The carcinogenic air pollutant 3-nitrobenzanthrone induces GC to TA transversion mutations in human p53 sequences

Abstract

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and a suspected human carcinogen present in particulate matter of diesel exhaust and ambient air pollution. Employing an assay with human p53 knock-in (Hupki) murine embryonic fibroblasts (HUFs), we examined p53 mutations induced by 3-NBA and its active metabolite, N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA). Twenty-nine immortalized cultures (cell lines) from 89 HUF primary cultures exposed at passage 1 for 5 days to 2 microM 3-NBA harboured 22 different mutations in the human DNA-binding domain sequence of the Hupki p53 tumour suppressor gene. The most frequently observed mutation was GC to TA transversion (46%), corroborating previous mutation studies with 3-NBA, and consistent with the presence of persistent 3-NBA-guanosine adducts found in DNA of exposed rodents. Six of the transversions found solely in 3-NBA-treated HUFs have not been detected thus far in untreated HUFs, but have been found repeatedly in human lung tumours. (32)P-post-labelling adduct analysis of DNA from HUF cells treated with 2 microM 3-NBA for 5 days showed a pattern similar to that found in vivo, indicating the metabolic competence of HUF cells to metabolize 3-NBA to electrophilic intermediates. Total DNA binding was 160 +/- 56 per 10(7) normal nucleotides with N(2)-guanosine being the major adduct. In contrast, identical treatment with N-OH-3-ABA resulted in a 100-fold lower level of specific DNA adducts and no carcinogen-specific mutation pattern in the Hupki assay. This indicates that the level of DNA adduct formation by the mutagen is critical to obtain specific mutation spectra in the assay. Our results are consistent with previous experiments in Muta Mouse and are compatible with the possibility that diesel exhaust exposure contributes to mutation load in humans and to lung cancer risk.

Linking environmental carcinogen exposure to TP53 mutations in human tumours using the human TP53 knock-in (Hupki) mouse model

“…3) [54][55][56][57][58][59]. In each case, a unique TP53 mutation pattern was generated in the HUF immortalization assay, which differed from that found in control HUFs that had undergone spontaneous immortalization.…”

“…B[a]P is metabolically activated by cytochrome P450 (CYP) enzymes (e.g. CYP1A1, CYP1B1) and epoxide hydrolase to the ultimately reactive metabolite B[a]P-7,8-diol-9,10-epoxide (BPDE) [62], which reacts primarily at the N [59] or AAI [54,56,58]. Also shown is the mutation pattern in spontaneous immortalized HUFs (controls) [53].…”

“…It has been proposed that methylation at CpG sites may increase the potential for planar carcinogen compounds to intercalate prior to covalent binding, although the precise mechanism still remains to be determined. Furthermore, the majority of G fi T transversions occur on the nontranscribed DNA strand, particularly at hotspot codons 157, 158 and 273, which may be linked to the fact that [59]. Also shown is the mutation pattern and spectra of TP53 mutations in spontaneously immortalized HUFs (controls) (E) [53].…”

“…Using the HUF immortalization assay, 19 cell lines carrying a total of 29 TP53 mutations were derived from 3-NBA treatment [59]. The major mutation type induced by 3-NBA was G fi T transversion (38%), followed by A fi G (24%) and G fi C (17%) mutations (Fig.…”

Nutlin‐3a selects for cells harbouring TP53 mutations

“…C > T and CC > TT mutations in head and neck squamous carcinomas, associated with ultraviolet (UV)‐radiation exposure; G > T mutations in smokers' lung cancers, associated with exposure to tobacco carcinogens, such as benzo[ a ]pyrene (BaP); A > T mutations in urothelial carcinomas, associated with exposure to aristolochic acid (AA) 2. Some of these signatures have been replicated experimentally using embryo fibroblasts from the partial human TP53 knock‐in (Hupki) mouse, in which exons 4–9 of human TP53 replace the corresponding exons of murine Trp53 5, 6, 7…”