Clonal xenobiotic resistance during pollution-induced toxic injury and hepatocellular carcinogenesis in liver of female flounder (Platichthys flesus (L.))

Juvenile and adult female flounder (Platichthys flesus (L.)) were caught either in the estuary of the most polluted European river, the Elbe, or as controls in a reference site to study pollution-induced xenobiotic resistance in their livers in relation to pathological alterations. In juvenile fish, livers displayed reversible and irreversible degenerative toxipathic lesion types but never showed (pre)neoplastic changes. Tumour frequencies up to 70% were found macroscopically in livers of adult female flounder which had progressed to adenomas and carcinomas in the most polluted site. Because male adult flounder show only up to 50% of livers containing early preneoplastic foci but never malignancies, we focussed our study on female individuals. (Pre)neoplastic changes ranged from early eosinophilic foci to basophilic foci, adenomas and hepatocellular carcinomas. Adenomas were generally eosinophilic whereas carcinomas were mainly basophilic. These phenotypical sequential changes strongly resemble those found in chemically-induced liver carcinogenesis in mammals. Characteristic mutations known from mammalian cancers have not been found so far in these flounder livers. Therefore, we investigated whether epigenetic events had induced a metabolic "resistant phenotype" of (pre)malignant cancer cells during hepatocellular carcinogenesis. With a quantitative immunohistochemical approach, we studied expression of P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR), cytochrome P4501A1, glutathione-S-transferase-A which are key proteins in xenobiotic metabolism and elimination. Glucose-6-phosphate dehydrogenase (G6PDH) activity, the major source of the reducing power NADPH which is needed for biotransformation, oxyradical scavenging and biosynthesis, was detected as well. We observed upregulation of G6PDH activity already in early preneoplastic eosinophilic foci and subsequent further upregulation in basophilic foci and carcinomas. P-gp started to become overexpressed in basophilic foci and was overexpressed even more strongly in carcinomas and their invasively-growing protrusions (satellites). In carcinomas, P-gp protein was predominantly present in membranes of lysosomes which are the intracellular sites of deposition of xenobiotics. CYP450 was reduced whereas GST-A was increased in these carcinomas. Progression towards malignancy was positively correlated with levels of mitogenic organochlorines in these livers which are "fingerprint contaminants" of the river Elbe. We conclude that (pre)neoplastic hepatocytes in female flounder acquire growth advantages over normal hepatocytes by epigenetic metabolic adaptations during liver carcinogenesis as a result of chronic exposure to (pro)carcinogens in the polluted habitat.