BPDE-induced genotoxicity: relationship between DNA adducts, mutagenicity in the in vitro PIG-A assay, and the transcriptional response to DNA damage in TK6 cells

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 N 2-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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-017-2003-0) contains supplementary material, which is available to authorized users.

Mode of action-based risk assessment of genotoxic carcinogens

“…Within a recent study we specifically addressed the question on dose-response relationship in the low dose range for BaP-induced stable DNA adducts in the N 2 -position of guanine, their repair and the induction of mutations, and compared it to the onset of the DNA damage response on the transcriptional level (Piberger et al 2017). All endpoints were investigated in the same cell line, namely TK6 cells.…”

“…16 BPDE-induced DNA adduct levels determined via HPLC/Fluorescence detection (a), mutation frequencies determined by PIG-A assay (b), and the correlation between DNA adducts and mutation frequencies (c). For details see Piberger et al (2017) GG-NER on the other hand is slower and may be incomplete, leading to an accumulation of mutations in poorly repaired regions (Fousteri and Mullenders 2008;Mullenders et al 1991). Accordingly, three levels of repair efficiencies have been identified in human fibroblasts after treatment with BPDE: The transcribed strand of the active HPRT gene was repaired fastest, followed by the non-transcribed strand, while only a small fraction of BPDE adducts were removed from the inactive locus 754 within 20 h (Chen et al 1992).…”

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

“…The data points represent the means 6 SD from at least 3 independent experiments and * indicates p < .05 for a treatment group versus the corresponding DMSO control. support of this notion, a recent study showed that following direct exposure to BPDE, a B[a]P metabolite, a 60% relative repair of DNA adducts was observed in TK6 cells after 24 h (Piberger et al, 2018), and functional p53 is required for nucleotide excision repair of DNA adducts (Akyü z et al, 2002;Seo and Jung, 2004). Because we used relatively low concentrations of the parent compound (B[a]P), p53-related DNA repair machinery may have efficiently removed any damage induced following metabolism of B[a]P by CYP2C19 and 3A4.…”

PARP1 protects from benzo[a]pyrene diol epoxide-induced replication stress and mutagenicity

“…Benzo[a]pyrene is a potent and widespread environmental carcinogen and serves as lead model substance to study the response of cells to chemical-induced, bulky DNA adducts that are repaired by NER (Angerer et al 1997 , EPA 2006 ; Kim et al 2013 ; Madureira et al 2014 ; Piberger et al 2017 ). While there is ample evidence that PARP1 participates in the removal of UV-induced DNA damage during NER (Fischer et al 2014 ; Pines et al 2012 ; Purohit et al 2016 ; Robu et al 2013 , 2017 ; Vodenicharov et al 2005 ), the understanding of how PARP1 participates in genotoxic stress response to chemical-induced, bulky DNA adducts is understood incompletely.…”