2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a possible human carcinogen formed in cooked fish and meat. PhIP is bioactivated by cytochrome P450 enzymes to form 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N–OH-PhIP), a genotoxic metabolite that reacts with DNA leading to the mutation-prone DNA adduct N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP). Here, we studied N–OH-PhIP-induced whole genome mutagenesis in human TP53 knock-in (Hupki) mouse embryo fibroblasts (HUFs) immortalised and subjected to whole genome sequencing (WGS). In addition, mutagenicity of N–OH-PhIP in TP53 and the lacZ reporter gene were assessed. TP53 mutant frequency in HUF cultures treated with N–OH-PhIP (2.5 μM for 24 h, n = 90) was 10% while no TP53 mutations were found in untreated controls (DMSO for 24 h, n = 6). All N–OH-PhIP-induced TP53 mutations occurred at G:C base pairs with G > T/C > A transversions accounting for 58% of them. TP53 mutations characteristic of those induced by N–OH-PhIP have been found in human tumours including breast and colorectal, which are cancer types that have been associated with PhIP exposure. LacZ mutant frequency increased 25-fold at 5 μM N–OH–PHIP and up to ~350 dG-C8-PhIP adducts/108 nucleosides were detected by ultra-performance liquid chromatography-electrospray ionisation multistage scan mass spectrometry (UPLC-ESI-MS3) at this concentration. In addition, a WGS mutational signature defined by G > T/C > A transversions was present in N–OH-PhIP-treated immortalised clones, which showed similarity to COSMIC SBS4, 18 and 29 signatures found in human tumours.
Bibliographical noteFunding Information:
Lisa Hölzl-Armstrong was supported by a PhD studentship from the MRC Centre for Environment and Health. Work at King's College London and the Wellcome Trust Sanger Institute was supported by the Cancer Research UK Grand Challenge Award “Mutographs of Cancer” (grant C98/A24032 ). Other funding was from the National Cancer Institute of the National Institutes of Health ( R01CA122320 to R.J.T.); Mass spectrometry was carried out in the Analytical Biochemistry Shared Resource of the Masonic Cancer Center, University of Minnesota , funded in part by Cancer Center Support ( CA-077598 ).
© 2020 Elsevier Ltd
- DNA adducts
- Dietary carcinogen
- Whole genome sequencing
PubMed: MeSH publication types
- Journal Article