Ability of structurally related polycyclic aromatic carcinogens to induce homologous recombination between duplicated chromosomal sequences in mouse L cells

Abstract

To investigate the role of DNA damage in the induction of homologous recombination in mammalian cells, a series of structurally related, polycyclic aromatic carcinogens, i.e., 1-nitrosopyrene (1-NOP), N-acetoxy-2-acetylaminofluorene (N-AcO-AAF), and 4-nitroquinoline 1-oxide (4-NQO), were compared for their ability to cause intrachromosomal homologous recombination between two herpes simplex virus thymidine kinase (Htk) genes stably integrated in the genome of a tk- mouse L cell strain 333 M. Each Htk gene contains an 8-bp XhoI linker inserted at a unique site so that expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. Each carcinogen caused a dose-dependent increase in the frequency of recombination. The results were compared to what had been found previously for a structurally related carcinogen, (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). As a function of concentration, BPDE was the most active agent, followed by 4-NQO, and 1-NOP, and then N-AcO-AAF. When compared on the basis of equal cell killing, the most efficient carcinogen was 1-NOP, followed by N-AcO-AAF and BPDE, and then 4-NQO. Use of tritium-labeled compounds to determine the frequency of recombination as a function of the number of adducts initially bound to DNA showed that the most effective agent was BPDE, followed by 1-NOP and 4-NQO, and then N-AcO-AAF (ratio, 6.6:2.5:1.8:1.0). To determine if these differences in recombinagenic effectiveness reflected different rates of removal of the adducts from DNA, we measured the percentage of DNA adducts removed during the 24-h period post treatment and found that 1-NOP, 4-NQO and N-AcO-AAF residues were removed at approximately the same rate, i.e., 25%-30% off. Cellular analysis of a series of independent recombinants indicated that approximately 82% of the recombinational events induced by each agent were consistent with gene conversion. DNA-DNA hybridization analysis confirmed this, and showed that each recombinant tested contained an XhoI-resistant (wild-type) Htk gene; with the majority retaining the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids.