Dissimilar patterns of promotion by di(2-ethylhexyl)phthalate and phenobarbital of hepatocellular neoplasia initiated by diethylnitrosamine in B6C3F1 mice

Abstract

Potentially preneoplastic hepatocellular hyperplastic foci and hepatocellular neoplasms were studied in weanling male B6C3F1 mice that received a single i.p. injection (80 mg/kg) of diethylnitrosamine (DEN) at 4 weeks of age, followed by oral administration of phenobarbital (PB) or di(2-ethylhexyl)-phthalate (DEHP) that began 2 weeks after DEN injection and continued for up to 6 months. PB was administered in drinking water at 500 p.p.m. and DEHP in the feed at 3000, 6000 or 12 000 p.p.m. Groups of mice were sacrificed at 2, 4 and 6 months after DEN exposure; formalin-fixed liver samples were evaluated histologically. Hepatocellular neoplasms and foci of hyperplasia were quantified with the aid of an image analysis computer. Few foci were seen at 2, 4 or 6 months in mice exposed to DEN, PB or DEHP alone, while numerous foci and neoplasms were seen in mice given DEHP or PB after DEN. Area-perimeter measurements for each hepatocellular focus or neoplasm transection revealed that foci and neoplasms in PB-exposed mice increased both in size (area and volume) and in number throughout the study. In DEHP-exposed mice the pattern of response was different in that the numbers of foci did not increase between 4 and 6 months, but the foci increased in mean diameter and volume throughout the experiment. Foci and tumors appeared earlier in mice given higher dietary levels of DEHP than in those given lower doses. By the end of the study the number of foci per unit volume of liver was similar in mice given any dose of DEHP, but their volume was dose-related. Hepatocellular foci and neoplasms in PB-exposed mice were composed predominantly of eosinophilic hepatocytes, while in DEHP-exposed mice, basophilic foci and neoplasms predominated; the latter were more malignant in appearance than neoplasms in PB-exposed mice. At 6 months, the neoplasms in high dose DEHP-exposed mice were significantly larger than those in PB exposed mice. Histochemistry, however, revealed similarities between lesions in mice exposed to PB or DEHP. PB given continuously for 6 months revealed no initiating activity of DEHP given once by gavage and followed by PB in drinking water. Both morphology and biology of hepatocellular foci and neoplasms, which develop in mice after a single exposure to a carcinogen with initiating activity, thus depend, in part, on the subsequent promoting agent. More than one process of tumor promotion, as characterized by a specific sequence of morphologic and biochemical changes, is possible for the mouse hepatocyte.