Gene- and strand-specific damage and repair in Chinese hamster ovary cells treated with 4-nitroquinoline 1-oxide

Abstract

4-Nitroquinoline 1-oxide (4NQO) is a model chemical carcinogen that has often been referred to as a UV mimetic agent. Previous studies have indicated that UV-induced pyrimidine dimers are repaired preferentially and strand-specifically in actively transcribing genes. In the current study we have examined the gene-specific and strand-specific repair of 4NQO in Chinese hamster ovary B-11 cells treated with 2.5 microM 4NQO. The methodology used for detecting adducts involved the treatment of DNA from 4NQO-exposed cells with uvrABC excinuclease, which incises DNA at adduct sites, followed by denaturing gel electrophoresis of DNA, Southern hybridization, and probing for the sequence of interest. We examined the active and inactive coding regions of the DHFR gene, the active adenine phosphoribosyltransferase gene, relatively inactive c-fos oncogene, and the mitochondrial genome for 4NQO adducts. Initial 4NQO adduct levels found in these genes varied from 1.10 to 1.52 adducts/10 kilobases. Little difference in repair was found between active coding and inactive regions of the DHFR gene, or between DHFR, adenine phosphoribosyltransferase, and c-fos genes, which are transcribed at different levels. Approximately 71% of 4NQO adducts were repaired within 24 h in all gene sequences examined. During this same time period, approximately 51% of adducts were repaired from the genome overall, as determined by comparing the removal of bound radiolabeled 4NQO to total DNA. The results indicate that 4NQO adducts, unlike UV light-induced cyclobutane pyrimidine dimers (UV dimers), are not preferentially repaired in transcriptionally active genes. However, there may be regions of the genome that are not repaired with the same efficiency as the specific genes examined here. In addition, little to no difference was observed in the repair of 4NQO adducts in the transcribed and nontranscribed strands of the DHFR gene, a finding which is also in contrast to results with UV dimers. Interestingly, 4NQO adducts, unlike UV dimers, were removed from the mitochondrial genome, suggesting that repair of select lesions occurs in this organelle. Thus, there appear to be some differences in the repair pathways operating for 4NQO adducts and UV dimers, particularly with respect to gene- and strand-specific DNA repair.