Effect of the separate and combined administration of mestranol and phenobarbital on the development of altered hepatic foci expressing placental form of glutathione S-transferase in the rat

Abstract

The stages in the carcinogenesis process include initiation, promotion and progression. Although many characteristics of tumor promotion and promoting agents have been reported, relatively few studies on the effects of combinations of promoting agents have been detailed. For study of the combined effects of phenobarbital (PB) and mestranol (MS) in multistage rat hepatocarcinogenesis, an initiation-promotion protocol was developed. Female rats were injected i.p. at 5 days of age with either diethylnitrosamine (DEN) (10 mg/kg) or the solvent tricaprilyn. At weaning, ~10 rats from both the DEN-initiated and the solvent control groups were provided basal diet alone, PB (10, 100 or 500 mg/kg diet), MS (0.02 or 0.2 mg/kg diet) or various combinations of both PB and MS in the basal diet. At 8 months of age, the rats were killed and the livers removed, sectioned and fixed in ice-cold acetone. Sections of 5 μm thickness were stained for placental glutathione S-transferase (PGST) expression, and the volume fraction of liver occupied by altered hepatic foci was determined by stereology. In addition, incorporation of bromodeoxyuridine into nuclei of PGST-expressing (focal) and non-PGST-expressing (non-focal) hepatocytes was determined. Administration of the highest dose of PB resulted in a significant decrease in non-focal hepatocyte labeling index, with a 4-fold differential between the focal and non-focal hepatocyte labeling index. Administration of 0.2 mg/kg diet MS resulted in effective promotion. The non-focal labeling index was increased and the focal labeling index was further enhanced (3-fold) relative to the non-focal index by this dose of MS. Combination of the lower MS dose with PB resulted in at least an additive promoting effect; however, a lower volume fraction was noted for the combination of low MS dose plus the highest PB dose. Combination of the higher MS dose with PB resulted in an elevation of volume fraction only for the middle PB dose. These findings indicate that the potency of promotion by mixtures is modulated by the dose of each component as well as by pharmacodynamic and pharmacokinetic properties of each component of the mixture.