DNA repair synthesis in isolated rainbow trout liver cells

Isolated trout liver cells were treated with lysolecithin to produce an in situ system for characterizing DNA repair in teleosts. In this preparation, the integrity of the plasma membrane is altered, nuclei remain intact, and the concentrations of dNTPs and nucleotide analogs, which normally do not penetrate intact plasma membranes, can be controlled. Following lysolecithin treatment, 50% of the total cellular protein and nearly 75% of total lactate dehydrogenase activity was released from the liver cells. Microscopic examination indicated that the integrity of the plasma membrane of trout hepatocytes was disrupted by lysolecithin; however, smaller nonhepatocytic liver cells were resistant to the disrupting effects of this detergent. Bleomycin induced DNA repair synthesis in lysolecithin-treated cells, as demonstrated by CsCl gradient analysis of 5-bromo, 2'-deoxyuridine, 5'-triphosphate-labeled DNA. Optimal conditions for bleomycin-induced DNA repair synthesis in lysolecithin-treated trout liver cells were considerably different from that in lysolecithin-treated mammalian cells. Bleomycin-induced DNA repair synthesis was lower in lysolecithin-treated trout liver cells than in lysolecithin-treated mammalian cells at identical concentrations of 2'-deoxyribonucleoside, 5'-triphosphates (dNTPs), suggesting the decreased sensitivity of trout cells in unscheduled DNA synthesis assays can be attributed to factors other than differences in dNTP pools. Bleomycin-induced DNA repair synthesis in trout hepatocytes was shown to be very sensitive to inhibition by 2', 3'-dideoxythymidine, 5'-triphosphate and was resistant to inhibition by cytosine arabinoside, 5'-triphosphate, butylphenyldeoxyguanosine, 5'-triphosphate and aphidicolin. These observations indicate repair of bleomycin-induced DNA damage in trout cells occurs through a mechanism similar to that in mammalian cells, utilizing DNA polymerase beta.