Comparative Effects of Dietary Administration of 2(3)-ferf-Butyl-4- hydroxyanisole and 3,5-di-ferf-Butyl-4-hydroxytoluene on Several Hepatic Enzyme Activities in Mice and Rats1

Effects of feeding mice and rats with 2(3)-ferf-butyl-4-hydroxyanisole (BHA) and 3,5-di-ferf-butyl-4-hydroxytoluene (BHT), the two most commonly used food-additive phenolic antioxidants with known anticarcinogenic properties but with only minor differences in their chemical structures, have been compared to search for common effects between the two agents in two different rodent species and then applied toward better understanding of the mechanisms involved in their pro tective actions. In liver microsomes of treated mice, both BHA and BHT enhanced the relative activity of aniline ring hydroxylation but decreased the relative benzo(a)pyrene monooxidase activities. However, in rats, although aniline ring hydroxylation activity was decreased by both compounds, the de crease of benzo(a)pyrene monooxidase activity was observed only with BHT. Thus, common effects could not be recognized at the microsomal mixed-function oxidase level. Contrary to expectations based on chemical structures, BHT feeding elevated the epoxide hydrolase activity to an even greater extent than that produced by BHA, especially in rats. However, enzyme activities involved in the glucuronide conju gation system (uridine diphosphate:glucuronyl transferase, uridine diphosphate:glucose dehydrogenase, and quinone reductase) are all elevated by both antioxidants in both rodent species. With BHA treatment, the levels of acid-soluble thiols were increased in both rats and mice. However, with BHT, the level was increased only in mice but not in rats. Similar trends were produced for glucose-6-phosphate dehydrogenase activ ity, but glutathione reducÃ-ase activity was increased even for BHT-treated rats. Additionally, the glutathione S-transferase activities were also increased by both antioxidant treatments and in both rodent species. Based on these results, the eleva tions of epoxide hydrolase activity along with the enhanced glucuronide conjugation and glutathione oxidation and reduc tion conjugation system enzyme activities were common to both compounds in both rodent species. This suggests their involvement in anticarcinogenic mechanisms. Increases of these detoxification enzyme activities appeared to be all de signed to accelerate the elimination of administered antioxi dants but, inadvertantly, conferring protective effects from xenobiotics such as carcinogens. INTRODUCTION Dietary administrations of BHA,3 BHT, and some other food Received November 5, 1981 ; accepted April 8, 1982. ' Supported by NIH Grants CA 27594 and CA 18251. 2 To whom requests for reprints should be addressed, at Department of Pathobiology, The Johns Hopkins University, 615 North Wolfe Street, Baltimore, Md. 21205. 3 The abbreviations used are: BHA, 2{3)-ferf-butyl-4-hydroxyanisole; BHT, 3,5-di-fe/t-butyl-4-hydroxytoluene; GSH, glutathione; QR, quinone reducÃ-ase; additive antioxidants protect against tumor induction by chem ical carcinogens of diverse structures (i.e., benzo(a)pyrene, dimethylaminoazobenzene, fluorenylacetamide) in a variety of rodent tissues (42, 48, 49). Recent studies dealing particularly with BHA have demonstrated (a) reduction of mutagenic activ ity arising from benzo(a)pyrene and several drugs (3); (b) reduction in the binding of benzo(a)pyrene metabolites arising from liver microsomes of treated mice to purified calf thymus DNA (44) and to hepatocyte DMA (15); (c) alterations in meta bolic profile of benzo(a)pyrene by liver microsomes (16, 24) and by isolated hepatocytes (15) from treated mice; (d) eleva tions of various enzyme activities catalyzing the inactivation of reactive electrophiles (4-6, 8-10, 16, 17, 31, 33); (e) altera tions of microsomal mixed-function oxidase activities with var ious substrates (8, 16) which may support the observed changes of metabolic profiles; and ( f) increased concentration of nonprotein thiol compounds in liver and other tissues (3, 5). Therefore, the protective effects of BHA could be accounted for, at least in part, by its ability to enhance the activities of a variety of detoxification enzymes or by shifting the metabolic profile in such a manner that lowers the intracellular concen tration of reactive products. However, the multiplicity of the effects of BHA treatments does not indicate which or what combination of altered enzyme activities is responsible for the anticarcinogenic properties of this antioxidant. In an effort to understand the biochemical basis of BHA-dependent protective action, we have selected another anticarcinogenic phenolic antioxidant BHT and com pared its effects with those of BHA. The strategy involved was to search for common effects. Previous studies dealing with this principle by comparing with the effects of few well-known inducers of hepatic drug metabolism have uncovered some differences.4 Another purpose for selecting BHT for this study was to test the hypothesis that, because of the absence of free doublebond carbon atoms in this compound, it could not form arene oxide and, thus, should not enhance epoxide hydrolase activity. On the other hand, since the BHA has such carbon atoms, it suggested the possibility that BHA epoxide could have formed and caused marked elevation of the enzyme activity. If this were the case, the increase of this enzyme activity would be unique to BHA feeding and most probably would not be in volved in anticarcinogenic mechanism of this antioxidant. As was mentioned earlier, the anticarcinogenic effects were also observed with rats (48). However, our previous experi ences with this rodent species have indicated either a complete lack of or much suppressed responses to BHA treatments (10). UDPGDH, UDPrglucose dehydrogenase; GST. glutathione S-transferases; UDPGA, UDP:glucuronic acid. 4 Y-N. Cha, H. S. Heine, and S. Ansher, unpublished observation.