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

Abstract

While the experimental data upon which current concepts in mechanistically based risk assessment and molecular epidemiology are grounded derive almost entirely from rodent models, fish models have several attributes (e.g., low background incidence, extremely low cost tumor studies, nonmammalian comparative status for extrapolation of mechanisms to humans) that make them valuable adjuncts for addressing these concepts. This report provides an initial characterization of the dose dependency of dietary N-nitrosodiethylamine (DEN) hepatocarcinogenicity in Shasta strain rainbow trout (Oncorhynchus mykiss) and the potential of DEN to elicit ras proto-oncogene activation in this species. Carcinogen was administered in the diet at five concentrations for 12 months. Necropsies were per formed at 9, 12, and 18 months, the latter on fish maintained on control diet for 6 months after cessation of DEN exposure. The incidence of hepatic neoplasms at the lower dietary concentrations (≤70 ppm) did not consistently exceed that for control groups, which were higher in this particular study (2%) than expected (historically 0.1%). For the higher DEN concentrations, a linear relationship between the hepatic tumor incidence (expressed as log odds, log [p/(1-p)1, where p = proportion of fish bearing tumors), and the logarithm of total cumulative dose was observed, with response being independent of the length of time (9 or 12 months) during which the dose was accumulated. The dose-response curve for fish maintained an additional 6 months postexposure was shifted toward higher incidence but was parallel to the curve for fish killed at cessation of exposure. The model predicts that doubling the dose will produce some what more than a doubling of the odds (pl(100 - p) for tumor incidence and that the odds for lesions 6 months postexposure will be approximately double those at cessation of exposure. Comparison of these results with previous studies using rats suggests an overall similarity in dose-response curves, with trout being somewhat less sensitive than rats to DEN hepato carcinogenesis. To examine the molecular basis for DEN carci nogenesis in this species, seven liver tumors induced separately by short-term DEN treatment were probed by 3'-mismatch primer polymerase chain reaction analysis for evidence of Ki-ras proto-oncogene activating point mutations. A very high proportion (6/7) of tumors was found to carry codon 12 GGA - AGA mutations, whereas no codon 61 mutants were detected in this sample. These initial results differ from those reported using hepatic tumors from DEN-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 DEN do not suggest a simple, consistent oncogenic pathway or mutational spectrum useful in the molecular epidemiology of human cancers.