title: Evaluation of the potential health effects of the atmospheric reaction products of polycyclic aromatic hydrocarbons.
abstract: The genotoxic risks from exposure to polycyclic aromatic hydrocarbons (PAHs) have long been recognized. Less well understood are the potential genotoxic risks of the atmospheric reaction products of this class of compounds. In this investigation, we have utilized several human cell assays to evaluate the genotoxicity of naphthalene, phenanthrene, and their atmospheric reaction products 1-nitronaphthalene, 2-nitronaphthalene (2NN), 1-hydroxy-2NN, 2-hydroxy-1-nitronaphthalene, 1,4-naphthoquinone, and 2-nitrodibenzopyranone (2NDBP). In addition, simulated atmospheric reaction products of naphthalene were generated in a 6,700 liter (L) Teflon environmental chamber, collected on a solid adsorbent, extracted, and fractionated by normal-phase high-performance liquid chromatography (HPLC). Individual fractions were then analyzed using gas chromatography/mass spectrometry (GC/MS), and tested for genotoxic effects. Genotoxicity was primarily determined using the human B-lymphoblastoid cell line, MCL-5, which expresses several transfected P450 and epoxide hydrolase genes. Mutagenicity was evaluated at both the heterozygous thymidine kinase (tk) locus and the hemizygous hypoxanthine phosphoribosyl transferase (hprt) locus, permitting detection of both intragenic and chromosomal scale mutational events. Test compounds were also screened using the CREST modified micronucleus assay. The results indicate that 2NN and 2NDBP possess greater mutagenic potency than their parent compounds, and, interestingly, both compounds induced significant increases in mutation frequency at the tk but not the hprt locus. These findings suggest a mechanistic difference in human cell response to 2NN and 2NDBP as compared to bacteria, where both compounds were previously shown to induce point mutations in the Salmonella typhimurium reversion assay. The genotoxicity of 2NN and 2NDBP in human cells, together with their high concentrations in ambient air relative to nitro-PAHs directly emitted from combustion sources, emphasizes the need to consider atmospheric reaction products of PAHs in assessments of the genotoxicity of air pollutants. We also investigated whether transfected cytochrome P450 monooxygenase activities were required to activate 2NN and 2NDBP to genotoxic species, and whether a single enzyme could be sufficient for metabolic activation. Three directly related cell lines with multiple (MCL-5), single (AHH-1 1A1), or no (L3) transfected cytochrome P450 genes were used. AHH-1 is additionally distinguished by elevated mutagenic response at the tk locus, a heterozygous mutation in p53, and apoptosis capacity. The effect of these metabolic and genetic differences on genotoxicity of 2NN, 2NDBP, and beta-naphthylamine (beta NA) was also investigated. The results indicated that 2NN and 2NDBP were not activated to genotoxic species through nitroreduction pathways. Mutagenicity induced at the tk locus was dependent on oxidative metabolism, provided by transfected cytochrome P450 enzymes in MCL-5 and AHH-1 1A1. Mutagenicity was not observed in the L3 cell line, which does not carry transfected cytochrome P450 activities. The negative response of beta NA in all cell lines indicates that, contrary to previous hypotheses, 2NN and beta NA are not activated by similar metabolic pathways in these human cell lines. Taken as a whole, these results suggest that the genotoxicity of nitro-PAHs in human cells requires oxidative metabolism.
mesh_terms: D000393:Air Pollutants / Q000009:adverse effects* / Q000737:chemistry / Q000378:metabolism*; D001402:B-Lymphocytes / Q000166:cytology; D002460:Cell Line; D004249:DNA Damage / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D009152:Mutagenicity Tests; D009153:Mutagens / Q000009:adverse effects* / Q000737:chemistry / Q000378:metabolism*; D009281:Naphthalenes / Q000009:adverse effects* / Q000737:chemistry / Q000378:metabolism*; D010616:Phenanthrenes / Q000009:adverse effects* / Q000737:chemistry / Q000378:metabolism*; D011083:Polycyclic Compounds / Q000009:adverse effects* / Q000737:chemistry / Q000378:metabolism*; D001335:Vehicle Emissions