title: TP53 mutations induced by BPDE in Xpa-WT and Xpa-Null human TP53 knock-in (Hupki) mouse embryo fibroblasts.
abstract: Somatic mutations in the tumour suppressor gene TP53 occur in more than 50% of human tumours; in some instances exposure to environmental carcinogens can be linked to characteristic mutational signatures. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalization assay (HIMA) is a useful model for studying the impact of environmental carcinogens on TP53 mutagenesis. In an effort to increase the frequency of TP53-mutated clones achievable in the HIMA, we generated nucleotide excision repair (NER)-deficient HUFs by crossing the Hupki mouse with an Xpa-knockout (Xpa-Null) mouse. We hypothesized that carcinogen-induced DNA adducts would persist in the TP53 sequence of Xpa-Null HUFs leading to an increased propensity for mismatched base pairing and mutation during replication of adducted DNA. We found that Xpa-Null Hupki mice, and HUFs derived from them, were more sensitive to the environmental carcinogen benzo[a]pyrene (BaP) than their wild-type (Xpa-WT) counterparts. Following treatment with the reactive metabolite of BaP, benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), Xpa-WT and Xpa-Null HUF cultures were subjected to the HIMA. A significant increase in TP53 mutations on the transcribed strand was detected in Xpa-Null HUFs compared to Xpa-WT HUFs, but the TP53-mutant frequency overall was not significantly different between the two genotypes. BPDE induced mutations primarily at G:C base pairs, with approximately half occurring at CpG sites, and the predominant mutation type was G:C>T:A in both Xpa-WT and Xpa-Null cells. Further, several of the TP53 mutation hotspots identified in smokers' lung cancer were mutated by BPDE in HUFs (codons 157, 158, 245, 248, 249, 273). Therefore, the pattern and spectrum of BPDE-induced TP53 mutations in the HIMA are consistent with TP53 mutations detected in lung tumours of smokers. While Xpa-Null HUFs exhibited increased sensitivity to BPDE-induced damage on the transcribed strand, NER-deficiency did not enhance TP53 mutagenesis resulting from damage on the non-transcribed strand in this model.
authors.affiliation: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, King's College London, Franklin-Wilkins Building, 150 Stamford Street, 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: 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: Environmental Health Science and Research Bureau, Health Canada, 50 Colombine Driveway, Ottawa, Ontario K1A 0K9, Canada.
authors.forename: Paul A
authors.initials: PA
authors.lastname: White
authors.affiliation: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment & Health, King's College London, Franklin-Wilkins Building, 150 Stamford Street, 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, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, United Kingdom.
authors.forename: Volker M
authors.initials: VM
authors.lastname: Arlt
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003062:Codon; D018736:DNA Adducts / Q000378:metabolism; D004260:DNA Repair; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D016158:Genes, p53; D006801:Humans; D051379:Mice; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D010100:Oxygen / Q000494:pharmacology; D051760:Xeroderma Pigmentosum Group A Protein / Q000502:physiology*
chemical_list: D003062:Codon; D018736:DNA Adducts; D009153:Mutagens; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D001564:Benzo(a)pyrene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D010100:Oxygen
keywords: Benzo[a]pyrene; DNA adducts; Environmental carcinogen; Mutation; Nucleotide excision repair; TP53
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