damage is associated with increased cancer risk in rare genetic chromo somal instability syndromes (19). However, there has been no known example of a genetic polymorphism in human carcino gen metabolism that both is associated with increased cancer risk and has been shown to lead to increased cellular sensitivity to mutagens. In this study, we used the induction of SCEs in peripheral blood lymphocytes to study the relationship between concentra tion-dependent increases in cytogenetic damage and genetic GSH 5-transferase n deficiency. The SCE method was used as an endpoint of cytogenetic damage because it is rapid and highly sensitive for determining the actions of mutagens and carcino gens (20). To test the effects of GSH 5-transferase ^ deficiency on the induction of SCEs by epoxide mutagens, peripheral blood cultures were treated with TSO or CSO, and the number of SCEs induced was measured after 72 h. TSO was used because it is an excellent substrate for GSH 5-transferase Min human lymphocytes (14-18). To test the specificity of GSH 5transferase ^ deficiency on SCE induction, we also treated lymphocytes from some of the subjects with CSO, a poor substrate for GSH 5-transferase M(16). We found that persons whose lymphocytes are deficient in GSH 5-transferase n were more sensitive to the induction of SCEs by TSO. GSH 5transferase ^ deficiency, however, had no effect on the sensitiv ity of lymphocytes to SCEs induced by CSO. MATERIALS

AND METHODS

Study Population. The study group consisted of 45 unrelated normal adult volunteers (19 males; 26 females). Venous blood samples were drawn into 10-ml heparinized Vacutainer tubes (Becton-Dickenson) with the participants' informed consent. Information regarding the subjects' age, sex, smoking history, and occupation was collected. Eight of the 45 subjects were current smokers. Aside from cigarette smoking, none of the subjects was known to have been exposed to agents that might affect SCE frequencies. Cell Culture and Cytogenetics. Whole heparinized blood (0.5 ml) was added to 4.5 ml of RPMI 1640 culture medium containing 2 HIMLglutamine, 10% fetal calf serum, 100 Mg/m' penicillin, 100 units/ml streptomycin, and 2% phytohemagglutinin (Difco, PHA-M). Lympho cytes were cultured for 21 h at 37°C,at 5% CO2 in 1-oz glass prescrip tion bottles. Cells were treated from 21 to 72 h with TSO or CSO (Aldrich Chemical Co., Milwaukee, WI) dissolved in ethanol (0.4% v/ v). Control cultures were treated with ethanol alone. At 24 h of culture 5-bromo-2-deoxyuridine (50 JIM) was added and cells were incubated an additional 48 h. Two h before fixation colcemid (2 x 10~7M; CIBA Pharmaceuticals, Summit, NJ) was added. The cells were collected by centrifugation, exposed to 0.075 M KC1 for 8 min to spread the chromosomes and hemolyze the RBC, and fixed 3 times in methanolacetic acid (3:1). Drops of a concentrated cell suspension were placed on microslides and air dried. The cells were stained by a modification of the fluorescence-plus-giemsa technique (21) to obtain harlequin chromosomes. The slides were immersed for 15 min in a solution of 5 MgHoechst 33258 (Riedel-De Haen AG, Hannover, FRG)/ml in So-

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GSH S-TRANSFERASE

DEFICIENCY AFFECTS SCE INDUCTION

rensen's buffer, pH 6.8, then washed, dried, mounted with buffer under the coverslip, and exposed to a black light 2 cm from two BLB GE tubes at 55"C for 8 min. Finally, the slides were stained for 4 min in a 3% giemsa solution made in the same Sorensen's buffer. SCEs were analyzed in 50 second-division metaphases for each point. GSH S-Transferase Assay. Lymphocytes were isolated by gradient separation with Ficoll-Paque (22), counted, pelleted by centrifugation at 400 x g, and frozen at —¿20"C. GSH 5-transferase n was assayed by a variation of a published procedure (13). Briefly, frozen lymphocytes were sonicated. Approximately 3 x IO6 cells were incubated in a final volume of 100 ^1 containing 176 mM sodium phosphate, pH 7.2, 4 mM reduced glutathione (final concentration), and 250 pM [3H]TSO (spe cific activity, 1.4 Ci/mmol; Chemsyn Science Laboratories, Lenexa, KA). The reaction mixture was incubated at 37'C for 30 min. The reaction was terminated by extraction with 200 p\ n-hexyl alcohol. The tritium content of the aqueous phase was determined by liquid scintil lation spectrometry. Protein concentration was estimated by the mod ified Lowry procedure described by Peterson (23); enzyme activity was expressed as pmol conjugate formed/min/mg protein. The purity of the TSO substrate was 98.2% by thin layer chromatography. The maximum rate of nonenzymatic reaction (boiled lymphocytes) as a percentage of native activity was 24%. Variability in GSH 5-transferase activity was ±5%(SE). The data presented are the means of three determinations. Statistical Methods. To compute mean SCE/cell, 50 second-division mitotic cells were scored for each culture. Comparisons of group mean SCE/cell were made by Student's t test. Analysis of covariance for homogeneity of slopes was carried out with an established computer software program (24).

RESULTS

the isozyme-deficient group, there were six individuals (subjects 25, 33, 35, 36, 38, and 44) whose slope estimates appeared to be greater than the overall group mean slope. To test whether the slopes in this subgroup were comparable to the slopes in the other isozyme-deficient individuals, we performed an analy sis of covariance for homogeneity of slopes. Analysis of covariance showed slopes for the nondeficient group to be homogeneous, but there was significant nonhomogeneity of slopes within the GSH S-transferase ^-deficient group (F = 3.631; P < 0.001). When the slope estimates for these six individuals were removed from the data set and the analysis of covariance was carried out again, the slopes for deficient individuals were homogeneous (F = 0.597; P = 0.845). Consequently, to compare the slopes of isozyme-deficient and isozyme-positive groups, the population was divided into three groups (Table 2), each of which had slope estimates judged homogeneous by analysis of covariance. The mean slope of each of the three groups differed. The mean slope of the moderately sensitive isozyme-deficient group was significantly greater than the mean slope of the resistant group with measurable GSH Stransferase p activity (t = 11.624; P < 0.001). The mean slope of the highly sensitive isozyme-deficient group was significantly greater than that of the moderately sensitive deficient group (t = 10.400; P < 0.001). These results indicate a trimodal distri bution of TSO-induced SCEs in this study group. The three types of dose-response relatinships for each group are shown in Fig. 3. To further test the relationship of enzyme activity and SCE induction, the absolute activity of GSH S'-transferase was cor related with the slope of the SCE dose-response curve within the three groups and for all combinations of groups. There was no correlation of slope with isozyme activity in the nondeficient group (i = 0.455; P = 0.654) or in the deficient group (f = -0.041; P = 0.960). When the data from both groups were combined, however, there was a significant correlation of iso zyme activity with SCE induction (t = -6.971; P < 0.001). Hence, the presence or absence of measurable isozyme activity had a marked effect on the sensitivity of human lymphocytes to SCE induction by TSO; variations in absolute levels of enzyme activity within either deficient or nondeficient groups had no detectable effect on SCE inducibility. Linear correlation analysis of age and the slope of the SCE dose-response curve indicated that age did not influence the sensitivity of cells to the induction of SCEs by TSO. However, five of the six isozyme-deficient persons who were also highly sensitive to SCE induction by TSO were women, whereas based on the number of women in the study, only two would have been expected. cis-Stilbene Oxide-induced SCEs in GSH S-transferase itDeficient Lymphocytes. To test the specificity of GSH S'-trans

GSH .V-Transit-rase p Activity in Human Mononuclear Cells. GSH S-transferase p activity was measured in each of the 45 individuals (Table 1). Twenty-four subjects (53%) had measur able levels of the enzyme (subjects 1-24), and 21 subjects (47%) had negligible levels (less than twice the background) (subjects 25-45). The mean age of persons deficient in the isozyme was 38 ±12 years (median, 35 years); for persons with measurable levels of GSH S-transferase p the mean age was 39 ±11 (median, 35 years). Linear regression analyses indicated that age had no effect on enzyme levels in either the deficient or nondeficient groups or in both groups combined. The sex of an individual had no effect on either the proportion of isozymepositive persons or on the absolute enzyme activity measured. Correlation of TSO-induced SCEs with Expression of GSH 5-Transferase p. Background and TSO-induced SCE frequen cies were measured in each subject (Table 1). The background SCE frequencies of GSH 5-transferase /^-deficient individuals did not differ from those of persons with normal isozyme levels (t = 0.399; P = 0.692). At each TSO concentration tested (50, 100, and 200 pM TSO), lymphocytes from persons with defi cient isozyme activity contained increased numbers of SCEs (P < 0.001). The distributions of SCE scores for ¡sozyme-deficient ferase p deficiency on SCE induction by alkene oxide mutagens, and nondeficient persons were distinct, showing no overlap at lymphocyte cultures were established from five isozyme-defi cient and five nondeficient individuals and treated with 50, 100, either the 100 pM or the 200 pM TSO concentration (Fig. 1). TSO induced linear concentration-dependent increases in the or 200 pM CSO, a poor substrate for GSH 5-transferase p in human lymphocytes. CSO induced SCEs in a concentrationSCE frequencies of lymphocyte cultures from each individual studied. To summarize the sensitivity of an individual to SCE dependent fashion in cells from each of the subjects (Fig. 4). Linear correlation coefficients were computed for each experi induction with TSO, we calculated the slope of the linear regression of SCEs as a function of concentration for each ment. The mean slope of the CSO SCE dose-response curve in nondeficient individuals (7.8 ±2.54 SCE/cell//iM x 10~2) was person. An overall 3.7-fold variation in SCE dose-response slopes was observed (5.7-21.2 SCEs induced/cell/MM TSO x not significantly different from the slope for isozyme-deficient 10~2). The slopes of the dose-response curves for each person individuals (10.34 ±1.6; t = 1.914; P = 0.092). The mean tested were highly reproducible, varying only by 5-10% within slopes for these same individuals when TSO was used as an inducer of SCE were 10.23 ±1.07 and 17.54 ±2.9 SCE/cell/ an individual on repeated blood sampling. pM TSO x 10~2for nondeficient and deficient subjects, respecPlotting the distribution of slopes (Fig. 2) showed that, within 1586 Downloaded from cancerres.aacrjournals.org on January 11, 2016. © 1990 American Association for Cancer Research.

GSH S-TRANSFERASE DEFICIENCY AFFECTS SCE INDUCTION

Table 1 GSH-S-transferase n activity and trans-slilbene oxide-induced sister chromatid exchanges in lymphocyte cultures from 45 individuals

SCE/cell at indicated S-transferase concentration08.047.029.288.347.9610.408.489.849.428.027.726.947.6610.3810.628.129.327.288.68 TSO Subject 11activity (SCE/cellAiM X10~2)9.010.410.710.111.28.310.27 no.123456789101112131415161718192021222324252627282930313233343536373839404142434445Age(yr)3543463332363566356427365132343547362931342636603336362445476 smokerYesNoNoNoNoNoYesNoNoNoNoNoNoYesYesNoNoNoNoNoNoNoNoYesNoNoYesNoNoNoNoNoYesNoNoNoNoNoNoNoNoNoNoNo (pmol/min/mg)24.222.527.317.59.919.415.411.914.012.213.211.011.012.313.914.312.021.821.012.430.615.022.227.64.31.54.92 liM17.5017.2819.5421.7020.0214.4616.1415.1218.5014.7015.8217.1219.3419.8 »IM26.1528.1830.4628.1629.5426.8128.9025.1628.5

DN D3.93.11.6Mean

" ND, not detected, i.e., no detectable activity.

lively, and were different from each other (t = 4.735; P = 0.002). The slope estimates for CSO in human lymphocytes were similar to those for TSO in nondeficient individuals (Table 2); CSO and TSO were roughly equipotent inducers of SCE in these experiments. Therefore, the failure of GSH 5-transferase n deficiency to affect CSO-induced SCEs cannot be attributed to differences in the numbers of SCEs induced by the two isomers.

with previous estimates (12-17). Our data also show that enzyme-deficient individuals can be differentiated from normal individuals by analysis of TSO-induced SCEs. Relative insensitivity to TSO induction of SCEs was observed only in individ uals who expressed GSH S-transferase ß.Individuals who lacked this isozyme activity showed moderate or high sensitivity to SCE induction. Thus, our data indicate that GSH S-transferase n deficiency is a marker of susceptibility to SCE induction by TSO. The contrasting results with c/s-stilbene oxide illus trate that the stereoselectivity of the ^-isozyme can determine DISCUSSION whether enzyme deficiency will differentially affect the sensitiv ity of a population to specific epoxide mutagens. These observations represent the first demonstration in a Humans are exposed to epoxides directly from environmental human population of a common genetic deficiency in carcino sources and via metabolic oxidation of exogenous and endoge nous alkene and arene compounds (25-28). Many epoxides are gen metabolism that is associated with increased susceptibility to mutagen-induced cytogenetic damage. The epoxide used to potent mutagens and carcinogens (29-31). The detoxification demonstrate this effect is a specific substrate for GSH Sof epoxides is mediated by conjugation with GSH, a reaction catalyzed by GSH transferases, or through hydrolysis by epoxtransferase p, but it has not been shown to be a constituent of cigarette smoke. In future studies of the relationship between ide hydrolases (32, 33). In humans, the existence of a genetic deficiency in the ^-isozyme of GSH transferase provides a GSH S-transferase n deficiency and lung cancer, it will be important to identify substrates of this isozyme that are derived unique opportunity to test the relative importance of GSH from exposure to cigarette smoke itself. For example, it is conjugation in the induction of genetic damage by epoxide known that benzo(a)pyrene, which is found in cigarette smoke, mutagens. can be metabolized in human cells to benzo(a)pyrene 4,5-oxide Our observation that approximately half of the test popula tion was deficient in GSH S-transferase n activity agrees well and anti-benzo(a)pyrene 7,8-diol 9,10-oxide, both substrates 1587 Downloaded from cancerres.aacrjournals.org on January 11, 2016. © 1990 American Association for Cancer Research.

GSH S-TRANSFERASE

15

DEFICIENCY AFFECTS SCE INDUCTION

PANEL A Control

10

PANEL B ISO 50(jM

PANEL C TSCMOOÃ-/M

10

15

20

25

Linear Regression Slope (SCE/Cell/0Mx10-2) Fig. 2. Population distribution of linear regression slopes for SCE doseresponse curves for TSO-treated lymphocyte cultures from 45 subjects. Slope estimates are based on mean SCE/cell at 0. 50, 100. or 200 MMTSO for each individual. The units for the slopes are SCEs/cell/MM x IO"2. •¿. slope estimates for individuals with measurable GSH S-transferase Mactivity; El, slope estimates for persons deficient in GSH 5-transferase M.

including two subgroups of GSH 5-transferase /u-deficient in dividuals, one with moderate sensitivity to TSO-induced dam age and one with high sensitivity. Although the mechanisms responsible for this variation in mutagen sensitivity among isozyme-deficient groups are as yet unknown, it is possible that the highly sensitive individuals carry a second genetic deficiency that further increases susceptibility to epoxide-induced DNA 5 10 15 20 25 30 35 40 45 50 damage. It has been proposed that double genetic polymor Mean SCE Cell phism in drug metabolism can lead to greatly exaggerated drug Fig. 1. Population distribution of mean SCE/cell for lymphocyte cultures toxicity (40). from 45 subjects. Cultures for each individual were treated with ethanol alone Epoxide hydrolases, which catalyze the conversion of epox(A), 50 MMTSO (fi), 100 MMTSO (C), or 200 MMTSO (D). •¿ SCE scores for individuals with measurable GSH 5-transferase M activity; O, SCE scores for ides to dihydrodiols, could also modify the genotoxic effects of persons deficient in GSH S-transferase M.Note that the ordinate scale is different TSO. Although genetic deficiency in epoxide hydrolases has for each panel. Where the solid and hatched bars are at the same SCE value, the not been reported in humans, cytosolic epoxide hydrolase activ hatched bars should be read as beginning at the top of the black bars. ity toward TSO varies widely in liver (41) and mononuclear cells (42) from different individuals. A microsomal epoxide for GSH 5-transferase M (34, 35). Other potential substrates associated with smoking include styrène oxide (16, 36) and hydrolase with activity toward TSO has also been described in mice (43). Methods have been developed that allow the meas ethylene oxide (16, 37). Although the genotoxic effects of these and other epoxide substrates for GSH S-transferase ft would be urement of both GSH S-transferase n and cytosolic epoxide hydrolase sequentially, using the same cell homogenate (44). It expected to be greater in isozyme-deficient cells, other factors, is likely that the induction of SCEs by TSO, like epoxideincluding competing pathways for epoxide degradation, GSH concentration, and the subcellular compartmentalization of induced mutagenicity (45), is the product of several enzymes that control the concentrations of reactive metabolites capable GSH 5-transferase isozymes (38, 39), may also affect genotox of damaging DNA (46). A full understanding of the significance icity. Consequently, the degree of increased susceptibility to of various enzymes in the control of TSO-induced damage will genotoxicity in GSH 5-transferase ^-deficient cells will prob ably vary for each mutagen substrate and will have to be likely require a correlation of various enzyme activities with measures of genotoxicity. In addition, it may also be important determined experimentally. GSH 5-transferase \i activity toward TSO is inherited as a to consider the potential role of individual variations in DNA repair in TSO induction of SCEs. simple autosomal dominant trait (15). Consequently, the pop The increased frequency of GSH S-transferase n deficiency ulation distribution of sensitivities to TSO-induced cytogenetic observed in lung cancer patients has led to the proposal that damage should be bimodal: resistant individuals with enzyme activity and sensitive individuals deficient in the enzyme. How this isozyme deficiency is a genetic marker of cancer suscepti bility in humans (18). As we have shown, GSH-transferase ¿< ever, we found a trimodal distribution of sensitivities to TSO, 1588 Downloaded from cancerres.aacrjournals.org on January 11, 2016. © 1990 American Association for Cancer Research.

GSMS--TRANSFERASE DEFICIENCY AFFECTS SCEINDUCTION Table 2 Induction of sister chromatid exchanges in lymphocyte cultures by trans-stilbene oxide from 24 individuals with measurable (iSH-S-transferase ¡iactivity and 21 isozyme-deflcient individuals

«activity (pmol/min/mg)17.2

SCE inducibility byTSOResistant

SCE/cell at concentration08.87 indicated TSO MM28.49

MM18.39

IHM13.13

±1.34 ±6.1 ±1.22 Sensitive 18.87± 1.94 1.8 ±1.8 9.02 ±1.30 21 Intermediate sensitivity 1.6 ±1.7 8.95 ±1.02 18.13 ±1.58 15 High sensitivityn24 2.4 ±1.9Mean 9.18 ±1.9650 20.73 ±1.50100 6GSH-S-transferase °The mean ±SD of the individual linear regression coefficients for each group indicated. Units

slope"9.9

±2.12 ±3.01 28.26 ±3.24 42.06 ±5.07 16.4 39.19 ±2.33 15.0 26.68 ±2.06 31.95 ±2.37200 49.22 ±1.20Mean19.9 for the slope estimates are SCE/cell/jiM

of slopes5.7-11.3

±1.4 13.0-21.1 ±2.5 13.0-16.5 ±1.0 ±0.9Range18.8-21.1 x 10

35

50

30 40

CD O

111 30 O

LU Q CO

20

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Human Glutathione S-Transferase Deficiency as a Marker of Susceptibility to Epoxide-induced Cytogenetic Damage John K. Wiencke, Karl T. Kelsey, Rosito A. Lamela, et al. Cancer Res 1990;50:1585-1590.

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