Contents lists available at ScienceDirect Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/taap Capsaicin reduces genotoxicity, colonic cell proliferation and preneoplastic lesions induced by 1,2-dimethylhydrazine in rats Brunno Felipe Ramos Caetanoa, Mariana Baptista Tablasb, Natália Elias Ferreira Pereirab, Nelci Antunes de Mourab, Robson Francisco Carvalhob, Maria Aparecida Marchesan Rodriguesa, Luis Fernando Barbisanb,⁎ a Department of Pathology, Medical School, São Paulo State University (UNESP), Botucatu, 18618-687, Brazil bDepartment of Morphology, Institute of Biosciences, São Paulo State University (UNESP), Botucatu, 18618-689, Brazil A R T I C L E I N F O Keywords: Capsaicin Colon Cancer Chemoprevention A B S T R A C T Capsaicin (8-Methyl-N-vanillyl-(trans)-6-nonenamide) is the major pungent ingredient found in chili peppers consumed worldwide. Most reports on capsaicin potential carcinogenicity have yielded inconsistent findings. Some studies have shown that capsaicin exerts anti-proliferative and pro-apoptotic effects on different cancer cell lines, while others have reported an association between capsaicin at high doses with mutagenicity and carcinogenicity. Thus, this study aimed at assessing the effects of capsaicin administration on 1,2-dimethyl- hydrazine (DMH)-induced colon carcinogenesis in male Wistar rats. Our results show that capsaicin adminis- tration, before and during carcinogen exposure, modified DMH-induced cytotoxicity and genotoxicity, pro- moting anti-proliferative and pro-apoptotic responses through the expression of the genes involved in apoptosis, cell cycle suppression and cell/tissue differentiation. Furthermore, capsaicin reduced aberrant crypt foci (ACF) number and multiplicity, although there were no differences in tumor incidence and multiplicity among the groups. Taken together, the results suggest that capsaicin may have a preventive effect against DMH-induced colorectal carcinogenesis. 1. Introduction Colorectal cancer (CRC) is the third most common type of cancer, and a leading cause of death worldwide (Torre et al. 2015). World Health Organization (WHO) GLOBOCAN estimates for 2015 showed that CRC burden represents up to 9.7% of all incident malignancies, accounting for 746,000 new cases in men and 614,000 in women (Ferlay et al. 2015). The incidence and mortality rates of CRC vary greatly across the world (Kamangar et al. 2006). However, CRC more frequently occurs in developed countries, indicating a correlation with western dietary habits and lifestyle patterns, such as smoking, alcohol consumption, obesity and physical inactivity (Gingras and Béliveau 2011). These are known risk factors that are potentially modifiable and avoidable through specific public strategies for cancer prevention. Re- ducing consumption of refined starches, saturated fat, and processed or red meat, as well as increasing the intake of fruits and vegetables has been associated with lower CRC risk (Carr et al. 2016; Dahham and Majid 2016). Natural anticancer bioactive compounds have been lately ac- knowledged with great public enthusiasm. Indeed, several bioactive compounds found in vegetables and medicinal plants can reduce the risk of developing chronic diseases such as cancer (Sales et al. 2014). Fruits and vegetables play an essential role in human nutrition and health, providing natural fibers, antioxidants, and a broad range of bioactive phytochemicals (Liu 2013). Evidence from many pre-clinical and clinical studies support that dietary interventions stand as a pro- mising strategy for CRC prevention (Baena and Salinas 2015; Hou et al. 2013). Capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide) is the major pungent alkaloid ingredient found in chili peppers (Bosland et al. 2012). The chili pepper is the fruit of herbaceous plants of the genus Capsicum, members of the family Solanacea, native to the Americas. Chilies have long been domesticated by Mesoamerican civilizations and are appreciated worldwide for both culinary and medicinal purposes (Heiser and Smith 1953). In fact, chili peppers represent a fair amount of total vegetables daily consumed around the world (Kantar et al. 2016). Capsaicin has emerged as a potential therapeutic drug to treat a number of human diseases, including chronic pain, obesity, diabetes, cardiovascular conditions, airway diseases and cancer (Fattori et al. 2016). https://doi.org/10.1016/j.taap.2017.11.008 Received 14 August 2017; Received in revised form 20 October 2017; Accepted 10 November 2017 ⁎ Corresponding author at: Rua Prof. Dr. Antonio Celso Wagner Zanin s/n, 18618-689, Botucatu, SP, Brazil. E-mail address: barbisan@ibb.unesp.br (L.F. Barbisan). Toxicology and Applied Pharmacology 338 (2018) 93–102 Available online 16 November 2017 0041-008X/ © 2017 Elsevier Inc. All rights reserved. T http://www.sciencedirect.com/science/journal/0041008X https://www.elsevier.com/locate/taap https://doi.org/10.1016/j.taap.2017.11.008 https://doi.org/10.1016/j.taap.2017.11.008 mailto:barbisan@ibb.unesp.br https://doi.org/10.1016/j.taap.2017.11.008 http://crossmark.crossref.org/dialog/?doi=10.1016/j.taap.2017.11.008&domain=pdf Scientific reports on capsaicin potential carcinogenicity have yielded inconsistent findings (Bode and Dong 2011). Some studies have shown that capsaicin exerts anti-proliferative and pro-apoptotic effects on different cancer cell lines (Brown et al. 2010; Díaz-Laviada 2010; Garufi et al. 2016; Lau et al. 2014) and might inhibit the metabolism of chemical carcinogens by interacting with a number of cytochrome P450 enzymes (CYPs) (Zhang et al. 2012). On the other hand, Lee and Park have reported an association between capsaicin at high doses with mutagenicity and carcinogenicity (Lee and Park 2003). Furthermore, several preclinical studies have suggested that the chili extract or cap- saicin alone can have co-carcinogenic effects on the stomach, liver, colon and skin in different chemically-induced carcinogenesis models (Agrawal et al. 1986; Díaz Barriga Arceo et al. 1995; Johnson 2007; Liu et al. 2015). Considering that the molecular mechanisms underlying the putative effects of capsaicin on colon carcinogenesis are largely un- known, this study aimed at assessing the effects of capsaicin oral ad- ministration on DNA damage, cell proliferation, and apoptosis, as well as on the expression of the genes involved in oxidative metabolism, antioxidant activity, cell cycle, DNA repair and cell death pathways during the early stages of colon carcinogenesis induced by 1,2-di- methylhydrazine (DMH) in rats. 2. Material and methods 2.1. – Chemicals Capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide, purity ≥95%, PubChem CID:1,548,943) and DMH (1,2-dimethylhydrazine hydro- chloride, PubChem CID: 1322) were purchased from Sigma-Aldrich (Darmstadt, Germany). All other reagents were of the highest grade available commercially. 2.2. – Study design Four-week-old male Wistar rats weighing 125 g (ANILAB, Paulínia- SP, Brazil) were housed in polypropylene cages under standard condi- tions (21 ± 2 °C temperature, 55 ± 10% humidity, and 12 h/12 h light-dark cycle) with food (NUVILAB-CR-1, Curitiba, Brazil) and tap water ad libitum. The animals used in this study were handled in ac- cordance with the principles of laboratory animal care adopted by the Brazilian College of Animal Experimentation (COBEA). This study was approved by the institution's Ethics Review Board (1153/2015-CEUA). After a 3-week acclimation period, the animals (7-week old) were randomly assigned into six experimental groups with 16 animals each. Intragastric doses of corn oil (capsaicin vehicle, G1 and G6), capsaicin at 5 mg/kg body weight (bw) (G2 and G4) and 50 mg/kg bw (G3 and G5) were administered three times a week for four weeks. The capsaicin dosages used were determined based on previous reports (Saito and Yamamoto 1996). Either a subcutaneous injection of DMH (G1, G2 and G3, 40 mg/kg bw) or disodium ethylenediamine tetraacetic acid (Na2EDTA, DMH vehicle, G4, G5 and G6) was given twice a week over weeks 3 and 4. DMH was dissolved in 1 mM Na2EDTA in order to en- sure stability (Rubio 2017). Body weight and food consumption were recorded weekly throughout the experiment. By the end of week 4, 6 animals from each group were sacrificed (short-term assays, n = 6). The remaining animals were sacrificed at 22 weeks (mid-term assays, n = 10) (Fig. 1). 2.3. Short-term assays Leukocyte genotoxicity. Capsaicin anti-genotoxic potential was assessed in peripheral blood leukocytes 24 h after the last DMH injection using the single cell gel electrophoresis (comet) assay under alkaline conditions as previously described (Nandhakumar et al. 2011). Peripheral blood samples, col- lected by retroorbital venipuncture, were mixed with 100 μL of low melting point agarose (0.75% in PBS, Invitrogen, USA.), spread on slides pre-coated with normal point agarose (1.5% in PBS, Invitrogen, USA), and coverslipped. Following agarose solidification (4 °C for 10 min), coverslips were carefully removed and the slides were in- cubated with cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 1% sarkosyl, pH 10) overnight, at 4 °C. Subsequently, the slides were washed three times in PBS and immersed in fresh cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH > 13) for 20 min. Electrophoresis was conducted at a room tem- perature of 21 °C for 20 min at 1 V/cm (300 mA) for 20 min. The slides were then neutralized with 0.4 M Tris (pH 7.5), dehydrated in 100% ethanol, and stained with Sybr Gold (Invitrogen, USA). An epi-fluor- escence microscope (Olympus BX-50, Japan) coupled to a CCD camera was used to score fifty random nucleoid/sample using the Comet Assay IV Image Analysis System (Perceptive Instruments, UK). All experi- ments were performed in duplicate. Fecal water genotoxicity. Cecal feces were collected at sacrifice and kept frozen at −20 °C prior to use. Fecal water was prepared as described elsewhere (Klinder et al. 2007) with minor modifications. Briefly, fecal slurry was prepared by mixing feces with ice-cold PBS at a 1:1 rate (1 g of fecal content +1 mL of PBS). This mixture was homogenized for 3 min. Fecal debris were removed by centrifuging homogenates at 35,000g for 30 min. The supernatant was filtered with an Ø 0.22 μM sterile filter unit (Millipore, Germany), aliquoted and frozen until analysis. Caco-2 (human colon adenocarcinoma) cells were obtained from the Rio de Janeiro Cell Bank (BCRJ, Brazil) and grown in 75-cm2 culture flasks with DMEM high-glucose medium supplemented with 10% fetal bovine serum, 0.1 nM non-essential amino acids, 50 μg/mL strepto- mycin, in a humid 5% CO2 atmosphere at 37 °C. CaCO-2 cells between passages 38 and 39 were used in the analysis of fecal water genotoxi- city. Upon reaching confluence, cells were harvested with Accutase cell detachment solution (Sigma Aldrich, USA), split into 1-mL centrifuged tubes and spun at 1200g for 1 min. The supernatant was removed and cells were directly incubated with 100% fecal water at 37 °C for 30 min. Cell viability was determined by the trypan blue exclusion assay. The remaining cell pellet was then mixed with 100 μL of low melting point agarose (0.75% in PBS), spread on slides pre-coated with normal point agarose (1.5% in PBS), and coverslipped. Fecal water genotoxicity in CaCO-2 cells was determined by the comet assay as described above. Serum biochemistry and tissue collection. Six animals from each group were sacrificed 24 h after the last DMH injection. Blood samples were collected by cardiac puncture under xy- lazine and ketamine anesthesia (10 mg/kg and 80 mg/kg bw, respec- tively). Serum alanine aminotransferase (ALT) and aspartate amino- transferase (AST) activity was determined using the COBAS 6000 (Roche Diagnostics, USA) with commercial kits. After laparotomy, colon and liver tissue fragments were collected and either stored at −80 °C for RNA extraction, or fixed in 4% buffered formalin and stored in 70% ethanol for histopathology and immunohistochemistry analyses. Immunohistochemistry analysis. Paraffin-embedded 5-μm-thick colon sections were deparaffinized and rehydrated in a graded xylene-alcohol series. Antigen retrieval was performed using a 10 nM sodium citrate buffer solution by pressure- cooker heating (Pascal, Dako). Endogenous peroxidase was quenched with 10% hydrogen peroxide solution for 10 min. Tissues sections were incubated with blocking solution (7% skimmed milk in PBS) for 1 h and then immunostained overnight with primary antibodies for Ki-67 (Abcam no. 15580) and active Caspase-3 (Abcam no. ab2302). Sections were washed three times in PBS and incubated with one-step universal HRP polymer (Easy Path, USA) for 25 min. Tissue sections were stained for 5 min using DAB as chromogen and counter-stained with Harry's hematoxylin for 1 min. Six rats from each group were analyzed and 25 crypts were scored per animal. Ki-67 and active Caspase-3 labeling indexes (LI) were scored by the number of positive-stained cells/ number of cells per crypt ratio. B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 94 RNA isolation and reverse transcription. Total RNA was extracted from frozen colon and liver samples using the Rneasy Mini kit (Qiagen, Hilden, Germany). Following on-column DNA digestion, RNA samples were solubilized in nuclease-free water (Qiagen, Hilden, Germany). RNA concentration and integrity were evaluated by spectrophotometry (NanoVue™ Plus, GE Healthcare Bio- Sciences Corp, Piscataway, NJ, EUA) and capillary electrophoresis (Agilent 2100 bioanalyzer, Agilent Technologies, Boeblingen, Germany), respectively. Total RNA (60 ng/μl) was reverse-transcribed to first-strand cDNA using SuperScript IV First Strand SuperMix (Invitrogen™, Life Tech, USA) according to the manufacturer's instruc- tion. Quantitative real-time PCR. RNA expression assessment was performed using a 96-well TaqMan® Array Cards (TAC)-based real-time polymerase chain reaction (PCR). A total of 96 genes involved in the oxidative metabolism, pro- and antioxidant activity, cell proliferation, DNA damage, DNA repair and apoptosis were assessed (Supplementary Material, SM1 and SM2). β-Actin, Gapdh, Gusb and Hprt1 were used as housekeeping genes to normalize mRNA expression. Target genes were amplified with TaqMan® Universal Mastermix II (Life Technologies, USA) using the following cycling protocol: heat activation at 50 °C for 1 min and de- naturation at 95 °C for 10 min followed by 40 cycles (95 °C for 15 s and 60 °C for 1 min). Fluorescence was detected using the QuantStudio™ 12 K Flex Real-Time PCR System (Life Technologies, USA). The relative expression of target genes was analyzed by the comparative Ct method (ExpressionSuite™ software, Life Technologies, USA). Functional en- richment analysis was conducted using the Gene Ontology annotation tool (Ashburner et al. 2000). This study was conducted according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR experiments) guidelines (Bustin et al. 2009). 2.4. – Mid-term assays Tumor volume and histopathology. Ten animals from each group were sacrificed 22 weeks after the last DMH administration. Colon specimens were removed, opened longitudinally, and pinned flat. The specimens were fixed in 10% phosphate-buffered formalin for 24 h and kept in ethanol 70% prior to analysis. Macroscopic tumors were counted, removed, and measured ex vivo using a digital caliper. Tumor volumes were calculated using the following prolate spheroid formula: 4/3 × 3.14 x (length/2) x (width/ 2) x (depth/2) (Schiavon et al. 2012). Colon specimens were paraffin embedded and sectioned for histopathological analysis. Adenocarci- nomas were classified into invasive (tubular or mucinous) or non-in- vasive (carcinoma in situ) according to the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (Nolte et al. 2016). Identification and quantification of ACF. Aberrant crypt foci (ACF) pre-neoplastic lesions were identified in formalin-fixed colon specimens (proximal, medial and distal) stained with 0.2% methylene blue. The total number of ACF and the number of aberrant crypts (AC) were counted under light microscopy. The ACF were identified topographically according to Bird's morphological cri- teria (Bird 1987): (i) increased size; (ii) thickened epithelial cell lining; (iii) increased pericryptal space and (iv) irregular lumens. Since ACF size is closely related to the risk of developing colon tumors, ACF were divided into 3 categories: 1–3 crypts/focus, 4–8 crypts/focus and ≥ 9 crypts/focus (Corpet and Taché 2002). 2.5. – Statistical analysis Data were statistically evaluated using the Prism v6 software (GraphPad). One-way ANOVA analysis followed by post hoc Tukey's test was used to compare groups. Fisher's exact test was used to compare tumor incidence and histopathological categories. To identify sig- nificant differences in gene expression, normalized expression means were compared using Student's t-test. Significance was set at p < 0.05. 3. Results 3.1. - Short-term assays Leukocyte and fecal water genotoxicity. Fig. 1. Schematic diagram of the experimental protocol. G1: DMH + corn oil (capsaicin vehicle); G2: DMH + capsaicin at 5 mg/kg bw; G3: DMH+ capsaicin at 50 mg/kg bw; G4: Na2EDTA (DMH vehicle) + capsaicin at 5 mg/kg bw; G5: Na2EDTA + capsaicin at 50 mg/kg bw; G6: Na2EDTA + corn oil. S1: sacrifice, 24 h after DMH initation; S2: sacrifice, 22 weeks after DMH initiation; DMH: 1,2-dimethylhidrazine; Na2EDTA: disodium ethylenediamine tetraacetic acid; IHC: immunohistochemistry; ACF: aberrant crypt foci. B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 95 Fig. 2 (A and B) shows the levels of DNA damage in peripheral blood leukocytes from all groups. DNA damage levels were significantly higher in the DMH-treated groups (G1-G3) than in their respective control groups (G4-G6) (p = 0.0001). DMH-induced genotoxicity was significantly reduced in the group receiving capsaicin at 50 mg/kg (G3) when compared to other DMH-treated groups (G1 and G2) (p = 0.0001). Capsaicin treatment per se (G5 and G6) did not induce DNA damage in comparison to the control group (G6). Fig. 2 (C and D) shows the effects of capsaicin oral administration on fecal water genotoxicity in all groups. CaCO-2 cell viability re- mained unchanged after exposure to fecal water from the DMH-treated and control groups (data not shown). Fecal water genotoxicity was significantly higher in the DMH-treated groups (G1-G3) than in their respective controls (G4-G6) (p = 0.004). DMH-induced fecal water genotoxicity was significantly reduced by capsaicin at 50 mg/kg (G3) (p = 0.004). Fecal water genotoxicity levels in capsaicin at 5 and 50 mg/kg groups (G4 and G5) remained similar to the control group (G6). Body weight, liver weight, food intake, biochemical and histopathological analyses. Table 1 shows body weight, liver weight and serum biochemical parameters in all groups at weeks 4 and 22. No differences in body weight and relative liver weight were found among the groups by the end of weeks 4 and 22 (Table 1). Over the first four weeks, there was a significant elevation in ALT and AST serum levels in the DMH-treated groups (G1-G3) (p < 0.0005). No differences in food intake and liver relative weight were observed among groups. In the DMH-treated groups (G1-G3), the colonic mucosa showed toxic lesions characterized by crypt distortions, depletion of goblet cells and increased apoptosis (Fig. 3A). Capsaicin oral administration alone (G4 and G5) did not in- duce colonic toxicity when compared to the control group (G6) (Fig. 3B). Ki-67 and active caspase-3 labeling indexes. As shown in Fig. 3C, Ki-67 proliferation index was significantly reduced (20%) with capsaicin at 50 mg/kg (G3) (p = 0.0001) when compared to DMH-treated groups (G1 and G2). Caspase-3 labeling in- dexes were similar among groups (Fig. 3D). Differential gene expression evaluation. Table 2 compares differential gene expression in colonic mucosa from the capsaicin-treated (G2 to G5) and control groups (G1 and G6). Three genes were differentially expressed in both groups receiving capsaicin 5 mg/kg (G2 and G4, Table 4) when compared to control groups (G1 and G6). Capsaicin at 50 mg/kg (G3) also induced the dif- ferential expression of 15 genes on colonic mucosa from the DMH- treated group (Table 2). Functional enrichment analysis demonstrated that these upregulated genes belong to functional categories involved in the adaptive response to chemicals, as well as apoptosis and tissue development (Table 3). No genes were found to be differentially ex- pressed in the liver in all groups (Supplementary Material, SM3 and SM4). 3.2. - Mid-term assays Tumor volume and histopathological analysis. In group receiving capsaicin at 50 mg/kg, (G3) the rate of small tumors (35%) was higher than those in the DMH-treated groups (G1, G2), but the statistical difference among groups was not significant (Fig. 4A and B). The average tumor volume was 105 mm3 in DMH- treated group (G1) whereas in the groups receiving capsaicin at 5 and 50 mg/kg (G2 and G3) it was 34 and 59 mm3, respectively. By the end of week 22, nearly all rats in the DMH-treated groups (G1-G3) devel- oped colorectal tumors. The tumor incidence and multiplicity were si- milar among groups as shown in Table 4. A trend towards the reduction of invasive tumors was observed in the group given capsaicin at 50 mg/ Figure 2. Detection of DNA damage by the comet assay1. (A) Suppressing effects of capsaicin administration on DMH-induced genotoxicity in peripheral blood leukocytes. (B) Representative comet images showing different levels of DNA damage in peripheral blood leukocytes. (C) Suppressing effects of capsaicin administration on DMH- induced fecal water genotoxicity in CaCO-2 tumor cells. (D) Representative comet images showing different levels of DNA damage in CaCO-2 cells. 1Data are presented as box plot with median and interquartile ranges, compared by One-way ANOVA followed by post hoc Tukey's test. G1: DMH + corn oil (capsaicin vehicle); G2: DMH+ capsaicin at 5 mg/kg bw; G3: DMH+ capsaicin at 50 mg/kg bw; G4: Na2EDTA (DMH vehicle) + capsaicin at 5 mg/kg bw; G5: Na2EDTA + capsaicin at 50 mg/kg bw; G6: Na2EDTA + corn oil. S: sacrifice; DMH: 1,2-dimethylhi- drazine; Na2EDTA: disodium ethylenediamine tetraacetic acid. B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 96 kg (G3) (Table 4). Histopathological analysis showed that most tumors were either well-differentiated tubular adenocarcinomas (Fig. 4C and D) or poorly-differentiated mucinous adenocarcinomas (Fig. 4E and F). ACF formation. Table 5 summarizes the effects of capsaicin on DMH-induced ACF formation. All DMH-treated animals (G1-G3) developed colon ACF 22 weeks after the last DMH administration. No ACF was observed in the control groups (G4-G6). Capsaicin at 50 mg/kg (G3) significantly reduced (0.0008 < p < 0.0209) the number of ACF consisting of 1–3, and ≥10 crypts per focus, as well as the total number of AC and ACF (0.0209 < p < 0.0244), when compared to DMH-treated group (G1). Fig. 4 shows light-micrographs of normal crypts (4G) and an ACF stained with methylene blue (4H). 4. Discussion In this study, capsaicin anti-genotoxicity, anti-proliferative and pro- apoptotic effects were investigated in rats, before and during DMH administration (short-term), as well as pre-neoplastic lesions and tumors 22 weeks after the last DMH injection (mid-term). The results obtained in the short-term assays show that capsaicin at 50 mg/kg suppressed DMH-induced cytotoxicity and genotoxicity, promoting anti-proliferative and pro-apoptotic responses through the expression of the genes involved in apoptosis, cell cycle suppression and cell/tissue differentiation on the colonic mucosa. In the mid-term assays, capsaicin at 50 mg/kg reduced ACF number and multiplicity. Our findings indicate that capsaicin at 50 mg/kg decreased both DMH-induced genotoxicity in the leukocytes and fecal water geno- toxicity in CaCO-2 cells. Both leukocyte and fecal water comet assay analyses demonstrate that capsaicin alone did not increase genotoxi- city. Previous studies have shown that capsaicin has substantial anti- mutagenic and anti-genotoxic effects on different chemical mutagens (Fernández-Bedmar and Alonso-Moraga 2016; Hassan et al. 2012; Huynh and Teel 2005). Most studies on capsaicin genotoxicity and mutagenicity have used capsaicin of different levels of purity or chili extracts (Bley et al. 2012). However, some studies have reported cap- saicin contamination with organic phosphates, pesticides, fusarium and aflatoxin, which can seriously affect genotoxicity assessment (Johnson Table 1 Body weight, liver weight, food intake and serum biochemical parameters in controls and capsaicin-treated rats1. 4 weeks (n= 6) G1 G2 G3 G4 G5 G6 Parameters DMH DMH + CAP 5 DMH + CAP 50 CAP 5 CAP 50 Control Initial body weight (g) 232.25 ± 19.34 225.25 ± 21.08 243.75 ± 20.65 220.75 ± 20.75 222.88 ± 24.17 236.63 ± 34.90 Final body weight (g) 299.94 ± 21.75 281.75 ± 35.05 311.88 ± 39.95 304.54 ± 20.65 307.31 ± 27.85 325.75 ± 41.16 Weight gain (g) 67.69 ± 10.06 61.29 ± 21.41 68.13 ± 28.22 83.92 ± 20.15 89.00 ± 17.02 89.13 ± 21.38 Food intake (g/rat/day) 19.91 ± 4.66 19.04 ± 4.82 21.50 ± 5.29 21.51 ± 2.70 21.79 ± 6.65 22.75 ± 2.66 Liver relative weight (g) 2.91 ± 0.25 2.99 ± 0.20 2.96 ± 0.44 2.91 ± 0.31 3.06 ± 0.28 2.75 ± 0.07 ALT (IU/L) 99.80 ± 28.69† 72.80 ± 18.77 79.20 ± 12.73 47.40 ± 9.48 55.60 ± 8.31 51.20 ± 16.34 AST (IU/L) 269.40 ± 85.95† 207.60 ± 28.88 262.80 ± 99.61† 131.80 ± 6.05 119.60 ± 24.18 126.20 ± 28.10 22 weeks (n= 10) Parameters DMH DMH + CAP 5 DMH + CAP 50 CAP 5 CAP 50 Control Initial body weight (g) 226.40 ± 18.22 223.20 ± 21.98 244.70 ± 25.51 221.57 ± 24.08 212.71 ± 15.93 241.33 ± 28.04 Final body weight (g) 445.60 ± 30.62 449.50 ± 51.08 468.70 ± 51.97 446.00 ± 35.02 438.00 ± 26.58 467.40 ± 34.66 Weight gain (g) 218.22 ± 19.88 220.38 ± 38.39 221.75 ± 37.21 198.40 ± 26.54 230.00 ± 34.91 228.00 ± 22.83 Liver relative weight (g) 1.97 ± 0.15 2.27 ± 0.37 2.04 ± 0.15 2.21 ± 0.28 2.11 ± 0.13 2.12 ± 0.25 1 Values represent the mean ± SD for 6–10 rats/group. Differences between groups were determined using one-way ANOVA followed by Tukey's test. †Different from G4, G5 and G6, p < 0.0005. ALT: alanine aminotransferase; AST: aspartate aminotransferase; DMH: 1,2-dimethylhydrazine; CAP 5: capsaicin 5 mg/kg bw; CAP 50: capsaicin 50 mg/kg bw. Fig. 3. Histopathology and immunohistochemistry of co- lonic mucosa in the short-term (4 weeks) assay (A) DMH- induced toxic lesions in the colonic mucosa, exhibiting crypt distortions (*), depletion of goblet cells, and increased apoptosis. (C) Ki-67 proliferation labeling indexes. (B) Normal histology features of the colon in control group. (D) Active caspase-3 apoptosis labeling indexes. 1Data are presented as mean ± SD for 6 rats/group. Differences be- tween groups were determined using by One-way ANOVA followed by post hoc Tukey's test. G1: DMH+ corn oil (capsaicin vehicle); G2: DMH+ capsaicin at 5 mg/kg bw; G3: DMH + capsaicin at 50 mg/kg bw; G4: Na2EDTA (DMH vehicle) + capsaicin at 5 mg/kg bw; G5: Na2EDTA + capsaicin at 50 mg/kg bw; G6: Na2EDTA + corn oil. S: sacrifice; DMH: 1,2-dimethylhi- drazine; Na2EDTA: disodium ethylenediamine tetraacetic acid. B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 97 2007; Kuzma et al. 2014; Proudlock et al. 2004). Together, our results suggest that capsaicin has an anti-genotoxic effect and may inhibit the DNA damage induced by DMH, as previously demonstrated by other (De et al. 1995; Melgar-Lalanne et al. 2017; Proudlock et al. 2004). This study showed that oral administration of capsaicin increased the expression of the genes NF-κB and Ikbkg, which is a regulatory subunit of the kappaB kinase (NEMO/IKKγ) complex that phosphor- ylates and activates NF-κB (Salminen et al. 2012). Methyldiazonium ion is the ultimate DMH carcinogenic metabolite responsible for the me- thylation of DNA bases that induces genotoxic stress and trigger NF-κB activation in colonic epithelial cells (Perše and Cerar 2011; Tanwar et al. 2009). Nuclear factor kappa B (NF-κB), an important mediator of cell response to DNA damage, has been shown to facilitate cell escape from the letal effects of DNA damage, stimulate cell growth, and induce cell proliferation (Hoesel and Schmid 2013). Conversely, capsaicin also induced the expression of NF-κB inhbitors, such as the genes Mapk3 Mapk14, and Smad4. Mapk14, also known as p38α, is a serine/threo- nine stress-activated protein kinase that is activated in response to a variety of extracellular stimuli, including genotoxic stress induced by chemicals (Igea and Nebreda 2015), promoting apoptosis and NF-κB regulation (Gil-Araujo et al. 2014; Igea and Nebreda 2015; Olson et al. 2007). In the colon, Smad4 downregulation leads to uncontrolled cell proliferation (Dienstmann et al. 2017; Handra-Luca et al. 2011). Ac- cording to our results, capsaicin 50 mg/kg suppressed Ki-67 prolifera- tion indexes under carcinogen insult. This finding is consistent with the concomitant expression of the Mapk3, Mapk14 and Smad4 genes that are involved in the suppression of NF-κB activation, cell growth and proliferation (Aggarwal and Shishodia 2004; Brown et al. 2010; Qian et al. 2016). The oral administration of capsaicin in this study markedly induced the expression of apoptosis-related genes in the colonic mucosa, in- cluding Casp4, Sp1, Aifm1 and Dffb. Capsaicin-induced apoptosis has been reported to cause ER calcium release and to increase the tran- scriptional activation of pro-apoptotic genes (O'Neill et al. 2012; Srivastava 2013; Thomas et al. 2011) such as Aifm, an important ef- fector for caspase-independent cell death (Tica Sedlar et al. 2016). Aifm encodes an apoptosis-inducing factor (AIF) that functions as an oxi- doreductase in the inner mitochondrial membrane (Sun et al. 2016). Upon cell death stimuli, increased cytoplasmic calcium concentration causes the disruption of the mitochondrial membrane, leading to AIF translocation to the nucleus (Daugas et al. 2000). AIF is binds to the DNA, causing chromatin condensation and DNA fragmentation re- gardless of caspase activation (Cregan et al. 2004). Both doses of cap- saicin also increased the expression of the Dffb gene. Dffb encodes the active subunit of the apoptotic nuclease DNA fragmentation factor (DFF), a heterodimeric protein that triggers both DNA fragmentation and chromatin condensation during apoptosis (Samejima and Earnshaw 2005). DNA fragmentation factors such as DFF, greatly contribute to genomic stability by ensuring the removal of DNA-damaged cells (Ohyashiki et al. 2017; Yan et al. 2006). The functional enrichment analysis revealed that capsaicin oral administration up-regulated the expression of the genes associated with tissue development and cell differentiation. This finding may be the molecular clue to the chemopreventive effect of capsaicin on DMH-in- duced colonic mucosa toxicity. Histopathological analysis showed that DMH exposure induced apoptosis, loss of goblet cell differentiation and crypt distortions on the colonic mucosa. Indeed, tissue loss is replaced via compensatory cell proliferation following chemical insult and sig- nificant cell death, (Meier and Banreti 2016). Moreover, capsaicin oral administration has been shown to modulate DMH-induced cell pro- liferation by increasing the expression of anti-proliferative and cell differentiation genes. Our findings indicate an increased expression of the Foxa1 and Cdh1 genes in the group receiving capsaicin at 50 mg/kg. This is consistent with a cellular response towards cell differentiation because Foxa1 is known to play a pivotal role in postnatal development and cell differ- entiation (Bernardo and Keri 2012). In the colon, Foxa1 modulates the secretory activity and controls the differentiation of goblet cells (Ye and Kaestner 2009). Another important gene associated with cell differ- entiation is Cdh1. Cdh1 encodes E-cadherin, a cell-cell adhesion gly- coprotein that plays a leading role in the suppression of cell growth and Table 2 Differential gene expression in the colon samples of capsaicin-treated rats1. Comparisons Gene Ensembl ID Fold Change P value G2 vs G1 Dffb ENSRNOG00000025030 1.532 0.022 Gsk3b ENSRNOG00000002833 1.627 0.041 Raf1 ENSRNOG00000010153 1.781 0.021 G3 vs G1 Dffb ENSRNOG00000025030 1.548 0.016 Casp4 ENSRNOG00000033697 2.094 0.010 Aifm1 ENSRNOG00000006067 1.689 0.030 Wee1 ENSRNOG00000010017 1.553 0.006 Sp1 ENSRNOG00000014084 1.769 0.004 Foxa-1 ENSRNOG00000009284 1.761 0.002 Cdh1 ENSRNOG00000020151 1.951 0,032 Smad4 ENSRNOG00000051965 1.549 0.034 Grb2 ENSRNOG00000037360 1.940 0.006 Raf1 ENSRNOG00000010153 2.046 0.036 Mapk3 ENSRNOG00000053583 2.229 0.028 Mapk14 ENSRNOG00000000513 1.867 0.032 Nfkb1 ENSRNOG00000023258 1.851 0.032 Stat5b ENSRNOG00000019075 1.583 0.020 Ikbkg ENSRNOG00000060936 1.786 0.016 G4 vs G6 Igfr1 ENSRNOG00000014187 0.569 0.032 Akt1 ENSRNOG00000028629 0.652 0.019 CdkN1a ENSRNOG00000000521 1.933 0.018 1 Relative expression levels were determined by normalization to beta-actin (Actb), glyceraldeyde-3-phosphate dehydrogenase (Gapdh), beta-glucuronidase (Gusb) and hy- poxanthine-guanine phosphoribosyltransferase (Hprt1). Experimental groups were com- pared using the Student's t-test. Fold change boundary of 1.5 (1.5-fold change) and a P value of< 0.05 were used. G1: DMH+ corn oil (capsaicin vehicle); G2: DMH + cap- saicin 5 mg/kg bw; G3: DMH + capsaicin at 50 mg/kg bw; G4: Na2EDTA (DMH vehicle) + capsaicin at 5 mg/kg bw; G5: Na2EDTA + capsaicin at 50 mg/kg bw; G6: Na2EDTA + corn oil. S: sacrifice; DMH: 1,2-dimethylhidrazine; Na2EDTA: disodium ethylenediamine tetraacetic acid. Table 3 Significantly enriched gene ontology (GO) annotated terms in up-regulated genes of rats treated with capsaicin at 50 mg/kg (G3). S. No. GO term Fold Enrichment No. of Genes P value 1 GO:0070887 - cellular response to chemical stimulus 7.68 13 0.00002 2 GO:0033554 - cellular response to stress 9.10 10 0.00016 3 GO:0006915 - apoptotic process 17.43 9 0.00004 4 GO:0050790 - regulation of catalytic activity 7.00 11 0.00310 5 GO:0070848 - response to growth factor 23.13 9 0.00001 6 GO:0080134 - regulation of response to stress 9.02 8 0.00812 7 GO:0006974 - cellular response to DNA damage 13.32 6 0.02930 8 GO:0010941 - regulation of cell death 9.30 11 0.00018 9 GO:0009888 - tissue development 7.30 9 0.00760 10 GO:0030154 - cell differentiation 5.57 14 0.00015 B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 98 invasion. E-cadherin loss is an integral step in the epithelial-mesench- ymal transition (EMT), that is associated with tumor progression, in- vasion and metastasis in CRC (Heerboth et al. 2015; Yun et al. 2014). Conversely, increased E-cadherin expression has been shown to de- crease ERK1/2 phosphorylation, suggesting a suppressor role in the Kras oncogenic pathway (Satow et al. 2014). Our results show that capsaicin failed to modulate the expression of the genes involved in liver oxidative metabolism, pro- and anti-oxida- tive activity, cell proliferation, DNA damage, DNA repair and apoptosis (Supplementary Data, SM2). Although capsaicin has been hypothesized to interact with a number of cytochrome P450 enzymes (CYPs) in the liver (Zhang et al. 2012), studies have demonstrated that the in vitro inhibition of cytochrome P450 enzymes by capsaicin can be observed only at very high doses, suggesting that capsaicin inhibitory effect on Figure 4. Tumor volume, histopathology and aberrant crypt foci (ACF) induced by DMH in control and capsaicin- treated rats. (A) Percentage of small, medium and large tumor volumes in the DMH-treated groups. (B) Macroscopic image of colon carcinomas in the medial region of the co- lonic mucosa. (C) Sessile, exophytic tumor mass arising from the colonic mucosa. (D) Tubular adenocarcinoma. (E) Endophytic tumor with extensive submucosal spread. (F) Mucinous adenocarcinoma with signet ring cells. (G) Normal-appearing colonic mucosa stained with methylene blue. (H) Methylene blue-stained aberrant crypt foci (ACF) consisting of seven large, elliptical crypts with thickened epithelial cell lining and increased pericryptal space. G1: DMH + corn oil (capsaicin vehicle); G2: DMH+ capsaicin at 5 mg/kg bw; G3: DMH+ capsaicin at 50 mg/kg bw; DMH: 1,2-dimethylhidrazine. (For interpretation of the re- ferences to colour in this figure legend, the reader is re- ferred to the web version of this article.) B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 99 drug metabolism is minimal (Chanda et al. 2008; Babbar et al. 2010). Thus, the protective effects of the capsaicin regimen adopted did not affect DMH metabolism via either CYP induction or inhibition. In this study, capsaicin reduced total AC and ACF development, as well as ACF multiplicity, in agreement with other report (Yoshitani et al. 2001). ACF have been adopted as biomarkers for the screening of preventive agents and has been correlated with tumor growth in dif- ferent models of colon carcinogenesis (Rodrigues et al. 2002; Wargovich et al. 2010). ACF with a high number of aberrant crypts are more likely to progress to adenomas and adenocarcinomas during col- orectal carcinogenesis (Takahashi et al. 2012). In this regard, we ob- served that capsaicin at 50 mg/kg trend to reduce the tumor size as well as the number of invasive tumors in the colon. Despite these differ- ences, however, tumor incidence and multiplicity rates were similar in the capsaicin-treated groups. These results demonstrate that capsaicin has a weak protective effect for colon tumors induced by DMH. Therefore, chemopreventive potential of capsaicin was evidenced only shortly after DMH administration and latter on ACF development. 5. Conclusion Capsaicin administration reduced cell proliferation as well as modulated the genes involved in cell proliferation, apoptosis, tissue development and differentiation, suppressing ACF development. Thus, our results indicate that capsaicin may have a chemopreventive effect against DMH-induced colorectal carcinogenesis. Acknowledgments This work was supported by the Sao Paulo Research Foundation (FAPESP) and the National Council for Scientific and Technological Development (CNPq) under grants 2014/21951-6, 2014/24762-0 and 304128/2015-5, respectively. We thank Paulo Cesar Georgete for technical support and Mariza Branco da Silva for English editing of this paper. Author contributions All authors contributed equally to this work. Conflicts of interest The authors declare no conflict of interest. Supplementary data. Supplementary material 1. Supplementary material 2. Supplementary material 3. Supplementary material 4. References Aggarwal, B.B., Shishodia, S., 2004. Suppression of the nuclear factor-κB activation pathway by spice-derived phytochemicals: reasoning for seasoning. Ann. N. Y. Acad. Sci. 1030, 434–441. http://dx.doi.org/10.1196/annals.1329.054. Agrawal, R.C., Wiessler, M., Hecker, E., Bhide, S.V., 1986. Tumour-promoting effect of chilli extract in BALB/c mice. Int. J. Cancer 38, 689–695. Ashburner, M., Ball, C.A., Blake, J.A., Botstein, D., Butler, H., Cherry, J.M., Davis, A.P., Dolinski, K., Dwight, S.S., Eppig, J.T., Harris, M.A., Hill, D.P., Issel-Tarver, L., Kasarskis, A., Lewis, S., Matese, J.C., Richardson, J.E., Ringwald, M., Rubin, G.M., Sherlock, G., 2000. Gene ontology: tool for the unification of biology. Nat. Genet. 25, 25–29. http://dx.doi.org/10.1038/75556. Babbar, S., Chanda, S., Bley, K., 2010. Inhibition and induction of human cytochrome P450 enzymes in vitro by capsaicin. Xenobiotica 40, 807–816. http://dx.doi.org/10. 3109/00498254.2010.520044. Baena, R., Salinas, P., 2015. Diet and colorectal cancer. Maturitas 80, 258–264. http://dx. doi.org/10.1016/j.maturitas.2014.12.017. Bernardo, G.M., Keri, R.A., 2012. FOXA1: a transcription factor with parallel functions in development and cancer. Biosci. Rep. 32, 113–130. http://dx.doi.org/10.1042/ BSR20110046. Bird, R.P., 1987. Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen: preliminary findings. Cancer Lett. 37, 147–151. Bley, K., Boorman, G., Mohammad, B., McKenzie, D., Babbar, S., 2012. A comprehensive review of the carcinogenic and Anticarcinogenic potential of capsaicin. Toxicol. Pathol. 40, 847–873. http://dx.doi.org/10.1177/0192623312444471. Bode, A.M., Dong, Z., 2011. The two faces of capsaicin. Cancer Res. 71, 2809–2814. http://dx.doi.org/10.1158/0008-5472.CAN-10-3756. Bosland, P.W., Votava, E.J., Votava, E.M., 2012. Peppers: Vegetable and Spice Capsicums. CABI. Brown, K.C., Witte, T.R., Hardman, W.E., Luo, H., Chen, Y.C., Carpenter, A.B., Lau, J.K., Dasgupta, P., 2010. Capsaicin displays anti-proliferative activity against human small Table 4 Incidence and multiplicity of various tumors induced by DMH in control and capsaicin-treated rats1. Groups/Treatments Number of animals Number of tumors Multiplicity Incidence (%) Tubular Adenocarcinoma Carcinoma in situ Mucinous Adenocarcinoma (G1) DMH 10 15 1.67 ± 1.32 72.72 9.10 18.18 (G2) DMH + CAP 5 10 11 2.20 ± 0.84 61.53 15.40 23.07 (G3) DMH + CAP 50 10 13 1.86 ± 1.07 71.43 28.57 0 1 Multiplicity is the average number of all tumors in each tumor-bearing mouse. Multiplicity values are represented as the mean ± SD. Tumor incidence is the percentage of mice bearing the indicated type of tumor. Incidence values are represented as percentage, compared by the Fisher's exact test (p = 0.20). DMH: 1,2-dimethylhydrazine; CAP 5: capsaicin 5 mg/ kg bw; CAP 50: capsaicin 50 mg/kg bw. Table 5 Inhibitory effects of capsaicin treatment on the number of aberrant crypt foci pre-neoplastic lesions1. Groups/Treatments2 No. of animals Number of ACF Total Number 1–3 crypts 4–9 crypts ≥ 10 crypts AC3 ACF AC/ACF (G1) DMH 10 170.10 ± 55.47 110.70 ± 43.47 17.90 ± 13.54 1230.00 ± 375.74 311.30 ± 57.80 3.91 ± 0.71 (G2) DMH+ CAP 5 10 156.70 ± 54.05 140.10 ± 39.63 13.40 ± 7.99 1260.60 ± 370.90 309.90 ± 89.91 4.06 ± 0.35 (G3) DMH+ CAP 50 10 106.90 ± 35.04a 75.00 ± 18.98b 2.60 ± 1.65a,b 660.60 ± 147.52a,b 184.60 ± 44.92a,b 3.62 ± 0.36 (G4) CAP 5 7 0 0 0 0 0 0 (G5) CAP 50 7 0 0 0 0 0 0 (G6) Control 7 0 0 0 0 0 0 1 Values represent the mean ± SD for 7–10 rats/group. Differences between groups were determined using one-way ANOVA followed by Tukey's test. aDifferent from G1, 0.0008 < p < 0.0209. bDifferent from G2, 0.0008 < p < 0,0244. ACF: aberrant crypt foci; AC: aberrant crypt; DMH: 1,2-dimethylhydrazine; CAP 5: capsaicin 5 mg/kg bw; CAP 50: capsaicin 50 mg/kg. B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 100 http://dx.doi.org/10.1196/annals.1329.054 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0010 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0010 http://dx.doi.org/10.1038/75556 http://dx.doi.org/10.3109/00498254.2010.520044 http://dx.doi.org/10.3109/00498254.2010.520044 http://dx.doi.org/10.1016/j.maturitas.2014.12.017 http://dx.doi.org/10.1016/j.maturitas.2014.12.017 http://dx.doi.org/10.1042/BSR20110046 http://dx.doi.org/10.1042/BSR20110046 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0035 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0035 http://dx.doi.org/10.1177/0192623312444471 http://dx.doi.org/10.1158/0008-5472.CAN-10-3756 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0050 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0050 cell lung cancer in cell culture and nude mice models via the E2F pathway. PLoS One 5. http://dx.doi.org/10.1371/journal.pone.0010243. Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622. http://dx.doi.org/10.1373/clinchem.2008. 112797. Carr, P.R., Walter, V., Brenner, H., Hoffmeister, M., 2016. Meat subtypes and their as- sociation with colorectal cancer: systematic review and meta-analysis. Int. J. Cancer 138, 293–302. http://dx.doi.org/10.1002/ijc.29423. Chanda, S., Bashir, M., Babbar, S., Koganti, A., Bley, K., 2008. In vitro hepatic and skin metabolism of capsaicin. Drug Metab. Dispos. Biol. Fate Chem. 36, 670–675. http:// dx.doi.org/10.1124/dmd.107.019240. Corpet, D.E., Taché, S., 2002. Most effective colon cancer chemopreventive agents in rats: a systematic review of aberrant crypt foci and tumor data, ranked by potency. Nutr. Cancer 43, 1–21. Cregan, S.P., Dawson, V.L., Slack, R.S., 2004. Role of AIF in caspase-dependent and caspase-independent cell death. Oncogene 23, 2785–2796. http://dx.doi.org/10. 1038/sj.onc.1207517. Dahham, S.S., Majid, A.M.A., 2016. The impact of life style and nutritional components in primary prevention of colorectal cancer. J. Appl. Pharm. Sci. 6, 237–244. Daugas, E., Nochy, D., Ravagnan, L., Loeffler, M., Susin, S.A., Zamzami, N., Kroemer, G., 2000. Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis. FEBS Lett. 476, 118–123. http://dx.doi.org/10.1016/S0014- 5793(00)01731-2. De, A.K., Agarwal, K., Mukherjee, A., Sengupta, D., 1995. Inhibition by capsaicin against cyclophosphamide-induced clastogenicity and DNA damage in mice. Mutat. Res. 335, 253–258. Díaz Barriga Arceo, S., Madrigal-Bujaidar, E., Calderón Montellano, E., Ramírez Herrera, L., Díaz García, B.D., 1995. Genotoxic effects produced by capsaicin in mouse during subchronic treatment. Mutat. Res. 345, 105–109. Díaz-Laviada, I., 2010. Effect of capsaicin on prostate cancer cells. Future Oncol. Lond. Engl. 6, 1545–1550. http://dx.doi.org/10.2217/fon.10.117. Dienstmann, R., Vermeulen, L., Guinney, J., Kopetz, S., Tejpar, S., Tabernero, J., 2017. Consensus molecular subtypes and the evolution of precision medicine in colorectal cancer. Nat. Rev. Cancer advance online publication. http://dx.doi.org/10.1038/nrc. 2016.126. Fattori, V., Hohmann, M.S.N., Rossaneis, A.C., Pinho-Ribeiro, F.A., Verri, W.A., 2016. Capsaicin: current understanding of its mechanisms and therapy of pain and other pre-clinical and clinical uses. Molecules 21, 844. http://dx.doi.org/10.3390/ molecules21070844. Ferlay, J., Soerjomataram, I., Dikshit, R., Eser, S., Mathers, C., Rebelo, M., Parkin, D.M., Forman, D., Bray, F., 2015. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int. J. Cancer 136, E359–386. http://dx.doi.org/10.1002/ijc.29210. Fernández-Bedmar, Z., Alonso-Moraga, A., 2016. vivo and in vitro evaluation for nu- traceutical purposes of capsaicin, capsanthin, lutein and four pepper varieties. Food Chem. Toxicol. 98, Part B 89–99. http://dx.doi.org/10.1016/j.fct.2016.10.011. Garufi, A., Pistritto, G., Cirone, M., D'Orazi, G., 2016. Reactivation of mutant p53 by capsaicin, the major constituent of peppers. J. Exp. Clin. Cancer Res. CR 35, 136. http://dx.doi.org/10.1186/s13046-016-0417-9. Gil-Araujo, B., Toledo Lobo, M.-V., Gutiérrez-Salmerón, M., Gutiérrez-Pitalúa, J., Ropero, S., Angulo, J.C., Chiloeches, A., Lasa, M., 2014. Dual specificity phosphatase 1 ex- pression inversely correlates with NF-κB activity and expression in prostate cancer and promotes apoptosis through a p38 MAPK dependent mechanism. Mol. Oncol. 8, 27–38. http://dx.doi.org/10.1016/j.molonc.2013.08.012. Gingras, D., Béliveau, R., 2011. Colorectal cancer prevention through dietary and lifestyle modifications. Cancer Microenviron. 4, 133–139. http://dx.doi.org/10.1007/ s12307-010-0060-5. Handra-Luca, A., Olschwang, S., Fléjou, J.-F., 2011. SMAD4 protein expression and cell proliferation in colorectal adenocarcinomas. Virchows arch. Int. J. Pathol. 459, 511–519. http://dx.doi.org/10.1007/s00428-011-1152-4. Hassan, M.H., Edfawy, M., Mansour, A., Hamed, A.-A., 2012. Antioxidant and anti- apoptotic effects of capsaicin against carbon tetrachloride-induced hepatotoxicity in rats. Toxicol. Ind. Health 28, 428–438. http://dx.doi.org/10.1177/ 0748233711413801. Heerboth, S., Housman, G., Leary, M., Longacre, M., Byler, S., Lapinska, K., Willbanks, A., Sarkar, S., 2015. EMT and tumor metastasis. Clin. Transl. Med. 4, 6. http://dx.doi. org/10.1186/s40169-015-0048-3. Heiser, C.B., Smith, P.G., 1953. The cultivated capsicum peppers. Econ. Bot. 7, 214–227. http://dx.doi.org/10.1007/BF02984948. Hoesel, B., Schmid, J.A., 2013. The complexity of NF-κB signaling in inflammation and cancer. Mol. Cancer 12, 86. http://dx.doi.org/10.1186/1476-4598-12-86. Hou, N., Huo, D., Dignam, J.J., 2013. Prevention of colorectal cancer and dietary man- agement. Chin. Clin. Oncol. 2, 13. http://dx.doi.org/10.3978/j.issn.2304-3865.2013. 04.03. Huynh, H.T., Teel, R.W., 2005. Vitro Antimutagenicity of Capsaicin toward Heterocyclic Amines in Salmonella Typhimurium Strain TA98. Anticancer Res. Vol. 25. pp. 117–120. Igea, A., Nebreda, A.R., 2015. The stress kinase p38α as a target for cancer therapy. Cancer Res. 75, 3997–4002. http://dx.doi.org/10.1158/0008-5472.CAN-15-0173. Johnson, W.J., 2007. Final report on the safety assessment of capsicum Annuum extract, capsicum Annuum fruit extract, capsicum Annuum resin, capsicum Annuum fruit powder, capsicum Frutescens fruit, capsicum Frutescens fruit extract, capsicum Frutescens resin, and capsaicin. Int. J. Toxicol. 26, 3–106. http://dx.doi.org/10. 1080/10915810601163939. Kamangar, F., Dores, G.M., Anderson, W.F., 2006. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J. Clin. Oncol. 24, 2137–2150. http:// dx.doi.org/10.1200/JCO.2005.05.2308. Kantar, M.B., Anderson, J.E., Lucht, S.A., Mercer, K., Bernau, V., Case, K.A., Le, N.C., Frederiksen, M.K., DeKeyser, H.C., Wong, Z.-Z., Hastings, J.C., Baumler, D.J., 2016. Vitamin variation in capsicum Spp. provides opportunities to improve nutritional value of human diets. PLoS One 11, e0161464. http://dx.doi.org/10.1371/journal. pone.0161464. Klinder, A., Karlsson, P.C., Clune, Y., Hughes, R., Glei, M., Rafter, J.J., Rowland, I., Collins, J.K., Pool-Zobel, B.L., 2007. Fecal water as a non-invasive biomarker in nutritional intervention: comparison of preparation methods and refinement of dif- ferent endpoints. Nutr. Cancer 57, 158–167. http://dx.doi.org/10.1080/ 01635580701274848. Kuzma, M., Past, T., Mozsik, G., Perjesi, P., 2014. Pharmacobotanical analysis and reg- ulatory qualification of Capsicum fruits and Capsicum extracts. A survey. doi:https:// doi.org/10.5772/58812. Lau, J.K., Brown, K.C., Dom, A.M., Witte, T.R., Thornhill, B.A., Crabtree, C.M., Perry, H.E., Brown, J.M., Ball, J.G., Creel, R.G., Damron, C.L., Rollyson, W.D., Stevenson, C.D., Hardman, W.E., Valentovic, M.A., Carpenter, A.B., Dasgupta, P., 2014. Capsaicin induces apoptosis in human small cell lung cancer via the TRPV6 receptor and the calpain pathway. Apoptosis Int. J. Program. Cell Death 19, 1190–1201. http://dx.doi.org/10.1007/s10495-014-1007-y. Lee, B.M., Park, K.-K., 2003. Beneficial and adverse effects of chemopreventive agents. Mutat. Res. 523–524, 265–278. Liu, R.H., 2013. Health-promoting components of fruits and vegetables in the diet. Adv. Nutr. Int. Rev. J. 4, 384S–392S. http://dx.doi.org/10.3945/an.112.003517. Liu, Z., Zhu, P., Tao, Y., Shen, C., Wang, S., Zhao, L., Wu, H., Fan, F., Lin, C., Chen, C., Zhu, Z., Wei, Z., Sun, L., Liu, Y., Wang, A., Lu, Y., 2015. Cancer-promoting effect of capsaicin on DMBA/TPA-induced skin tumorigenesis by modulating inflammation, Erk and p38 in mice. Food Chem. Toxicol. 81, 1–8. http://dx.doi.org/10.1016/j.fct. 2015.04.002. Meier, P., Banreti, A., 2016. Tissue repair: how to inflame your Neighbours. Curr. Biol. 26, R192–R194. http://dx.doi.org/10.1016/j.cub.2016.01.033. Melgar-Lalanne, G., Hernández-Álvarez, A.J., Jiménez-Fernández, M., Azuara, E., 2017. Oleoresins from capsicum spp.: extraction methods and bioactivity. Food Bioprocess Technol. 10, 51–76. http://dx.doi.org/10.1007/s11947-016-1793-z. Nandhakumar, S., Parasuraman, S., Shanmugam, M.M., Rao, K.R., Chand, P., Bhat, B.V., 2011. Evaluation of DNA damage using single-cell gel electrophoresis (comet assay). J. Pharmacol. Pharmacother. 2, 107–111. http://dx.doi.org/10.4103/0976-500X. 81903. Nolte, T., Brander-Weber, P., Dangler, C., Deschl, U., Elwell, M.R., Greaves, P., Hailey, R., Leach, M.W., Pandiri, A.R., Rogers, A., Shackelford, C.C., Spencer, A., Tanaka, T., Ward, J.M., 2016. Nonproliferative and proliferative lesions of the gastrointestinal tract, pancreas and salivary glands of the rat and mouse. J. Toxicol. Pathol. 29, 1S–125S. http://dx.doi.org/10.1293/tox.29.1S. O'Neill, J., Brock, C., Olesen, A.E., Andresen, T., Nilsson, M., Dickenson, A.H., 2012. Unravelling the mystery of capsaicin: a tool to understand and treat pain. Pharmacol. Rev. 64, 939–971. http://dx.doi.org/10.1124/pr.112.006163. Ohyashiki, K., Kuroda, M., Ohyashiki, J.H., 2017. Chromosomes and Chromosomal Instability in Human Cancer. In: Coleman, W.B., Tsongalis, G.J. (Eds.), The Molecular Basis of Human Cancer. Springer New York, pp. 241–262. http://dx.doi.org/10. 1007/978-1-59745-458-2_15. Olson, C.M., Hedrick, M.N., Izadi, H., Bates, T.C., Olivera, E.R., Anguita, J., 2007. p38 mitogen-activated protein kinase controls NF-kappaB transcriptional activation and tumor necrosis factor alpha production through RelA phosphorylation mediated by mitogen- and stress-activated protein kinase 1 in response to Borrelia burgdorferi antigens. Infect. Immun. 75, 270–277. http://dx.doi.org/10.1128/IAI.01412-06. Perše, M., Cerar, A., 2011. Morphological and molecular alterations in 1,2 Dimethylhydrazine and Azoxymethane induced colon carcinogenesis in rats. J Biomed Biotechnol 2011. http://dx.doi.org/10.1155/2011/473964. Proudlock, R., Thompson, C., Longstaff, E., 2004. Examination of the potential geno- toxicity of pure capsaicin in bacterial mutation, chromosome aberration, and rodent micronucleus tests. Environ. Mol. Mutagen. 44, 441–447. http://dx.doi.org/10.1002/ em.20072. Qian, K., Wang, G., Cao, R., Liu, T., Qian, G., Guan, X., Guo, Z., Xiao, Y., Wang, X., 2016. Capsaicin suppresses cell proliferation, induces cell cycle arrest and ROS production in bladder cancer cells through FOXO3a-mediated pathways. Mol. Basel Switz. 21. http://dx.doi.org/10.3390/molecules21101406. Rodrigues, M. a. M., Silva, L. a. G., Salvadori, D.M.F., de Camargo, J.L.V., Montenegro, M. R., 2002. Aberrant crypt foci and colon cancer: comparison between a short- and medium-term bioassay for colon carcinogenesis using dimethylhydrazine in Wistar rats. Braz. J. Med. Biol. Res. 35, 351–355. doi:https://doi.org/10.1590/S0100- 879X2002000300010. Rubio, C.A., 2017. Corrupted colonic crypt fission in carcinogen-treated rats. PLoS One 12. http://dx.doi.org/10.1371/journal.pone.0172824. Saito, A., Yamamoto, M., 1996. Acute oral toxicity of capsaicin in mice and rats. J. Toxicol. Sci. 21, 195–200. Sales, N.M.R., Pelegrini, P.B., Goersch, M.C., 2014. Nutrigenomics: definitions and ad- vances of this new science. J. Nutr. Metab. 2014, e202759. http://dx.doi.org/10. 1155/2014/202759. Salminen, A., Kauppinen, A., Kaarniranta, K., 2012. Emerging role of NF-κB signaling in the induction of senescence-associated secretory phenotype (SASP). Cell. Signal. 24, 835–845. http://dx.doi.org/10.1016/j.cellsig.2011.12.006. Samejima, K., Earnshaw, W.C., 2005. Trashing the genome: the role of nucleases during apoptosis. Nat. Rev. Mol. Cell Biol. 6, 677–688. http://dx.doi.org/10.1038/nrm1715. B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 101 http://dx.doi.org/10.1371/journal.pone.0010243 http://dx.doi.org/10.1373/clinchem.2008.112797 http://dx.doi.org/10.1373/clinchem.2008.112797 http://dx.doi.org/10.1002/ijc.29423 http://dx.doi.org/10.1124/dmd.107.019240 http://dx.doi.org/10.1124/dmd.107.019240 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0075 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0075 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0075 http://dx.doi.org/10.1038/sj.onc.1207517 http://dx.doi.org/10.1038/sj.onc.1207517 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0085 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0085 http://dx.doi.org/10.1016/S0014-5793(00)01731-2 http://dx.doi.org/10.1016/S0014-5793(00)01731-2 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0095 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0095 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0095 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0100 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0100 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0100 http://dx.doi.org/10.2217/fon.10.117 http://dx.doi.org/10.1038/nrc.2016.126 http://dx.doi.org/10.1038/nrc.2016.126 http://dx.doi.org/10.3390/molecules21070844 http://dx.doi.org/10.3390/molecules21070844 http://dx.doi.org/10.1002/ijc.29210 http://dx.doi.org/10.1016/j.fct.2016.10.011 http://dx.doi.org/10.1186/s13046-016-0417-9 http://dx.doi.org/10.1016/j.molonc.2013.08.012 http://dx.doi.org/10.1007/s12307-010-0060-5 http://dx.doi.org/10.1007/s12307-010-0060-5 http://dx.doi.org/10.1007/s00428-011-1152-4 http://dx.doi.org/10.1177/0748233711413801 http://dx.doi.org/10.1177/0748233711413801 http://dx.doi.org/10.1186/s40169-015-0048-3 http://dx.doi.org/10.1186/s40169-015-0048-3 http://dx.doi.org/10.1007/BF02984948 http://dx.doi.org/10.1186/1476-4598-12-86 http://dx.doi.org/10.3978/j.issn.2304-3865.2013.04.03 http://dx.doi.org/10.3978/j.issn.2304-3865.2013.04.03 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0175 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0175 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0175 http://dx.doi.org/10.1158/0008-5472.CAN-15-0173 http://dx.doi.org/10.1080/10915810601163939 http://dx.doi.org/10.1080/10915810601163939 http://dx.doi.org/10.1200/JCO.2005.05.2308 http://dx.doi.org/10.1200/JCO.2005.05.2308 http://dx.doi.org/10.1371/journal.pone.0161464 http://dx.doi.org/10.1371/journal.pone.0161464 http://dx.doi.org/10.1080/01635580701274848 http://dx.doi.org/10.1080/01635580701274848 https://doi.org/10.5772/58812 https://doi.org/10.5772/58812 http://dx.doi.org/10.1007/s10495-014-1007-y http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0210 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0210 http://dx.doi.org/10.3945/an.112.003517 http://dx.doi.org/10.1016/j.fct.2015.04.002 http://dx.doi.org/10.1016/j.fct.2015.04.002 http://dx.doi.org/10.1016/j.cub.2016.01.033 http://dx.doi.org/10.1007/s11947-016-1793-z http://dx.doi.org/10.4103/0976-500X.81903 http://dx.doi.org/10.4103/0976-500X.81903 http://dx.doi.org/10.1293/tox.29.1S http://dx.doi.org/10.1124/pr.112.006163 http://dx.doi.org/10.1007/978-1-59745-458-2_15 http://dx.doi.org/10.1007/978-1-59745-458-2_15 http://dx.doi.org/10.1128/IAI.01412-06 http://dx.doi.org/10.1155/2011/473964 http://dx.doi.org/10.1002/em.20072 http://dx.doi.org/10.1002/em.20072 http://dx.doi.org/10.3390/molecules21101406 https://doi.org/10.1590/S0100-879X2002000300010 https://doi.org/10.1590/S0100-879X2002000300010 http://dx.doi.org/10.1371/journal.pone.0172824 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0280 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0280 http://dx.doi.org/10.1155/2014/202759 http://dx.doi.org/10.1155/2014/202759 http://dx.doi.org/10.1016/j.cellsig.2011.12.006 http://dx.doi.org/10.1038/nrm1715 Satow, R., Hirano, T., Batori, R., Nakamura, T., Murayama, Y., Fukami, K., 2014. Phospholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells. Proc. Natl. Acad. Sci. U. S. A. 111, 13505–13510. http://dx. doi.org/10.1073/pnas.1405374111. Schiavon, G., Ruggiero, A., Schöffski, P., van der Holt, B., Bekers, D.J., Eechoute, K., Vandecaveye, V., Krestin, G.P., Verweij, J., Sleijfer, S., Mathijssen, R.H.J., 2012. Tumor volume as an alternative response measurement for imatinib treated GIST patients. PLoS One 7, e48372. http://dx.doi.org/10.1371/journal.pone.0048372. Srivastava, S.K., 2013. Role of Capsaicin in Oxidative Stress and Cancer. (Springer Science & Business Media). Sun, H., Yang, S., Li, J., Zhang, Y., Gao, D., Zhao, S., 2016. Caspase-independent cell death mediated by apoptosis-inducing factor (AIF) nuclear translocation is involved in ionizing radiation induced HepG2 cell death. Biochem. Biophys. Res. Commun. 472, 137–143. http://dx.doi.org/10.1016/j.bbrc.2016.02.082. Takahashi, H., Yamada, E., Ohkubo, H., Sakai, E., Higurashi, T., Uchiyama, T., Hosono, K., Endo, H., Nakajima, A., 2012. Relationship of human rectal aberrant crypt foci and formation of colorectal polyp: one-year following up after polypectomy. World J. Gastrointest. Endosc. 4, 561–564. http://dx.doi.org/10.4253/wjge.v4.i12.561. Tanwar, L., Vaish, V., Sanyal, S.N., 2009. Chemoprevention of 1,2-Dimethylhydrazine- Induced Colon Carcinogenesis by a Non-steroidal Anti-Inflammatory Drug, Etoricoxib. In: Rats: Inhibition of Nuclear Factor kappaB. Asian Pac. J. Cancer Prev. APJCP 10, pp. 1141–1146. Thomas, K.C., Ethirajan, M., Shahrokh, K., Sun, H., Lee, J., Cheatham, T.E., Yost, G.S., Reilly, C.A., 2011. Structure-activity relationship of capsaicin analogs and transient receptor potential Vanilloid 1-mediated human lung epithelial cell toxicity. J. Pharmacol. Exp. Ther. 337, 400–410. http://dx.doi.org/10.1124/jpet.110.178491. Tica Sedlar, I., Petricevic, J., Saraga-Babic, M., Pintaric, I., Vukojevic, K., 2016. Apoptotic pathways and stemness in the colorectal epithelium and lamina propria mucosae during the human embryogenesis and carcinogenesis. Acta Histochem. 118, 693–703. http://dx.doi.org/10.1016/j.acthis.2016.08.004. Torre, L.A., Bray, F., Siegel, R.L., Ferlay, J., Lortet-Tieulent, J., Jemal, A., 2015. Global cancer statistics, 2012. CA Cancer J. Clin. 65, 87–108. http://dx.doi.org/10.3322/ caac.21262. Wargovich, M.J., Brown, V.R., Morris, J., 2010. Aberrant crypt foci: the case for inclusion as a biomarker for colon cancer. Cancers 2, 1705–1716. http://dx.doi.org/10.3390/ cancers2031705. Yan, B., Wang, H., Peng, Y., Hu, Y., Wang, H., Zhang, X., Chen, Q., Bedford, J.S., Dewhirst, M.W., Li, C.-Y., 2006. A unique role of the DNA fragmentation factor in maintaining genomic stability. Proc. Natl. Acad. Sci. 103, 1504–1509. http://dx.doi. org/10.1073/pnas.0507779103. Ye, D.Z., Kaestner, K.H., 2009. Foxa1 and Foxa2 control the differentiation of goblet and Enteroendocrine L- and D-cells in mice. Gastroenterology 137, 2052–2062. http://dx. doi.org/10.1053/j.gastro.2009.08.059. Yoshitani, S.I., Tanaka, T., Kohno, H., Takashima, S., 2001. Chemoprevention of azox- ymethane-induced rat colon carcinogenesis by dietary capsaicin and rotenone. Int. J. Oncol. 19, 929–939. Yun, J.-A., Kim, S.-H., Hong, H.K., Yun, S.H., Kim, H.C., Chun, H.-K., Cho, Y.B., Lee, W.Y., 2014. Loss of E-cadherin expression is associated with a poor prognosis in stage III colorectal cancer. Oncology 86, 318–328. http://dx.doi.org/10.1159/000360794. Zhang, Q.-H., Hu, J.-P., Wang, B.-L., Li, Y., 2012. Effects of capsaicin and dihy- drocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo. J. Asian Nat. Prod. Res. 14, 382–395. http://dx.doi.org/10.1080/ 10286020.2012.656605. B.F.R. Caetano et al. Toxicology and Applied Pharmacology 338 (2018) 93–102 102 http://dx.doi.org/10.1073/pnas.1405374111 http://dx.doi.org/10.1073/pnas.1405374111 http://dx.doi.org/10.1371/journal.pone.0048372 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0310 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0310 http://dx.doi.org/10.1016/j.bbrc.2016.02.082 http://dx.doi.org/10.4253/wjge.v4.i12.561 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0325 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0325 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0325 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0325 http://dx.doi.org/10.1124/jpet.110.178491 http://dx.doi.org/10.1016/j.acthis.2016.08.004 http://dx.doi.org/10.3322/caac.21262 http://dx.doi.org/10.3322/caac.21262 http://dx.doi.org/10.3390/cancers2031705 http://dx.doi.org/10.3390/cancers2031705 http://dx.doi.org/10.1073/pnas.0507779103 http://dx.doi.org/10.1073/pnas.0507779103 http://dx.doi.org/10.1053/j.gastro.2009.08.059 http://dx.doi.org/10.1053/j.gastro.2009.08.059 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0360 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0360 http://refhub.elsevier.com/S0041-008X(17)30445-3/rf0360 http://dx.doi.org/10.1159/000360794 http://dx.doi.org/10.1080/10286020.2012.656605 http://dx.doi.org/10.1080/10286020.2012.656605 Capsaicin reduces genotoxicity, colonic cell proliferation and preneoplastic lesions induced by 1,2-dimethylhydrazine in rats Introduction Material and methods – Chemicals – Study design Short-term assays – Mid-term assays – Statistical analysis Results - Short-term assays - Mid-term assays Discussion Conclusion Acknowledgments Author contributions Conflicts of interest References