title: TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts.
abstract: 3-Nitrobenzanthrone (3-NBA) is a highly mutagenic compound and possible human carcinogen found in diesel exhaust. 3-NBA forms bulky DNA adducts following metabolic activation and induces predominantly G:CT:A transversions in a variety of experimental systems. Here we investigated the influence of nucleotide excision repair (NER) on 3-NBA-induced mutagenesis of the human tumour suppressor gene TP53 and the reporter gene lacZ. To this end we utilised Xpa -knockout (Xpa-Null) human TP53 knock-in (Hupki) embryo fibroblasts (HUFs). As Xpa is essential for NER of bulky DNA adducts, we hypothesized that DNA adducts induced by 3-NBA would persist in the genomes of Xpa-Null cells and lead to an increased frequency of mutation. The HUF immortalisation assay was used to select for cells harbouring TP53 mutations following mutagen exposure. We found that Xpa-Null Hupki mice and HUFs were more sensitive to 3-NBA treatment than their wild-type (Xpa-WT) counterparts. However, following 3-NBA treatment and immortalisation, a similar frequency of TP53-mutant clones arose from Xpa-WT and Xpa-Null HUF cultures. In cells from both Xpa genotypes G:CT:A transversion was the predominant TP53 mutation type and mutations exhibited bias towards the non-transcribed strand. Thirty-two percent of 3-NBA-induced TP53 mutations occurred at CpG sites, all of which are hotspots for mutation in smokers' lung cancer (codons 157, 158, 175, 245, 248, 273, 282). We also examined 3-NBA-induced mutagenesis of an integrated lacZ reporter gene in HUFs, where we again observed a similar mutant frequency in Xpa-WT and Xpa-Null cells. Our findings suggest that 3-NBA-DNA adducts may evade removal by global genomic NER; the persistence of 3-NBA adducts in DNA may be an important factor in its mutagenicity.
authors.affiliation: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, King's College London, London SE1 9NH, United Kingdom. Electronic address: jill.kucab@kcl.ac.uk.
authors.forename: Jill E
authors.initials: JE
authors.lastname: Kucab
authors.affiliation: Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven 3721 MA, The Netherlands.
authors.forename: Edwin P
authors.initials: EP
authors.lastname: Zwart
authors.affiliation: Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven 3721 MA, The Netherlands.
authors.forename: Harry
authors.initials: H
authors.lastname: van Steeg
authors.affiliation: Center for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven 3721 MA, The Netherlands.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Luijten
authors.affiliation: Division of Radiopharmaceutical Chemistry (E030), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
authors.forename: Heinz H
authors.initials: HH
authors.lastname: Schmeiser
authors.affiliation: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, King's College London, London SE1 9NH, United Kingdom.
authors.forename: David H
authors.initials: DH
authors.lastname: Phillips
authors.affiliation: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, King's College London, London SE1 9NH, United Kingdom.
authors.forename: Volker M
authors.initials: VM
authors.lastname: Arlt
mesh_terms: D000818:Animals; D001551:Benz(a)Anthracenes / Q000494:pharmacology*; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts; D004249:DNA Damage; D004260:DNA Repair; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000187:drug effects / Q000235:genetics; D007763:Lac Operon / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008817:Mice, Mutant Strains; D016296:Mutagenesis; D009153:Mutagens / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects / Q000235:genetics*; D051760:Xeroderma Pigmentosum Group A Protein / Q000235:genetics*
chemical_list: D001551:Benz(a)Anthracenes; D018736:DNA Adducts; D009153:Mutagens; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D051760:Xeroderma Pigmentosum Group A Protein; C497315:Xpa protein, mouse; D004247:DNA; C117220:3-nitrobenzanthrone
keywords: 3-Nitrobenzanthrone; DNA adduct; Environmental carcinogen; Mutation; Nucleotide excision repair; TP53