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Effect of Butylated Hydroxyanisole Pretreatment on in Vitro Hepatic Aflatoxin B₁-DNA Binding and Aflatoxin B₁-Glutathione Conjugation in Rats¹

Fels Research Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

The effect of 3(2)-tert-butyl-4-hydroxyanisole (BHA) pretreatment of rats on both in vitro hepatic aflatoxin B₁ (AFB₁)-DNA binding and AFB₁-glutathione (AFB₁-SG) conjugation has been examined. For these studies, young male F344 rats were fed AIN-76 A diet with or without 0.75% BHA for 2 weeks. There were no significant differences either in microsomal cytochrome P-450 content or microsome-mediated exogenous DNA binding to AFB₁ with cytochrome P-450 from control or BHA-treated animals. There were large differences in reduced glutathione S-transferase activity with treated cytosols showing 2.5-fold higher activity than the controls. Hepatic reduced glutathione levels were 25% higher in treated than in controls. Kinetics of cytosolic inhibition of microsome-mediated AFB₁-DNA binding and formation of AFB₁-SG conjugate when examined at two levels of AFB₁ (2 and 10 µM) and a 4-fold range of cytosolic concentrations showed that inhibition of AFB₁-DNA binding was greater with cytosol from the treated compared to the controls. However, AFB₁-SG conjugation was 3- to 4-fold greater in treated than in controls. Inhibition of AFB₁-DNA binding by cytosol was reversed in the presence of 1 mM level of various epoxides with concomitant inhibition of AFB₁-SG conjugation. In reconstitution studies with 2 µM AFB₁, intact nuclei alone from either group did not yield significant amounts of either DNA binding or AFB₁-SG conjugation. However, addition of microsomes from either group to these nuclei generated a large amount of AFB₁-DNA binding (82–111 pmol) and a smaller amount of AFB₁-SG conjugate (9–28 pmol). The presence of cytosols from the control group reduced AFB₁-DNA binding to a much lesser extent than the cytosols from the treated group. However, AFB₁-SG conjugation was much higher with the cytosol from treated than with the controls. These reconstitution studies with endogenous DNA show more AFB₁-DNA binding with the control than with BHA-treated animals and are in agreement with the studies in vivo. It appears that induced levels of cytosolic reduced glutathione S-transferase modulate AFB₁-DNA binding and AFB₁ hepatocarcinogenesis.

¹ Supported by Grants CA-12227 and CA-40885 from the National Cancer Institute, Department of Health and Human Services, by Grant SIG-6 from the American Cancer Society, and by the Samuel S. Fels Fund of Philadelia. A preliminary report on this work has appeared recently (45).

² On leave from the Department of Biochemistry, School of Dentistry, Chonbuk National University, Chonju, Chonbuk 560-20, South Korea.

³ To whom requests for reprints should be addressed.

Received 11/18/87. Revised 2/16/88. Accepted 2/19/88.