Mutagenicity of N‐hydroxy‐4‐aminobiphenyl in human TP53 knock‐in (Hupki) mouse embryo fibroblasts

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 knock‐in 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 32P‐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/108 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.

TP53 is commonly mutated in human tumors and specific mutation spectra linked to environmental carcinogens have been observed (Hölzl-Armstrong et al., 2019). For instance, aristolochic acid I (AAI) induces A>T/T>A transversion mutations at a variety of codons and this pattern is also found in urothelial carcinomas associated with aristolochic acid I exposure. In contrast, the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) leads to G>T/C>A transversions amongst TP53 codons 157,158,175,245,248,273, which is well reflected in smokers' lung cancer (Kucab et al., 2010). The effect of environmental carcinogens on TP53 mutagenesis can be modeled using the Human TP53 knock-in (Hupki) mouse embryo fibroblast (HUF) immortalization assay (HIMA), where TP53 mutations can be created and selected experimentally by carcinogen treatment. The Hupki mouse has human exons 4-9 in place of the corresponding mouse exons, including the codons where most mutations are found in human tumors (Hölzl-Armstrong et al., 2019). The information acquired in the HIMA can be compared to the TP53 mutation database curated by IARC that currently lists around 30,000 mutations (www.p53.iarc.fr). Some characteristic mutations of environmental carcinogens (e.g., BaP and AAI) were successfully replicated in the HIMA (Liu et al., 2005;Feldmeyer et al., 2006;Nedelko et al., 2009). Bladder tumors often harbor TP53 mutations, which have three distinct characteristics: mutations are (a) distributed evenly across exons 5-8 of TP53; (b) not more frequent at methylated CpG sites; and (c) distributed among five hotspot   codons, namely R175, R248, R273, R280, E285, with R280 and E285 being unique to bladder tumors (Feng et al., 2002a). Mutations at these hotspots are responsible for 2.9, 5.8, 3.5, 4.8 and 5.6% of all mutations in bladder cancer, respectively. Proximate metabolites of 4-ABP have been shown to bind to all bladder tumor TP53 hotspots except R273 in human bladder cells suggesting an involvement of 4-ABP in the TP53 mutation distribution in bladder cancer (Feng et al., 2002a;Feng et al., 2002b). No study has yet utilized an experimental in vitro model to analyze TP53 mutagenesis after 4-ABP exposure. In order to understand if 4-ABP is responsible for the codon distribution and mutation pattern found in TP53 in human bladder tumors the present study used primary HUFs to study N-OH-4-ABP-induced TP53 mutagenesis. It was hypothesized that immortalized clones created in the HIMA would carry TP53 mutations like those found in human bladder tumors and that these mutations would be found mainly at the hotspots described above. To find the optimal treatment conditions, cell viability, DNA adduct formation, induction of the DNA damage response (DDR) and lacZ reporter gene mutagenicity were assessed in primary HUFs exposed to N-OH-4-ABP.

| Carcinogens
N-OH-4-ABP was purchased from Toronto Research Chemicals (#H767500, Toronto, Ontario, Canada). Stock solutions were prepared at 100 mM by dissolving carcinogen in water-free DMSO and aliquots were stored under nitrogen gas at −80 C. 3-Nitrobenzanthrone (3-NBA) was synthesized as described previously (Arlt et al., 2002) and stored in aliquots at −20 C as a 2 mM stock solution in DMSO.
When confluence was reached, cells were detached with 0.05% trypsin-EDTA (Thermo Fisher Scientific #25300054) for 2-10 min and, if not otherwise specified, reseeded at 16,000 cells/cm 2 into flasks or multiwell plates.

| Crystal violet staining assay for cell survival
Cell viability was assessed by crystal violet staining as described previously (Hölzl-Armstrong et al., 2020) for concentrations up to 150 μM N-OH-4-ABP diluted in growth medium. In addition to a 24 hr time-point a 24 hr + 24 hr time-point was included in which the treatment medium was replaced by fresh growth medium after 24 hr.
Following treatment cells were washed with 180 μl PBS and stained with 30 μl 0.1% (wt/vol) crystal violet dye (Sigma #C3886) in 10% ethanol (Sigma-Aldrich #32221) for at least 10 min. After removal of excess dye by washing twice with PBS, plates were then air-dried at room temperature. At the time of measurement, 100 μl 50% ethanol per well were added and absorbance determined at 595 nm using a plate reader. Data are expressed as the percentage of absorbance per well relative to control cells and are representative of at least three independent experiments.

| DNA adduct analysis by 32 P-postlabelling
For DNA adduct analysis primary HUFs were seeded into 75-cm 2 flasks and exposed the next day to cytotoxic and sub-cytotoxic concentrations of N-OH-4-ABP with and without a 24 hr recovery period in fresh medium or solvent control (DMSO) diluted in growth medium. After treatment cells were harvested and stored as pellets at −20 C until DNA was isolated using a standard phenol-chloroform extraction method as described previously (Kucab et al., 2015). DNA adducts were determined using the butanol enrichment version of the thin-layer chromatography (TLC) 32 P-postlabelling method as described previously . Briefly, samples were digested using 4 μl micrococcal nuclease (288 mU/sample; Sigma-Aldrich #N3755) and bovine spleen phosphodiesterase (1.2 mU/samples; Worthington Biochemical Corp. #LS003603) and enriched by 1-butanol extraction. Adducts were then labeled with 50 μCi [γ-32 P]ATP (Hartmann-Analytic #HP601PE), followed by TLC as previously reported . The TLC sheets were scanned and adduct spots visualized using an Amersham Typhoon Biomolecular Imager (GE Healthcare). The signal intensity measured is proportional to the level of radioactivity present on an area of the TLC plates. Levels of DNA adducts were calculated as relative adduct labeling (RAL), which is the ratio of signal intensity of adducted nucleotides (adducts) over signal intensity of total (adducted plus normal) nucleotides in the DNA samples analyzed. A blank area on the TLC plates was used to determine the background level of radioactivity. A deoxyadenosine-3 0 -monophosphate (dAp) standard was labeled in each experiment to estimate the signal intensity of total nucleotides (normals).

| LacZ mutation assay
To assess lacZ mutagenicity pellets of treated HUFs were prepared and the mutation assay performed as described previously (Hölzl-Armstrong et al., 2020). Briefly, primary HUFs were seeded into 75-cm 2 flasks and treated for 24 hr. After 48 hr cells 175-cm 2 flasks were reseeded with 2 × 10 6 cells and allowed to grow for 4 days.
Pellets were prepared and DNA isolated using a standard phenolchloroform extraction. DNA was digested using HindIII, incubated with magnetic beads coated with lacI fusion protein and eluted from the beads using isopropyl-β-D-thiogalactopyranoside (IPTG). Plasmids were then circularized with T4 DNA ligase and electroporated into Escherichia coli lacking β-galactosidase (lacZ − ) and galactose epimerase ( galE − ). One-thousand of the transformed bacteria were plated on nonselective, titer plates containing 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal) and the remainder on mutant selective plates containing the lactose analogue phenyl-β-D galactosidase (P-Gal). The ratio of the number of mutant colonies on the selective plates to the number of colonies formed on the nonselective titer plate (× dilution factor 1,000) equals the mutant frequency.

| HUF immortalization assay (HIMA) and TP53 mutation analysis
The HIMA, Nutlin-3a counter-screen and TP53 mutation analysis were performed as described previously (Hölzl-Armstrong et al., 2019). Exons 4-9 of Nutlin-3a-selected cultures were amplified and subjected to Sanger sequencing and mutations analyzed as described previously (Hölzl-Armstrong et al., 2020). Mutations were confirmed by sequencing DNA from an independent sample of cells from the same culture.

| Statistical analysis
Results are shown as mean values ± SD. The sample size is indicated in each section. Using GraphPad Prism version 8.2.0 (GraphPad F I G U R E 1 Optimization of treatment conditions for the HIMA. (a) Cell viability assessment: Primary HUFs were treated with indicated concentrations of N-OH-4-ABP for 24 hr and cell viability (% control) was assessed by staining with crystal violet. 24 hr + 24 hr refers to treatment for 24 hr followed by 24 hr in fresh growth medium. Cells treated with 0.1% DMSO served as controls. Shown are mean values ± SD (n > 3). AUCs were calculated and significance was determined using unpaired t test assuming unequal variances (n.s., nonsignificant p > .05). (b) Western blot analysis of various DDR proteins (p-p53, p-Chk1, p21, and γ-H2ax) in primary HUFs exposed to indicate concentrations of N-OH-4-ABP for 24 hr with or without a 24 hr recovery period in fresh medium. Gapdh was used as a loading control and 3-NBA (2 μM, 48 hr) included as positive control. Representative images of Western blot analysis are shown. Analysis was performed in duplicate from independent experiments. (c-e) DNA adduct analysis: (c) Primary HUFs were treated with N-OH-4-ABP as indicated for 24 hr + 24 hr and formation of the dG-C8-4-ABP adduct was quantified using 32 P-postlabelling. Control cells were treated with 0.1% DMSO only. Shown are mean values ± SD (n = 4). n.d. (nondetected). (d) Representative autoradiographic profiles of DNA adducts formed in primary HUFs after N-OH-4-ABP exposure. The arrow indicates the dG-C8-4-ABP adduct. The bottom left corners (=origins) were cut off prior to imaging. (e) Additionally, DNA adduct levels were compared in HUFs treated with 40 μM N-OH-4-ABP for 24 hr with and without a 24 hr recovery period. Significance was determined using unpaired t test (***p < .001). (f) Induction of lacZ mutants in primary HUFs treated with N-OH-4-ABP for 24 hr + 24 hr. Cells treated with 0.1% DMSO served as controls. After a total of 48 hr cells were passaged to a new flask and allowed to double six times to fix DNA mutations. LacZ mutant frequencies were calculated as the number of mutant colonies per number of recovered transformants. Shown are mean values ± SD (n > 3) Software Inc., La Jolla, CA), groups of two were compared by twosample t-test assuming unequal variances (***p < .001). For the cell viability data, the area under the curve (AUC) and half-maximal inhibitory concentration (IC 50 ) values were calculated.

| DDR in primary HUFs after N-OH-4-ABP treatment
Induction of DDR proteins (p-p53, p-Chk1, γ-H2ax, and p21) in primary HUFs was assessed by Western blotting in response to N-OH-4-ABP treatment for 24 hr or 24 hr + 24 hr. As shown in

| Formation of DNA adducts in primary HUFs after N-OH-4-ABP treatment
For DNA adduct analysis, cells were treated with the three N-OH-4-ABP concentrations selected above for 24 hr + 24 hr; additionally cells were treated for 24 hr with 40 μM reflecting 60-40% cell viability. One major DNA adduct spot was detected by 32 P-postlabelling analysis in primary HUFs after exposure to N-OH-4-ABP (Figure 1c).
Based on previous studies this adduct spot was tentatively identified as dG-C8-4-ABP (Beland et al., 1999;Torino et al., 2001). No DNA adducts were detectable in untreated (control) HUFs (data not shown). Formation of the dG-C8-4-ABP adduct was concentrationdependent, with levels starting at 2 adducts/10 8 nucleotides after treatment with 30 μM N-OH-4-ABP ( Figure 1d); this value tripled to 7 adducts/10 8 nucleotides when cells were treated with 40 μM N-OH-4-ABP and finally was 12-fold higher compared to the lowest treatment concentration at 24 adducts/10 8 nucleotides after treatment with 80 μM N-OH-4-ABP. In addition, adduct levels were also examined after 24 hr exposure to 40 μM N-OH-4-ABP, in order to observe the extent of DNA damage at an earlier time-point and the ability of primary HUFs to repair pre-mutagenic DNA adducts.
Approximately 44 adducts/10 8 nucleotides were detected at 24 hr, which was six-fold more adducts than detected following the 24 hr recovery period (Figure 1e). This indicates that the HUF cells were able to repair the DNA adducts (dG-C8-4-ABP) extensively within 24 hr.

| Determination of lacZ mutant frequency in primary HUFs after N-OH-4-ABP treatment
Before initiating the HIMA the induction of lacZ mutants by N-OH-4-ABP was assessed in an E. coli host system (Figure 1f). Background mutant levels of 9 × 10 −5 mutants were calculated, which did not change at any of the treatment conditions used, with lacZ mutant levels in the range 9-10 × 10 −5 after exposure to N-OH-4-ABP.
Thus, no increase in mutant frequency was identified in the lacZ reporter gene.

| Nutlin-3a counter-screen and sequence analysis in N-OH-4-ABP-treated HUFs
Following the Nutlin-3a counter-screen 9 out of 150 cultures (6%) were classified as TP53-mutant HUFs. No TP53 mutations were found in spontaneously immortalized cultures (i.e., controls) which is consistent with historic controls (Hölzl-Armstrong et al., 2020). TP53mutant cultures were expanded into cell lines from which DNA was isolated. Exons 4-9 of the TP53 gene were amplified by PCR and subjected to Sanger dideoxy sequencing. There was a distinctive pattern of 56% G>C/C>G and 33% G>T/C>A transversions with 22% (2/9) based at CpG sites. The distribution of mutations on the transcribed and non-transcribed strand was the same. The observed mutant frequency and pattern are summarized in Table 1. It was not possible to establish the mutation type for one culture (i.e., NOA-59), which has been classified as "complex". It was still included as a definite TP53mutant for several reasons: (a) the Nutlin-3a counter-screen showed a strong resistant response; (b) p53 and its related pathway proteins p21 and Mdm2 were not expressed after treatment with Nutlin-3a (see Figure 2); and (c) after PCR amplification, sequencing of exon 7 was not successful. Therefore, it is likely that clone NOA-59 harbors a complex deletion in exon 7 that prevented primer annealing during sequencing.

| Exon and codon distribution of N-OH-4-ABP-induced mutations
A detailed overview of all N-OH-4-ABP-induced TP53 mutations can be found in Table 2

| Expression and induction of p53 pathway proteins in N-OH-4-ABP-treated TP53 mutants following treatment with Nutlin-3a
To further understand the cellular response towards Nutlin-3a, TP53mutant cultures were exposed to 10 μM Nutlin-3a for 24 hr and analyzed by Western blotting for the expression of p53 and its pathway proteins p21 and Mdm2. The effect of the mutation and Nutlin-3a on p53, p21 and Mdm2 expression is shown in Figure 2 and summarized in the supporting materials (Table S1). A TP53-WT culture was included to demonstrate a typical response towards Nutlin-3a treatment, which is reflected by an induction of all three proteins. In TP53-  Note: Mutations were detected by Sanger dideoxy sequencing and mutation data acquired from the IARC TP53 mutation database (R20, July 2019). Nutlin-3a response means cellular response to 10 μM Nutlin-3a treatment for 5 days. Activity refers to the activity of the respective mutation found in the yeast promotor assay according to Kato et al. (2003): IARC TP53 mutation database (R20, July 2019) and are summarized in Table 3. The seven different mutations identified in N-OH-4-ABPtreated HUFs were found in a total of 155 human tumors with numbers ranging from 14-43 tumor samples. Further, all codons and splice sites targeted by N-OH-4-ABP were found to be mutated in other human cancers. As 4-ABP is a known bladder carcinogen (IARC, 2010), the occurrence of each mutation and targeted codon was further compared specifically with TP53 mutations found in human bladder cancer (Table 3). The exact mutations have been reported in 10 human bladder tumors.
As discussed previously, there are specific TP53 mutation hotspots in bladder cancer, so the codon distribution in bladder cancer of non-smokers and smokers was compared with this study, the historical HUF control, and all cancer types (Figure 3a N-OH-4-ABP no G>A/C>T transitions were detected. However, the hallmark mutations were identified as 56% G>C/C>G and 33% G>T/ C>A transversions, which are also common TP53 mutation types in bladder tumors (Figure 3g). The predominant mutation type in historical HUF controls is 50% G>C/C>G transversions followed by 24% A>C/T>G transitions (Figure 3h).

| Mutations induced by 4-ABP and its metabolites in the literature
Finally, the mutation pattern observed in the TP53 gene of N-OH-4-ABP-exposed HUFs was compared with mutations observed in other studies examining 4-ABP or its metabolites. As shown in Figure 4, studies found mutations predominantly at G:C base pairs (Besaratinia et al., 2002;Chen et al., 2005;Yoon et al., 2012). In the cII T A B L E 3 Number of TP53 mutations in bladder and all tumors at TP53 mutation sites found in immortalized HUFs exposed to N-OH-4-ABP Note: Occurrence refers to the number of human tumors harboring the exact indicated mutation. In addition, the total number of times the respective codon is mutated in human tumors is listed. Total count of mutations is 28,866 in all tumors and 1,522 in bladder tumors (IARC TP53 mutation database, R20, July 2019). Studies recommended to be excluded by IARC were not considered.
gene of Big Blue ® MEFs treated with N-hydroxy-4-acetylaminobiphenyl (N-OH-4-AABP) 43% were G>T/C>A mutations, followed by 21% G>A/ C>T and 9% G>C/C>G (Figure 4c) (Besaratinia et al., 2002). Another study examined the cII gene in livers of Big Blue ® mice treated by intraperitoneal injection with 4-ABP and found no difference in the mutation spectrum between treated and untreated (i.e., controls) tissues; both spectra mostly carried G>A/C>T transitions. However, when the liver cII gene of neonatal mice treated in the same way was sequenced, mainly G>T/C>A transversions (41%) and an equal amount of 16% each G>A/C>T and G>C/C>G substitutions were found (Figure 4a) (Chen et al., 2005). The mutation pattern of the cII gene in bladders of Big Blue ® mice exposed for 6 weeks to various doses of 4-ABP administered by intraperitoneal injection was defined by 42% G>T/C>A and 25% G>A/C>T substitutions. In addition, 12% G>C/C>G transversions were induced in the bladder cII gene (Figure 4b) (Yoon et al., 2012).
F I G U R E 3 Codon distribution and pattern of TP53 mutations found in N-OH-4-ABP-treated immortalized HUFs (a,g), the historical HUF control (b,h), bladder (c,i), smoker's bladder (d,j), nonsmoker's bladder (e,k) and all tumors (f,l). Shown are exons 4-9. Reference for human tumor data is the IARC TP53 mutation database (R20, July 2019). Studies recommended to be excluded by IARC were not considered. References for the historical control data are from Whibley et al. (2010) and Kucab et al. (2015) and Kucab et al. (2016). Mutation hotspots are indicated in gray

| DISCUSSION
The objective of this study was to assess the mutagenicity of N-OH-4-ABP in primary HUFs. It was hypothesized that N-OH-4-ABP induces a specific mutation spectrum in the human TP53 gene of immortalized HUFs reflecting the mutation spectrum observed in human bladder cancer. Human bladder cancer shows an even distribution of mutations amongst exons 5-8 of TP53 that is characterized by five hotspot codons (R175, R248, R273, R280, E285), while mutations are not biased towards CpG sites (Feng et al., 2002a). When the results acquired in the HIMA are compared with human bladder cancer, the first difference is that the codon distribution of TP53 muta-  (Feng et al., 2002a). However, codon D281, which has also been shown to be a target for dG-C8-4-ABP adduct formation in human uroepithelial cells (Feng et al., 2002a), was mutated in two immortalized HUF clones after N-OH-4-ABP treatment. Further, TP53 mutations in immortalized HUFs were harbored in other bladder hotspot codons in this study, namely R248 and R273. In previous HIMAs TP53 mutations in R273 have been observed (Table S2), but finding a mutation at this codon after N-OH-4-ABP treatment was unexpected as dG-C8-4-ABP adduct formation did not occur at this codon in previous work (Feng et al., 2002a;Feng et al., 2002b). Thus, it could be speculated that the observed differences between HUFs and human uroepithelial cells are due to variations of enzymatic capabilities (e.g., N-acetyltransferases or sulfotransferases) and/or differences in DNA repair. Furthermore, while in human bladder tumors mutations are evenly distributed amongst the TP53 sequence, most TP53 mutations in HUFs were harbored within exons 7 and 8. However, only 20% of TP53 mutations occurred at CpG sites, which agrees with lack of bias for CpG mutations in bladder cancer (Feng et al., 2002a).
The fact that G>C/C>G transversions are amongst the predominant mutation types in spontaneously immortalized HUFs (Figure 3h) could lead to the conclusion that the mutations found in the present study are induced spontaneously and not due to N-OH-4-ABP treatment. Importantly, three mutations (C135, G245 and D281) have previously been predominantly observed in untreated spontaneously immortalized HUF clones, and it cannot be ruled out that these mutations are unspecific to N-OH-4-ABP treatment but rather happened due to spontaneous mutation events. In addition, even though the spontaneous TP53 mutation frequency is very low (0-3.7%) in primary HUFs prepared and treated at 3% oxygen (Kucab et al., 2015;Kucab et al., 2016;Hölzl-Armstrong et al., 2020), some mutations could be caused spontaneously and not due to N-OH-4-ABP treatment. However, several reasons argue that most of the observed mutations are indeed caused by N-OH-4-ABP and not spontaneous: First, three of the TP53 mutations induced by N-OH-4-ABP have not been observed previously in carcinogen-treated or spontaneously immortalized HUFs, so they appear to be related to N-OH-4-ABP exposure. Secondly, guanine adducted with 4-ABP (i.e., dG-C8-4-ABP) can, F I G U R E 4 Comparison of mutation patterns induced by 4-ABP and its metabolites in different experimental systems. Mutation pattern induced by 4-ABP in the cII gene of neonatal Big Blue ® mouse livers (a, Chen et al., 2005), the cII gene Big Blue ® mouse bladders of (b, Yoon et al., 2012), in the cII gene of Big Blue ® MEFs treated with N-OH-4-AABP (c, Besaratinia et al., 2002) and in the TP53 gene of N-OH-4-ABP-treated immortalized HUFs (d, present study) depending on the conformation of the DNA, mispair with either adenine or guanine to result in G>T/C>A and G>C/C>G transversions, respectively (Besaratinia and Tommasi, 2013). Therefore, the predominant mutation type detected in the present study agrees with dG-C8-4-ABP adduct formation as it was detected in N-OH- is insufficient to make a firm conclusion. Finally, 4-ABP has been linked to the formation of reactive oxygen species (Wang et al., 2006) resulting in oxidative damage to DNA, including 8-oxo-7,8-dihydro-2 0 -deoxyguanosine (8-oxo-dG) (Murata et al., 2001), which can result in the induction of G>T/C>A mutations (Marnett, 2000). Thus, the high number of G>T/C>A transversions could also be linked to oxidative damage.
Additionally, most other studies examining 4-ABP or its metabo- The results of the lacZ mutation assay (Figure 1f), in which mutations were not induced in N-OH-4-ABP-treated primary HUFs, were surprising. dG-C8-4-ABP adducts were clearly formed in primary HUFs and other studies have observed mutagenicity after exposure to 4-ABP or its metabolites in reporter gene assays (e.g., cII, Tk, HGPRT) in other cultured mammalian cells (Bookland et al., 1992;Besaratinia et al., 2002;Guo et al., 2016). In other studies lacZ mutagenicity has been a good predictor of TP53 mutagenicity (Kucab et al., 2016;Hölzl-Armstrong et al., 2020). It is puzzling that there is a lack of lacZ mutants after treatment with N-OH-4-ABP, although the TP53 mutant frequency of 6% obtained in this study is also low compared to the mutant frequencies induced by other agents in previous HIMAs, ranging from 9 to 33% (Feldmeyer et al., 2006;vom Brocke et al., 2009;Kucab et al., 2015;Hölzl-Armstrong et al., 2020). It seems likely that the dG-C8-4-ABP adduct levels in N-OH-4-ABP-treated primary HUFs were not high enough to induce mutagenicity in the lacZ reporter gene or that the majority of adducts were repaired before mutation fixation, as suggested by the greatly decreased dG-C8-4-ABP adduct levels in HUFs after adding a 24 hr recovery period.
Other studies have also shown that the dG-C8-4-ABP adduct can be repaired. For instance, in human urinary bladder transitional cell carcinoma cell lines treated with 15 μM N-OH-4-AABP for 8 hr, 75% of adducts were removed within 25 hr (Torino et al., 2001). Another explanation for the lack of lacZ mutants and low rate of TP53 mutants could be efficient error-free translesion synthesis past the dG-C8-4-ABP adduct. In fact it has been shown that the efficiency of translesion synthesis is affected by the sequence surrounding the dG-C8-4-ABP adduct (Yagi et al., 2017), which could explain why mutations were still induced in the TP53 gene. This observation could also be linked to differences in the timing of the two assays. As shown by the DNA adduct experiments many adducts were repaired within 24 hr. Thus, remaining unrepaired adducts resulting in mutations are very rare and the 6 days allowed for mutation fixation in the lacZ assay might not be sufficient for recognition because lacZ mutant cells are too diluted amongst lacZ WT cells. In contrast, as the HIMA cultures are sometimes grown for up to 3 months the TP53-mutants HUFs have a chance to appear amongst the WT cells due to clonal expansion.
In summary, we have performed the first comprehensive in vitro study assessing N-OH-4-ABP-induced mutagenesis in the human TP53 gene, which is often mutated in human bladder cancer. N-OH-4-ABP induced a characteristic TP53 pattern in immortalized HUFs, which agrees with the formation of the main dG-C8-4-ABP adduct that leads to mutations at G:C base pairs. However, the observed mutation spectrum of TP53 mutations was different to that observed in human bladder tumors, which is mostly likely due to other factors also playing a role in human bladder cancer development. High repair efficiency together with a possible error-free translesion synthesis could be responsible for the absence of lacZ mutants and low TP53 mutation frequencies.