Arthrospira (Spirulina) platensis feeding reduces the early stage of chemically induced rat colon carcinogenesis

Accepted manuscript This peer-reviewed article has been accepted for publication but not yet copyedited or typeset, and so may be subject to change during the production process. The article is considered published and may be cited using its DOI 10.1017/S0007114522001350 The British Journal of Nutrition is published by Cambridge University Press on behalf of The Nutrition Society Arthrospira (Spirulina) platensis feeding reduces the early stage of chemically-induced rat colon carcinogenesis Simone Oliveira Amadeu1#; Luis Manuel Sarmiento-Machado2#; Ariane Rocha Bartolomeu2, María Angel García Chaves3; Guilherme Ribeiro Romualdo2, 4; Nelci Antunes de Moura4; Luis Fernando Barbisan4* 1Graduate Program of General and Applied Biology, Biosciences Institute, Sao Paulo State University (UNESP), Botucatu-SP, Brazil 2Graduate Program of Pathology, Botucatu Medical School, Sao Paulo State University (UNESP), Botucatu-SP, Brazil 3Department of Oncology, Biosanitary Research Institute of Granada (ibs.GRANADA), University Hospitals of Granada-University of Granada, Granada, Spain 4Department of Structural and Functional Biology, Institute of Biosciences, Sao Paulo State University (UNESP), Botucatu-SP, Brazil *Address correspondence to: Luís Fernando Barbisan, Ph.D. Departamento de Biologia Estrutural e Funcional, Instituto de Biociências, Universidade Estadual Paulista (UNESP), E-mail: [email protected] # Authors share first authorship https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript Abstract Colorectal cancer is the third most diagnosed cancer worldwide and linked to dietary/lifestyle factors. Arthrospira (Spirulina) platensis (AP) contains bioactive compounds with beneficial effects in vivo/in vitro. Thus, we evaluated the preventive effects of AP feeding against 1,2dimethylhydrazine (DMH)-induced colon carcinogenesis. Male Sprague Dawley rats were given subcutaneous injections of DMH (4×40 mg/kg body weight) (G1-G3) or vehicle (G4G5) twice a week (weeks 3-4). During weeks 1-4, animals were fed a diet containing 1% (G2) or 2% (G3-G4) AP powder (w/w) as a chemopreventive agent. After this period, all groups received a balanced diet until week 12. Some animals were euthanized after the last DMH injection (week 4) for histological, immunohistochemical (Ki-67, γ-H2AX and caspase-3) and molecular analyses (RT-PCR for 91 genes), while other animals were euthanized at week 12 for preneoplastic aberrant crypt foci (ACF) analysis. Both AP treatments (G2-G3) significantly decreased the DMH-induced increase in γ-H2AX (DNA damage) and caspase 3 (DNA damage–induced cell death) in colonic crypts at week 4. In addition, Cyp2e1 (Drug metabolism), Notch1, Notch2, and Jag1 genes (Notch pathway) and Atm, Wee1, Chek2, Mgmt, Ogg1 and Xrcc6 genes (DNA repair) were also down-regulated by 2% AP feeding (G3) at week 4. A significant reduction in ACF development was observed in both AP-treated groups (G2-G3) at week 12. In conclusion, findings indicate that AP feeding reduced acute colonic damage after DMH, resulting in fewer preneoplastic lesions. Our study provided mechanistic insights on dietary AP-preventive effects against early colon carcinogenesis. Keywords: Arthrospira (Spirulina) platensis; tumor initiation; Notch and DNA repair genes; colonic preneoplastic lesions; colon cancer prevention. https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript 1. Introduction Colorectal cancer (CRC) is one of the most commonly diagnosed gastrointestinal malignancies in western industrialized countries in both men and women aged > 50, but a dramatic increase among those younger people (20 to 49) has been reported (Siegel et al., 2020; Sung et al., 2021). Globally, CRC is the third most commonly diagnosed neoplasia, and the second leading cause cancer-related deaths (Siegel et al., 2020; Sung et al., 2021). About 55–70% of cases are classified as sporadic, and more than 50% of these cases are related to modifiable risk factors. In fact, epidemiological and animal evidence indicate that a sedentary lifestyle and “westernized” dietary habits, including a high intake of red and processed meat, saturated fats and refined starches, associated with a low consumption of fresh fruit/vegetables and fiber, are risk factors for CRC development (Lofano et al. 2013; Bouvard et al. 2015; Pan et al., Wang, 2018; Keum & Giovannucci 2019; Siegel et al. 2020; Sung et al. 2021). In order to identify potential dietary agents against sporadic CRC development, carcinogen-induced preclinical models in rodents have been widely used for the screening of new preventive strategies (Corpet & Pierre, 2005; Raju, 2008; Fleet, 2014). In these translational rodent models, 1,2-dimethylhydrazine (DMH) hydrochloride and its main metabolite azoxymethane (AOM) are potent genotoxic agents to induce colonic preneoplastic (i.e., aberrant crypt foci, ACF) and neoplastic (i.e., adenomas and adenocarcinomas) lesions that resemble human CRC in most morphological and molecular aspects (Rosenberg et al., 2009; Perše & Cerar, 2011; Ward & Treuting 2014). In this context, the photosynthetic cyanobacteria Arthrospira spp., an edible bluegreen microalga, has been widely studied and commercialized worldwide. This cyanobacteria contains high protein levels that can reach between 60-70% of the its dry-weight biomass (Lupatini et al., 2017; Lafarga et al., 2020; Barros de Medeiros et al., 2021). Other bioactive compounds have been described including antioxidants [β-carotene and C-Phycocyanin (CPC)], minerals (P, K, Na, Ca, Mg, Fe, Zn), vitamins (E, B1, B2, B3, B9), essential amino acids, PUFAs [especially γ-linolenic acid (ALA, 18:3 n-6)] and some phenolic compounds (Lupatini et al., 2017; Papalia et al., 2019; Lafarga et al., 2020; Barros de Medeiros et al., 2021). In special, C-PC is a natural light-blue pigment used in food, cosmetic and pharmaceuticals products or formulations (Park et al., 2018, Pez et al., 2021). Nowadays, Arthrospira spp. powder is used as a nutraceutical and functional food supplement, and is clinically recommended for patients with higher risk factors for cardiovascular diseases or https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript metabolic syndrome, including arterial hypertension, insulin resistance, hypercholesterolemia and obesity (Martínez-Sámano et al., 2018; Moradi et al., 2019; Ramos-Romero et al., 2021). Some in vivo studies have demonstrated that Arthrospira spp. has preventive properties against oral, liver, skin and breast carcinogenesis, exerting anti-inflammatory, antioxidant and pro-apoptotic properties (Grawish et al., 2010; Yogianti et al., 2014; Ouhtit et al., 2014; Mahmoud et al., 2021). Álvarez-González et al. (2015) demonstrated a potential preventive effect of oral administration of high concentrations of Arthrospira maxima (100, 400, and 800 mg/kg) on the initial stages of AOM-induced colon carcinogenesis. Nonetheless, the mechanistic landscape of this preventive effect is not fully unveiled. Thus, in the light of the potential beneficial properties of Arthrospira (Spirulina) platensis (AP) (Wu et al., 2016), we evaluated the protective effects of AP feeding, before and during DMH administrations, against carcinogen-induced colonic mucosal damage and preneoplastic lesion development in rodents. 2. Materials and Methods 2.1 – Chemicals, AP composition, and chow preparation 1,2-dimethylhydrazine hydrochloride (PubChem CID: 1322) was obtained from Sigma–Aldrich (USA) and AP powder was originated and generously donated by Fazenda Tamanduá (Brazil) with the following composition per gram: proteins (66.7%), total carbohydrates (20.0%), total lipids (9.33%) and fiber (6.0%) expressed on dry weight. According to Pereira et al. (2020) findings, C-PC levels in AP powder are 0.492 (± 0.086) mg/g (in triplicate). AP powder was incorporated into a standard rodent chow (Nuvilab-CR1, Nuvital - Brazil), achieving the final concentration of 1 or 2% (10 or 20 g of AP powder/kg chow). Thus, C-PC concentrations in chows were estimated: 4.92 (AP 1%) and 9.84 (AP 2%) mg/kg. The balanced diet was standardized according to the National Research Council’s recommendation affording the rodent nutritional necessities (National Research Council, 1995). The chow was complete homogenized and humidified into an industrial mixer (model M60, CAF, Brazil), then pelleted (7.5 CV model, Chavantes, Brazil), dried trough ventilation, stored into identified plastic bags, and finally kept under refrigeration (−4°C). Representative photos of each chow are displayed in Figure 1. The nutritional composition of the experimental chow can be found in Table 1. https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript 2.2 - Study design and sampling For the sample size calculation, the total number of animals (n = 68) was estimated using the G*Power 3.1118 software, considering a significance level (α) of 5%, power (1-β) close to 95% and effect (f) corresponding to 0.55 (large effect), as previously recommended (Charan & Kantharia, 2013). The calculation can be found in Supplementary Data 1. Following a 2-week acclimation period, seventy-six four-week-old male Sprague Dawley rats - obtained from Multidisciplinary Center for Biological Research (CEMIB) at the State University of Campinas (UNICAMP, Campinas, SP, Brazil) - were randomly distributed into five groups (G1 – G5, n = 10 or 16 rats/group) (Figure 1). Chows were offered to the groups as it follows: standard chow (G1 and G5), or standard chow containing AP at 1 (G2) or 2% (G3 and G4) for four weeks. AP powder concentrations in chow were based on European Food and Safety Authority Scientific Committee’s recommendations, indicating that feeding interventions should not exceed 5% in order to avoid nutritional imbalances in rodents (EFSA, 2011). Indeed, AP was safely consumed - considering body weight evolution and histopathological endpoints - up to 5% supplemental level in both male and female mice (Yang et al., 2011). Injections of DMH (G1 – G3; 40 mg/kg body weight) or DMH vehicle (G4 and G5; Na2EDTA, 37 mg/L, 5 mL/kg body weight) were subcutaneously applied twice a week at weeks 3 and 4 (Figure 1), as previously established by our research group for male rats (Caetano et al., 2020). At the week 4, 24 hours after the last DMH administration, five animals of each group were euthanized. The remaining rats had their AP-supplemented chows (G2 – G4) replaced for standard chow (as G1 and G5) for more 8 weeks, until they were euthanized at week 12 (Figure 1). In both procedures, rats were euthanized by exsanguination under ketamine/xylazine anesthesia (160 and 10 mg/kg body weight, respectively). At necropsy (weeks 4 and 12), colon samples were opened longitudinally, cleaned with 0.9% NaCl, pinned flat, fixed in 10% phosphate-buffered formalin during 24 hours, and then kept in 70% ethanol for histological analysis. Distal portions of the colon mucosa (week 4) were also sampled, snap frozen in nitrogen, and stored at -80°C for molecular analysis. Body weight, water and chow consumption were weekly registered during the experiment. Animals were accommodated in polypropylene cages under accepted conditions (22 ± 2°C temperature, 55 ± 10% humidity, 12h light/12h dark cycle) with free access to drinking water and chow. The study was developed according to the principles of the institution’s Ethics Committee Board under the protocol CEUA-1128/2019. https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript 2.3Immunohistochemistry for Ki-67, caspase-3, and γ-H2AX and at week 4 Ki-67 (i.e., proliferation), active caspase-3 (i.e., apoptosis) and γ-H2AX (i.e., DNA damage) markers were detected in colon sections. Briefly, deparaffinated 5 μm colon histological sections were placed onto silanized slides and sequentially treated with 0.01 M citrate buffer (pH 6.0) at 120ºC for 5 min in a Pascal Pressure Chamber (Dako Cytomation Denmark A/S), 10% H2O2 in phosphate-buffered saline (PBS) for 10 min, skim milk for 60 min, anti-Ki-67 (1:200 dilution, MA5-14520, Thermo Fisher Scientific, USA) anti-active caspase-3 (1:100 dilution, ab179517, 1:200, Abcam, UK) and anti-γ-H2AX (1:100 dilution, MA5-27753, Thermo Fisher Scientific, USA) antibodies overnight at 4°C, a one-step horseradish peroxidase (HRP)-polymer (EasyPath - Erviegas, Brazil) for 20 min at room temperature. Chromogen color was accomplished with 3,3-diaminobenzidine tetrahydrochloride (Sigma–Aldrich, USA). The slides were counterstained with Harris hematoxylin for 1 min. For each group (n = 5 each group), 20 randomly entire colonic crypts were scored per animal. Ki-67, caspase-3 and γ-H2AX labeling indexes (LI%) were scored by the number of positive-stained cells/number of cells per crypt ratio. 2.4Low Density Gene Array, functional and network analysis at week 4 Total RNA was isolated from distal colon samples (n = 5 each group) with RNeasy Mini kit (Qiagen, Germany) following the manufacturer’s instructions. The quantification of RNA was performed by NanoVue Plus (GE HealthCare, UK) and the quality was evaluated using Bioanalyzer 2100 plataform (Agilent Technologies, USA). The cDNA synthesis was performed using SuperScript VILO cDNA Synthesis Kit and Master Mix (Thermo Scientific, USA) following the manufacturer’s instructions. TaqMan Low Density Array cards (TLDA, Life Technologies, USA) were used for quantitative real-time polymerase chain reaction (RTqPCR) following the manufacturer’s instructions. TLDA cards comprised 94 genes involved in DNA repair, anti/pro-oxidant metabolism, cell proliferation, death, and differentiation (Supplementary Data 2). Briefly, 100 µl of cDNA template (422 ng mRNA) was added to 100 μL of TaqMan Fast Advanced Master Mix (Life Technologies, USA) and dispensed into loading wells on the TLDA card. The cycling protocol included heat activation at 50°C for 1 min and denaturation at 95°C for 10 min, followed by 40 cycles of 95 °C for 15s and 60 °C for 1 min. Fluorescence detection was performed on a QuantStudio 12 K Flex Real-Time PCR System (Life Technologies, USA). Relative quantitation was calculated based on the 2-(ΔΔCt) method (Livak & https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript Schmittgen, 2001), using Expression Suite Software v1.1 (Life Technologies, USA). Actb, Gapdh, Gusb and Hprt housekeeping genes were used for normalization. The output list of differentially expressed mRNA (≥1.5fold change) was analyzed on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) database via the DAVID tools website (https://david.ncifcrf.gov/) in order to identify significantly enriched biological pathways [P<0.05; enrichment score = -log (p value)] (Huang et al., 2009). Network confidence analysis was carried out using the STRING database (https://string-db.org/). 2.5Screening of colonic preneoplastic lesions at week 12 All the segments (distal, medial, and proximal) from each animal were stained with 0.2% methylene blue solution. Aberrant crypt foci (ACF) are clusters of abnormal crypts (AC) displaying oval-like lumens and a thicker epithelial cell lining (Bird & Good, 2000). The total number of ACF, AC, and the number of ACF with ≤ 4 AC or > 4 AC were counted according to Bird’s criteria, under light microscopy at 20× magnification (Bird & Good, 2000). 2.6Statistical analysis Data from body weight gain, water and chow consumption, immunohistochemistry and ACF analysis were compared among the groups using one-way analysis of variance (ANOVA) or Kruskal Wallis followed by post hoc Tukey test. The analyses were performed using the GraphPad Prism software (V4.03, GraphPad, USA). The pairwise comparisons of gene expression data were performed and analyzed by using the Student t-test. Correlations were performed using Pearson’s coefficient (r). The differences among groups were considered significant when p < 0.05. 3. Results 3.1 – Both weight, water and chow consumption at weeks 4 and 12 During the 4-week AP feeding period, no significant alteration in body weight, body weight gain or chow and water consumption was observed with exception of absolute liver weight that was lower in DMH-initiated groups (G1-G3) when compared to the non-initiated groups (G5 and G6) (Table 2). The average consumption of AP powder during the 4 weeks was 236.4± 25.5 (G2), 489.4 ± 52.1 (G3) and 470.1 ± 42.8 (G4) mg/rat/day (data are mean ± standard deviation). Based on average AP intake, the estimated C-PC consumption was 0.11 https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript ± 0.01 (G2), 0.24 ± 0.02 (G3), and 0.23 ± 0.02 (G4) mg/rat/day (data are mean ± standard deviation). In addition, the mean body weight and liver absolute and relative weights did not differ among groups from weeks 5 to 12 (Supplementary Data 3). At week 12, survival rates were 100% in both DMH and vehicle groups. 3.2Colonic cell proliferation, apoptosis and DNA damage at week 4 Twenty-four hours after the last DMH administration, the γ-H2Ax and caspase-3associated to DNA insult LI%, but not Ki-67 LI%, were significantly increased in the colonic mucosa from DMH-initiated groups (G1-G3, n=8 each) in comparison to non-initiated groups (G4 and G5, n=5 each) (Figures 2, 3 and 4). Noteworthy, a significant (p < 0.001, for both) reductions in γ-H2AX and caspase-3 LI% were observed in the colonic mucosa from DMHinitiated fed with AP at 1% and 2% groups (G2 and G3) when compared to only control DMH-initiated group (G1) (Figures 2 and 3). However, colonic cell proliferation LI% did not significantly differ among groups (Figure 3). 3.3 - Gene expression analysis at week 4 Twenty-four hours after the last DMH administration, the levels of mRNA encoding target proteins involved in the cell cycle, DNA repair and apoptosis pathways were measured in colonic mucosa samples at week 4 (Caetano et al., 2020). Gene expression analysis revealed 16 downregulated and 1 upregulated gene in the colonic mucosa of DMHinitiated and AP 2%-fed rats (G3) compared to the DMH counterpart (G1) (Table 3). Genes involved in the Notch pathway were decreased – as Notch 1, Notch 2 and Jag1 – as well as cell cycle/DNA repair-related ones – including Atm, Wee1, Chek2, Mgmt, Ogg1 and Xrcc6. Of note, Cyp2e1 gene encoding DMH metabolizing cytochrome P450 CYP2E1 enzyme was downregulated in AP 2%-fed rats. Raw data of the gene expression profile from and DMHinitiated fed 2% AP group (G3) was included in Supplementary Data 2. As expected, functional enrichment analyses of the differentially expressed genes in AP 2%-fed mice (G3) indicated a significant correlation between the downregulation of these genes and “Notch signaling pathway”, “Cell cycle” and, “Base excision repair” functional annotations (Figure 5). STRING network confidence analysis was in keeping with these functional correlations, as Notch1, Notch2 and Jag1 network nodes are strongly functionally correlated, a featured also observed among Xrcc6, Atm, Chek2 and Wee1. https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript 3.4Colonic preneoplastic lesion analysis at week 12 Classical ACF were evaluated in methylene blue-stained whole-mount colon samples (Bird & Good, 2000) at the end of week 12 (Supplementary Data 4). No ACF were observed in non-initiated groups (G4 and G5). Data from the number and multiplicity (AC/ACF) of stereoscopically-analyzed ACF in the different DMH-initiated groups are summarized in Table 4. The mean number of ACF with ≤4 crypts, and > 4 AC, and the total number of ACF and AC were significantly (0.008 <p< 0.01) lower in the group fed AP at 1.0 or 2.0% (G2 and G3) when compared to the DMH-initiated group (G1). 3.5 Correlations We evaluated whether the 2% AP-mediated alterations in DNA damage and Notch pathway at week 4 are correlated to AC/ACF development at week 12. The reduction of γH2AX was positively correlated to the reductions in AC (p=0.047, r=0.63) and ACF (p=0.007, r=0.77) (Figure 6). Only the downregulation of Notch 1 was positively correlated to the reductions in AC (p=0.089, r=0.56) and ACF (p=0.04, r=0.64) (Figure 6), not Notch2. 4. Discussion In this feeding study, we investigated the beneficial effects of dietary AP interventions, before and during carcinogen administration, on the colonic mucosal acute damage as well as on the late development of putative preneoplastic lesions using a mediumterm bioassay for rat colon carcinogenesis. Twenty-four hours after the last DMH administration, the results showed that both dietary AP interventions significantly reduced carcinogen-induced DNA damage and apoptosis in epithelial cells of the colonic crypts. In addition, AP at 2% feeding significantly reduced expression of Notch 1, Notch 2 and Jag1 genes (Notch pathway), Atm, Wee1, Chek2, Mgmt, Ogg1 and Xrcc6 genes (DNA repair pathway) and Cyp2e1 (Metabolism) in the colonic mucosa in comparison to the only DMHinitiated group. As AP feeding reduced the noxious effect of DMH on the colonic mucosa, a significant reduction in mean number of AC and ACF development was also detected for both AP interventions groups at the end of week 12. The estimated AP intake was 236 (G2) and ~470-490 (G3 and G4) mg/rat/day, corresponding to a daily dose of 0.81 (G2) and 1.6 (G3 and G4) g/kg/day (Supplementary Data 5). Using the Human Equivalent Dose (HED) allometric dose translation formula (Reagan-Shaw et al., 2008), the estimated animal dose used herein corresponds to a dose of 130 (AP at 1%) and 250 (AP at 2%) mg/kg/day in humans (Supplementary Data 5). Although there are no clinical studies on the https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript EFSA. (2011). Guidance on conducting repeated-dose 90-day oral toxicity study in rodents on whole food/feed. EFSA Journal, 9(12), 2438. Fradique M, Batista AP, Nunes MC, Gouveia L, Bandarra NM, Raymundo A. 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DMH=1,2dimethylhydrazine hydrochloride, EDTA= Ethylenediamine tetraacetic acid, ACF= Aberrant crypt foci, RT-qPCR= Quantitative Real Time, IHC= Immunohistochemistry and E= euthanasia. https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript Figure 2. Effects of Arthrospira (Spirulina) platensis feeding on γ-H2AX labeling index (LI%) in the different experimental groups at week 4. Representative photomicrographs of phospho-H2A.X-immunostained sections of colonic crypts are presented (scale bar: 50 µm). Data are mean + standard deviation (n= 5-8 rats each group). Different letters correspond to statistical difference by ANOVA followed by Tukey’s test (p < 0.05). AP= Arthrospira (Spirulina) platensis powder at 1% or 2% in the chow (w/w), DMH= 1,2 dimethylhydrazine dihydrochloride (4×40mg/kg body weight by subcutaneous injections). https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript Figure 3. Effects of Arthrospira (Spirulina) platensis feeding on caspase-3 labeling index (LI%) in the different experimental groups at week 4. Representative photomicrographs of γH2AX-immunostained sections of colonic crypts are presented (scale bar: 25 µm). Data are mean + standard deviation (n= 5 rats each group). Different letters correspond to statistical difference by ANOVA followed by Tukey’s test (p < 0.05). AP= Arthrospira (Spirulina) platensis powder at 1% or 2% in the chow (w/w), DMH= 1,2 dimethylhydrazine dihydrochloride (4×40mg/kg body weight by subcutaneous injections). https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press Accepted manuscript Figure 4. Effects of Arthrospira (Spirulina) platensis feeding on Ki-67 labeling index (LI%) in the different experimental groups at week 4. Representative photomicrographs of Ki-67immunostained sections of colonic crypts are presented (scale bar: 50 µm). Data are mean + standard deviation (n= 5 rats each group). Different letters correspond to statistical difference by ANOVA followed by Tukey’s test (p < 0.05). AP= Arthrospira (Spirulina) platensis powder at 1% or 2% in the chow (w/w), DMH= 1,2 dimethylhydrazine dihydrochloride (4 x 40mg/kg body weight by subcutaneous injections). https://doi.org/10.1017/S0007114522001350 Published online by Cambridge University Press