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. 2024 Jan;34(1):47–56. doi: 10.1101/gr.278326.123

Oxidative stress accelerates intestinal tumorigenesis by enhancing 8-oxoguanine-mediated mutagenesis in MUTYH-deficient mice

Mizuki Ohno 1,✉, Noriko Takano 1, Kyoko Hidaka 1,2, Fumiko Sasaki 1, Kazumi Yamauchi 1,3, Yasunobu Aoki 4, Takehiko Nohmi 5, Yusaku Nakabeppu 6,7, Yoshimichi Nakatsu 1, Teruhisa Tsuzuki 1
PMCID: PMC10904009  PMID: 38290979

Abstract

Oxidative stress–induced DNA damage and its repair systems are related to cancer etiology; however, the molecular basis triggering tumorigenesis is not well understood. Here, we aimed to explore the causal relationship between oxidative stress, somatic mutations in pre-tumor-initiated normal tissues, and tumor incidence in the small intestines of MUTYH-proficient and MUTYH-deficient mice. MUTYH is a base excision repair enzyme associated with human colorectal cancer. Mice were administered different concentrations of potassium bromate (KBrO3; an oxidizing agent)–containing water for 4 wk for mutagenesis studies or 16 wk for tumorigenesis studies. All Mutyh−/− mice treated with >0.1% KBrO3 developed multiple tumors, and the average tumor number increased dose dependently. Somatic mutation analysis of Mutyh−/−/rpsL transgenic mice revealed that G:C  > T:A transversion was the only mutation type correlated positively with KBrO3 dose and tumor incidence. These mutations preferentially occurred at 5′G in GG and GAA sequences in rpsL. This characteristic mutation pattern was also observed in the genomic region of Mutyh−/− tumors using whole-exome sequencing. It closely corresponded to signature 18 and SBS36, typically caused by 8-oxo-guanine (8-oxoG). 8-oxoG-induced mutations were sequence context dependent, yielding a biased amino acid change leading to missense and stop-gain mutations. These mutations frequently occurred in critical amino acid codons of known cancer drivers, Apc or Ctnnb1, known for activating Wnt signal pathway. Our results indicate that oxidative stress contributes to increased tumor incidence by elevating the likelihood of gaining driver mutations by increasing 8-oxoG-mediated mutagenesis, particularly under MUTYH-deficient conditions.

Oxidative stress reflects a cellular consequence of an imbalance between reactive oxygen species (ROS) generation and a biological system's ability to detoxify the resulting damage (Sies 2015). It is considered an important etiologic factor in tumor initiation (Ames 1983; Waris and Ahsan 2006). ROS are constantly produced in vivo through normal cellular metabolism; however, their production is further enhanced during infection, inflammation, aging, or exposure to environmental factors, including ionizing radiation or chemicals (Sauer et al. 2001). Various types of oxidatively damaged DNA are produced when ROS attack nucleic acids, resulting in mutations (Maynard et al. 2009). However, molecular mechanisms underlying ROS-triggered tumorigenesis remain unknown.

Of the four DNA nucleobases, guanine is most susceptible to oxidation, and 8-oxo-7,8-dihydroguanine (8-oxoguanine [8-oxoG]) is its major oxidized form (Kasai and Nishimura 1984). 8-oxoG is a potent premutagenic lesion because it can form a stable base pair with adenine and cytosine, resulting in the G:C > T:A transversion mutation (Shibutani et al. 1991). The nuclear DNA of human and mouse normal somatic cells have more than 10,000 8-oxoG residues (Ohno et al. 2006). The amount of 8-oxoG in the DNA of steady-state living cells is determined based on a dynamic equilibrium between its generation through oxidation and removal by the DNA repair system. Therefore, exposure of cells to excess oxidative stress or dysregulations in the repair system results in biological consequences of 8-oxoG, including an increase in mutation frequency (MF) in both somatic and germline cells (Ohno et al. 2014; Ohno 2019).

Common molecular mechanisms for preventing 8-oxoG-mediated mutagenesis have been reported across a range of organisms, from bacteria to mammals (Tsuzuki et al. 2007; Boiteux et al. 2017; Nohmi 2018). In mice and humans, three enzymes, namely, NUDT1 (also known as MTH1), OGG1, and MUTYH, act in coordination to reduce mutations. NUDT1 hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP to prevent the incorporation of oxidized nucleotides into DNA during replication. OGG1 is a DNA glycosylase that recognizes the 8-oxoG:C pair in double-stranded DNA and initiates base excision repair by excising the 8-oxoG base (Radicella et al. 1997; Rosenquist et al. 1997). MUTYH is an adenine DNA glycosylase, which excises misincorporated adenine opposite of 8-oxoG in the template strand during DNA replication as the first step of base excision repair (McGoldrick et al. 1995). The deficiency in the enzymatic activities of NUDT1 or/and OGG1 increases 8-oxoG levels in the DNA, resulting in MUTYH repair target 8-oxoG:A mispairs. Mice lacking one of these enzymes are cancer-prone later in age (Sakumi et al. 2003; Xie et al. 2004; Sakamoto et al. 2007), whereas TOY mice, which lack all three of the genes Nudt1, Ogg1, and Mutyh, develop spontaneous tumors at a younger age and have a short lifespan (Ohno et al. 2014). TOY mice show a high spontaneous germline mutation rate, showing significantly increased G:C > T:A mutations. The administration of potassium bromate (KBrO3), an oxidizing reagent, increases tumor frequency in the small intestines of Mutyh-deficient mice (Sakamoto et al. 2007). MUTYH-associated polyposis (MAP) is a human colorectal cancer predisposition syndrome caused by biallelic germline mutations in MUTYH (Al-Tassan et al. 2002; Poulsen and Bisgaard 2008). MAP tumors show distinct molecular features, including frequent G:C > T:A mutations in cancer-related genes or genomic regions (Al-Tassan et al. 2002; Poulsen and Bisgaard 2008; Rashid et al. 2016; Pilati et al. 2017; Viel et al. 2017). MUTYH may play an important role in the prevention of oxidative stress–induced tumors in humans.

Human somatic cells acquire many mutations during development and aging. Recent studies have reported the prevalence of cancer-associated mutations in normal tissues, which accumulate with aging (Risques and Kennedy 2018; Evans and Degregori 2021). Somatic MF in normal tissues is influenced by internal or external mutagens from a young age and can be an important predictor of subsequent tumor incidence in the same organ. Therefore, we aimed to examine whether the increase in somatic MF in normal tissues of oxidatively stressed mice preceded tumor development and whether it was correlated to tumor incidence at a later age in a mouse model of an intestinal tumor. However, quantitative and qualitative analyses of somatic mutations in nontumor normal tissues are challenging because spontaneous MF is extremely low for detection using simple massively parallel sequencing. Therefore, herein, we used rpsL transgenic (Tg) mice, which harbor multiple copies of reporter genes, for an in vivo mutagenesis assay (Gondo et al. 1996; Egashira et al. 2002).

Here, we examined correlations among oxidative stress level, somatic MF, and spectra in pre-tumor-initiated normal tissues and tumor frequency at the later stage. First, we analyzed effects of oxidative stress levels on tumor incidence in Mutyh+/+, Mutyh+/−, and Mutyh−/− mice. Next, we analyzed the somatic MF and spectra in normal precancerous tissues using Mutyh+/+/rpsL-Tg and Mutyh−/−/ rpsL-Tg mice. Finally, we conducted whole-exome sequencing (WES) analysis to identify unique somatic mutations acquired during tumor development and explored mutation patterns and driver mutations in Mutyh−/− tumors.

Results

Analysis of oxidative stress–induced tumors

To assess the causal effect of oxidative stress on tumorigenesis, we analyzed tumor frequency in small intestines of Mutyh+/+, Mutyh+/−, and Mutyh−/− mice after chronic administration of KBrO3. Mice were administered 0.5%, 0.1%, 0.15%, or 0.2% KBrO3-containing water or regular water for the nontreated control for 16 wk from 4 wk of age (Fig. 1A). No tumors were observed in nontreated control groups and 0.5% KBrO3-treated groups regardless of the Mutyh genotype. Notably, Mutyh−/− mice showed a remarkable elevation in tumor number with an increase in KBrO3 dose (Fig. 2A; Table 1). For instance, the average tumor number per mouse was 8.8, 41.6, and 61.9 in the 0.1%, 0.15%, and 0.2% KBrO3-treated groups, respectively, whereas those in Mutyh+/+ and Mutyh+/− mice were 0.9 ± 0.6 and 3.2 ± 1.3, respectively, even with treatment with the highest KBrO3 dose. Although Mutyh+/+ and Mutyh+/− mice showed substantially lower tumor numbers than Mutyh−/− mice, a significant positive correlation between tumor number and KBrO3 dose was observed in all genotypes (P < 0.0001, ρ = 0.9028, 0.5865, and 0.5815 in Mutyh−/−, Mutyh+/−, and Mutyh+/+, respectively) (Supplemental Table S1). Most tumors were detected predominantly in regions corresponding to the duodenum and jejunum, showing nonpolypoid growth with almost the same height as around normal villi (Fig. 1B–F; Supplemental Fig. S1), consistent with the findings of our previous studies (Sakamoto et al. 2007; Isoda et al. 2014).

Figure 1.

Figure 1.

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Experimental design and tumor analysis. (A) Experimental design of KBrO3 administration. Schedule for tumor analysis (upper) for rpsL mutation analysis (lower). Blue and magenta lines indicate the administration period of regular water or KBrO3-containing water, respectively. (B) Formalin-fixed small intestines (duodenum to ileum, right to left) collected from 0.15% KBrO3-treated mice. Upper panel: specimens from Mutyh−/− mouse. Multiple tumors (white spot-like parts) are observed. Lower panel: specimens from Mutyh+/+ mouse. No visible tumor was observed. Scale bars indicate 1 cm. (C) Magnified images of B. The scale bar indicates 1 mm. (D–F) HE-stained section of StR of the small intestine (Supplemental Methods) of 0.15% KBrO3-treated Mutyh−/− mice. The scale bar indicates 1 mm. (D) Multiple tumors (asterisks) at the outer side correspond to the region of the duodenum and jejunum. (E) Magnified view of the marked area in D, with >1-mm sized tumors (polyp-like form) among normal villi. (F) Magnified view of the marked area in E.

Figure 2.

Figure 2.

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Dose-dependent increase in intestinal tumors in KBrO3-treated mice. Magenta, green, and blue dots and lines indicate the data of Mutyh−/−, Mutyh+/−, and Mutyh+/+, respectively. (A) The number of tumors per mouse is plotted based on genotype and KBrO3 dose. The line is drawn through the mean values. The pale-colored bands represent ± SD. (B) Tumor prevalence. The percentage of mice with tumor(s) in each study group is plotted against KBrO3 dose.

Table 1.

Results of tumor analysis

Mutyh KBrO3 concentrations
0% 0.05% 0.10% 0.15% 0.20%
+/+ Mean tumor number ± SD 0 0 0.4 ± 0.6 0.9 ± 1.0 0.9 ± 0.6
Tumor prevalence (%) 0 0 35.7 59.1 76.5
Number of mice analyzed 17 11 14 22 17
+/− Mean tumor number ± SD 0 0 1.4 ± 0.9 2.2 ± 1.2 3.2 ± 1.3
Tumor prevalence (%) 0 0 89.3 95.6 100
Number of mice analyzed 3 3 29 23 6
−/− Mean tumor number ± SD 0 0 8.8 ± 4.7 41.6 ± 12.7 61.9 ± 24.9
Tumor prevalence (%) 0 0 100 100 100
Number of mice analyzed 11 9 12 20 19
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When focusing on the tumor prevalence, namely, percentage of mice having at least one tumor, we observed the obvious KBrO3 dose effect in all genotypes (Table 1; Fig. 2B); 100% of Mutyh−/− mice developed tumors at doses of ≥0.1%. In Mutyh+/+ mice, it gradually increased with KBrO3 dose; however, ∼20% of mice treated with the highest dose did not develop tumors during the experimental period. In Mutyh+/− mice, although the average tumor number was small, the plot pattern of tumor prevalence by KBrO3 dose was close to that of Mutyh−/− mice.

To evaluate effects of longer exposure to oxidative stress on tumorigenesis in wild-type mice, Mutyh+/+ mice were administered 0.15% KBrO3 for 40 wk. Eleven out of 12 mice (92%) developed one to four tumors (2.42 ± 1.38); the mean tumor number per mouse was 2.69 times higher than that of the 16-wk treated group (Wilcoxon test, P = 0.003) (Supplemental Fig. S2). Thus, the dose and administration period of oxidative stress may affect tumor incidence even under the MUTYH-proficient condition.

Analysis of somatic mutations in the normal precancer tissues by reporter gene assay

To evaluate the MF during the pre-tumor-initiation phase, we conducted a rpsL reporter gene assay using normal small intestinal tissues obtained from mice treated with KBrO3 for 4 wk. Here, MF was defined as the fraction of mutated rpsL gene among the total rpsL gene loci analyzed, namely, per locus MF.

Total MF in Mutyh+/+ and Mutyh−/− mice in the control groups was 1.41 × 10−5± 0.71 and 2.07 × 10−5± 0.47, respectively (Table 2; Fig. 3A; Supplemental Table S2). The basal level MF in Mutyh−/− mice was approximately 1.5 times higher than that in Mutyh+/+ mice; however, there was no statistically significant difference. All the KBrO3-treated groups showed relatively higher MF compared with those in the matched control group in both genotypes. The mean total MF in Mutyh+/+ and Mutyh−/− mice in the 0.2% KBrO3-treated groups was 2.34 × 10−5± 1.61 and 4.67 × 10−5± 0.34, respectively. However, using Steel's test, no statistically significant difference was detected in any pairs compared with the matched control.

Table 2.

Mutation frequency based on spectra

Mutyh genotype +/+ −/−
MF (×10−5)
KBrO3 (%) 0 0.05 0.1 0.15 0.2 0 0.05 0.1 0.15 0.2
Transition
 G:C > A:T 0.16 0.12 0.92 0.63 − 0.19 0.58 0.74 0.18 −
 A:T > G:C − 0.06 0.04 0.11 0.11 0.13 − 0.16 0.05 −
Transversion
 G:C > T:A 0.08 0.31 0.34 0.28 0.47 0.16 1.02 2.25 2.54 2.73
 G:C > C:G − − 0.06 − − 0.04 − 0.03 − −
 A:T > T:A 0.05 − − − − 0.1 − − − −
 A:T > C:G − − 0.03 − − − − − − −
Indel
 1-bp del
(poly(A))		 0.49
(0.20)		 0.39 0.31
(0.06)		 0.7
(0.06)		 0.43
(0.14)		 0.57
(0.19)		 1.33
(0.06)		 0.48
(0.05)		 0.78
(0.06)		 0.49
 1 bp ins
(poly(A))		 − − 0.02
(0.02)		 0.13 − − − 0.1
(0.1)		 0.1
(0.1)		 −
−
 >2-bp indel 0.64 1.04 0.63 0.83 1.33 0.93 0.87 0.94 1.04 1.46
 Others − − − 0.1 − 0.05 − − 0.07 −
 Total 1.41 1.92 2.36a 2.78a 2.34 2.17a 3.79 4.69a 4.75a 4.67
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aTotal MF including more than two mutations detected in a single Km/Sm-resistant colonies. (−) Not detected.

Figure 3.

Figure 3.

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Somatic mutations in the normal intestinal tissues detected using the rpsL assay. (A) Mutation frequency (MF) in Mutyh−/− mice (magenta) and Mutyh+/+ mice (blue) based on the KBrO3 dose. Mean ± SD. (B) The relative ratio of mutation type is shown in the stacked bar chart. Mutations in the Mutyh+/+ mice. Green, magenta, and blue indicate indels, transversions, and transitions, respectively. (C) Same chart as B for Mutyh−/− mice. (D–G) MF is plotted based on the mutation type. Mean ± SD. Mutyh−/− mice (magenta) and Mutyh+/+ mice (blue). Steel's test (vs. KBrO3 0%; control) was performed, and P-values are described in the plot if significant. (D) G:C > T:A transversion. (E) G:C > A:T transition. (F) One-base-pair deletion. (G) More than 2-bp indel.

To determine the site and spectra for each mutation, rpsL coding regions from all mutant colonies were sequenced (Table 2). In Mutyh+/+ mice, insertion/deletion (indel) mutations were more frequently observed than base substitutions, accounting for ∼70% of all mutations (Table 2; Fig. 3B). In Mutyh−/− mice, the relative ratio of transversion mutations gradually increased with KBrO3 dose, which accounted for >50% of the total mutations in the 0.1%, 0.15%, and 0.2% KBrO3-treated groups (Fig. 3C). This was largely attributed to the increase in G:C > T:A substitutions. Only G:C > T:A substitutions increased with KBrO3 dose in Mutyh−/− mice (Table 2; Fig. 3D; Supplemental Fig. S3). The MF of G:C > T:A in 0.2% KBrO3-treated Mutyh−/− mice was 2.73 × 10−5, which was approximately 17 times higher than that of the nontreated control group. A significant positive correlation was detected between the KBrO3 dose and MF of G:C > T:A in Mutyh−/− mice (Spearman's rank correlation coefficient, ρ = 0.765, P < 0.0001) (Supplemental Table S1). Furthermore, an elevation in MF of G:C > T:A was observed in KBrO3-treated Mutyh+/+ mice. However, it was not statistically significant. No other mutation types showed a positive correlation with KBrO3 dose in Mutyh+/+ or Mutyh−/− mice, except for the total MF in Mutyh−/− mice (Fig. 3E–G; Supplemental Table S1). We next ascertained the correlations between each mutation type and tumor frequency. Notably, only the MF of G:C > T:A showed a positive correlation with tumor frequency in Mutyh−/− mice (Supplemental Table S1).

To analyze the site distribution and sequence context around mutated sites, base substitution mutations were mapped onto the rpsL gene sequence (Supplemental Fig. S4A–D). G:C > T:A mutations detected in KBrO3-treated Mutyh−/− mice tended to occur at 5′ guanine in GAA/TTC or GG/CC sites (Supplemental Fig. S4B). In this experiment, G:C > T:A mutations were found at 19 sites, of which six were regarded as hot spots because G:C > T:A mutations were identified multiple times in different mice. All six hot spots contained GAA/TTC or GG/CC sequences. Mutations identified in those sites constituted >70% of all G:C > T:A mutations detected in Mutyh−/− mice (Supplemental Fig. S4B). In Mutyh+/+ mice, G:C > T:A mutations also tended to occur at the same six hot spots (Supplemental Fig. S4D). In contrast, G:C > A:T mutations were predominantly detected at two other hot spots, containing CG/GC and CC/GG sites, regardless of genotype or KBrO3 treatment (Supplemental Fig. S4D).

Analysis of somatic mutations in KBrO3-induced Mutyh−/− tumors by WES

To explore whether the characteristic mutation pattern seen in the rpsL reporter genes was also observed in the genomic region of Mutyh−/− mice, we performed WES analysis. Four tumor samples and two matched normal samples derived from two KBrO3-treated Mutyh−/− mice were used. In total, 1073 single-nucleotide variants (SNVs), an average of 268 per tumor, 17 indels, and six multinucleotide substitutions were identified from four tumors (Table 3; Supplemental Table S3). Consistent with the rpsL assay, the most frequently observed mutation type was GC > T:A, accounting for 80.5% of total SNVs, followed by G:C > A:T (9.4%), and A:T > G:C (5.7%) (Table 3). The transition/transversion ratio was 0.2.

Table 3.

Number of variants detected in Mutyh−/− tumors using WES

Tumor ID Total
RY1114 T6 RY1114 T7 RY1115 T2 RY1115 T6
Transition
 G:C > A:T 34 29 26 12 101
 A:T > G:C 21 23 12 5 61
Transversion
 G:C > T:A 272 172 297 123 864
 G:C > C:G 3 4 3 4 14
 A:T > T:A 6 9 2 3 20
 A:T > C:G 3 5 2 3 13
SNV total 339 242 342 150 1073
Indel, MNS 5 9 2 8 23
Total 344 251 344 157 1096
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(MNS) Multinucleotide substitution.

To analyze the sequence context of the mutated sites, six types of base substitutions were broken down into 96 subtypes, which were all conservable trinucleotide sequences, including 5′ and 3′ flanking bases at mutated sites (Alexandrov et al. 2013). Note that bases of mutated sites were denoted by the pyrimidine bases of the Watson–Crick base pair in accordance with the convention of mutational signature analysis. C > A mutations were preferentially located at the center of trinucleotides of TCT, CCT, CCA, and ACA, followed by TCA, TCC, GCT, GCA, and CCC (Fig. 4A; Supplemental Fig. S5). SNVs were almost equally distributed on both transcribed and nontranscribed strands (Supplemental Fig. S6A,B). Indels and multinucleotide substitutions were less frequently detected than SNVs, with no evidence of instability at short tandem repeats (Supplemental Fig. S6C). In the 1536 pentanucleotide pattern, thymine was frequently located at 5′ and 3′ ends of trinucleotides such as TTCTT or TCCTT (Supplemental Fig. S7).

Figure 4.

Figure 4.

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Mutation pattern of KBrO3-induced tumor in Mutyh−/− mice. The mutation pattern is displayed based on the 96-base substitution classification defined by the substitution class and sequence context immediately 3′ and 5′ to the mutated base. The y-axis indicates the contribution of each mutation to the total mutation number. The x-axis indicates 96 trinucleotide types. (A) Somatic mutations in the tumor samples of KBrO3-treated Mutyh−/− mice detected using WES. (B) Somatic mutations were detected in the normal intestinal tissues (pre-tumor-initiation stage) of KBrO3-treated Mutyh−/− mice using the rpsL assay. (C) Mutation pattern of de novo germline mutations detected in TOY mice (Ohno et al. 2014). (D) Mutation pattern of tumor samples from human patients with MAP (see Methods). (E) Signature 18 in COSMIC mouse ver.2. (F) SBS 36 in COSMIC mouse ver.3. (G) SBS 45 in COSMIC mouse ver.3.

The mutation pattern of the Mutyh−/− tumor detected using WES was consistent with results of the rpsL reporter gene assay and the TOY germline mutation pattern (Fig. 4A–C; Supplemental Tables S4, S5). Among the known COSMIC signatures, SBS36, SBS18, and signature 18 (Fig. 4E–G) showed higher values of cosine similarity against the mutation pattern of Mutyh−/− mice tumor (0.858, 0.851, and 0.835, respectively) (Supplemental Table S4). These signatures are often found in human cancers with defective MUTYH. Indeed, the mutation pattern of Mutyh−/− tumor showed a significant positive correlation with that of human MAP tumors (r = 0.828, P < 0.0001) (Fig. 4D; Supplemental Table S5), suggesting a functional commonality of MUTYH in mice and humans.

Functional analysis of the detected mutations revealed that the ratio of nonsynonymous/synonymous was 2.5. In the nonsynonymous mutations, 82% were missense and 12% were stop-gain mutations. This bias toward nonsynonymous mutations could be attributed to the preferred sequence contexts of 8-oxoG. To examine whether the characteristic mutation pattern owing to unrepaired 8-oxoG:A yields any biases in amino acid change, we analyzed the codon and amino acid changes that occurred in the tumors of Mutyh−/− mice. The occurrence of base substitutions in each codon sequence largely differed among 64 trinucleotide types, which resulted in a distinct pattern of amino acid change (Supplemental Figs. S8, S9). E > stop (glutamic acid to stop codon) was the most frequently detected type of amino acid change. This was attributed to frequent G > T mutations at the first G in GAA and GAG sequences, both of which code glutamic acid. Following this, K > N, L > I, and D > Y were positioned at the top of the list.

Next, we searched for driver mutations in each tumor. We found G:C > T:A mutations within TCT/AGA sequences in exon 3 of Ctnnb1 in three out of four tumors (Supplemental Fig. S10; Supplemental Table S3). These mutations led to S > Y change at the 33rd and 37th amino acids. S > Y was attributed to TCC > TAC and TCT > TAT mutations; this amino acid change was frequently detected here (Supplemental Figs. S8, S9). In one tumor (RY1115-T2), with no Ctnnb1 mutation, an E > stop mutation in Hydin, a candidate cancer driver (Viel et al. 2017), was found at 15% allelic frequency. No other pathogenic mutations were observed in known driver genes frequently mutated in human MAP tumors, such as Apc, Amer1, Fat1, Fat4, Kdm6a, Kras, Pik3ca, Trp53, Smad2, Smad4, and Braf (Viel et al. 2017).

Finally, we re-evaluated the somatic mutations from the tumor WES using the newly released mouse reference genome mm39 (Supplemental Table S3). The number and pattern of these mutations were nearly identical to those obtained using the mm10 reference genome (Supplemental Fig. S11).

Discussion

Here, we provided compelling evidence that chronic exposure to oxidative stress accelerates tumorigenesis by evoking excessive somatic mutations, particularly under MUTYH-deficient conditions (Tables 1–3; Figs. 1, 2). When treated with 0.2% KBrO3, Mutyh−/− mice developed approximately 69 times more tumors than Mutyh+/+ mice (Table 1). We observed positive correlations between the KBrO3 dose and MF of G:C > T:A, MF of G:C > T:A and tumor incidence, and KBrO3 dose and tumor incidence in Mutyh−/− mice. A strong tumor-suppressive role of MUTYH was attributed to preventing oxidative stress–induced mutagenesis, particularly in avoiding 8-oxoG-mediated G:C > T:A transversion (Fig. 3). Thus, we concluded that 8-oxoG:A is the key molecular initiating event triggering intestinal tumorigenesis in Mutyh−/− mice. Moreover, sequence context–dependent G:C > T:A mutagenesis yielded biased amino acid change to missense and stop-gain mutations (Supplemental Fig. S9). Our results and human MAP studies (Al-Tassan et al. 2002; Viel et al. 2017) suggest the importance of MUTYH repair function in the prevention of oxidative stress–induced tumorigenesis in both mice and humans.

We previously showed that Mutyh−/− mice did not show a remarkable phenotype at a younger age (i.e., before 12 mo) but showed increased spontaneous tumor susceptibility after aging (i.e., after 18 mo) (Sakamoto et al. 2007). For example, eight of 121 mice developed one or two tumors in the small intestines at 18 mo of age. However, no intestinal tumors were observed in 109 age-matched wild-type control mice. In the present study, 100% of Mutyh−/− mice treated with >0.1% KBrO3 for 16 wk developed multiple tumors at 5 mo of age; the tumor number per mouse increased with an increase in KBrO3 concentration. This finding suggests that when Mutyh−/− mice experience oxidative stress from a younger age, they show a moderate-to-high mutator phenotype and high tumor susceptibility (Tables 2, 3). Furthermore, additional disruption of Ogg1 in Mutyh−/− background increased spontaneous tumor incidence in multiple organs and shortened lifespan compared with Mutyh single-knockout mice (Russo et al. 2009; Ohno et al. 2014). Defective OGG1 repair activity leads to the accumulation of 8-oxoG in the genomic DNA (Ohno et al. 2014), subsequently increasing 8-oxo-G:A mismatches during DNA replication. This is a possible reason for the severe phenotype of double-knockout mice. These observations indicate that adverse health effects caused by MUTYH deficiency appear when 8-oxo-G:A mismatch, the repair target of MUTYH, is generated beyond a certain critical level. The risk of colon cancer in patients with MAP increases with age, rapidly elevating after age 50 (Lubbe et al. 2009). An age-dependent increase in oxidative stress could be the cause of this. Indeed, MUTYH-deficient human cells showed high MF when exposed to KBrO3 (Ruggieri et al. 2013; Grasso et al. 2014). Additionally, MFs in the normal intestinal tissues of patients with MAP are higher compared with those in healthy controls (Robinson et al. 2022). Minimizing oxidative stress from a younger age may help in reducing the induction of 8-oxoG and the subsequent formation of 8-oxo-G:A-induced somatic mutations, thereby potentially lowering the risk of tumorigenesis in MAP.

Currently, genotoxic carcinogens are regulated assuming no threshold for their action (Nohmi 2018). We attempted to determine whether this is the case for KBrO3-induced oxidative stress. Our results suggest that a practical threshold for mutagenicity and tumorigenicity existed at doses of 0.05%–0.1% KBrO3 regardless of mice genotype. This is consistent with a previous report showing a practical threshold of mutagenicity in gpt delta mice of 0.02%–0.06% (Aoki et al. 2020). Although MUTYH plays a significant role in the molecular mechanisms underlying the threshold for KBrO3-induced mutagenesis and tumorigenesis in wild-type mice, although it not the only factor involved.

G:C > T:A frequency was affected by the surrounding sequence context. The mutation pattern from rpsL and WES indicated that the 5′ G at GG and GAA was frequently mutated. Similar sequence contexts were also preferred in G:C > T:A mutations in the mutY− Escherichia coli mutator strain (Tajiri et al. 1995; Foster et al. 2015) or wild-type E. coli strain harboring supF plasmid (Watanabe et al. 2001). Molecular mechanisms underlying the sequence context tendency of G:C > T:A mutation are common among bacteria, mice, and humans. G:C > T:A mutations tended to occur at the same hot spots as in rpsL even in KBrO3-treated wild-type mice; therefore, these mutations may not be because of a MUTYH defect. OGG1 repair activity of 8-oxoG in DNA is affected by the surrounding sequence or presence of DNA lesions (Sassa et al. 2012); however, the same mutation pattern was observed in mice lacking both OGG1 and MUTYH (Ohno et al. 2014), possibly because of a physicochemical feature of 8-oxoG in DNA, at least in part. At the 5′ guanine of GG, GA showed lower ionization potential (Sugiyama and Saito 1996), thereby forming preferential oxidation sites in the genome (Fleming and Burrows 2021).

We observed striking similarities between mutation patterns of Mutyh−/− tumors and germline mutation detected in TOY mice that lack Nudt1, Ogg1, and Mutyh (Ohno et al. 2014); COSMIC signature 18 and SBS36; and human MAP tumors (Pilati et al. 2017; Viel et al. 2017). Our results provided direct experimental evidence for these signatures being caused by 8-oxoG-induced mutation and for a commonality of MUTYH repair function in mice and humans. SBS45 and SBS4 showed a higher cosine similarity (r > 0.8) (Supplemental Table S4) against the mutation pattern of Mutyh−/− mice tumor but not of TOY mice. Thus, SBS45 and SBS4 might be related to KBrO3 administration.

We considered the sequence context of G:C > T:A mutations may elicit some specific amino acid changes or nonsense mutations, possibly resulting in more adverse outcomes, particularly when they occur in particular cancer-related genes. According to a human cancer genome study, the ratio of nonsynonymous to synonymous mutations was approximately 1:1 (Hu et al. 2020). However, nonsynonymous mutations were more frequently observed than synonymous mutations in Mutyh−/− tumors. Among detected SVNs, 63%, 8.6%, and 28.3% were nonsynonymous, nonsense mutations, and synonymous mutations, respectively. Thus, high-impact mutations for tumor initiation are more likely to be induced by oxidative stress causing defective cell repair for 8-oxoG-mediated mutagenesis. Interestingly, the most frequently observed amino acid change was E > stop, which was often found in Apc/APC or KRAS in the tumor genome of Mutyh−/− mice or MAP (Venesio et al. 2012; Isoda et al. 2014; Viel et al. 2017). Previously, we showed that 87% of tumors derived from KBrO3-treated Mutyh−/− mice had nonsense mutations in Apc or missense mutations in Ctnnb1 and that catenin beta 1 (also known as beta-catenin) was accumulated in tumor cell nuclei (Isoda et al. 2014). Herein, three of four tumors had G:C > T:A mutations in specific codons of exon 3 of Ctnnb1, which are well-known driver mutations of human colorectal cancer. These mutations change critical amino acids and abrogate GSK3B phosphorylation sites, resulting in constitutively activated growth signaling by altering the Wnt signaling pathway (Krausova and Korinek 2014; Gao et al. 2018). These observations could explain why MUTYH-defective humans and mice predominantly developed intestinal tumors.

We also showed that chronic exposure to oxidative stress significantly induced intestinal tumors in wild-type mice. When the amount of repair target was exceeded or MUTYH repair ability was inhibited, MF and tumor incidence may increase even in individuals with no MUTYH mutations.

In conclusion, we showed that oxidative stress increases the risk of intestinal tumor development by promoting somatic mutation generation via 8-oxoG-mediated mutagenesis, particularly under MUTYH-deficient conditions. However, further studies are needed to investigate how common these observations are in other types of cancer.

Methods

Mice and KBrO3 administration

The Mutyh gene knockout mouse line was previously established (Sakamoto et al. 2007) and maintained by backcrossing with C57BL/6Jc, purchased from CLEA Japan. Mutyh heterozygous female and male mice were mated to generate congenic Mutyh+/+, Mutyh+/−, and Mutyh−/− mice for the tumor experiments. The rpsL transgenic (Tg) mice (Gondo et al. 1996; Egashira et al. 2002) were used for the somatic mutation assay. Mutyh+/+/rpsL-Tg and Mutyh−/−/rpsL-Tg mice were obtained by mating Mutyh+/−/rpsL-Tg female and male mice. For tumor analysis, mice were fed regular water (reverse osmosis water) or KBrO3-containing water (0.05%, 0.1%, 0.15%, and 0.2%) from 4 wk of age and until 16 wk. For mutation analysis, mice were given regular water for 2 wk after 4 wk KBrO3 administration (Fig. 1A). KBrO3 (Merck) was dissolved in distilled water at appropriate concentrations (g/vol) and stirred for 30 min. Freshly prepared KBrO3 solution in a bottle was set on a cage once a week. All mice were maintained in a specific-pathogen-free facility at controlled temperature and humidity (20°C–26°C, 60%). Animal care and procedures were approved by the institutional animal care and use committee of Kyushu University (approval nos. A22-027, A20-089, A30-147, A28-112). All experiments were performed according to the guidelines for proper conduct of animal experiments of the Science Council of Japan.

Pathological analysis

Pathological samples of full-length small intestinal tissue were prepared using our in-house stretch-and-roll (StR) protocol (for details, see Supplemental Methods). Briefly, the resected small intestine was cleansed to remove contents and then placed in a long plastic tube filled with 10% formalin to straighten it. After 16 h of fixation, the specimen was incised longitudinally, rolled up from one side, and embedded in a paraffin block. These samples were sectioned into 4-µm-thick slices and stained with hematoxylin and eosin (HE). The tumor frequency was evaluated using the flattened specimens fixed in this manner, observed under a stereomicroscope. Tumors with a diameter >1 mm were counted.

Mutation assay by rpsL-Tg mice

Tissue specimens resected from rpsL-Tg mice were immediately frozen in liquid nitrogen and stored in a −80°C deep freezer until genomic DNA extraction. The rpsL mutation assay was performed following methods described previously (Egashira et al. 2002; Isoda et al. 2014), with some modifications (for details, see Supplemental Methods).

WES and detection of somatic mutations

Intestinal tumors and heart tissues isolated from two KBrO3-treated Mutyh−/− mice (two tumors and one heart from each mouse) were subjected to WES. Genomic DNA was extracted from the tissue samples using the QIAamp fast DNA tissue kit (Qiagen). The target exon regions (46.9 Mb) were captured using the SureSelectXT mouse all exon kit (Agilent Technologies). Samples were sequenced on the HiSeq 2000 platform (Illumina) with 100-bp paired ends or on the NovaSeq 6000 platform (Illumina) with 150-bp paired ends. Sequenced reads were mapped to the mouse reference genome (UCSC mouse GRCm38/mm10, GRCm39/mm39) using BWA-MEM ver.0.7.17 (Li 2013). After applying base quality score recalibration (Lange et al. 2020), somatic mutations were detected using GATK Mutect2 ver.4.2.0.0 (McKenna et al. 2010) in the tumor-normal mode using the heart as the normal reference. Following FilterMutectCalls, an additional filter (total reads 10 or more for normal and tumor, variant reads three or more for tumor, zero for normal) was applied (Lange et al. 2020). Mutations located within Chr M or in the dbSNP database were excluded. Mutations shared by two or more tumors were also excluded. The called variants, along with their filtering information, are detailed in Supplemental Table S3, and VCF files are in Supplemental Files. The prediction of the genetic effects of called variants, including site information, gene annotation, codon change, and amino acid change, was performed using SnpEff ver.5.1 (Cingolani et al. 2012). The scripts used in the analysis are available in Supplemental Code.

Mutation pattern analysis

The mutation pattern based on 96 trinucleotides type was analyzed using MutationalPatterns ver.3.8.1 (Fleming and Burrows 2023) and Sigprofiler MatirixGenerator ver.1.2 (Islam et al. 2022). The lists of somatic mutations in the human MAP tumor were adapted from Pilati et al. (2017). The list of germline mutations identified from the TOY mice line was adapted from our previous report (Ohno et al. 2014). Mutational signature data (mm10, Mus musculus, version 3.3 and v2.0) were downloaded from COSMIC website (https://cancer.sanger.ac.uk/signatures/downloads/). The mutation pattern of tumor WES, human MAP tumor, and rpsL assay data were normalized to the mouse genomic trinucleotide frequency using SigsPack ver.12.0 (Schumann et al. 2019).

Statistical analysis

Statistical analysis and data plotting were performed using JMP Pro 15 (SAS Institute) and Microsoft Excel. Mean tumor number, namely, tumor frequency, was calculated as the number of tumors detected in each experimental group divided by the number of mice used. The correlation between tumor frequency or MF and KBrO3 dose was determined using the Spearman's rank correlation test (Supplemental Table S1). Tumor frequency, total MF, and by-type MF in each mouse were used. For tumor prevalence, the ratio of the number of tumor-bearing mice to the total number of mice in each experimental group was used. To test differences in MFs in the control and KBrO3-treated groups in Mutyh+/+ and Mutyh−/− mice, the Steel's test was performed using total MF or by-type MF (Fig. 3A–G).

Data access

All raw and processed sequencing data generated in this study have been submitted to the NCBI BioProject database (https://www.ncbi.nlm.nih.gov/bioproject/) under accession number PRJDB15815.

Supplementary Material

Supplement 1
Supplemental_Code.zip (6.4KB, zip)
Supplement 2
Supplement 3
Supplemental_Method.pdf (821.7KB, pdf)
Supplement 4
Supplement 5
Supplement 6
Supplemental_Table_S4.xlsx (146.4KB, xlsx)
Supplement 7
Supplemental_Table_S5.xlsx (220.3KB, xlsx)

Acknowledgments

This work was partly supported by Grant-in-Aid for Scientific Research (Japan Society for the Promotion of Science) grant numbers JP25241012, JP16H05109, JP20012037, JP26281022, JP22K11733, and JP20H03254. We thank Kosuke Teshima and Yuichiro Hara for their variable advice for sequence data analysis. We thank Kuniko Ishihara for technical support. Computations were partially performed on the National Institute of Genetics supercomputer at the Research Organization of Information and Systems, National Institute of Genetics.

Author contributions: M.O., Yo.N., and T.T. designed the research. Yu.N. provided mice strain information. M.O., N.T., F.S., K.Y., and K.H. performed the experiments and data analyses. M.O. wrote the paper. Y.A. and T.N. provided critical suggestions during manuscript preparation. All authors read and approved the final manuscript.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.278326.123.

Competing interest statement

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1
Supplemental_Code.zip (6.4KB, zip)
Supplement 2
Supplemental_Materials.pdf (1.7MB, pdf)
Supplement 3
Supplemental_Method.pdf (821.7KB, pdf)
Supplement 4
Supplemental_Table_S1_S2.xlsx (15.9KB, xlsx)
Supplement 5
Supplemental_Table_S3.xlsx (18.5MB, xlsx)
Supplement 6
Supplemental_Table_S4.xlsx (146.4KB, xlsx)
Supplement 7
Supplemental_Table_S5.xlsx (220.3KB, xlsx)

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