Dose-Dependent Carcinogenicity and Frequent Ki-ras Proto-oncogene Activation by Dietary N-Nitrosodiethylamine in Rainbow Trout

While the experimental data upon which current concepts in mechanisticallybased risk assessment and molecular epidemiology are groundedderive almost entirely from rodent models, fish models haveseveral attributes (e.g., low background incidence, extremelylow cost tumor studies, nonmammalian comparative status forextrapolation of mechanisms to humans) that make them valuableadjuncts for addressing these concepts. This report providesan initial characterization of the dose dependency of dietaryN-nitrosodiethylamine (DEN) hepatocarcinogenicity in Shastastrain rainbow trout (Oncorhynchus mykiss) and the potentialof DEN to elicit ras proto-oncogene activation in this species.Carcinogen was administered in the diet at five concentrationsfor 12 months. Necropsies were per formed at 9, 12, and 18 months,the latter on fish maintained on control diet for 6 months aftercessation of DEN exposure. The incidence of hepatic neoplasmsat the lower dietary concentrations (70 ppm) did not consistentlyexceed that for control groups, which were higher in this particularstudy (2%) than expected (historically 0.1%). For the higherDEN concentrations, a linear relationship between the hepatictumor incidence (expressed as log odds, log [p/(1-p)1, wherep = proportion of fish bearing tumors), and the logarithm oftotal cumulative dose was observed, with response being independentof the length of time (9 or 12 months) during which the dosewas accumulated. The dose-response curve for fish maintainedan additional 6 months postexposure was shifted toward higherincidence but was parallel to the curve for fish killed at cessationof exposure. The model predicts that doubling the dose willproduce some what more than a doubling of the odds (pl(100 -p) for tumor incidence and that the odds for lesions 6 monthspostexposure will be approximately double those at cessationof exposure. Comparison of these results with previous studiesusing rats suggests an overall similarity in dose-response curves,with trout being somewhat less sensitive than rats to DEN hepatocarcinogenesis. To examine the molecular basis for DEN carcinogenesis in this species, seven liver tumors induced separatelyby short-term DEN treatment were probed by 3'-mismatch primerpolymerase chain reaction analysis for evidence of Ki-ras proto-oncogeneactivating point mutations. A very high proportion (6/7) oftumors was found to carry codon 12 GGA - AGA mutations, whereasno codon 61 mutants were detected in this sample. These initialresults differ from those reported using hepatic tumors fromDEN-treated mice, which exhibit frequent Ha-ras codon 61 mutations[Richardson et al., Carcinogenesis 13, 1277–1279 (1992)]and rats, which appear not to carry DEN-activated ras alleles[Bauer-Hoffman et al., Carcinogenesis 11, 1875–1877 (1990)].Thus the available oncogene data for the common carcinogen DENdo not suggest a simple, consistent oncogenic pathway or mutationalspectrum useful in the molecular epidemiology of human cancers.