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o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea, which are mutagenic but not carcinogenic in the colon, rapidly induce colonic tumors in mice with dextran sulfate sodium-induced colitis

o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea, which are mutagenic... Background: Several rodent models with chemically induced colon cancer have been developed. Among these models, dextran sulfate sodium (DSS), a colitis inducer, combined with azoxymethane as a colon mutagenic carcinogen, is commonly used. We previously reported that although benzo [a] pyrene (BP) is mutagenic but not carcinogenic in the colon, it rapidly develops colon tumors at a high incidence/multiplicity after treatment with DSS. In the present study, we examined whether other colon-mutagenic non-carcinogens (CMNCs) induced colon tumors after treatment with DSS. Results: o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea were selected as CMNCs. Male CD2F1 mice were orally administered CMNC for 5 consecutive days. After a 9-day dose-free period, mice were treated with 4% DSS in drinking water for 1 week. Three months after DSS treatment, colon samples were collected for histopathology and β-catenin immunohistochemistry analyses. All CMNCs in combination with DSS induced colonic adenocarcinomas at a high incidence/multiplicity in the distal and middle parts of the colon, coinciding with the location of colitis. Unlike in normal cells where β-catenin is exclusively located on the cell membrane, in adenocarcinoma cells, it was translocated to both the nucleus and cytoplasm or only to cytoplasm. The translocation of β-catenin is closely associated with colon carcinogenesis in rodents and humans. No colonic tumors or dysplastic lesions were found after exposure to either CMNC or DSS alone. Conclusion: We provided further evidence clearly showing that CMNCs can rapidly induce colonic tumors in mice with DSS-induced colitis, even if they are not colonic carcinogens. Keywords: Colon, Cancer, Dextran sulfate sodium, Inflammation, o-Aminoazotoluene, 7,12- Dimethylbenz[a]anthracene, N-Ethyl-N-nitrosourea, Mutagenic non-carcinogen * Correspondence: a-hakura@hhc.eisai.co.jp Global Drug Safety, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hakura et al. Genes and Environment (2022) 44:11 Page 2 of 10 Introduction thymus in mice [13, 29]; murine tumors were generated in Colorectal cancer is one of the most common human the vascular and nervous systems, skin, and malignant cancers in the Western world [1]. Rodent colon cancer lymphoma tissues [30, 31]. N-Ethyl-N-nitrosourea (ENU) models have been developed to understand the mecha- induces mutations in numerous organs of mice, such as nisms underlying colon carcinogenesis and to investigate the colon, small intestine, bone marrow, mammary gland, potential chemotherapy or chemoprevention regimens. and liver [13, 32, 33]. Multiple tissues are known to be the These models include carcinogen-induced, genetically site of tumor induction in mice, including the liver, Hard- modified, and transplant models [1–3]. Carcinogen- erian glands, stomach, ovaries, lymphoreticular system, induced models are highly reproducible and can be read- kidneys, mammary gland, uterus, nervous system, and ily tested on non-genetically modified animals with dif- lungs [34, 35]. For these three chemicals, the colon is not ferent genetic backgrounds. In addition, the processes recognized as a target organ for murine carcinogenesis. involved in the pathogenesis recapitulate human colon In this study, we showed that all these three CMNCs cancer, particularly, the early stages of this disease [4–6]. induced colonic cancer in the presence of colitis. In Among carcinogen-induced models, some models addition, we performed the immunohistochemical ana- using a colon mutagenic carcinogen in combination with lysis of tumors with β-catenin, given that the nuclear ac- dextran sulfate sodium (DSS), a colitis inducer, are often cumulation of β-catenin is well documented to be used [4]. Azoxymethane (AOM) [4, 7], 1,2-dimethylhy- closely associated with colon carcinogenesis in rodents drazine (DMH) [8, 9] and 2-amino-1-methyl-6-phenyli- and humans [4–6]. midazo[4,5-b]pyridine (PhIP) [10–12] are used as colon mutagenic carcinogens (referred to as CMCs) to initiate Materials and methods carcinogenesis. These CMCs can induce colonic cancer Chemicals by themselves [13, 14]; however, colonic tumors are in- o-Aminoazotoluene (AAT, CAS No. 97–56-3, purity > duced at varying incidences (0–100%), and it takes 3 97.0%) was purchased from Tokyo Chemical Industry months or longer to induce cancer, depending on the Co., Ltd. (Tokyo, Japan). 7,12-Dimethylbenz[a]anthra- species/strains or dosing regimens [15, 16]. For these cene (DMBA, CAS No. 57–97-6, purity > 95%) and N- reasons, DSS is used, in combination, to cause colitis in ethyl-N-nitrosourea (ENU, CAS No. 759–73-9, content: the colon (well documented as a DSS-induced colitis 43.2% in water with 1.7% acetic acid to prevent decom- model), thereby accelerating the development and pro- position) were obtained from Sigma-Aldrich Co. LLC gression of colonic tumors. DSS is not mutagenic in bac- (St. Louis, MO, USA). Dextran sulfate sodium (DSS, teria [17] and in murine mutagenicity tests [18] but is CAS No. 9011-18-1, molecular weight: 36,000–50,000) weakly carcinogenic in the rodent colon [19, 20], indicat- was purchased from MP Biochemicals, LLC (Aurora, ing that DSS potently promotes colonic carcinogenesis. OH, USA). However, even in such “short-term” colon cancer For administration, ENU was dissolved in water for in- models, the induction of cancer generally takes approxi- jection at a concentration of 1.1 mg/mL, and AAT and mately 10–20 weeks [4–6]. DMBA were dissolved in salad oil (Nisshin Oillio Group, We previously showed that benzo[a]pyrene (BP), a co- Ltd., Tokyo, Japan) at concentrations of 12.5 and 2.5 lonic mutagen [21, 22], induced colonic cancer at a high mg/mL, respectively. DSS was dissolved in water at a incidence/multiplicity in a shorter or comparable period, concentration of 40 mg/mL (4%). required for cancer induction in models using CMC and DSS after the treatment of mice with DSS, despite it be- ing a non-colonic carcinogen [23–25]. There is no evi- Animals dence showing that CMNCs (colon-mutagenic non- Male Crj: CD2F1 (BALB/c × DBA/2) mice were obtained carcinogens), except for BP, clearly induce the formation from Charles River Japan, Inc., Tokyo. All mice were of colonic tumors at a high multiplicity/incidence in the housed in metal cages (one mouse per cage) and were DSS-induced colitis model. The purpose of the present fed a basal diet (Oriental CRF-1, Oriental Yeast Co., study was to examine whether other CMNCs also act as Tokyo, Japan) and tap water ad libitum, under controlled initiators for carcinogenesis in a DSS-induced colitis conditions of temperature (23 ± 3 °C), humidity (55 ± model. 20%), and light (12-h light/12-h dark cycle). They were o-Aminoazotoluene (AAT) induces mutations in the quarantined and acclimatized for 1 week. The animals colon, liver, kidney, and urinary bladder [13, 26, 27]; only were assigned by stratified randomization to the two hepatocellular adenoma/carcinoma and hemangioen- groups according to their body weights; mice from both dothelioma in the lung have been generated in mice [14, the DSS- and CMNC/DSS-groups and both the non- 28]. 7,12-Dimethylbenz[a] anthracene (DMBA) induces treatment and CMNC groups were assigned to the heavy mutations in the colon, bone marrow, liver, skin, and and light groups, respectively, to minimize the mortality Hakura et al. Genes and Environment (2022) 44:11 Page 3 of 10 of light mice, given that they are likely to be more sensi- Tissue collection and histopathology tive to DSS-induced colitis than heavy mice. During necropsy, the large intestine was immediately excised, flushed with saline, infused with 10% neutral buffered formalin, cut open longitudinally along the anti- Experimental procedures mesenteric border, and grossly observed. Thereafter, the Figure 1 shows the outline of the protocol for the experi- tissues were stored in 10% neutral buffered formalin, cut ment. The number of mice, established upon initiation into four parts of equal length from the proximal to dis- of the experiment, were as follows: eight for the non- tal ends, processed, and embedded in paraffin. Each treatment group, six for each CMNC group, 16 (eight colon sample was embedded to expose both longitudin- for water for injection and eight for salad oil, vehicles ally cleaved edges. All longitudinal sections were stained with which CMNCs were dissolved) for the DSS group, with hematoxylin and eosin (H&E) and histopathologic- and eight for each CMNC/DSS-group. Three of 16 mice ally observed. The lesions of dysplasia, adenoma, and in the DSS-group and one of seven mice in the DMBA/ adenocarcinoma were classified according to the criteria DSS-group died 4 or 5 days after the last DSS treatment, that were described in detail in our previous report [24], due to severe colitis, resulting in 13 surviving mice in which were originally reported by Riddell et al. [36], Pas- the DSS group and 6 surviving mice in the DMBA/DSS- cal [37] and Ward [38]. Dysplasia or dysplastic foci were group. The numbers of surviving mice per group are characterized by irregular branching, distorted architec- shown in parentheses in Fig. 1 and Table 1. ture with cellular and nuclear pleomorphism, nuclear For each CMNC/DSS-group, mice (7-week old) were enlargement and hyperchromatism, and paucity of gob- orally (by gavage) treated with each CMNC for 5 con- let cells. secutive days, and starting 10 days after the last dose, mice were administered 4% DSS in drinking water for 7 Immunohistochemistry days. Twelve weeks later, the mice were necropsied Paraffin-embedded sections of mouse colons with under anesthesia to collect the colorectum from the adenocarcinomas from each CMNC/DSS group were cecocolic junction to anus for histopathology and subjected to immunohistochemical staining of β-catenin. immunohistochemistry. Immunohistochemical staining was performed according The in-life phase of the experiment was performed at to the procedure described in our previous report [24]. Sunplanet Co., Ltd. of the Eisai Co., Ltd. group, and the Monoclonal mouse anti-mouse β-catenin (clone 14, BD protocol was approved by the Institutional Animal Care Transduction Laboratories, Lexington, KY, USA) was and Use Committee and carried out according to the used at a concentration of 1/1000. After antigen re- Sunplanet animal experimentation regulations. trieval, the Envision™ + Dual Link System or a Fig. 1 Outline of the protocol for the experiment for histopathology and immunohistochemistry of the murine colon. The experiment consisted of Non-treatment-, CMNC-, DSS-, and CMNC/DSS-groups. Male CD2F mice were treated with each CMNC at an oral dose of either 125 (AAT), 25 (DMBA), or 11 (ENU) mg/kg/day for 5 consecutive days, and starting 10 days after the last dose, mice were given 4% DSS in drinking water for 7 days. All surviving mice were necropsied 12 weeks after the last treatment with DSS. The number of surviving animals is shown in parentheses. Salad oil was used as the vehicle for AAT and DMBA, and water for injection was used as the vehicle for ENU Hakura et al. Genes and Environment (2022) 44:11 Page 4 of 10 Table 1 Incidence of colonic dysplastic foci and tumors induced by a CMNC in combination with DSS Treatment Dysplasia Adenoma Adenocarcinoma Tumor Non-treatment 0% (0/8) 0% (0/8) 0% (0/8) 0% (0/8) Vehicle + 4% DSS 0% (0/13) 0% (0/13) 0% (0/13) 0% (0/13) AAT (125 mg/kg/day) 0% (0/6) 0% (0/6) 0% (0/6) 0% (0/6) AAT (125 mg/kg/day) + 4% DSS 75% (6/8)*** 100% (8/8)*** 100% (8/8)*** 100% (8/8)*** DMBA (25 mg/kg/day) 0% (0/5) 0% (0/5) 0% (0/5) 0% (0/5) DMBA (25 mg/kg/day) + 4% DSS 50% (3/6)** 50% (3/6)** 50% (3/6)** 83% (5/6)*** ENU (11 mg/kg/day) 0% (0/6) 0% (0/6) 0% (0/6) 0% (0/6) ENU (11 mg/kg/day) + 4% DSS 100% (7/7)*** 100% (7/7)*** 100% (7/7)*** 100% (7/7)*** The numbers in parentheses indicate the number of mice with dysplasias, adenomas, or adenocarcinomas per the number of mice CMNC; colon-mutagenic non-carcinogen : adenoma + adenocarcinoma : water for injection for 7 mice and salad oil for 6 mice **, *** ; significantly different from the DSS alone group at P < 0.05, 0.01 in Fisher’s exact probability test streptavidin biotin-peroxidase complex method (DAKO, parts of the colon. Neoplasms were nodular, papillary, or Glostrup, Denmark) was used to examine their expression polypoid masses composed of tubular and papillary prolif- and localization. These sections were counterstained with eration of epithelium that protruded into the intestinal Mayer’s hematoxylin solution for microscopic examination. lumen, extended into the lamina propria, and compressed the adjacent mucosa. Tumors induced by these three Dose setting for treatment with CMNCs CMNC/DSS were tubular adenomas or well- to moder- The doses of AAT (125 mg/kg/day), DMBA (25 mg/kg/ ately differentiated adenocarcinomas. No submucosal in- day), and ENU (11 mg/kg/day) tested in this study were de- vasion or metastasis were observed. Pre-neoplastic and termined based on our preliminary study (data not shown) neoplastic lesions were not observed in the colon in any or the studies to measure the mutant frequency in mice; CMNC-, DSS-, or non-treatment-group. for AAT, a single i.p. dose at 300 mg/kg, corresponding to In the CMNC/DSS- and DSS-groups, the large intes- 40% of LD (median lethal dose) [26, 27], for DMBA, a tine was shorter in length, and the intestinal wall showed single i.p. dose at 20 mg/kg [29]and forENU, five i.p. irregular thickening. Histopathologically, colitis with or doses at 22.2 mg/kg/day (once a week for 5 weeks) [39]. without erosion or ulceration was noted in the distal and middle parts of the colon. In animals with colitis, there Statistical analysis was occasional accumulation of foamy macrophages in The incidences and multiplicities were compared using the lamina propria. Fisher’s exact probability test and Welch’s t-test for paired samples. Incidence and multiplicity of dysplastic foci, adenomas, and adenocarcinomas Results Table 1 shows the incidences of dysplastic foci, aden- Clinical findings omas, and adenocarcinomas observed in the colon, In DSS-treated mice, with or without CMNC, bloody and Table 2 shows their multiplicity (= the number and/or soft stools were observed for 1 or 2 days before of tumors/mouse). All CMNCs rapidly produced neo- the last DSS treatment. Stool changes resolved by 5 days plastic lesions after combined treatment with DSS after DSS treatment cessation and were attributed to under the experimental conditions employed in this colitis. In the CMNC/DSS groups, three mice that devel- study. The combined treatments of ENU (11 mg/kg/ oped tumors showed bloody stools from 9 weeks after day)/4% DSS and AAT (125 mg/kg/day)/4% DSS the last DSS treatment. showed tumorigenicity in 100% mice, and the multi- plicities are 8.6 ± 1.1 and 7.9 ± 2.0, respectively. The Gross findings and histopathology combination of DMBA (25 mg/kg/day)/4% DSS in- The gross findings are shown in Fig. 2, and histopatho- duced the formation of tumors in 83% of mice with a logical findings are shown in Figs. 3, 4 and 5. In the sam- multiplicity of 3.0 ± 1.9. ples from the mice in each CMNC/DSS-group, there were multiple masses in the middle to distal parts of the colon Immunohistochemistry of β-catenin at necropsy (12 weeks after completion of DSS treatment). The expression of the β-catenin protein in adenocarcin- Histopathological examination revealed dysplastic foci, ad- omas is shown in Fig. 5. In adenocarcinoma cells, whose enomas, and adenocarcinomas in the distal and middle formation was induced by AAT/DSS, DMBA/DSS, or Hakura et al. Genes and Environment (2022) 44:11 Page 5 of 10 Fig. 2 Macroscopic view of the colons of mice from Non-treatment- (A), DSS- (B), AAT- (C), AAT/DSS- (D), DMBA- (E), DMBA/DSS- (F), ENU- (G) and ENU/DSS- (H) groups. Bar: 1 cm Fig. 3 Histopathology of the colons of mice from Non-treatment- (A), DSS- (B), AAT- (C), AAT/DSS- (D), DMBA- (E), DMBA/DSS- (F), ENU- (G) and ENU/DSS- (H) groups. H& E stain Hakura et al. Genes and Environment (2022) 44:11 Page 6 of 10 Table 2 Multiplicity of colonic dysplastic foci and tumors induced by a CMNC in combination with DSS Treatment Dysplasia Adenoma Adenocarcinoma Tumor Non-treatment 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Vehicle +4% DSS 0± 0 0 ±0 0± 0 0 ±0 AAT (125 mg/kg/day) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 AAT (125 mg/kg/day) + 4% DSS 0.9 ± 0.6*** 3.8 ± 1.2*** 4.1 ± 1.7*** 7.9 ± 2.0*** DMBA (25 mg/kg/day) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 DMBA (25 mg/kg/day) + 4% DSS 1.0 ± 1.1** 1.5 ± 2.0* 1.5 ± 1.6** 3.0 ± 1.9*** ENU (11 mg/kg/day) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 ENU (11 mg/kg/day) + 4% DSS 1.6 ± 0.5*** 4.9 ± 1.9*** 3.7 ± 1.5*** 8.6 ± 1.1*** Multiplicity indicates the number of dysplasias, adenomas, or adenocarcinomas per mouse, mean ± standard deviation CMNC; colon-mutagenic non-carcinogen : adenoma + adenocarcinoma : water for injection for 7 mice and salad oil for 6 mice *, **, ***; significantly different from the DSS alone group at P < 0.1, 0.05, 0.01 in the Welch’s t-test ENU/DSS, β-catenin was translocated predominantly to tubular adenomas or well- to moderately differentiated both the nucleus and cytoplasm or only cytoplasm from adenocarcinomas. These tumors predominantly ap- cell membrane, where it is exclusively expressed in nor- peared in the middle and distal parts of the colon, co- mal colon epithelial cells. inciding with the location of DSS-induced colitis. These findings were consistent with those reported for DSS in combination with BP or CMCs such as AOM, Discussion DMH, and PhIP [4, 7–12]. In adenocarcinoma cells, We previously reported that in mice administered BP, a whose formation was induced by CMC in combination CMNC, the formation of colon tumors was rapidly in- with or without DSS, or BP plus DSS, β-catenin was duced after DSS treatment [23, 24]. In the present study, expressed in both the nucleus and cytoplasm or cyto- we clearly showed that three more CMNCs (AAT, plasm [4, 7, 8, 10, 24]. Such translocation and accumu- DMBA, and ENU) can rapidly induce the formation of lation of β-catenin in the nucleus from cell membrane colon tumors at a high multiplicity/incidence. has been shown to be closely associated with the de- All tumors whose formation was induced by the velopment or progression of colon tumors through three CMNC/DSS were histologically diagnosed as Fig. 4 Histopathology results of H&E staining of colonic dysplasia (A, B, E, F, I, and J) and adenoma (C, D, G, H, K, and L) induced by AAT/DSS, DMBA/DSS or ENU/DSS. Figs. B, D, F, H, J, and L are enlarged magnifications of those in A, C, E, G, I, and K, respectively (the areas surrounded by the yellow frames). Yellow bar: 100 μm Hakura et al. Genes and Environment (2022) 44:11 Page 7 of 10 mutations in the β-catenin or APC genes or activation In rodent chemical-induced colon cancer models, CMCs, of the Wnt/β-catenin signaling pathway [4, 40–43]. particularly AOM, are commonly used in combination with MeIQx, a heterocyclic amine found in well-cooked DSS. Our present and previous studies [23, 24]provide evi- meat, is mutagenic but not carcinogenic in the colon of dence showing that CMNCs can also induce colonic tumors mice [44]. One study reported that MeIQx is carcinogenic after posttreatment with DSS, and that colonic mutagens in the mouse colon after DSS treatment [12]. However, can induce colonic tumors in the presence of DSS-induced the carcinogenicity was weak in that study (for adenomas colitis whether they are colonic carcinogens or not. To in- plus adenocarcinomas, the incidence was 22% and multi- duce colonic cancer, CMNCs may play a key role in the in- plicity was 0.30 ± 0.61), and another study reported duction of gene mutations responsible for tumorigenesis via that MeIQx was not carcinogenic after DSS treatment their characteristic mutational spectrum. [18]. Thus, the effect of DSS treatment on MeIQx- Figure 6 shows our hypothesis for possible mecha- induced carcinogenicity is not clear and may be mar- nisms of the induction of colonic tumors in the mouse ginal. Another heterocyclic amine, IQ, was carcino- colitis-associated model in combination with a colonic genic at a low incidence/multiplicity (for adenomas mutagen/carcinogen. Colon epithelial cells are mutated plus adenocarcinomas, the incidence was 14% and by colonic mutagens, resulting in the generation of mu- multiplicity was 0.34 ± 0.72) in mice after DSS treat- tated epithelial cells (probably stem cells or progenitor ment [12], although it is not reported to be a colonic cells, because cell turnover of colon epithelial cells is carcinogen in itself [44]. IQ has been reported to very fast, i.e., 2–3 days [32, 33]). Mutated epithelial cells show mutagenicity in the cecum, but not in the develop tumors via non-genetic effects when mice are colon, at 20 mg/kg for 5 days [45]. In that study, only treated with DSS, a colitis inducer and a potent pro- one dose was tested. moter of carcinogenesis. In the absence of DSS Fig. 5 Histopathology results of H&E staining (A, B, D, E, G and H) and β-catenin-immunostaining (C, F, and I) of colonic adenocarcinomas induced by AAT/DSS, DMBA/DSS or ENU/DSS. Figs. B, E and H are enlarged magnifications of those in A, D and G, respectively (the areas surrounded by the yellow frames). Immunohistochemical analysis of β-catenin was performed on serial sections used for H & E staining. The inserts are figures in enlarged magnification. Figs. J and K show the results of H & E staining and β-catenin-immunostaining, respectively, of normal colonic epithelial cells. Yellow bar: 100 μm Hakura et al. Genes and Environment (2022) 44:11 Page 8 of 10 treatment, mutated epithelial cells do not develop tu- related differences in the DNA repair system, may de- mors. Non-genetic effects include cell regeneration ac- crease the number of cells initiated by ENU [51, 52]. Min/+ companied by cell necrosis and inflammation in Colon tumors are induced when adult APC mice response to cell injury or microenvironment disruption, are treated with ENU [52]. This finding supports a canon- and thereby alterations in signal transduction [40–42, ical mechanism of colon tumorigenesis: the induction of 46] or epigenetics (DNA methylation or histone modifi- mutations in the dominant tumor-suppressor gene. In cation) [47, 48]. These effects are affected by intestinal contrast, our study showed that DSS post-treatment pro- bacteria [49, 50]. Many studies have shown that CMCs vides the same output of tumor induction as mutation in- rapidly induce colon cancer after treatment with DSS, duction in tumor-related genes in tumorigenesis. and that colitis (inflammation) caused by DSS contrib- Since the present study with the three colonic muta- utes to the rapid progression of colon carcinogenesis [4– gens was performed under the same experimental condi- 6]. Therefore, inflammation may be a requisite factor for tions, the potency of their carcinogenicity in this model colon carcinogenesis and one of the discriminating fac- may be correlated and can be compared. The most tors of colon carcinogens from CMNCs. However, fur- powerful initiation activity was estimated to be that of ther studies are required for verification. ENU (0.33), followed by that of DMBA (0.060) and AAT The colon becomes the site of tumor induction when (0.033), by calculating the multiplicity of adenocarcin- Min/+ juvenile APC mice (carrying a mutation in the Apc oma divided by the dose (mg/kg/day). gene to develop multiple intestinal adenomas) or even The purpose of the present study was to provide evi- +/+ APC mice (wild-type at the Apc locus) are intraperito- dence on the hypothesis that CMNCs act as initiators of neally treated with ENU, although the ENU does not in- carcinogenesis in a DSS-induced colitis model. Our duce colonic tumors when adult mice, generally used in study may help better understand the effects of environ- toxicity studies, are orally treated [34, 35, 51]. In both mental mutagens on inflammation-related cancer. In- strains of mice, the incidence of intestinal tumors age- creasing attention has been paid to the involvement of relatedly reduces during the periods from 5 to 14 days to inflammation in the initiation, promotion, and progres- 30–35 days of age at ENU treatment, with tumors being sion of tumors [53–56]. Patients with inflammatory induced at a very low incidence at 30–35 days of age bowel disease (IBD) are at a higher risk of developing [51]. The microenvironment of intestinal crypts dramat- colorectal cancer. The cumulative incidence of colorectal ically changes from infancy to early juvenile; when mice cancer in patients with IBD ranges from 7.6 to 18.4%, at are born, crypts are predominantly polyclonal, but 30 years post-diagnosis [5, 57]. A review paper by Rawla around two to 3 weeks of age, they become monoclonal. et al. [58] reported that patients with chronic IBD have a This phenomenon, known as crypt purification or age- two-fold higher risk of developing colorectal cancer, and Fig. 6 Hypothesis for possible mechanisms underlying the induction of colonic tumor formation after combined treatment with colonic mutagens and DSS. Colon epithelial cells are mutated by colonic mutagens, probably in stem or progenitor cells. Mutated epithelial cells develop tumors via non-genotoxic effects when mice are treated with DSS. When DSS is not treated, mutated epithelial cells do not develop tumors. Non-genotoxic effects of DSS include enhanced cell proliferation and inflammation in response to cell injury or microenvironment disruption, and thereby alterations in signal transduction or epigenetics. These effects are affected by intestinal bacteria. Colonic mutagens are not necessarily colonic carcinogens Hakura et al. Genes and Environment (2022) 44:11 Page 9 of 10 that ulcerative colitis increases the risk of colorectal can- 2. Jackstastadt R, Sansom OJ. Mouse models of intestinal cancer. J Pathol. 2016;238(2):141–51. https://doi.org/10.1002/path.4645. cer by 2.4 times. Our study suggests that colonic muta- 3. Nascimento-Gonçalves E, Mendes BAL, Silva-Reis R, Faustino-Rocha AI, Gama gens impose an increased risk of colon cancer in A, Oliveira PA. Animal models of colorectal cancer: From spontaneous to patients with IBD compared with healthy individuals. genetically engineered models and their applications. Vet Sci. 2021;8(4):59. https://doi.org/10.3390/vetsci8040059. 4. Rosenberg D, Giardina C, Tanaka T. Mouse models for the study of colon Conclusion carcinogenesis. Carcinogenesis. 2009;30(2):183–96. https://doi.org/10.1053/j. In our previous study, BP of a CMNC rapidly induced gastro.2008.12.049. 5. Tanaka T. Colorectal carcinogenesis: review of human and experimental the formation of colonic tumors in mice after DSS treat- animal studies. J Carcinog. 2009;8(5):1–19. https://doi.org/10.4103/1477-31 ment. In the present study, we clearly showed further 63.49014. evidence that the other three CMNCs (AAT, DMBA, 6. Tanaka T. Development of an inflammation-associated colorectal cancer model and its application for research on carcinogenesis and and ENU) also rapidly induced the formation of colonic chemoprevention. Int J Inflam. 2012;2012(1):1–16. https://doi.org/10.1155/2 tumors in mice after DSS treatment. These findings indi- 012/658786. cate that colonic mutagens can cause colonic tumors in 7. Suzuki R, Kohno H, Sugie S, Tanaka T. Sequential observations on the occurrence of preneoplastic and neoplastic lesions in mouse colon treated the presence of colitis due to DSS, regardless of whether with azoxymethane and dextran sodium sulfate. Cancer Sci. 2004;95(9):721– they are carcinogenic in the colon. 7. https://doi.org/10.1111/j.1349-7006.2004.tb03252.x. 8. Kohno H, Suzuki R, Sugie S, Tanaka T. β-Catenin mutations in a mouse Abbreviations model of inflammation-related colon carcinogenesis induced by 1,2- AAT: o-Aminoazotoluene; AOM: Azoxymethane; BP: Benzo[a]pyrene; dimethylhydrazine and dextran sodium sulfate. Cancer Sci. 2005;96(2):69–76. CMC: Colon-mutagenic carcinogen; CMNC: Colon-mutagenic non- https://doi.org/10.1111/j.1349-7006.2005.00020.x. carcinogen; DSS: Dextran sulfate sodium; DMBA: 7,12- 9. Onose J, Imai T, Hasumura M, Ueda M, Hirose M. Rapid induction of Dimethylbenz[a]anthracene; DMH: 1,2-Dimethylhydrazine; ENU: N-Ethyl-N- colorectal tumors in rats initiated with 1,2-dimethylhydrazine followed by nitrosourea; PhIP: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine dextran sodium sulfate treatment. Cancer Lett. 2003;198(2):145–52. https://doi.org/10.1016/S0304-3835(03)00316-1. Acknowledgements 10. Tanaka T, Suzuki R, Kohno H, Sugie S, Takahashi M, Wakabayashi K. Colonic We are grateful Dr. K. Nakano-Ito of Eisai Co., Ltd. for review of this manu- adenocarcinomas rapidly induced by the combined treatment with 2- script. We thank S. Tanahashi, E. Kawada, M. Ozeki, and M. Tanaka of Sunpla- amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and dextran sodium net Co., Ltd. for their skillful techniques of dosing or preparation of sulfate in male ICR mice possess β-catenin gene mutations and increases specimens for histopathology/immunohistochemistry. We would like to immunoreactivity for β-catenin, cyclooxygenase-2 and inducible nitric oxide thank Editage (www.editage.com) for English language editing. synthase. Carcinogenesis. 2005;26(1):229–38. https://doi.org/10.1093/carcin/ bgh292. Authors’ contributions 11. Cheung C, Loy S, Li GX, Liu AB, Yang CS. Rapid induction of colon AH designed the study and drafted the manuscript. YS and JS, both of carcinogenesis in CYP1A-humanized mice by 2-amino-1-methyl-6- whom had a diplomate of the Japanese Society of Toxicologic Pathology, phenylimidazo[4,5-b]pyridine and dextran sodium sulfate. Carcinogenesis. underwent histopathological examination. NK participated in the in-life 2010;32(2):233–9. https://doi.org/10.1093/carcin/bgq235. phase of the experiment. SA carefully reviewed the draft. All authors have 12. Nishikawa A, Imazawa T, Kuroiwa Y, Kitamura Y, Kanki K, Ishii Y, et al. reviewed and approved the final manuscript. Induction of colon tumors in C57BL/6J mice fed MeIQx, IQ, or PhIP followed by dextran sulfate sodium treatment. Toxicol Sci. 2005;84(2):243–8. Funding https://doi.org/10.1093/toxsci/kfi079. The entire study was funded by Eisai Co., Ltd. 13. Lambert IB, Singer TM, Boucher SE, Douglas GR. Detailed review of transgenic rodent mutation assays. Mutat Res. 2005;590(1–3):1–280. Availability of data and materials https://doi.org/10.1016/j.mrrev.2005.04.002. Data are available upon request. Material availability is not applicable. 14. The Carcinogenic Potency Database. http://toxnet.nlm.nih.gov/cpdb/. 15. Chang WWL. Histogenesis of symmetrical 1,2-dimethylhydrazine-induced Declarations neoplasms of the colon in the mouse. J Natl Cancer Inst. 1978;60(6):1405– 18. https://doi.org/10.1093/jnci/60.6.1405. Ethics approval and consent to participate 16. Nambiar PR, Girnun G, Lillo NA, Guda K, Whiteley HE, Rosenberg DW. Not applicable. Preliminary analysis of azoxymethane induced colon tumors in inbred mice commonly used as transgenic/knockout progenitors. Int J Oncol. 2003;22(1): Consent for publication 145–50. https://doi.org/10.3892/ijo.22.1.145. Not applicable. 17. Mori H, Ohbayashi F, Hirono I, Shimada T, Williams GM. 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o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea, which are mutagenic but not carcinogenic in the colon, rapidly induce colonic tumors in mice with dextran sulfate sodium-induced colitis

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Abstract

Background: Several rodent models with chemically induced colon cancer have been developed. Among these models, dextran sulfate sodium (DSS), a colitis inducer, combined with azoxymethane as a colon mutagenic carcinogen, is commonly used. We previously reported that although benzo [a] pyrene (BP) is mutagenic but not carcinogenic in the colon, it rapidly develops colon tumors at a high incidence/multiplicity after treatment with DSS. In the present study, we examined whether other colon-mutagenic non-carcinogens (CMNCs) induced colon tumors after treatment with DSS. Results: o-Aminoazotoluene, 7,12-dimethylbenz[a]anthracene, and N-ethyl-N-nitrosourea were selected as CMNCs. Male CD2F1 mice were orally administered CMNC for 5 consecutive days. After a 9-day dose-free period, mice were treated with 4% DSS in drinking water for 1 week. Three months after DSS treatment, colon samples were collected for histopathology and β-catenin immunohistochemistry analyses. All CMNCs in combination with DSS induced colonic adenocarcinomas at a high incidence/multiplicity in the distal and middle parts of the colon, coinciding with the location of colitis. Unlike in normal cells where β-catenin is exclusively located on the cell membrane, in adenocarcinoma cells, it was translocated to both the nucleus and cytoplasm or only to cytoplasm. The translocation of β-catenin is closely associated with colon carcinogenesis in rodents and humans. No colonic tumors or dysplastic lesions were found after exposure to either CMNC or DSS alone. Conclusion: We provided further evidence clearly showing that CMNCs can rapidly induce colonic tumors in mice with DSS-induced colitis, even if they are not colonic carcinogens. Keywords: Colon, Cancer, Dextran sulfate sodium, Inflammation, o-Aminoazotoluene, 7,12- Dimethylbenz[a]anthracene, N-Ethyl-N-nitrosourea, Mutagenic non-carcinogen * Correspondence: a-hakura@hhc.eisai.co.jp Global Drug Safety, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba, Ibaraki 300-2635, Japan Full list of author information is available at the end of the article © The Author(s). 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hakura et al. Genes and Environment (2022) 44:11 Page 2 of 10 Introduction thymus in mice [13, 29]; murine tumors were generated in Colorectal cancer is one of the most common human the vascular and nervous systems, skin, and malignant cancers in the Western world [1]. Rodent colon cancer lymphoma tissues [30, 31]. N-Ethyl-N-nitrosourea (ENU) models have been developed to understand the mecha- induces mutations in numerous organs of mice, such as nisms underlying colon carcinogenesis and to investigate the colon, small intestine, bone marrow, mammary gland, potential chemotherapy or chemoprevention regimens. and liver [13, 32, 33]. Multiple tissues are known to be the These models include carcinogen-induced, genetically site of tumor induction in mice, including the liver, Hard- modified, and transplant models [1–3]. Carcinogen- erian glands, stomach, ovaries, lymphoreticular system, induced models are highly reproducible and can be read- kidneys, mammary gland, uterus, nervous system, and ily tested on non-genetically modified animals with dif- lungs [34, 35]. For these three chemicals, the colon is not ferent genetic backgrounds. In addition, the processes recognized as a target organ for murine carcinogenesis. involved in the pathogenesis recapitulate human colon In this study, we showed that all these three CMNCs cancer, particularly, the early stages of this disease [4–6]. induced colonic cancer in the presence of colitis. In Among carcinogen-induced models, some models addition, we performed the immunohistochemical ana- using a colon mutagenic carcinogen in combination with lysis of tumors with β-catenin, given that the nuclear ac- dextran sulfate sodium (DSS), a colitis inducer, are often cumulation of β-catenin is well documented to be used [4]. Azoxymethane (AOM) [4, 7], 1,2-dimethylhy- closely associated with colon carcinogenesis in rodents drazine (DMH) [8, 9] and 2-amino-1-methyl-6-phenyli- and humans [4–6]. midazo[4,5-b]pyridine (PhIP) [10–12] are used as colon mutagenic carcinogens (referred to as CMCs) to initiate Materials and methods carcinogenesis. These CMCs can induce colonic cancer Chemicals by themselves [13, 14]; however, colonic tumors are in- o-Aminoazotoluene (AAT, CAS No. 97–56-3, purity > duced at varying incidences (0–100%), and it takes 3 97.0%) was purchased from Tokyo Chemical Industry months or longer to induce cancer, depending on the Co., Ltd. (Tokyo, Japan). 7,12-Dimethylbenz[a]anthra- species/strains or dosing regimens [15, 16]. For these cene (DMBA, CAS No. 57–97-6, purity > 95%) and N- reasons, DSS is used, in combination, to cause colitis in ethyl-N-nitrosourea (ENU, CAS No. 759–73-9, content: the colon (well documented as a DSS-induced colitis 43.2% in water with 1.7% acetic acid to prevent decom- model), thereby accelerating the development and pro- position) were obtained from Sigma-Aldrich Co. LLC gression of colonic tumors. DSS is not mutagenic in bac- (St. Louis, MO, USA). Dextran sulfate sodium (DSS, teria [17] and in murine mutagenicity tests [18] but is CAS No. 9011-18-1, molecular weight: 36,000–50,000) weakly carcinogenic in the rodent colon [19, 20], indicat- was purchased from MP Biochemicals, LLC (Aurora, ing that DSS potently promotes colonic carcinogenesis. OH, USA). However, even in such “short-term” colon cancer For administration, ENU was dissolved in water for in- models, the induction of cancer generally takes approxi- jection at a concentration of 1.1 mg/mL, and AAT and mately 10–20 weeks [4–6]. DMBA were dissolved in salad oil (Nisshin Oillio Group, We previously showed that benzo[a]pyrene (BP), a co- Ltd., Tokyo, Japan) at concentrations of 12.5 and 2.5 lonic mutagen [21, 22], induced colonic cancer at a high mg/mL, respectively. DSS was dissolved in water at a incidence/multiplicity in a shorter or comparable period, concentration of 40 mg/mL (4%). required for cancer induction in models using CMC and DSS after the treatment of mice with DSS, despite it be- ing a non-colonic carcinogen [23–25]. There is no evi- Animals dence showing that CMNCs (colon-mutagenic non- Male Crj: CD2F1 (BALB/c × DBA/2) mice were obtained carcinogens), except for BP, clearly induce the formation from Charles River Japan, Inc., Tokyo. All mice were of colonic tumors at a high multiplicity/incidence in the housed in metal cages (one mouse per cage) and were DSS-induced colitis model. The purpose of the present fed a basal diet (Oriental CRF-1, Oriental Yeast Co., study was to examine whether other CMNCs also act as Tokyo, Japan) and tap water ad libitum, under controlled initiators for carcinogenesis in a DSS-induced colitis conditions of temperature (23 ± 3 °C), humidity (55 ± model. 20%), and light (12-h light/12-h dark cycle). They were o-Aminoazotoluene (AAT) induces mutations in the quarantined and acclimatized for 1 week. The animals colon, liver, kidney, and urinary bladder [13, 26, 27]; only were assigned by stratified randomization to the two hepatocellular adenoma/carcinoma and hemangioen- groups according to their body weights; mice from both dothelioma in the lung have been generated in mice [14, the DSS- and CMNC/DSS-groups and both the non- 28]. 7,12-Dimethylbenz[a] anthracene (DMBA) induces treatment and CMNC groups were assigned to the heavy mutations in the colon, bone marrow, liver, skin, and and light groups, respectively, to minimize the mortality Hakura et al. Genes and Environment (2022) 44:11 Page 3 of 10 of light mice, given that they are likely to be more sensi- Tissue collection and histopathology tive to DSS-induced colitis than heavy mice. During necropsy, the large intestine was immediately excised, flushed with saline, infused with 10% neutral buffered formalin, cut open longitudinally along the anti- Experimental procedures mesenteric border, and grossly observed. Thereafter, the Figure 1 shows the outline of the protocol for the experi- tissues were stored in 10% neutral buffered formalin, cut ment. The number of mice, established upon initiation into four parts of equal length from the proximal to dis- of the experiment, were as follows: eight for the non- tal ends, processed, and embedded in paraffin. Each treatment group, six for each CMNC group, 16 (eight colon sample was embedded to expose both longitudin- for water for injection and eight for salad oil, vehicles ally cleaved edges. All longitudinal sections were stained with which CMNCs were dissolved) for the DSS group, with hematoxylin and eosin (H&E) and histopathologic- and eight for each CMNC/DSS-group. Three of 16 mice ally observed. The lesions of dysplasia, adenoma, and in the DSS-group and one of seven mice in the DMBA/ adenocarcinoma were classified according to the criteria DSS-group died 4 or 5 days after the last DSS treatment, that were described in detail in our previous report [24], due to severe colitis, resulting in 13 surviving mice in which were originally reported by Riddell et al. [36], Pas- the DSS group and 6 surviving mice in the DMBA/DSS- cal [37] and Ward [38]. Dysplasia or dysplastic foci were group. The numbers of surviving mice per group are characterized by irregular branching, distorted architec- shown in parentheses in Fig. 1 and Table 1. ture with cellular and nuclear pleomorphism, nuclear For each CMNC/DSS-group, mice (7-week old) were enlargement and hyperchromatism, and paucity of gob- orally (by gavage) treated with each CMNC for 5 con- let cells. secutive days, and starting 10 days after the last dose, mice were administered 4% DSS in drinking water for 7 Immunohistochemistry days. Twelve weeks later, the mice were necropsied Paraffin-embedded sections of mouse colons with under anesthesia to collect the colorectum from the adenocarcinomas from each CMNC/DSS group were cecocolic junction to anus for histopathology and subjected to immunohistochemical staining of β-catenin. immunohistochemistry. Immunohistochemical staining was performed according The in-life phase of the experiment was performed at to the procedure described in our previous report [24]. Sunplanet Co., Ltd. of the Eisai Co., Ltd. group, and the Monoclonal mouse anti-mouse β-catenin (clone 14, BD protocol was approved by the Institutional Animal Care Transduction Laboratories, Lexington, KY, USA) was and Use Committee and carried out according to the used at a concentration of 1/1000. After antigen re- Sunplanet animal experimentation regulations. trieval, the Envision™ + Dual Link System or a Fig. 1 Outline of the protocol for the experiment for histopathology and immunohistochemistry of the murine colon. The experiment consisted of Non-treatment-, CMNC-, DSS-, and CMNC/DSS-groups. Male CD2F mice were treated with each CMNC at an oral dose of either 125 (AAT), 25 (DMBA), or 11 (ENU) mg/kg/day for 5 consecutive days, and starting 10 days after the last dose, mice were given 4% DSS in drinking water for 7 days. All surviving mice were necropsied 12 weeks after the last treatment with DSS. The number of surviving animals is shown in parentheses. Salad oil was used as the vehicle for AAT and DMBA, and water for injection was used as the vehicle for ENU Hakura et al. Genes and Environment (2022) 44:11 Page 4 of 10 Table 1 Incidence of colonic dysplastic foci and tumors induced by a CMNC in combination with DSS Treatment Dysplasia Adenoma Adenocarcinoma Tumor Non-treatment 0% (0/8) 0% (0/8) 0% (0/8) 0% (0/8) Vehicle + 4% DSS 0% (0/13) 0% (0/13) 0% (0/13) 0% (0/13) AAT (125 mg/kg/day) 0% (0/6) 0% (0/6) 0% (0/6) 0% (0/6) AAT (125 mg/kg/day) + 4% DSS 75% (6/8)*** 100% (8/8)*** 100% (8/8)*** 100% (8/8)*** DMBA (25 mg/kg/day) 0% (0/5) 0% (0/5) 0% (0/5) 0% (0/5) DMBA (25 mg/kg/day) + 4% DSS 50% (3/6)** 50% (3/6)** 50% (3/6)** 83% (5/6)*** ENU (11 mg/kg/day) 0% (0/6) 0% (0/6) 0% (0/6) 0% (0/6) ENU (11 mg/kg/day) + 4% DSS 100% (7/7)*** 100% (7/7)*** 100% (7/7)*** 100% (7/7)*** The numbers in parentheses indicate the number of mice with dysplasias, adenomas, or adenocarcinomas per the number of mice CMNC; colon-mutagenic non-carcinogen : adenoma + adenocarcinoma : water for injection for 7 mice and salad oil for 6 mice **, *** ; significantly different from the DSS alone group at P < 0.05, 0.01 in Fisher’s exact probability test streptavidin biotin-peroxidase complex method (DAKO, parts of the colon. Neoplasms were nodular, papillary, or Glostrup, Denmark) was used to examine their expression polypoid masses composed of tubular and papillary prolif- and localization. These sections were counterstained with eration of epithelium that protruded into the intestinal Mayer’s hematoxylin solution for microscopic examination. lumen, extended into the lamina propria, and compressed the adjacent mucosa. Tumors induced by these three Dose setting for treatment with CMNCs CMNC/DSS were tubular adenomas or well- to moder- The doses of AAT (125 mg/kg/day), DMBA (25 mg/kg/ ately differentiated adenocarcinomas. No submucosal in- day), and ENU (11 mg/kg/day) tested in this study were de- vasion or metastasis were observed. Pre-neoplastic and termined based on our preliminary study (data not shown) neoplastic lesions were not observed in the colon in any or the studies to measure the mutant frequency in mice; CMNC-, DSS-, or non-treatment-group. for AAT, a single i.p. dose at 300 mg/kg, corresponding to In the CMNC/DSS- and DSS-groups, the large intes- 40% of LD (median lethal dose) [26, 27], for DMBA, a tine was shorter in length, and the intestinal wall showed single i.p. dose at 20 mg/kg [29]and forENU, five i.p. irregular thickening. Histopathologically, colitis with or doses at 22.2 mg/kg/day (once a week for 5 weeks) [39]. without erosion or ulceration was noted in the distal and middle parts of the colon. In animals with colitis, there Statistical analysis was occasional accumulation of foamy macrophages in The incidences and multiplicities were compared using the lamina propria. Fisher’s exact probability test and Welch’s t-test for paired samples. Incidence and multiplicity of dysplastic foci, adenomas, and adenocarcinomas Results Table 1 shows the incidences of dysplastic foci, aden- Clinical findings omas, and adenocarcinomas observed in the colon, In DSS-treated mice, with or without CMNC, bloody and Table 2 shows their multiplicity (= the number and/or soft stools were observed for 1 or 2 days before of tumors/mouse). All CMNCs rapidly produced neo- the last DSS treatment. Stool changes resolved by 5 days plastic lesions after combined treatment with DSS after DSS treatment cessation and were attributed to under the experimental conditions employed in this colitis. In the CMNC/DSS groups, three mice that devel- study. The combined treatments of ENU (11 mg/kg/ oped tumors showed bloody stools from 9 weeks after day)/4% DSS and AAT (125 mg/kg/day)/4% DSS the last DSS treatment. showed tumorigenicity in 100% mice, and the multi- plicities are 8.6 ± 1.1 and 7.9 ± 2.0, respectively. The Gross findings and histopathology combination of DMBA (25 mg/kg/day)/4% DSS in- The gross findings are shown in Fig. 2, and histopatho- duced the formation of tumors in 83% of mice with a logical findings are shown in Figs. 3, 4 and 5. In the sam- multiplicity of 3.0 ± 1.9. ples from the mice in each CMNC/DSS-group, there were multiple masses in the middle to distal parts of the colon Immunohistochemistry of β-catenin at necropsy (12 weeks after completion of DSS treatment). The expression of the β-catenin protein in adenocarcin- Histopathological examination revealed dysplastic foci, ad- omas is shown in Fig. 5. In adenocarcinoma cells, whose enomas, and adenocarcinomas in the distal and middle formation was induced by AAT/DSS, DMBA/DSS, or Hakura et al. Genes and Environment (2022) 44:11 Page 5 of 10 Fig. 2 Macroscopic view of the colons of mice from Non-treatment- (A), DSS- (B), AAT- (C), AAT/DSS- (D), DMBA- (E), DMBA/DSS- (F), ENU- (G) and ENU/DSS- (H) groups. Bar: 1 cm Fig. 3 Histopathology of the colons of mice from Non-treatment- (A), DSS- (B), AAT- (C), AAT/DSS- (D), DMBA- (E), DMBA/DSS- (F), ENU- (G) and ENU/DSS- (H) groups. H& E stain Hakura et al. Genes and Environment (2022) 44:11 Page 6 of 10 Table 2 Multiplicity of colonic dysplastic foci and tumors induced by a CMNC in combination with DSS Treatment Dysplasia Adenoma Adenocarcinoma Tumor Non-treatment 0 ± 0 0 ± 0 0 ± 0 0 ± 0 Vehicle +4% DSS 0± 0 0 ±0 0± 0 0 ±0 AAT (125 mg/kg/day) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 AAT (125 mg/kg/day) + 4% DSS 0.9 ± 0.6*** 3.8 ± 1.2*** 4.1 ± 1.7*** 7.9 ± 2.0*** DMBA (25 mg/kg/day) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 DMBA (25 mg/kg/day) + 4% DSS 1.0 ± 1.1** 1.5 ± 2.0* 1.5 ± 1.6** 3.0 ± 1.9*** ENU (11 mg/kg/day) 0 ± 0 0 ± 0 0 ± 0 0 ± 0 ENU (11 mg/kg/day) + 4% DSS 1.6 ± 0.5*** 4.9 ± 1.9*** 3.7 ± 1.5*** 8.6 ± 1.1*** Multiplicity indicates the number of dysplasias, adenomas, or adenocarcinomas per mouse, mean ± standard deviation CMNC; colon-mutagenic non-carcinogen : adenoma + adenocarcinoma : water for injection for 7 mice and salad oil for 6 mice *, **, ***; significantly different from the DSS alone group at P < 0.1, 0.05, 0.01 in the Welch’s t-test ENU/DSS, β-catenin was translocated predominantly to tubular adenomas or well- to moderately differentiated both the nucleus and cytoplasm or only cytoplasm from adenocarcinomas. These tumors predominantly ap- cell membrane, where it is exclusively expressed in nor- peared in the middle and distal parts of the colon, co- mal colon epithelial cells. inciding with the location of DSS-induced colitis. These findings were consistent with those reported for DSS in combination with BP or CMCs such as AOM, Discussion DMH, and PhIP [4, 7–12]. In adenocarcinoma cells, We previously reported that in mice administered BP, a whose formation was induced by CMC in combination CMNC, the formation of colon tumors was rapidly in- with or without DSS, or BP plus DSS, β-catenin was duced after DSS treatment [23, 24]. In the present study, expressed in both the nucleus and cytoplasm or cyto- we clearly showed that three more CMNCs (AAT, plasm [4, 7, 8, 10, 24]. Such translocation and accumu- DMBA, and ENU) can rapidly induce the formation of lation of β-catenin in the nucleus from cell membrane colon tumors at a high multiplicity/incidence. has been shown to be closely associated with the de- All tumors whose formation was induced by the velopment or progression of colon tumors through three CMNC/DSS were histologically diagnosed as Fig. 4 Histopathology results of H&E staining of colonic dysplasia (A, B, E, F, I, and J) and adenoma (C, D, G, H, K, and L) induced by AAT/DSS, DMBA/DSS or ENU/DSS. Figs. B, D, F, H, J, and L are enlarged magnifications of those in A, C, E, G, I, and K, respectively (the areas surrounded by the yellow frames). Yellow bar: 100 μm Hakura et al. Genes and Environment (2022) 44:11 Page 7 of 10 mutations in the β-catenin or APC genes or activation In rodent chemical-induced colon cancer models, CMCs, of the Wnt/β-catenin signaling pathway [4, 40–43]. particularly AOM, are commonly used in combination with MeIQx, a heterocyclic amine found in well-cooked DSS. Our present and previous studies [23, 24]provide evi- meat, is mutagenic but not carcinogenic in the colon of dence showing that CMNCs can also induce colonic tumors mice [44]. One study reported that MeIQx is carcinogenic after posttreatment with DSS, and that colonic mutagens in the mouse colon after DSS treatment [12]. However, can induce colonic tumors in the presence of DSS-induced the carcinogenicity was weak in that study (for adenomas colitis whether they are colonic carcinogens or not. To in- plus adenocarcinomas, the incidence was 22% and multi- duce colonic cancer, CMNCs may play a key role in the in- plicity was 0.30 ± 0.61), and another study reported duction of gene mutations responsible for tumorigenesis via that MeIQx was not carcinogenic after DSS treatment their characteristic mutational spectrum. [18]. Thus, the effect of DSS treatment on MeIQx- Figure 6 shows our hypothesis for possible mecha- induced carcinogenicity is not clear and may be mar- nisms of the induction of colonic tumors in the mouse ginal. Another heterocyclic amine, IQ, was carcino- colitis-associated model in combination with a colonic genic at a low incidence/multiplicity (for adenomas mutagen/carcinogen. Colon epithelial cells are mutated plus adenocarcinomas, the incidence was 14% and by colonic mutagens, resulting in the generation of mu- multiplicity was 0.34 ± 0.72) in mice after DSS treat- tated epithelial cells (probably stem cells or progenitor ment [12], although it is not reported to be a colonic cells, because cell turnover of colon epithelial cells is carcinogen in itself [44]. IQ has been reported to very fast, i.e., 2–3 days [32, 33]). Mutated epithelial cells show mutagenicity in the cecum, but not in the develop tumors via non-genetic effects when mice are colon, at 20 mg/kg for 5 days [45]. In that study, only treated with DSS, a colitis inducer and a potent pro- one dose was tested. moter of carcinogenesis. In the absence of DSS Fig. 5 Histopathology results of H&E staining (A, B, D, E, G and H) and β-catenin-immunostaining (C, F, and I) of colonic adenocarcinomas induced by AAT/DSS, DMBA/DSS or ENU/DSS. Figs. B, E and H are enlarged magnifications of those in A, D and G, respectively (the areas surrounded by the yellow frames). Immunohistochemical analysis of β-catenin was performed on serial sections used for H & E staining. The inserts are figures in enlarged magnification. Figs. J and K show the results of H & E staining and β-catenin-immunostaining, respectively, of normal colonic epithelial cells. Yellow bar: 100 μm Hakura et al. Genes and Environment (2022) 44:11 Page 8 of 10 treatment, mutated epithelial cells do not develop tu- related differences in the DNA repair system, may de- mors. Non-genetic effects include cell regeneration ac- crease the number of cells initiated by ENU [51, 52]. Min/+ companied by cell necrosis and inflammation in Colon tumors are induced when adult APC mice response to cell injury or microenvironment disruption, are treated with ENU [52]. This finding supports a canon- and thereby alterations in signal transduction [40–42, ical mechanism of colon tumorigenesis: the induction of 46] or epigenetics (DNA methylation or histone modifi- mutations in the dominant tumor-suppressor gene. In cation) [47, 48]. These effects are affected by intestinal contrast, our study showed that DSS post-treatment pro- bacteria [49, 50]. Many studies have shown that CMCs vides the same output of tumor induction as mutation in- rapidly induce colon cancer after treatment with DSS, duction in tumor-related genes in tumorigenesis. and that colitis (inflammation) caused by DSS contrib- Since the present study with the three colonic muta- utes to the rapid progression of colon carcinogenesis [4– gens was performed under the same experimental condi- 6]. Therefore, inflammation may be a requisite factor for tions, the potency of their carcinogenicity in this model colon carcinogenesis and one of the discriminating fac- may be correlated and can be compared. The most tors of colon carcinogens from CMNCs. However, fur- powerful initiation activity was estimated to be that of ther studies are required for verification. ENU (0.33), followed by that of DMBA (0.060) and AAT The colon becomes the site of tumor induction when (0.033), by calculating the multiplicity of adenocarcin- Min/+ juvenile APC mice (carrying a mutation in the Apc oma divided by the dose (mg/kg/day). gene to develop multiple intestinal adenomas) or even The purpose of the present study was to provide evi- +/+ APC mice (wild-type at the Apc locus) are intraperito- dence on the hypothesis that CMNCs act as initiators of neally treated with ENU, although the ENU does not in- carcinogenesis in a DSS-induced colitis model. Our duce colonic tumors when adult mice, generally used in study may help better understand the effects of environ- toxicity studies, are orally treated [34, 35, 51]. In both mental mutagens on inflammation-related cancer. In- strains of mice, the incidence of intestinal tumors age- creasing attention has been paid to the involvement of relatedly reduces during the periods from 5 to 14 days to inflammation in the initiation, promotion, and progres- 30–35 days of age at ENU treatment, with tumors being sion of tumors [53–56]. Patients with inflammatory induced at a very low incidence at 30–35 days of age bowel disease (IBD) are at a higher risk of developing [51]. The microenvironment of intestinal crypts dramat- colorectal cancer. The cumulative incidence of colorectal ically changes from infancy to early juvenile; when mice cancer in patients with IBD ranges from 7.6 to 18.4%, at are born, crypts are predominantly polyclonal, but 30 years post-diagnosis [5, 57]. A review paper by Rawla around two to 3 weeks of age, they become monoclonal. et al. [58] reported that patients with chronic IBD have a This phenomenon, known as crypt purification or age- two-fold higher risk of developing colorectal cancer, and Fig. 6 Hypothesis for possible mechanisms underlying the induction of colonic tumor formation after combined treatment with colonic mutagens and DSS. Colon epithelial cells are mutated by colonic mutagens, probably in stem or progenitor cells. Mutated epithelial cells develop tumors via non-genotoxic effects when mice are treated with DSS. When DSS is not treated, mutated epithelial cells do not develop tumors. Non-genotoxic effects of DSS include enhanced cell proliferation and inflammation in response to cell injury or microenvironment disruption, and thereby alterations in signal transduction or epigenetics. These effects are affected by intestinal bacteria. Colonic mutagens are not necessarily colonic carcinogens Hakura et al. Genes and Environment (2022) 44:11 Page 9 of 10 that ulcerative colitis increases the risk of colorectal can- 2. Jackstastadt R, Sansom OJ. Mouse models of intestinal cancer. J Pathol. 2016;238(2):141–51. https://doi.org/10.1002/path.4645. cer by 2.4 times. Our study suggests that colonic muta- 3. Nascimento-Gonçalves E, Mendes BAL, Silva-Reis R, Faustino-Rocha AI, Gama gens impose an increased risk of colon cancer in A, Oliveira PA. Animal models of colorectal cancer: From spontaneous to patients with IBD compared with healthy individuals. genetically engineered models and their applications. Vet Sci. 2021;8(4):59. https://doi.org/10.3390/vetsci8040059. 4. Rosenberg D, Giardina C, Tanaka T. Mouse models for the study of colon Conclusion carcinogenesis. Carcinogenesis. 2009;30(2):183–96. https://doi.org/10.1053/j. In our previous study, BP of a CMNC rapidly induced gastro.2008.12.049. 5. Tanaka T. Colorectal carcinogenesis: review of human and experimental the formation of colonic tumors in mice after DSS treat- animal studies. J Carcinog. 2009;8(5):1–19. https://doi.org/10.4103/1477-31 ment. In the present study, we clearly showed further 63.49014. evidence that the other three CMNCs (AAT, DMBA, 6. Tanaka T. Development of an inflammation-associated colorectal cancer model and its application for research on carcinogenesis and and ENU) also rapidly induced the formation of colonic chemoprevention. Int J Inflam. 2012;2012(1):1–16. https://doi.org/10.1155/2 tumors in mice after DSS treatment. These findings indi- 012/658786. cate that colonic mutagens can cause colonic tumors in 7. Suzuki R, Kohno H, Sugie S, Tanaka T. Sequential observations on the occurrence of preneoplastic and neoplastic lesions in mouse colon treated the presence of colitis due to DSS, regardless of whether with azoxymethane and dextran sodium sulfate. Cancer Sci. 2004;95(9):721– they are carcinogenic in the colon. 7. https://doi.org/10.1111/j.1349-7006.2004.tb03252.x. 8. Kohno H, Suzuki R, Sugie S, Tanaka T. β-Catenin mutations in a mouse Abbreviations model of inflammation-related colon carcinogenesis induced by 1,2- AAT: o-Aminoazotoluene; AOM: Azoxymethane; BP: Benzo[a]pyrene; dimethylhydrazine and dextran sodium sulfate. Cancer Sci. 2005;96(2):69–76. CMC: Colon-mutagenic carcinogen; CMNC: Colon-mutagenic non- https://doi.org/10.1111/j.1349-7006.2005.00020.x. carcinogen; DSS: Dextran sulfate sodium; DMBA: 7,12- 9. Onose J, Imai T, Hasumura M, Ueda M, Hirose M. Rapid induction of Dimethylbenz[a]anthracene; DMH: 1,2-Dimethylhydrazine; ENU: N-Ethyl-N- colorectal tumors in rats initiated with 1,2-dimethylhydrazine followed by nitrosourea; PhIP: 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine dextran sodium sulfate treatment. Cancer Lett. 2003;198(2):145–52. https://doi.org/10.1016/S0304-3835(03)00316-1. Acknowledgements 10. Tanaka T, Suzuki R, Kohno H, Sugie S, Takahashi M, Wakabayashi K. Colonic We are grateful Dr. K. Nakano-Ito of Eisai Co., Ltd. for review of this manu- adenocarcinomas rapidly induced by the combined treatment with 2- script. We thank S. Tanahashi, E. Kawada, M. Ozeki, and M. Tanaka of Sunpla- amino-1-methyl-6-phenylimidazo [4,5-b] pyridine and dextran sodium net Co., Ltd. for their skillful techniques of dosing or preparation of sulfate in male ICR mice possess β-catenin gene mutations and increases specimens for histopathology/immunohistochemistry. We would like to immunoreactivity for β-catenin, cyclooxygenase-2 and inducible nitric oxide thank Editage (www.editage.com) for English language editing. synthase. Carcinogenesis. 2005;26(1):229–38. https://doi.org/10.1093/carcin/ bgh292. Authors’ contributions 11. Cheung C, Loy S, Li GX, Liu AB, Yang CS. Rapid induction of colon AH designed the study and drafted the manuscript. YS and JS, both of carcinogenesis in CYP1A-humanized mice by 2-amino-1-methyl-6- whom had a diplomate of the Japanese Society of Toxicologic Pathology, phenylimidazo[4,5-b]pyridine and dextran sodium sulfate. Carcinogenesis. underwent histopathological examination. NK participated in the in-life 2010;32(2):233–9. https://doi.org/10.1093/carcin/bgq235. phase of the experiment. SA carefully reviewed the draft. All authors have 12. Nishikawa A, Imazawa T, Kuroiwa Y, Kitamura Y, Kanki K, Ishii Y, et al. reviewed and approved the final manuscript. Induction of colon tumors in C57BL/6J mice fed MeIQx, IQ, or PhIP followed by dextran sulfate sodium treatment. Toxicol Sci. 2005;84(2):243–8. Funding https://doi.org/10.1093/toxsci/kfi079. The entire study was funded by Eisai Co., Ltd. 13. Lambert IB, Singer TM, Boucher SE, Douglas GR. Detailed review of transgenic rodent mutation assays. Mutat Res. 2005;590(1–3):1–280. 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Journal

Genes and EnvironmentSpringer Journals

Published: Mar 29, 2022

Keywords: Colon; Cancer; Dextran sulfate sodium; Inflammation; o-Aminoazotoluene; 7,12-Dimethylbenz[a]anthracene; N-Ethyl-N-nitrosourea; Mutagenic non-carcinogen

References