CARCINOGENICITY, METABOLISM AND KI-RAS PROTOONCOGENE ACTIVATION BY 7,12-DIMETHYLBENZ[A]ANTHRACENE IN RAINBOW-TROUT EMBRYOS

Field studies suggest that recent epizootics of hepatic neoplasms in some feral fish populations are associated with polycyclic aromatic hydrocarbon (PAH) exposure, but attempts to induce liver tumors in these species under laboratory conditions have been unsuccessful. Several studies have shown hepatic neoplasms to be inducible in laboratory fish species following PAH exposure at the free-swimming life stage. However, neither the susceptibility of the fish embryonic life stage to tumor induction by PAHs nor the potential of these carcinogens to induce oncogenic point mutations analogous to those reported in feral fish hepatic tumors have been clearly established. To address this, rainbow trout embryos were exposed by passive water uptake to 7,12-dimethylbenz[a]anthracene (DMBA), a potent model PAH in many mammalian tumor protocols. DMBA was rapidly absorbed by trout eggs and metabolized. The major non-polar metabolites identified were 12-hydroxymethyl-7-methylbenz[a]anthracene and 3,4-dihydroxy-3,4-dihydro-DMBA, whereas approximately 25% of the water soluble metabolites were identified as glucuronides by beta-glucuronidase treatment. Embryonic DNA adduction increased with time of DMBA exposure (2.2 +/- 0.3 pmol DM[BA-equivalents/mg DNA at 24 h). Liver tumor incidence nine months after DMBA treatment was found to increase with DMBA concentration and exposure period (3.8% at 1 p.p.m./2 h; 23% at 5 p.p.m./2 h; 85% at 5 p.p.m./24 h). Stomach adenomas and nephroblastomas also were observed at low incidence in the DMBA-treated trout. Among 11 hepatic tumors examined, nine carried Ki-ras alleles with activating point mutations in codon 12 (4/11 GGA --> AGA; 4/11 GGA --> GTA) or codon 61 (1/11 CAG --> CTG). This spectrum differs substantially from those reported for DMBA-initiated mouse skin papillomas or hepatic tumors. These results may have important environmental implications because they suggest that even a brief exposure to PAHs during a sensitive stage of development may adversely affect some fish populations. They also indicate considerable variation in DMBA ras gene mutations among species and target organs.