TP53 and lacZ mutagenesis induced by 3-nitrobenzanthrone in Xpa-deficient human TP53 knock-in mouse embryo fibroblasts.

Volker M. Arlt,Harry van Steeg,Heinz H. Schmeiser,David H. Phillips,Mirjam Luijten,Jill E. Kucab,Edwin P. Zwart

doi:10.1016/j.dnarep.2015.11.004

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.

Highlights

Lung cancer is the most common malignant disease worldwide
We recently showed that XpaNull Hupki mouse embryo fibroblast (HUF), compared with Xpa+/+ (Xpa-WT) HUFs, are extremely sensitive to the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (BaP) and exhibit increased TP53 mutagenesis on the transcribed strand when treated with the reactive intermediate of BaP, benzo[a]pyrene-7,8-diol-9,10epoxide (BPDE) [27]
In addition to TP53 mutagenesis, we examined 3-NBAinduced mutations in a lacZ reporter gene in Xpa-WT and Xpa-Null HUFs derived from human TP53 knock-in (Hupki); Xpa +/− mice, as the pUR288 plasmid locus has been integrated into this strain [30]

Summary

Introduction

Lung cancer is the most common malignant disease worldwide. While tobacco smoking is the predominant cause of lung cancer, vehicular exhaust and ambient air pollution are implicated [1,2]. In Great Britain diesel engine exhaust is the sixth most important occupational carcinogen [3]. Two recent epidemiological studies of miners provide strong evidence for a link between diesel exposure and lung cancer risk, finding a 3-fold increased risk for lung cancer overall and a 5-fold increased risk for miners most heavily exposed to diesel exhaust [4,5]. The International Agency for Research on Cancer (IARC) has classified diesel engine exhaust as Group 1 human carcinogen [2]. J.E. Kucab et al / DNA Repair 39 (2016) 21–33

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