Hepatocyte expression of tumor associated aldehyde dehydrogenase (ALDH-3) and p21 Ras following diethylnitrosamine (DEN) initiation and chronic exposure to di(2-ethylhexyl)phthalate (DHEP).

Abstract

Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP) either promote or inhibit rat liver tumorigenesis depending on the carcinogenesis protocol. In this study, we examined the expression of two histochemical markers, the tumor associated isozyme of aldehyde dehydrogenase (ALDH-3) and the oncoprotein p21 Ras, in the livers of male F344 rats. The rats were initiated with DEN and further treated with either DEHP (a known inhibitor of hepatocarcinogenesis), phenobarbital (PB, a known promoter of hepatocarcinogenesis), or a combination of DEHP and PB. The studies were designed to examine the expression of these markers in both normal appearing liver and hepatic hyperplastic and neoplastic lesions and to correlate the early expression of the markers at 26 weeks in the normal appearing liver to later tumor incidence at 52 weeks. The expression of each marker was detected by immunohistochemical methods on formalin-fixed paraffin embedded sections of normal appearing liver or liver lesions. We found that ALDH-3 and p21 expression were significantly enhanced in rats receiving PB after DEN initiation at 26 weeks and that the incidence of hepatocellular carcinomas was likewise increased compared to control or DEN only treated animals. DEN initiation followed by a combination of PB and either 0.1 or 0.5% DEHP significantly reduced ALDH-3 but not p21 Ras expression at 26 weeks compared to DEN plus PB only. These treatment regimens also reduced the incidence of hepatocellular carcinomas at 52 weeks. DEN followed by any of the three doses of DEHP without PB resulted in ALDH-3 expression similar to DEN alone. However, p21 Ras expression was significantly increased after these treatments. For all treatment groups, both the early (26 weeks) expression of p21 Ras and ALDH-3 correlated with hepatocellular carcinoma incidence at 52 weeks. However, the correlation between hepatocellular carcinoma and ALDH-3 expression was better than p21 Ras or the other markers we have studied. We concluded that ALDH-3 expression is significantly downregulated after DEHP treatment, and that expression of the isozyme correlated with later hepatocarcinoma incidence and may indicate a significant relationship between ALDH-3 expression and hepatocarcinogenesis during DEHP treatment.