Expression of stably transfected murine glutathione S-transferase A3-3 protects against nucleic acid alkylation and cytotoxicity by aflatoxin B1 in hamster V79 cells expressing rat cytochrome P450-2B1
Wanda R. Fields1,3,
Charles S. Morrow1,
Johannnes Doehmer2 and
Alan J. Townsend1,4
1 Biochemistry Department, Bowman Gray School of Medicine, Wake Forest University Comprehensive Cancer Center, Medical Center Boulevard, Winston-Salem, NC 27157, USA and
2 Institut für Toxikologie und Umwelthygiene, Technische Universität Muenchen, Lazarettstrasse 62, D-80636, München, Germany
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Abstract
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Aflatoxin B1 (AFB1) is activated to AFB1-8,9-oxide (AFBO), a potent mutagenic and carcinogenic metabolite of AFB1. In the mouse, AFBO has been shown to be most efficiently detoxified by a specific isozyme of
-class glutathione S-transferase (GST), mGSTA3-3 (mGST-Yc). A hamster V79 cell line (V79MZr2B1, originally designated V79/SD1) previously transfected with the rat cytochrome P450-2B1 was stably transfected with an mGSTA3-3 expression vector, to study the chemopreventive role of GST in protecting against cytotoxicity or genotoxicity of AFBO. Immunoblotting demonstrated strong expression of an
-class GST in the mGSTA3-3 transfected cell line, whereas no detectable
-class GST protein was observed in the control (empty vector-transfected) cells. Previous studies with the V79MZr2B1 cell line indicated that it can activate AFB1 to a mutagenic metabolite via a transfected rat P450-2B1 stably expressed in the cells. We examined the ability of the expressed mGSTA3-3 to protect against AFB1-induced cytotoxicity or [3H]-covalent adduct formation in cellular nucleic acids. Exposure of empty vector-transfected control cells and mGSTA3-3 expressing cells to up to 600 nM [3H]-AFB1 indicated that a 7080% reduction in DNA and RNA adducts was afforded by the expression of mGSTA3-3 in the transfected cells. Clonogenic survival assays showed that the mGSTA3-3 cell line was 4.6-fold resistant to AFB1 cytotoxicity as compared with the empty vector-transfected control SD1 cells, with IC50 values of 69 and 15 µM, respectively. The results of these studies demonstrate that mGSTA3-3 confers substantial protection against nucleic acid covalent modification and cytotoxicity by AFB1 in this transgenic cell model system.
Abbreviations: AFB1, aflatoxin B1; [3H]-AFB1, 3H-labelled AFB1; AFBO, aflatoxin B1-8,9-epoxide; CDNB, 1-chloro-2,4-dinitrobenzene; GST, glutathione S-transferase; hGSTM1, human glutathione S-transferase µ-1; hGSTT1, human glutathione S-transferase
-1; mGSTA3-3, murine glutathione S-transferase
-3 homodimer; rCYP2B1, rat cytochrome P450-2B1.
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Introduction
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The aflatoxins are produced by the mold Aspergillus, as a contaminant of improperly stored food. Aflatoxin exposure may cause hepatotoxicity, kwashiokor, Reye's syndrome and liver cancer (1). Rats develop hepatic tumors after chronic exposure to alflatoxin B1 (AFB1). In contrast, mice are much less susceptible to AFB1 toxicity and induction of liver carcinogenesis (2,3).
Carcinogenic consequences of AFB1 adduct formation include the activation of ras oncogenes in F344 rats (4) and the inactivation of p53 tumor suppressor genes in humans (5). A specific mutation at codon 249 of the p53 tumor suppressor gene in hepatocellular carcinomas is highly correlated with AFB1 exposure in humans (6,7). This mutation is found in individuals residing in areas of high aflatoxin exposure, and is also strongly associated with prior hepatitis B infection (8).
The hepatic toxicity and carcinogenicity of AFB1 is directly associated with its metabolic activation. The primary carcinogenic metabolite is aflatoxin exo-8,9-epoxide (AFBO) which is formed by the oxidation of the furan ring by microsomal cytochrome P450 isozymes (9). Another isoform of this metabolite has also been described (10). The endo-epoxide may play a role in AFB1 toxicity (11), since it is more efficient for formation of protein adducts, whereas the exo-epoxide is apparently the more efficient metabolite for DNA adduct formation (12). Hence, the exo-epoxide metabolite is generally considered most relevant to AFB1 carcinogenesis (5).
Rats fed dietary cruciferous vegetables such as broccoli, or fractionated extracts thereof, prior to AFB1 exposure exhibit a reduction in AFB1DNA adduct formation, and a reduction in the number of enzyme-altered hepatic foci or primary liver tumors (13,14). Increased AFBO conjugating activity was related to decreased AFB1DNA binding in animals fed a broccoli diet. Potent protection against aflatoxin-induced hepatocarcinogenesis and correlation with increased GST activity has also been described in animals fed diets supplemented with synthetic antioxidants or anticarcinogenic compounds (15,16). Resistance to AFB1 toxicity and carcinogenesis in rats maintained under chemopreventive regimens is associated with induction of an
-class GST (rGSTA10-10, previously designated as Yc2) that exhibits high activity for conjugation of AFBO (17). The murine ortholog of the rat inducible Yc2 (mGSTA3-3) is constitutively expressed in mouse liver, and appears likely to be responsible for much of the resistance of this species to AFB1 toxicity (18,19).
The 100-fold resistance to aflatoxin-induced hepatotoxicity and reduction in AFB1DNA adducts in mice as compared with rats is associated with a 50- to 100-fold higher constitutive GST activity toward AFBO in mice as compared with rats, while the GST activity towards 1-chloro-2,2-dinitrobenzene (CDNB) is comparable between the two species (20). Bacterial GST expression systems were used to determine AFBO conjugating activities of 3.3 nmol/min/mg for rat GSTA5-5 (GST-Yc1) and 144 nmol/min/mg for murine GSTA3-3 (GST-Yc) (21). These differences in specific activity may account for the inherent differences in species susceptibility. However, other studies also implicated the induction or overexpression of other relevant phase II detoxification pathways (11). Therefore, the goal of the current study was to determine the protection conferred against the genotoxic and cytotoxic effects of AFB1 solely by expression of mGSTA3-3.
A hamster V79 cell line (V79MZr2B1, previously designated V79/SD1) which had previously been stably transfected with the rat cytochrome P450-2B1 (rCYP2B1) was supertransfected with a mGSTA3-3 expression vector, to model the role of this GST isozyme in protecting against covalent modification of cellular nucleic acids by AFBO. Previous studies with V79MZr2B1 cells indicated that these cells are rendered more sensitive to AFBO-induced mutations due to the activation of AFB1 by the stably transfected rat CYP2B1 in the cells (22). Using this model system, we have found that expression of mGSTA3-3 confers substantial protection against nucleic acid adduct formation and cytotoxicity by AFB1 in this transgenic cell model.
[3H]-AFB1 (18 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). Hygromycin B was purchased from Calbiochem (San Diego, CA). Unlabeled AFB1 and other chemicals were reagent grade, purchased from Sigma (St Louis, MO), Aldrich (Milwaukee, WI) or Fisher Scientific (Raleigh, NC).
Establishment of the V79MZr2B1 line has been described previously (22). This cell line expresses a rCYP2B1 activity of 36 pmol/min/mg, or ~4-fold higher activity than hepatocytes from untreated rat liver (22). All cell lines were passaged as a monolayer in DMEM (Gibco, Long Island, NY) containing 5% fetal bovine serum (FBS; Gibco) and 0.7 mg/ml of hygromycin, and maintained at 37°C in a humidified 95% air/5% CO2 atmosphere. Cells were grown without hygromycin for 1618 h prior to experiments.
Murine GSTA3-3 (mGSTYc) cDNA was provided by Dr David Eaton (University of Washington, DC). The mGSTYc cDNA was subcloned into the
pCEP4 mammalian expression vector, downstream of a CMV early promoter. The
pCEP4 expression vector has been used successfully in previous studies to obtain stable high level expression of GST or aldehyde dehydrogenase isoenzymes in V79 cells (23,24). This vector was generated by deletion of the EBNA and the oriP origin sequences of the commercially available pCEP4 plasmid expression vector obtained from Invitrogen (Carlsbad, CA). These modifications allow selection with hygromycin for stable integration of the plasmid construct into the host cell genome. The V79MZr2B1 cell line was transfected by calcium phosphate transfection with either empty
pCEP4 vector, or the mGSTA3-3 expression vector, then selected with 0.7 mg/ml hygromycin.
Transfected clonal cell lines were grown in 100 mm tissue culture dishes, and subconfluent plates were harvested by scraping into PBS/5 mM EDTA and pelleted by centrifugation at 208 g for 5 min. Cells were resuspended in 34 vol 50 mM Tris/5 mM EDTA and mildly sonicated for 10 s using an Artek model 300 sonicator and microprobe tip at 25% power. The lysates were microcentrifuged for 10 min at 4°C and supernatants were assayed for GST activity with CDNB at 25°C by the method of Habig and Jakoby (25). Protein was determined with the bicinchoninic acid protein assay reagent (Pierce, Rockford, IL). Expression of mGSTA3-3 in the stably transfected cell line was 98 mU/mg using CDNB as substrate, as determined by subtraction of the average activity of the control S
P-1 cell line (153 ± 5.0 mU/mg) from that measured in the mGSTA3-3-expressing cell line mGSTYc1-4 (251 ± 6.5 mIU/mg).
Western blot analysis using an affinity-purified polyclonal antibody developed against human
-class GST demonstrated a strongly expressed protein of the expected size in the cell line transfected with the mGSTA3-3 expression vector, but not in the empty vector-transfected control line (Figure 1
). The V79MZr2B1 cell line is a hamster lung fibroblast line that also expresses a low level of
-class GST (~150 mU/mg) but no hamster
- or µ-class GST activity. The rat and human
-class GSTs have very little activity for conjugation of AFBO (26) and hence the low background hamster GSTP1 expression should not interfere with the results of these studies, particularly since it is present equally in control and mGSTA3-3-expressing lines.

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Fig. 1. Western blot analysis of control and mGSTYc-transfected SD1 lines. Cytosol (100 µg/lane) was fractionated on a 14% gel by SDSPAGE, and transferred to nitrocellulose membrane (Schleicher and Schuell) by semi-dry electroblotting. Blots were probed with affinity-purified rabbit polyclonal antibodies directed against human -class GST. Blots were washed and incubated further at 25°C in a 1:1000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG antisera (Cappel), then developed in TBS containing 15% methanol, 0.5 mg/ml 4-chloro-1-napthol (Sigma) and 0.015% H202. Lane 1, 50 ng human GSTA2-2 standard; lane 2, molecular weight ladder; lane 3, SD1 (parental); lane 4, mGSTA3 transfected clone mGSTYc1-4.
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The V79MZr2B1 cell line used as recipient for transfection with the mGSTA3 expression vector was previously stably transfected with a retroviral vector encoding the rat cytochrome P450-2B1. Expression of the rCYP2B1 increased the number of mutant colonies by at least 4-fold over a range of AFB1 exposure levels (22). In order to assess the effect of mGSTA3-3 expression on covalent modification of total nucleic acids by [3H]-AFB1, the amount of radiolabel in DNA was measured at 100, 300 and 600 nM in the control (empty vector-transfected) and mGSTA3-transfected V79MZr2B1 cell lines. The degree of total nucleic acid AFB1 adducts observed in the control cell line was reduced ~3-fold in the mGSTYc1-4 line due to expression of mGSTA3-3 in these cells (Figure 2
). Adduct formation was also measured for DNA and RNA separately following treatment with [3H]-AFB1 (600 nM) and CsCl gradient ultracentrifugation. This was done to determine if the protection against covalent modification differed for these targets; for example, due to nucleic acid structure or intracellular compartmentation. Similar protection against adduct formation was conferred by mGSTA3-3 in the mGSTYc1-4 cell line, with reductions in covalent modification of 3-fold for DNA and 4-fold for RNA (Figure 3
).

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Fig. 2. Sensitivity of mGSTYC-transfected and control SD1 cell lines to covalent attachment of [3H]-AFB1 to total nucleic acids. Subconfluent cells were exposed to 100, 300 or 600nM [3H]-AFB1, or solvent (DMSO) for just 4 h in serum-free media and subsequently supplemented with serum-containing media to a final concentration of 5% FBS for a total of 24 h. Nucleic acids were isolated from harvested cell pellets by digestion with proteinase K, phenolchloroform extraction and ethanol precipitation, then analyzed by scintillation counting. Labeling was normalized to nucleic acid concentration by measurement of the absorbance at 260 nM. Filled bars represent S P-1 (empty-vector control) and hatched bars represent mGSTA3 transfected clone mGSTYc1-4. Error bars indicate means ± SD of three or more separate determinations.
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Fig. 3. Comparison of [3H]-AFB1 adduct formation in DNA and RNA. Cells were exposed to [3H]-AFB1 (600 nM, 10.8 µCi/ml) in serum-free media for 4 h. Afterwards, serum-containing media was supplemented to a final concentration of 5% FBS and the incubation was continued for a total of 24 h. DNA (A) and RNA (B) were isolated from harvested cell pellets by cesium chloride gradient ultracentrifugation and analyzed by scintillation counting. The results were normalized to the amount of nucleic acid in each sample (n = 3). Filled bars represent S P-1 (empty-vector control) and hatched bars mGSTA3 transfected clone mGSTYc1-4. Error bars indicate means ± SD of three or more separate determinations.
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Clonogenic survival of the S
P-1 empty vector-transfected control line and the mGSTA3-transfected cells was determined by continuous exposure to AFB1 concentrations ranging from 10 to 100 µM. The profile of the survival curves was distinctive between the control and GST-transfected cell line (Figure 4
). The averaged IC50 values were 14 ± 4.7 µM in the control and 69 ± 9.2 µM in the mGSTYc1-4 cells. Therefore, these results indicate that the expression of mGSTA3-3 conferred ~5-fold resistance to AFB1 cytotoxicity.

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Fig. 4. Sensitivity of mGSTA3-transfected and control V79/SD1 lines to AFB1 cytotoxicity. Single cell suspensions were prepared after brief exposure to trypsin/EDTA, and 200 cells/well were plated into 12-well plates for 1824 h prior to experiments. Cells were exposed 1618 h after plating to the indicated concentrations of AFB1, then grown for 7 days in continuous exposure, and stained with methylene blue. Surviving colonies were counted and clonogenic survival was expressed as a percentage of the control (vehicle-treated) wells. Open circles denote S P-1 (empty-vector control) and filled circles denote mGSTA3 transfected clone mGSTYc1-4. Error bars indicate means ± SD of three or more separate determinations.
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Prior investigations have indicated that covalent reaction of AFBO at the N7 position of guanine is the most frequently induced DNA lesion related to aflatoxin-induced carcinogenesis. However, significant variability exists in the capacity of tissues from different species to catalyze the oxidation of AFB1, with 4-fold greater rate observed with rat than with human liver microsomes (27). Nevertheless, although mouse liver microsomes have a higher rate of AFBO production than rat microsomes, mice are resistant to the toxicity and hepatocarcinogenicity of aflatoxin. Mouse liver also contains a constitutively expressed
-class GST with high activity for the conjugation of aflatoxin to glutathione, whereas the rat ortholog of this isozyme is only expressed upon induction; for example, by the antioxidant ethoxyquin. Furthermore, protection of rats against AFB1 toxicity, DNA adduct formation and tumorigenesis by exposure to anticarcinogenic agents is correlated with induction of rGSTA10-10 (GST-Yc2) (15).
These results suggested that the rate of detoxification, rather than activation, might be the principal phenotypic determinant of relative susceptibility to AFB1 toxicity and carcinogenesis in rodents under normal conditions. A corollary inference is that GSTs may be primary mediators of the effects of some anticarcinogenic agents by protecting DNA against genotoxic exposure to carcinogenic electrophiles. The results presented here are consistent with this hypothesis for the role of mGSTA3-3 in conferring inherent AFB1 resistance in the mouse model. Conversely, a low capacity for AFB1 conjugation with GSH in trout liver (28), and the inability to induce expression of a GST having high activity for AFB1 conjugation in this species (29) appears to explain the high sensitivity to AFB1 hepatocarcinogenesis in this species. Humans also appear to lack a specific GST isozyme having the exceptionally high activity for AFBO observed with the mGSTA3-3 or rat 10-10 isozyme, although the µ-class hGSTM1-1 has significant activity (26).
This study is the first direct demonstration that expression of this murine GST isozyme in stably transfected cells can protect against AFB1 genotoxicity and cytotoxicity at the cellular level. The degree of protection observed was less than that reported in rats treated with anticarcinogenic agents prior to AFB1 exposure despite the several-fold greater specific activity of the murine GSTA3-3 isozyme with AFBO as substrate (15). This may be due to differences in the cellular GST expression levels, or to the different intracellular milieu in the different types of cells expressing mGSTA3-3. Certainly, the activation by the transfected rat CYP2B1 may differ from activation in mouse liver in terms of the ratio of the endo- and exo-epoxide stereoisomers. Alternatively, other anticarcinogen-induced factors may contribute to AFBO detoxification in vivo, such as the increased expression of an aldo-keto reductase which was found to detoxify a cytotoxic aldehyde-containing metabolite of AFB1 (11). Yet another possibility is that more efficient removal of the conjugated product (AFBSG) may occur in rat liver. Indeed, conjugate efflux function has been shown by our group to be essential for maximal protection against other genotoxic and cytotoxic GST substrates such as 4-nitroquinoline-1-oxide, chlorambucil and ethacrynic acid (3033).
Attenuation of carcinogenesis by prevention of initiating DNA damage is dependent upon the balance between activation and detoxification of carcinogenic compounds. The use of the V79MZr2B1 cells previously transfected with rat cytochrome P450-2B1 and supertransfected with mGSTA3-3 allowed investigation of the toxicologic outcome of competition between activation of AFB1 and subsequent conjugation of AFBO in intact cells. These studies suggest that GST can effectively detoxify AFBO generated via oxidation by rCYP2B1, and that expression of the
-class mGSTA3-3 isozyme can achieve a dominant detoxification phenotype that controls the outcome of AFB1 exposure in this model system. Furthermore, molecular epidemiologic evidence has supported a link between known human genotypes involving deletion of hGSTM1 or hGSTT1 genes and susceptibility to hepatocellular carcinoma among individuals with chronic AFB1 exposure, and most particularly among those also previously infected with the hepatitis B virus (34,35). The capacities of these or other human GST genes for reduction of AFB1 genotoxicity and their possible roles in prevention of AFB1 carcinogenesis are key issues for future investigation.
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Acknowledgments
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This work was supported by USPHS grant no. 1-RO1-ES-06006 from the National Institute for Environmental Health Sciences (A.J.T.), and grant no. Do 242/6-2 from the Deutsche Forschungsgemeinschaft (J.D.). The authors wish to thank Dr David Eaton (University of Washington, DC) for providing the mGSTA3 cDNA used to construct the expression vector.
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Notes
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3 Present address: RJR Research and Development, Cellular and Molecular Biology Division, Building 630-2, Winston-Salem, NC 27102, USA 
4 To whom correspondence should be addressed Email: atown{at}wfubmc.edu 
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Received July 22, 1998;
revised January 13, 1999;
accepted January 27, 1999.