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Keywords:

  • K-ras;
  • Pink salmon;
  • Mismatch polymerase chain reaction assay;
  • Crude oil;
  • Genetic damage

Abstract

Previous studies demonstrated reduced survivorship of pink salmon embryos from populations in Prince William Sound, Alaska, USA, that were exposed to Exxon Valdez–released oil compared with populations from matched nonoiled streams. Survivorship was also significantly decreased in embyros from lineages that were oiled in Prince William Sound and reared in clean water under controlled hatchery conditions compared with the descendants of nonoiled lineages. This suggests that the effect of oiling on pink salmon populations was persistent and could be transmitted intergenerationally. However, the ability of environmentally released oil to cause DNA sequence alterations in natural populations has yet to be demonstrated. We used polymerase chain reaction analysis to screen for alterations in the K-ras oncogene in DNA from pink salmon embryos that were exposed under controlled laboratory conditions to weathered Prudhoe Bay crude oil. Polymerase chain reaction and direct DNA sequence analyses were used to identify mutational hotspots within exons 1 and 2 of K-ras, and 3′ primer mismatch analysis was used to determine the frequency of mutations in the 40 offspring of two families of pink salmon that were experimentally exposed to oiled or clean gravel. Mutations were only observed at codons 12, 13, and 61 of K-ras, sites that are frequently mutated in animal and human tumors. All mutations resulted in deduced amino acid substitutions. As expected, in all individuals exhibiting mutations, the copy number of the normal allele exceeded that of the mutated allele. The frequencies of mutations in oiled embryos at K-ras exons 1 and 2 were 68 and 41%, respectively. K-ras mutations were not observed in siblings that were exposed to clean gravel or in the parents of the two experimental matings. These results indicate that exposure of pink salmon embryos to weathered Prudhoe Bay crude oil under controlled laboratory conditions can elicit somatic cell mutations in high frequency at mutational hotspots in genes such as K-ras. However, the frequency of these events in oiled natural populations of pink salmon and other vulnerable species in Prince William Sound and the heritability of these mutations within oiled lineages have yet to be evaluated.