N-nitrosodiethylamine initiation of carcinogenesis in Japanese medaka (Oryzias latipes):: Hepatocellular proliferation, toxicity, and neoplastic lesions resulting from short term, low level exposure

To investigate relationships among carcinogen exposure, cell proliferation, and carcinogenesis, 14-day post-hatch Japanese medaka (Oryzias latipes) were exposed to 0, 10, 25, 50, or 100 ppm N-nitrosodiethylamine (DEN) for 48 h under static renewal conditions. They were then held in clean water until sampling at 3 and 6 months. The frequencies of hepatic lesions and neoplasms were determined from hematoxylin/eosin-stained paraffin sections. A significant (p < 0.0001) concentration-related increase in hepatic vacuolated foci occurred in 3- and 6-month samples, with males having a significantly (p = 0.02) higher incidence than females. Concentration-related increases in degenerative lesions were documented for spongiosis hepatis at 3 months (p = 0.053) and hepatic vacuoles at 6 months (p = 0.005). There was a significant (p = 0.0001) concentration-related increase in macrophage aggregates at 6 months. Basophilic foci were significantly related (p < 0.0001) to DEN concentration at 3 months post-exposure and were unaffected by gender or age. At both 3 and 6 months, there were significant concentration-related increases in hepatocellular carcinoma (p less than or equal to 0.02), Hepatocyte proliferation in 3-month whole specimens was quantified using an immunohistochemical assay for proliferating-cell nuclear antigen. Trend tests and a probit dose-response model showed a significantly positive correlation (p = 0.015) between proliferating hepatocytes and DEN concentrations. These results confirm that short-term exposure to low and moderate levels of DEN initiates concentration-dependent carcinogenic effects in medaka that are apparent at 3 months post exposure. DEN could be an effective initiator in an initiation/promotion assay for medaka using a 48-h exposure period, DEN concentrations less than or equal to 10 ppm, and a 6-month sampling period.