ICANCERRESEARCH 38,4486-4495, December 1978] 0008-5472/78/0038-0000$02.00 Elevation of Hepatic Glutathione S-Transferase Activities and Protection against Mutagenic Metabolites of Benzo(a)pyrene by Dietary Antioxidants1 Ann M. Benson, Robert P. Batzinger, Suh-Yun L. Ou, Ernest Bueding, Young-Nam Cha, and Paul TalaIay2 Department of Pharmacology andExperimental Therapeutics, TheJohnsHopkinsUniversity School of Medicine (A.M. B.,E.B., P.T.J,andDepartment of Pathobiology, The Johns Hopkins University Schoolof Hygiene and Public Health (A. P. B., S-V. L. 0., E. B., V-N. CI, Baltimore, Maryland 21205 ABSTRACT Addition of either 2(3)-tert-butyl-4-hydroxyanlsole (BHA) or I ,2-dlhydro-6-ethoxy-2,2,4-trlmethylquinollne (eth oxyquin) to the diet greatly decreases the bevels of muta genicmetabobitesof benzo(a)pyrenein CD-I mice.(R. P. Batzinger, S-V. L. Ou, and E. Bueding, Cancer Res., 38: 000, 1978).The mutagenic activity of the urinary metabo lites of benzo(a)pyreneis markedlyreduced in the pres enceofglutathionetogetherwiththe livercytosobsof rats or micefed on a diet containingBHA.The livercytosolsof mice and rats maintainedon controldiets are much less effective in this respect. Dietary BHA causes increases in mouse and rat hepatic glutathione S-transferase (EC 2.5.1 .18) specIfic actIvities with 1,2-dichboro-4-nitroben zene, l-chboro-2,4-dlnitrobenzene, p-nitrobenzylchboride, and @5-androstene-3,l7-dione. Inthe mousethe increases are larger (5- to 10-fold)and are dependenton the dose anddurationof administrationof BHA.Increasesin these glutathione 5-transferase specific activfties were also observed In mouse hepatic cytosols after feeding of ethoxyquin. Direct addition of reduced glutathione and purifiedglutathione5-transferasesA and BobtaInedfrom rat liver to the mutagenicityassay systemmimickedthe effect of the rodent cytosobs. Since BHA and ethoxyquin are knownto reducethe neoplasticeffects of a varietyof potent carcinogens, we suggest that the protective effects of these antioxidantsmay be accountedfor, at least in part, by their ability to elevate the glutathlone S-transfer ases. These enzymes inactivate arene oxides and other hydrophobicelectrophilesbycatalyzingtheirconjugation with glutathione. INTRODUCTION The induction of experimental tumors in rodents by a variety of chemical carcinogens can be prevented by the administration of certain â€oeantioxidants.― (The term antioxi dant is used in this study to designate compounds that can act as biological antioxidants, although it is by no means clear as to whether the effects on enzyme activities de scribed in this study can be in fact ascribed to their antioxidant properties as such.) This phenomenon was first explored systematically by Wattenbergeta!. (41—44), whose observations have been extended by Weisburger et a!. (45), Grantham et a!. (14), and Ulland et a!. (38). Antioxidants reduce the production of tumors at various anatomical sites by a wide variety of chemical carcinogens, including aro matic polycyclic hydrocarbons, urethan, dimethylhydra zine, nitrosamines, azo dyes, and aminofluorenes. The principal compounds used for protection against carcino gens have been BHA,3 3,5-di-tert-butyb-4-hydroxytobuene, ethoxyquin, and bis(diethylthiocarbamyl)disulfide (disulfi ram). BHA and ethoxyquin are of special interest, since they are widely used as preservatives in the diets of humans and domestic animals, respectively. Various theories have been advanced to explain these effects of antioxidants, but as yet no mechanism has been established unequivocally. Poten tiab mechanisms include: (a) direct interaction of the carcin ogen or of its activated metabolic products with the antiox idant; (b) enhanced activities of enzymes that inactivate the proximate or ultimate carcinogens, thereby diverting them from the damaging interactions with critical macromolecu barcomponents, thus preventing the initiation of the malig nant process; (c) blockade of specific metabolic activation processes required to convert procarcinogens to their re active intermediates; and (d) more efficient mechanisms for repair ofdamage toDNA. Antioxidant-mediated protection of rodents against the neoplastic effects of carcinogens appears to show little specificity with respect to the chemical nature of the carcin ogen, the route of its administration, the site of tumor formation, or the chemical structure or route of administra tion of the antioxidant. Hence, the antineopbasticeffect is likely to depend on some generalized mechanisms, the nature of which has not been hitherto elucidated. Batzinger et a!. (3) have reported recently that the urine of mice receiving i.m. injections of BP is highly mutagenic for 2 of the sensitive tester strains of histidine-requiring Salmonella typhimurium developed by Ames et a!. (1) and McCann et a!. (30). The urine induced reversions of both tester strains TA98 and TA100. The mutagenic activities of these urine specimens were enhanced moderately by the presence of rat liver microsomes and cytosob (the so-cabled 59 fraction) derived from rats treated with a hydrocarbon inducer of microsomal drug-metabolizing enzymes. The mutagenic activities of the urine samples obtained from BP treated mice were also increased if these urine samples were subjected to the action of f3-glucuronidase,suggest ing that at least some of the mutagenic metabobites were present as conjugates. The mutagenic activity of the urinary I Supported by NIH Grants GM 16492, Al 08022, AM 07422, and CA 18251 and bythe EdnaMcConnelClark Foundation.A preliminaryaccountof this work has been published (4). 2 To whom requests for reprints should be addressed, at Department of Pharmacology and Experimental Therapeutics, The Johns Hopkins Liniver sity School of Medicine, 725 N. Wolfe Street, Baltimore, Md. 21205. Received April 26, 1978; accepted August 23, 1978. 3 The abbreviations used are: BHA, 2(3)-tert-butyl-4-hydroxyanisoie (a mixture containing approximately 4% of compound in which the tert-butyi group is meta to the phenolic hydroxyl group and containing 96% of compound in which the tert-butyl group is ortho to the phenolic hydroxyl group); BP, benzo(a)pyrene; GSH, glutathione; CDNB, 1-chloro-2,4-dinitro benzene; DCNB, 1,2.dichloro-4-nitrobenzene ; NBC, p-nitrobenzyichloride. 4486 CANCERRESEARCHVOL. 38