Abstract
Benzo[a]pyrene is a known human carcinogen. As underlying mechanism, the induction of stable DNA adducts and mutations have been repeatedly demonstrated. Also, the activation of cellular stress response on the transcriptional level has been described. Nevertheless, the interrelationship between these different events is less well understood, especially at low, for human exposure relevant concentrations. Within the present study, we applied the reactive metabolite benzo[a]pyrene diolepoxide (BPDE) in the nanomolar, non-cytotoxic concentration range in human TK6 cells and quantified the induction and repair of stable DNA adducts at the N2-position of guanine by HPLC with fluorescence detection. Significant levels of DNA lesions were detected even at the lowest concentration of 10 nM BPDE, with a linear increase up to 50 nM. Relative repair was similar at all damage levels, reaching about 30% after 8 h and 60% after 24 h. Mutation frequencies were quantified as GPI-deficient cells by the recently established in vitro PIG-A mutagenicity assay. Again, a linear dose–response-relationship in the before-mentioned concentration range was observed, also when plotting the number of GPI-deficient cells against the number of DNA adducts. Furthermore, we explored the time- and concentration-dependent DNA damage response on the transcriptional level via a high-throughput RT-qPCR technique by quantifying the impact of BPDE on the transcription of 95 genes comprising DNA damage response, DNA repair factors, oxidative stress response, cell cycle arrest, cell proliferation, and apoptosis. As expected, BPDE activated DNA damage signaling, p53 and AP-1 dependent signaling, oxidative stress response, and apoptosis. However, in contrast to DNA adducts and mutations, the onset of the transcriptional DNA damage response was restricted to higher concentrations, indicating that its respective activations require a certain level of DNA lesions. Altogether, the results indicate that in case of BPDE, DNA lesions and mutations were correlated at all concentrations, suggesting that repair is not complete even at low levels of DNA damage. Considering the ongoing discussion on potential thresholds also for genotoxic carcinogens, the results are of major relevance, both with respect to basic research as well as to risk assessment of chemical carcinogens.
Highlights
In spite of manifold precautions to reduce exposure towards hazardous chemicals, there are still many carcinogens present in the environment, at workplaces, and in food
While cells may be very well adapted to remove oxidatively induced DNA lesions and most types of DNA base damage induced by alkylating agents via base excision repair (BER), many classes of environmental mutagens such as polycyclic aromatic hydrocarbons (PAHs) induce DNA lesions which provoke DNA helix distortions repaired by nucleotide excision repair (NER)
The low, non-cytotoxic dose range up to 50 nM was applied for DNA adduct quantification and mutagenicity testing, while the whole concentration range was applied for gene expression profiling to ensure the detection of effects occurring in the non-cytotoxic range, and those effects restricted to cytotoxic concentrations
Summary
In spite of manifold precautions to reduce exposure towards hazardous chemicals, there are still many carcinogens present in the environment, at workplaces, and in food. Directly genotoxic, i.e., DNA-reactive agents or their DNA-reactive metabolites, are generally assumed to represent risk factors at any concentration, following a linear dose–response in the low concentration range, implying that even one or a few DNA lesions may result in mutations, and may increase tumor risk This assumption has repeatedly been challenged during the last years, due to observations that, for example, in case of alkylating compounds such as EMS DNA lesions are linear in the low dose range, while increases in mutation frequencies follow a non-linear dose– response relationship (Doak et al 2007; Gocke and Müller 2009; Jenkins et al 2010; Pottenger et al 2009; for recent review see Klapacz et al 2016). As compared to BER, the latter repair pathway is usually slower and not evenly efficient throughout the genome (Fousteri and Mullenders 2008)
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