The role of hyperplastic nodules in dichloroacetic acid-induced hepatocarcinogenesis in B6C3F1 male mice

Abstract

Dichloroacetic acid (DCA) has recently been shown to increase significantly the incidence of hepatic adenomas (HAs) and hepatocarcinomas (HCs) in male B6C3F1 mice. Although little is known about the mechanism of DCA carcinogenesis, chronic ingestion of the compound in drinking water induces primarily hyperplastic nodules (HNs) prior to the appearance of HAs and HCs. Given the putative preneoplastic potential of the HNs, we undertook this study to determine the role of the HNs in the progression of DCA-induced hepatocarcinogenesis. This role was assessed by detecting the expression of five different tumor markers: p21 ras, p39 c-jun, phosphotyrosine, tumor-associated aldehyde dehydrogenase and alpha-fetoprotein, all known from previous studies to be expressed more often in neoplastic liver lesions than in normal liver. Tumor marker expression was detected by immunohistochemical methods using formalin-fixed, paraffin-embedded sections of normal B6C3F1 mouse liver, and DCA-induced HNs, HAs and HCs. The results demonstrated that, except for the c-jun marker, HNs expressed the markers significantly less often than either HAs or HCs. Equal expression of c-jun occurred in any of the three lesion types. Although these results could be used to argue that no relationship existed between HNs and later-appearing HAs and HCs, those HNs that were marker positive contained small nests of marker-positive hepatocytes among a field of normally appearing unstained hepatocytes. No similar nests of marker-positive cells were detected in any area of normal liver outside the HNs. Also very few altered hepatic foci (AF) were detected with these markers or with hematoxylin and eosin, or with histochemical stains for ATPase or glucose-6-phosphatase deficiencies. These results suggested that these nests within some HNs were areas of transformed, or neoplastic hepatocytes. Phenotypic heterogeneity analysis, in which the number of tumor markers co-expressed by any given lesion was examined, confirmed a significantly greater percentage of HAs and HCs expressing multiple markers than HNs. Those HNs that expressed multiple markers, however, expressed at the same frequency as HAs and HCs and the expression was confined to the same nests of cells. Taken together, these data suggest that these nests of marker-positive cells within the HNs were neoplastic and could develop into later-appearing HAs and/or HCs. The absence of marker expression in normal liver and limited expression in the few AF indicates that the HNs may be the only significant preneoplastic lesion in DCA-induced hepatocarcinogenesis.