Acrylamide is a suspected human carcinogen formed during high-temperature cooking of starch-rich foods. It is metabolised by cytochrome P450 2E1 to its reactive metabolite glycidamide, which forms pre-mutagenic DNA adducts. Using the human TP53 knock-in (Hupki) mouse embryo fibroblasts (HUFs) immortalisation assay (HIMA), acrylamide- and glycidamide-induced mutagenesis was studied in the tumour suppressor gene TP53 . Selected immortalised HUF clones were also subjected to next-generation sequencing to determine mutations across the whole genome. The TP53 -mutant frequency after glycidamide exposure (1.1 mM for 24 h, n = 198) was 9% compared with 0% in cultures treated with acrylamide [1.5 ( n = 24) or 3 mM ( n = 6) for 48 h] and untreated vehicle (water) controls ( n = 36). Most glycidamide-induced mutations occurred at adenines with A > T/T > A and A > G/T > C mutations being the most common types. Mutations induced by glycidamide occurred at specific TP53 codons that have also been found to be mutated in human tumours (i.e., breast, ovary, colorectal, and lung) previously associated with acrylamide exposure. The spectrum of TP53 mutations was further reflected by the mutations detected by whole-genome sequencing (WGS) and a distinct WGS mutational signature was found in HUF clones treated with glycidamide that was again characterised by A > G/T > C and A > T/T > A mutations. The WGS mutational signature showed similarities with COSMIC mutational signatures SBS3 and 25 previously found in human tumours (e.g., breast and ovary), while the adenine component was similar to COSMIC SBS4 found mostly in smokers’ lung cancer. In contrast, in acrylamide-treated HUF clones, only culture-related background WGS mutational signatures were observed. In summary, the results of the present study suggest that glycidamide may be involved in the development of breast, ovarian, and lung cancer. Electronic supplementary material The online version of this article (10.1007/s00204-020-02878-0) contains supplementary material, which is available to authorized users.

DNA in dividing cells is prone to mutagenesis, with mutations making key contributions to human disease including cancer. The tumour suppressor gene TP53 is the most frequently mutated gene in human tumours. Here, we present a robust protocol for studying TP53 mutagenesis utilising human TP53 knock-in (Hupki) mouse embryonic fibroblasts (HUFs). In the HUF immortalisation assay (HIMA), primary HUFs are treated with known or suspected carcinogens at 3% oxygen and then transferred to 20% atmospheric oxygen to induce senescence. Cells containing mutations (e.g., in TP53) that allow bypassing of senescence eventually emerge as immortalised clonal cell lines after 2–3 months of serial passaging. As not all immortalised HUF cells contain TP53 mutations, we developed a Nutlin-3a counter-screen to select for TP53-mutated clones prior to sequencing. TP53 mutation spectra generated can be compared with those of human tumours recorded in the International Agency for Research on Cancer TP53 mutation database. Environmental mutagens that have demonstrated and validated the utility of the HIMA include ultraviolet radiation, aristolochic acid, and benzo[a]pyrene. The TP53 mutation patterns induced by these mutagens in the HIMA corresponded to those found in human tumours from patients exposed to these mutagens. The approach presented helps to deepen our understanding of human cancer aetiology.

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide −S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide −S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP −S9, 4-ABP ±S9 and N-OH-4-ABP −S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

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TP53 harbors somatic mutations in more than half of human tumors with some showing characteristic mutation spectra that have been linked to environmental exposures. In bladder cancer, a unique distribution of mutations amongst several codons of TP53 has been hypothesized to be caused by environmental carcinogens including 4-aminobiphenyl (4-ABP). 4-ABP undergoes metabolic activation to N-hydroxy-4-aminobiphenyl (N-OH-4-ABP) and forms pre-mutagenic adducts in DNA, of which N-(deoxyguanosin-8-yl)-4-ABP (dG-C8-4-ABP) is the major one. Human TP53 knockin mouse embryo fibroblasts (HUFs) are a useful model to study the influence of environmental carcinogens on TP53-mutagenesis. By performing the HUF immortalization assay (HIMA) TP53-mutant HUFs are generated and mutations can be identified by sequencing. Here we studied the induction of mutations in human TP53 after treatment of primary HUFs with N-OH-4-ABP. In addition, mutagenicity in the bacterial lacZ reporter gene and the formation of dG-C8-4-ABP, measured by 32 P-postlabelling analysis, were determined in N-OH-4-ABP-treated primary HUFs. A total of 6% TP53-mutants were identified after treatment with 40 μM N-OH-4-ABP for 24 hr (n = 150) with G>C/C>G transversion being the main mutation type. The mutation spectrum found in the TP53 gene of immortalized N-OH-4-ABP-treated HUFs was unlike the one found in human bladder cancer. DNA adduct formation (40 adducts/10 8 nucleotides) was detected after 24 hr treatment with 40 μM N-OH-4-ABP, but lacZ mutagenicity was not observed. Adduct levels decreased substantially (sixfold) after a 24 hr recovery period indicating that primary HUFs can efficiently repair the dG-C8-4-ABP adduct possibly before mutations are fixed. In conclusion, the observed difference in the N-OH-4-ABP-induced TP53 mutation spectrum to that observed in human bladder tumors do not support a role of 4-ABP in human bladder cancer development.