Kinetics of Glutathione Transferase, Glutathione ... - Cancer Research

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and Genetics [J. F. M.], TheJohns Hopkins University School of Medicine, Baltimore, Maryland ... ase] specific activities in several tissues, and elevated hepatic.
[CANCER RESEARCH 44, 5256-5261,

November 1984]

Kinetics of Glutathione Transferase, Glutathione Transferase Messenger RNA, and Reduced Nicotinamide Adenine Dinucleotide (Phosphate):Quinone Reductase Induction by 2(3)-ferf-Butyl-4-hydroxyanisole in Mice1 Ann M. Benson,2 Markus J. Hunkeler,3 and John F. Morrow Department of Pharmacology and Experimental Therapeutics [A. M. B., M. J. H.], and Howard Hughes Medical Institute Laboratory, Department of Molecular Biology and Genetics [J. F. M.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

genie aclivily in vivo (2, 6), alters microsomal metabolism of benzo(a)pyrene(13,18, 25, 35, 41, 42), increases Ihe activities The mechanisms by which 2(3)-fe/f-butyl-4-hydroxyanisole of several enzymes lhal have major roles in Ihe nonoxidalive (BHA) protects against chemical carcinogenesis and toxicity metabolism of a variely of reactive eleclrophiles (3-6, 8-10,13, include enhancement of the activities of several detoxification 32, 35, 43, 44), and elevates tissue concenlralions of GSH (2, enzymes. In previous studies, 14-day administration of BHA to 4). female CD-1 mice at 0.75% of the diet led to large increases in GSH Iransferases (EC 2.5.1.18) and NAD(P)H:quinonereduc cytosolic glutathione transferase (EC 2.5.1.18) and reduced nicÃ-ase(EC 1.6.99.2) are induced by BHA and have key roles in otinamide adenine dinucleotide (phosphate) dehydrogenase (qui- protection againsl deleterious chemicals (5, 11, 17, 19-23, 28, none) (EC 1.6.99.2) [NAD(P)H:quinone reducÃ-ase;DT-diaphor29, 31, 38, 50). The rates al which Ihese enzymes respond to ase] specific activities in several tissues, and elevated hepatic BHA can indicate Ihe time required for increases in detoxication glutathione transferase messenger RNA. In the present study, potential in organs that are targels for specific carcinogens and one day of dietary BHA significantly increased NAD(P)H:quinone in Ihose lhal have major roles in systemic metabolism of carcin reducÃ-aseand glutalhione Iransferase aclivilies in Ihe liver, kid ogens. ney, and proximal small inlesline, and NAD(P)H:quinonereduc We examined previously Ihe rales al which GSH Iransferase Ã-aseactivity in the forestomach and lung. In Ihe proximal small activities increased in liver cylosol in response lo dietary admin intestine, glutathione transferase specific aclivilies toward 1islralion of BHA lo mice for 3 to 12 days (3). These sludies have chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nilrobenzene rose been extended now to include hepatic GSH Iransferase and to 2.6 and 8 limes Ihose of conlrol, respeclively, and NAD(P)H:quinonereducÃ-aseal earlier lime poinls. Dietary BHA NAD(P)H:quinone reducÃ-asespecific activity doubled, wilhin 1 elevates mRNA for Ihe major GSH Iransferase of mouse liver day on Ihe BHA diet Six hr after a single p.o. dose of BHA (620 (33). To investigate further Ihe mechanism of Ihe increase in Ihis mg/kg), intestinal glutathione Iransferase specific aclivilies were enzyme, we have examined Ihe kinelics of increase of ils mRNA 30 to 50% above Ihose of conlrol mice. In liver, Ihe kinelics of accompanying BHA adminislralion. Dietary BHA enhances cy increase of glulalhione Iransferase messenger RNA were in tosolic GSH Iransferase and NAD(P)H:quinonereducÃ-aseaclivi accord wilh increased synlhesis as the mechanism of elevation lies also in extrahepatic tissues (4, 5, 39, 40). We now report of glulalhione Iransferase aclivily in response to BHA. Allhough Ihe rales of response of Ihese enzymes in Ihe lung and in changes in mixed-funclion oxygenase aclivilies have been re digestive and urinary Iracl tissues. ported to occur more rapidly, Ihe kinelics of Ihe response of glulalhione Iransferase and NAD(P)H:quinonereducÃ-asespecific MATERIALS AND METHODS aclivilies lo BHA indicates lhal nonoxidalive detoxification po Treatment of Animals and Tissues. Female CD-1 mice (Charles tential is substantially enhanced within 24 hr or less after initiation River Breeding Laboratories, Wilmington, MA) were 4 to 5 weeks old of BHA adminislralion. when received and were acclimatized for at least 20 days prior to the ABSTRACT

INTRODUCTION

BHA4prolecls againsl numerous slruclurally dissimilar carcin ogens (47-49). The mechanisms of protection are believed to include extensive effecls of BHA on enzymes involved in Ihe melabolism of such chemicals (6, 43, 49). BHA reduces mula1Supported by American Cancer Society Special Institutional Grant No. 3 and by NIH Grants CA 16519 and CA 38791. 2To whom requests for reprints should be addressed, at Department of Bio chemistry, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205. 3 Present address: Department of Biochemistry, University of Maryland, College Park, MD. 'The abbreviations used are: BHA, 2(3)-tert-butyl-4-hydroxyanisole (approxi mately 5% 2-fert-butyl-4-hydroxyanisote and 95% 3-fert-butyl-4-hydroxyanisote); GSH, glutathione; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene; BP, benzo(a)pyrene; GT-8.7, major glutathione transferase of murine liver, which has an ¡soelectricpH of 8.7. Received September 12, 1983; accepted August 2,1984.

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administration of BHA. The mice were housed in stainless steel wire cages and received ad libitum either a powdered diet of Purina laboratory chow (Ralston-Purina Co., St. Louis, MO) or the BHA diet. BHA (Sigma Chemical Co., St. Louis, MO) was incorporated into the powdered diet at a final concentration of 7.5 g/kg. The change to the BHA diet was made at 3:30 p.m., and the mice were killed by cervical dislocation between 2:30 p.m. and 4:30 p.m. on the days indicated below. Alterna tively, BHA was administered by intubation p.o. as a single dose of 620 mg/kg in 0.1 ml of Emulphor EL-620P (GAP Corporation, New York, NY). The collection and storage of tissues and the preparation of cytosol fractions were performed as described previously (4, 5). Enzyme Assays. Cytosol fractions were assayed promptly for GSH transferase and dicumarol-sensitive NAD(P)H:quinone reducÃ-ase activi ties. The quinone reducÃ-ase activities were measured with NADH (P-L Biochemicals, Milwaukee, Wl) and 2,6-dichloroindophenol (Sigma) as substrates, by a modification of the procedure of Ernster (15), as de scribed previously (5). GSH transferase activities were determined with CDNB or DCNB (both from Eastman Organic Chemicals, Rochester, NY), and GSH (Sigma) as substrates, according to the procedures of

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Induction of Enzymes and mRNA by BHA Habig et al. (16) except that the concentration of DCNB in the assay system was 0.5 HIM (rather than 1 mw) to facilitate solubility. All values have been corrected for nonenzymatic rates. Protein concentrations in the cytosol fractions were determined by the method of Lowry ef al. (30). RNA Purification and Cell-free Translation. Equal portions of 4 livers, making a total of 0.75 g, were homogenized in a Polytron (Brinkman Instruments) with 12 ml of guanidinium thiocyanate stock solution, and RNA was purified by a method described by Chirgwin et al. (12). After centrifugation for 10 min at 8000 rpm, 8.4 ml of supernatant was layered over 2.9 ml of 5.7 M CsCI, 0.1 M trisodium EDTA, pH 7, and centrifuged in an SW41 rotor (Beckman Instruments, Inc.) at 34,000 rpm for 24 hr at 20°.Each pellet was extracted overnight with 0.4 ml of 67% ethanol, 30 m«NaCI, at -20°, dissolved in 1.5 ml H2O, and extracted with 1.5 volumes of phenohchloroform (1:1, by volume). The RNA was precipi tated from 0.06 M potassium acetate (pH 5) in 67% ethanol, at -20°. It was dissolved in H2O and stored at -80°. Cell-free translation was carried out in a system derived from wheat germ, obtained from BRL, Gaithersburg, MD (36). Each translation mixture (25 /il) contained 2 /tg liver RNA and 12 //Ci of L-^SJmethionine (specific activity, 770 Ci/mmol). After incubation at 24°for 90 min, the polypeptides synthesized were analyzed by sodium dodecyl sul fate: poly aery lamide gel electrophoresis (24). An aliquot of a translation mixture containing 160,000 counts/min of incorporated L-^SJmethionine (about 12 /il) was heated to 90°for 10 min with 2-mercaptoethanol and sodium dodecyl sulfate (24). It was then applied to a sample well of a gel of 20% acrylamide:0.25% bisacrylamide of dimensions 1 mm x 14 cm x 17 cm high, with a stacking gel of 6% acrylamide:0.075% bisacry lamide. Electrophoresis was for 14 hr at a constant current of 10 ma. Quantitative fluorography of gels utilized the method of Laskey and Mills (26). The proteins which served as molecular weight standards applied to other samples of the same gel were bovine serum albumin (M, 68,000) ovalbumin (M, 43,000), a-chymotrypsinogen A (M, 25,700), and lysozyme (M, 14,300). The relative molecular weight of the polypeptides indicated as Mr 17,000 in Fig. ÌA was estimated from a graph of the logarithm of molecular weight versus electrophoretic mobility.

RESULTS Liver. Although GSH transferase and NAD(P)H:quinone reductase specific activities in the liver cytosol increased steadily throughout the experimental period, very substantial increases (p < 0.001) were observed already at the earliest time points (Chart 1). Within 1 day on the BHA diet, GSH transferase activities with both CDNB and DCNB rose to 1.7 times control levels, and the quinone reducÃ-ase activity more than doubled. The rates of increase in GSH transferase specific activities during the remainder of the experimental period were very similar to those reported previously (3) and are shown here for comparison with the rates of increase in GSH transferase mRNA (Fig. 1). In order to investigate the mechanisms by whiui BHA in creases hepatic GSH transferase activities, we examined the rate of increase of GSH transferase mRNA in response to BHA. At various times after initiation of the BHA diet, RNA was purified from livers by homogenization in guanidinium thiocyanate solu tion and sedimentation through CsCI solution (12). mRNA was translated in a cell-free system (derived from wheat germ) which is dependent upon added mRNA (36). The polypeptides synthe sized were analyzed by sodium dodecyl sulfate:polyacrylamide gel electrophoresis (Fig. 14). It has been shown that the major M, 24,000 polypeptide synthesized in vitro using template mRNA extracted after 9 days of the BHA diet is similar to or identical with the major GSH transferase of murine liver, GT-8.7: it comigrates with GT-8.7 in 2-dimensional electrophoresis; it is immunoprecipitable with antibody to GT-8.7; and the mRNA encoding NOVEMBER 1984

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DAYS ON BHA DIET Chart 1. Response of liver cytosol GSH transferase (GST) and NAD(P)H:quinone reducÃ-ase (Oft) specific activities to dietary BHA. Mice received either a diet containing BHA (0.75% by weight) or the control diet. Liver cytosol fractions were prepared and assayed as described in •Materialsand Methods." Bars, mean ± S.E. of the specific activities of 4 mouse livers. Control values were established at 1, 3, 9, and 14 days, and the means of these measurements were assigned the value of 100% for each substrate. The mean control specific activities (nmol/min/ mg) were as follows: GSH transferase with CDNB, 1396 ±92; GSH transferase with DCNB, 29.6 ±1.4; quinone reducÃ-ase, 132 ±6. The enzyme specific activities at each,100% time point level.are expressed as a percentage of the means for the controls;

it has nucleotide sequence homology to a complementary DNA plasmid encoding a hepatic GSH transferase (33). The cell-free synthesis of the M, 24,000 polypeptide provides a measure of the major GSH transferase mRNA. Quantitative fluorography (26) of the gel electrophoretograms of Fig. ÃŒA was carried out in order to measure the kinetics of the major GSH transferase mRNA during the period on BHA diet (Fig. 18). An increase to 3 times the control value in 1 day was observed. Additional in creases were seen at 2 and 4 days. The 9-day value was indistinguishable from the 4-day value. Lung. No significant increase in GSH transferase activity was observed in the lung within the first 2 days of BHA administration, but near-maximal levels of 1.5 to 1.6 times those of controls (p < 0.01 ) were reached within 4 days. Quinone reducÃ-aseactivity did rise significantly (p < 0.001) even within a single day of BHA diet, and increased to more than 3 times control levels within 4 days (Chart 2). Urinary Tract Tissues. In the kidney, BHA elicits a much greater elevation of quinone reducÃ-ase activity than of GSH transferase activities (4, 5). Although these enzyme activities increased gradually during 9 to 14 days of BHA administration, statistically significant (p < 0.01) changes relative to controls were observed at all time points (Chart 3). In the urinary bladders of untreated mice, GSH transferase specific activities were extremely high [CDNB, 5340 ±264 (S.E.) nmol/min/mg; DCNB, 118 ±4 nmol/min/mg; n = 8] in compari son with other tissues. These activities were only modestly elevated in bladders from mice that had received the BHA diet for 14 days (CDNB, 7614 ±282 nmol/min/mg; DCNB, 158 ±6 nmol/min/mg; n = 4, p < 0.001). The response to BHA was not 5257

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Chart 2. Response of lung GSH transferase (GST) and NAD(P)H:quinone reduc Ã-ase(OR) specific activities to dietary BHA. Conditions are as described in the legend to Chart 1. The mean control specific activities (nmol/min/mg) were as follows: GSH transferase with CDNB, 501 ±18; GSH transferase with DCNB, 9.33 ±0.32; quinone reducÃ-ase,96.0 ±2.4. Bars, S.E.

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DAYSON BHA DIET Fig. 1. Cell-free translation products of liver mRNA obtained at various times after initiation of BHA administration. RNA was purified from the livers of 4 mice fed a powdered diet without BHA (A, Lane 2). Similarly, RNA was purified from the livers of 4 mice fed the same diet containing 0.75% BHA for 1 day (Lane 3), 2 days (Lane 4), 4 days (Lane 5), or 9 days (Lane 6). The RNA was translated in a cell-free protein synthesizing system containing L-f*S]methionine, and the polypeptide products were analyzed by sodium dodecyl sullateipolyacrylamide gel electrophoresis and quantitative fluorography (see "Materials and Methods"). The products of the cell-free system with no RNA added are also shown (Lane 1). The scale on the right indicates the positions of porypeptides of M, 68,000, 43,000, 25,700, and 17,000. The fraction of radioactive polypeptide translation products which comigrated with the major GSH transferase of murine liver, M, 24,000, is shown (B).

rapid. Only 33 to 36% of the observed increases in GSH trans ferase activities occurred within 3 days of BHA administration (data not shown). Quinone reducÃ-aseactivity increased from 1127 ±44 nmol/min/mg (n = 8) to 1527 ±78 nmol/min/mg (n 5258

= 4) in 3 days and to 2102 ±101 nmol/min/mg (n = 4) in 14 days on the BHA diet. Digestive Tract Tissues. In the forestomach,quinonereduc Ã-aseactivity was substantially elevated after 1 day on the BHA diet (p < 0.05), whereas GSH transferase activity responded more slowly (Chart 4). GSH transferase activity with DCNB (not shown) rose very similarly to that with CDNB as a substrate. The intestinal mucosa exhibited the most rapid relative in creases in GSH transferase and quinone reductase specific activities in response to BHA administration. In the proximal half of the small intestine, GSH transferase specific activity toward DCNB increased to more than 8 times control levels within 1 day after initiation of the BHA diet (Chart 5). Maximal values of at least 20 times those of controls were attained within 3 days. Activity toward CDNB also responded very rapidly, rising to 2.6 times that of controls in 1 day and reaching near-maximal values of 6.6 times those of controls within 3 days. The difference in the relative increases in activities with CDNB and DCNB is due to differential induction of GSH transferase isozymes (32). The quinone reductase specific activity doubled in 1 day on the BHA diet and rose to 4.5 times that of control mice in 3 days (Chart CANCER

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Induction of Enzymes and mRNA by BHA DCNB was enhanced from 1.44 ±0.08 nmol/min/mg to 5.44 ± 0.41 nmol/min/mg (p < 0.001). No significant increases in these activities occurred after the initial 3 days of BHA administration (data not shown). Dietary BHA has little effect on GSH transfer ase activities in the colon and yields only modest increases in quinone reductase activities in the distal small intestine and the colon (4, 5). The rates of increase of these enzyme activities were not examined. DISCUSSION

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DAYS ON BHA DIET Chart 4. Response of forestomach GSH transferase (GST) and NAD(P)H:quinone reducÃ-ase(OR) specific activities to dietary BHA. Conditions are as described in the legend to Chart 1. The mean control specific activities (nmol/ min/mg) were as follows: GSH transferase with CDNB, 940 ±44; quinone reduc Ã-ase,1133 ±68. Bars, S.E.

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500tf 200nUPPER DAYS ON BHA DIET Chart 5. Response of GSH transferase (GST) and NAD(P)H:quinone reducÃ-ase (OR) specific activities in the mucosa of the proximal small intestine to dietary BHA. Conditions are as described in the legend to Chart 1. The mean control specific activities (nmol/min/mg) were as follows: GSH transferase with CDNB, 580 ±23; GSH transferase with DCNB, 3.11 ±0.14; quinone reducÃ-ase, 560 ±18. Bars, S.E.

5). The enzyme activities shown in Chart 5 were significantly different from those of controls (p < 0.001) at all time points. GSH transferase specific activities were elevated significantly in the proximal small intestine 6 hr after administration of a single dose of BHA. Six mice received BHA (620 mg/kg) by intubation p.o. in Emulphor EL-620P, and a control group of 6 mice received only the carrier. GSH transferase and quinone reducÃ-aseactivi ties were determined for the cytosol fractions prepared from the mucosa of the proximal 6 inches of the small intestine. The specific activities (nmol/min/mg) for the BHA-treated and control groups, respectively, were: GSH transferase, CDNB, 1011 ±79 and 775 ±67 (p < 0.1); DCNB, 11.02 ±1.25 and 7.40 ±0.75 (p < 0.05); quinone reducÃ-ase, 617 + 44 and 558 ±27 (p < 0.4). Thus, even within 6 hr after administration of BHA, GSH transferase activities increased by 30 to 50%, while quinone reductase activity did not change significantly. In contrast to the large increases in GSH transferase activities in the proximal small intestine, distal small intestine GSH trans ferase activity toward CDNB increased to only 1.7 times that of control mice (849 ±30 nmol/min/mg, n = 4, in 14 days on the BHA diet, in comparison to 512 ±23 nmol/min/mg, n = 8, for controls; p < 0.001). Under these conditions, activity toward NOVEMBER 1984

Although the relative magnitude of the response of GSH transferase and NAD(P)H:quinone reductase specific activities to BHA administration varied with both the particular enzyme activ ity and the anatomical site involved, the time required for attain ment of maximal levels of these enzyme activities was generally characteristic of the specific tissue or organ. Intestinal mucosal cells undergo rapid exfoliation (27), and this may account for the fact that GSH transferase and quinone reductase specific activ ities reached maximal levels most rapidly in the intestine. Induc tion of rat intestinal GSH transferases by phénobarbital also occurs rapidly (34). In the liver, GSH transferase mRNA activity increased more rapidly than did transferase enzyme activities following initiation of BHA administration. This difference diminished toward the end of the period investigated, as the mRNA level and the transferase activities reached plateaus. This time course of mRNA activity suggests increased synthesis as the major mech anism for the increase in GSH transferase protein and activities during BHA administration. In support of this, increased trans ferase synthesis and transferase mRNA have been demon strated after 9 to 14 days of BHA administration (33). The early increase in transferase mRNA in this study further supports the above conclusion concerning transferase synthesis. Lung and forestomach are major target tissues for BP-induced neoplasia and are protected by BHA (49). Speier et al. (41) showed that p.o. administration of BHA 4 hr before each of a series of p.o. doses of BP gave substantial, although suboptimal, protection against the pulmonary tumorigenicity of this carcino gen. Reduced liver microsome-mediated binding of BP to DNA was observed within 4 hr of administration of a single dose of BHA along with alteration of the BP metabolite pattern (25, 46). BHA treatment of mice has been reported to result in a variety of alterations in the profiles of BP metabolites produced in vitro by liver microsomes and isolated hepatocytes (13, 14, 18). Reduced binding of [3H]BP metabolites to DNA has also been observed in isolated hepatocytes from BHA-fed mice (14). Al though BP-diol-epoxide:DNA adduct formation was not signifi cantly different in incubations containing liver, lung, or foresto mach microsomes from BHA-treated verus control mice, BHA reduced markedly the amounts of BP diol-epoxide:DNA adducts formed from BP in vivo in mouse lung, forestomach, and liver (1, 18). One mechanism by which BHA inhibits BP-induced neoplasia may be to shift the metabolism of BP-7,8-did to products other than the diol-epoxides. Another mechanism may be to enhance inactivation of the diol-epoxides. These mechanisms would of course not be mutually exclusive. Both may occur. GSH transferases have been shown recently to protect against DNA-binding metabolites of BP, both by catalyzing the conju gation of reactive metabolites with GSH and by binding reactive species covalently (7, 17, 22, 31, 37). Substantial increases in

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A. M. Benson et al. GSH transferase activities occurred in the liver and small intestine mucosa within 24 hr after the initiation of the BHA diet. Even within 6 hr after a single p.o. dose of BHA, significantly elevated GSH transferase activities in the proximal small intestine may afford increased protection against a variety of reactive electrophiles. Although we found no significant change in GSH trans ferase specific activities in 2 major sites of BP-induced tumors, lung and forestomach, within 24 hr on the BHA diet, quinone reductase specific activity was significantly elevated within this time period. The liver has a major role in the metabolism of xenobiotics and is protected by BHA against the tumorigenic effects of several hepatocarcinogens (46-49). The gastrointestinal tract is also of particular interest since it serves as a portal of entry for exogenous chemicals and as a possible primary site of metabo lism of p.o. ingested carcinogens (45). Thus, the small intestine, which is not generally a site at which tumors are formed, may protect other tissues by metabolizing ingested toxic and carcin ogenic substances to inactive products. We conclude that the rates of increase in the specific activities of GSH transferase and quinone reductase in the liver and in the intestinal mucosa, and of quinone reductase in the forestomach, lung, and kidney, suggest that significant enhancement of the potential for inactivation of a wide variety of reactive electrophiles occurs within 24 hr or less after initiation of BHA administration. REFERENCES 1. Anderson, M. W., Boroujerdi, M., and Wilson, A. G. E. Inhibition in vivo of the formation of adducts between metabolites of benzo(a)pyrene and DNA by butylated hydroxyanisote. Cancer Res., 41: 4309-4315,1981. 2. Batzinger, R. P., Ou, S-Y. L, and Bueding, E. Antimutagente effects of 2(3)ferf-butyl-4-hydroxyanisote and of antimicrobial agents. Cancer Res., 38: 4478-4485,1978. 3. Benson, A. M., Batzinger, R. P., Ou, S-Y. L., Bueding, E., Cha, Y-N., and Talalay, P. Elevation of hepatic glutathinone S-transferase activities and pro tection against mutagenic metabolites of benzo(a)pyrene by dietary antioxidants. Cancer Res., 38:4486-4495,1978. 4. Benson, A. M., Cha, Y-N., Bueding, E., Heine, H. S., and Talalay, P. Elevation of extrahepatic glutathione S-transferase and epoxide hydratase activities by 2(3Hert-butyl-4-hydroxyanisole. Cancer Res., 39: 2971-2977,1979. 5. Benson, A. M., Hunkeler, M. J., and Talalay, P. Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc. Nati. Acad. Sci. USA, 77: 5216-5220,1980. 6. Bueding, E., Batzinger, R. P., Cha, Y.-N., Talalay, P., and Molineaux, C. J. Protection from mutagenic effects of antischistosomal and other drugs. Pharmacol. Rev., 30: 547-554, 1979. 7. Burke, M. D., Vadi, H., Jemström, B., and Orrenius, S. Metabolism of benzo