The effect of butylated hydroxytoluene on the growth of enzyme-altered foci in male Fischer 344 rat liver tissue.

Butylated hydroxytoluene (BHT) is a synthetic, food-use, phenolic antioxidant. It has previously been demonstrated to be operationally non-genotoxic and, in addition, failed to induce biologically significant increases in cellular proliferation in the liver, urinary bladder and thyroid gland on feeding to young adult Wistar rats. Nevertheless, it has been reported to enhance the yield of liver tumors when fed to rats or mice that developed an appreciable background incidence of these tumors without treatment. In order to resolve this situation, cell proliferation in response to BHT treatment was studied in enzyme-altered foci (EAF) induced in male Fischer 344 rats using the Solt-Farber procedure. It was demonstrated that feeding 0.5% dietary BHT for 30 days after the induction of EAF led to a 20- to 30-fold increase in the gamma-glutamyltranspeptidase-positive areas in both DEN- and saline-initiated rat livers, but to no major effects in glutathione S-transferase placental form (GSTP)-positive foci. Cell proliferation rates within EAF and surrounding normal liver were measured using different histological techniques. Nuclear labeling with [3H]thymidine and proliferating cell nuclear antigen (PCNA) over the total hepatocyte population indicated that BHT approximately doubled nuclear labeling in rats initiated with DEN. PCNA labeling in GSTP-positive foci was not affected by BHT. In GSTP-positive foci, evaluation of nucleolar organizer regions (AgNOR), which reflect cell proliferative in addition to transcriptional activity of ribosomal RNA, was achieved using a novel double staining technique. BHT diet did not affect the number of AgNOR per nucleus or the percentage AgNOR area/nucleus. Nevertheless, both PCNA labeling and the AgNOR area per nucleus were significantly greater in GSTP-positive foci compared with non-focal regions in rats fed either BHT or control diets. These results are discussed in the light of further experimental work required to determine the relevance of these data to possible human risk assessment for BHT.