Induction of ras oncogene mutations and hepatocarcinogenesis in medaka (Oryzias latipes) exposed to diethylnitrosamine

Medaka (Oryzias latipes), a fish model for assessing the carcinogenic impact of environmental exposure to genotoxic chemicals, were subjected to 390 ppm of diethylnitrosamine (DEN) in an aqueous bath for 48 h. Histological examinations, at 5 and 7 months post-exposure, revealed that 11 of 17 fish developed hepatic neoplasms (hepatocellular carcinomas, cholangiomas and mixed hepato-cholangiocellular carcinomas). Genomic DNA extracted from the livers of normal medaka was used to obtain and sequence the structure of ras gene exon 1 and exon 2. Comparing PCR-amplified products from exon 1 and 2 by hybridization with specific mutation containing probes and/or by sequencing of liver DNA front control and exposed fish revealed an association of ras oncogene mutations and DEN-induced tumor formation. While none of the control fish showed any mutations, three of 11 fish with tumors had ras gene mutations. All three fish had mutations at codon 12 replacing glycine with aspartic acid, while one fish had an additional mutation at codon 16 replacing lysine with threonine. In addition to identifying DEN-induced mutations at codon 12 of the medaka ras oncogene, the present study represents a first report of an in vivo association of hepatocarcinogenesis, in any species, with a mutation at codon 16. Mutation at codon 16 and tumorigenesis has been previously predicted and artificially produced. Our study also supports the relevance of the medaka fish model for in vivo detection of the carcinogenic potential of environmental pollutants in sediments and biota of ambient water systems.

Duke Scholars

Author David E. Hinton Environmental Sciences and Policy