Modulation of Aflatoxin Metabolism, Aflatoxin-N⁷-guanine Formation, and Hepatic Tumorigenesis in Rats Fed Ethoxyquin: Role of Induction of Glutathione S-Transferases

Abstract

The effects of dietary administration of ethoxyquin (EQ) on aflatoxin B₁ (AFB₁) metabolism, DNA adduct formation and removal, and hepatic tumorigenesis were examined in male Fischer rats. Rats were fed a semipurified diet containing 0.4% EQ for 1 wk, gavaged with 250 µg of AFB₁ per kg 5 times a wk during the next 2 wk, and, finally, restored to the control diet 1 wk after cessation of dosing. At 4 mo, focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for γ-glutamyl transpeptidase. Treatment with EQ reduced by >95% both area and volume of liver occupied by γ-glutamyl transpeptidase-positive foci. Utilizing the same multiple dosing protocol, patterns of covalent modifications of DNA by AFB₁ were determined. EQ produced a dramatic reduction in the binding of AFB₁ to hepatic DNA: 18-fold initially and 3-fold at the end of the dosing period. Although binding was detectable at 3 and 4 mo postdosing, no effect of EQ was observed, suggesting that these persistent adducts are not of primary relevance to AFB₁ carcinogenesis. Analysis of nucleic acid bases by high-performance liquid chromatography revealed no qualitative differences in adduct species between treatment groups. The inhibitory effect of EQ on AFB₁ binding to DNA and tumorigenesis appears related to induction of detoxication enzymes. Rats fed 0.4% EQ for 7 days showed a 5-fold increase in hepatic cytosolic glutathione S-transferase (GST)-specific activities. Multiple molecular forms of GST were induced, and concomitant elevations in messenger RNA levels coding for the synthesis of GST subunits were observed. Correspondingly, biliary elimination of AFB₁-glutathione conjugate was increased 4.5-fold in animals on the EQ diet during the first 2 h following p.o. administration of 250 µg of AFB₁ per kg. Thus, induction by EQ of enzymes important to AFB₁ detoxication, such as GST, can lead to enhanced carcinogen elimination, as well as reductions of AFB₁-DNA adduct formation and subsequent expression of preneoplastic lesions, and, ultimately, neoplasia.