Toxicology 312 (2013) 123–131
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Colon preneoplasia after carcinogen exposure is enhanced and colonic serotonergic system is suppressed by food deprivation Vinicius Kannen a,∗ , Cleverson R. Fernandes b , Helga Stopper a , Dalila L. Zanette c , Frederico R. Ferreira c , Fernando T. Frajacomo b , Milene C. Carvalho d , Marcus L. Brandão d , Jorge Elias Junior e , Alceu Afonso Jordão Junior f , Sérgio Akira Uyemura g , Ana Maria Waaga-Gasser h , Sérgio B. Garcia b a
Department of Toxicology, University of Wuerzburg, Germany Department of Pathology, University of São Paulo, Ribeirão Preto, Brazil c Department of Genetics, National Institute of Science and Technology in Stem Cell and Cell Therapy, CNPq/FAPESP. University of São Paulo, Ribeirão Preto, Brazil d Laboratory of Psychobiology, Faculty of Philosophy, Sciences and Letter, University of São Paulo, Ribeirão Preto, Brazil e Radiology Divisio, University of São Paulo, Ribeirão Preto, Brazil f Nutrition Division, University of São Paulo, Ribeirão Preto, Brazil g Ribeirão Preto School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, Brazil h Department of Surgery I, Molecular Oncology and Immunology, University of Wuerzburg, Wuerzburg, Germany b
a r t i c l e
i n f o
Article history: Received 23 July 2013 Received in revised form 8 August 2013 Accepted 15 August 2013 Available online 23 August 2013 Keywords: Food deprivation Lipid peroxidation Adipose tissue Serotonin Aberrant crypt foci
a b s t r a c t Calorie restriction regimens usually promote health and extend life-span in mammals. This is partially related to their preventive effects against malignancies. However, certain types of nutritional restriction failed to induce beneficial effects. The American Institute of Nutrition defines calorie restriction as diets which have only 40% fewer calories, but provide normal amounts of necessary food components such as protein, vitamins and minerals; whereas, food restriction means 40% less of all dietary ingredients plus 40% less calories. Our study aimed to test the hypothesis that the latter type of food deprivation (40% less food than consumed by standard fed rats) might increase cancer risk instead of reducing it, as is generally assumed for all dietary restrictive regimens. Since the endogenous modulation of the colon serotonergic system has been observed to play a role during the early steps of carcinogenesis we also investigated whether the serotoninergic system could be involved in the food intake modulation of cancer risk. For this, rats were exposed to a carcinogen and subjected to food deprivation for 56 days. Triglyceride levels and visceral adipose tissue were reduced while hepatic and colonic lipid peroxidation was increased. This dietary restriction also decreased serotonin levels in colon, and gene expression of its intestinal transporter and receptors. Finally, the numbers of preneoplastic lesions in the colon tissue of carcinogen-exposed rats were increased. Our data suggest that food deprivation enhances formation of early tumorigenic lesions by suppressing serotonergic activity in colon tissue. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The relationship between the amount of food intake and health is a fundamental issue, but the current literature data remain controversial. It is well known that calorie restriction regimens are often associated with increased health and life-span in mammals, which is at least partially related to their preventive effects against
∗ Corresponding author at: Department of Toxicology, University of Wuerzburg, Versbacher Strasse 9, D-97078 Wuerzburg, Germany. Tel.: +49 931 201 48777; fax: +49 931 201 48446. E-mail address:
[email protected] (V. Kannen). 0300-483X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tox.2013.08.014
malignancies (Colman et al., 2009). On the other hand some regimens were found to induce stress in animals and humans (Adam and Epel, 2007; Guarnieri et al., 2012), and no increase in longevity was observed in rhesus monkeys maintained under long-term calorie restriction regimen (Mattison et al., 2012). As experienced by the Dutch population during World War II, food restriction may even increase tumor risk in humans (Elias et al., 2004, 2005, 2007). In support of this observation, the restriction of food in some animal experiments enhanced early carcinogenic steps (Birt et al., 1991; Premoselli et al., 1996, 1998). Overall, benefits of moderate calorie restriction regimens are beyond any doubt protective against cancer when applied early in life and throughout the whole life span in experimental mammalian models. However, effects of shorter
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Table 1 Experimental design. Groups
Description
Treatment/procedures Standard control SD/C SD/D Carcinogen control DR/C Food deprivation control Food deprivation and carcinogen DR/D
Isotonic saline
DMH
Food ad libitum
Food deprivation (56 days)
x − x −
− x − x
x x − −
− − x x
Isotonic saline (single intraperitoneal injection, 5 ml/kg body weight); 1,2 dimethylhydrazine (DMH; single intraperitoneal injection, 125 mg/kg body weight); SD/C, standard diet control group; SD/D, standard diet carcinogen control group; DR/C, food deprivation control group; DR/D, food deprivation carcinogen group.
lasting and more severe food deprivation in the context of cancer development are largely unknown. Colon cancer is one of the most important causes of death worldwide, and much effort has been made to understand its development (Bird and Good, 2000; Kannen et al., 2011a). The carcinogen 1,2-dimethylhydrazine (DMH) is the most well-known and used model substance to induce and study colon tumor formation in rats. DMH is known to impair hepatic and colonic antioxidant capacity (Baskar et al., 2012) while promoting lipid peroxidation and therefore preneoplastic lesions (Sangeetha et al., 2012). Besides, DMH disrupted serotonin (5-HT) levels in the central nervous and gastrointestinal systems (Arutjunyan et al., 2001; Kannen et al., 2011a, 2012). We previously reported that the serotonin metabolite melatonin reduced colon preneoplastic lesions in animals exposed to highly stressful environments, such as constant light (Kannen et al., 2011b). Moreover, a high-fat diet which promoted colon tumorigenesis (Garcia et al., 2006), enhanced the formation of preneoplastic lesions and suppressed the colon serotonergic system (Kannen et al., 2012). Finally, application of the selective serotonin reuptake inhibitor fluoxetine revealed that endogenously upregulated 5-HT levels reduced the development of colon dysplasia (Kannen et al., 2011a). In the present study we investigated whether a food deprivation regimen combined with the application of a carcinogen would reduce the development of colon preneoplastic lesions as known from long-term calorie restriction regimens, or whether it might have opposite effects. We further aimed to verify whether the serotoninergic system is involved in the food intake modulation of cancer risk, since the endogenous modulation of the colonic serotonergic system has been observed to play a role during the early steps of carcinogenesis. For this purpose, we used an in vivo rat model to study the development of preneoplastic lesions. Several concerns arise from our findings regarding the effects of food deprivation regimens on the hepatic and colonic systems. 2. Materials and methods 2.1. Animal care and experimental design Male Wistar rats (150–160 g) were purchased from the Medical School of Ribeirao Preto, University of Sao Paulo (FMRP, USP, Brazil). All experimental protocols were approved by the Animal Care and Use Committee at the FMRP-USP (No. 137/2010). After adaptation period (1 week), 32 rats were randomly assigned to one of four groups (n = 8/group). Experimental design is given in Table 1. One day before starting the dietary regimens, the carcinogen-treated rats were exposed to DMH (single i.p. injection, 125 mg/kg). Food intake from the SD groups (control; feeding ad libitum) was analyzed three times per week prior to the experimental starting point; 40% less food than the average of these measurements was given to the DR groups (food deprivation). This protocol was based on previous murine feeding models named either by “calorie restriction” or “dietary restriction” (Moura et al., 2012; Tomita, 2012). Considering that these models did not include nutritional supplementation as advised for “calorie restrictive regimens” (Colman et al., 2009; Lasko and Bird, 1995; Mattison et al., 2012; Raju and Bird, 2003; Yang and Newmark, 1987), we used the termination “food deprivation” to describe our model. All groups were fed standard Purina rat chow (Nestlé Purina, Brazil) and had free access to water during the entire experimental period. Further nutritional information is given in Supplementary Material (Table 1). Five rats were housed per plastic cage, with softwood chips used as bedding. Cages were maintained at 22 ± 2 ◦ C with
55% humidity and a 12 h light/dark cycle. Because feeding restrictive models might induce dispute for chow among rats housed at same cage impairing the equal distribution of food into the group, all rats were weighed three times weekly, and body weight gain (BWG; g/day) was calculated as (FW [final weight] − IW (initial weight)/TD [total days]). No large intra-group differences were observed during the current experimental period. Full-data are shown in the results section. Rats were sacrificed after 8 weeks. Details of euthanasia procedures and sample collection are given in Supplementary Material (section I). 2.2. Histopathological analysis of colonic epithelia As we previously reported (Kannen et al., 2011a,b, 2012), relative values for preneoplastic lesions dysplasia are given as the total number of lesions per m2 . Briefly, dysplastic lesions were assessed according to previous description (Paulsen et al., 2005) by a pathologist who examined transverse colon-rectal sections stained with H&E, and enumerated lesions (from mild to severe dysplasia). Then, the entire area of the analyzed sections (in m2 ) was determined at 100× magnification using a graduated lens (Carl Zeiss, Heidelberg, Germany). Further information is given in Supplementary Material (section II). 2.3. Immunohistochemistry (IHC) and proliferation analysis in colon tissue Standard IHC was performed as previously described (Kannen et al., 2011a, 2012). The primary anti-proliferative cellular nuclear antigen (PCNA; clone PC 10) antibody was acquired from Novocastra® (Buffalo Grove, IL, USA). Positive reactions were detected in longitudinal sections as a brown precipitate in the nucleus. Cryptal cell proliferative index (PCNA-Li, labeling index) was expressed as the number of positive cells out of the total number of cells for each sample. Analyses were performed by two independent observers to avoid intraobserver bias. 2.4. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) According to our previous description (Kannen et al., 2011a, 2012), data were continuously collected and analyzed using the ABI-7500 SDS software package. Primer sequences were designed with DNASTAR software (version 3.0) for serotonin reuptake transporter (SERT), serotonin receptor 5-HT1A, and serotonin receptor 5HT2C, and were amplified using the SYBRGreen Master Mix (Applied Biosystems). mRNA encoding caspase-3 (CASP-3; Assay ID Rn00563902 m1; Applied Biosystems) and caspase-8 (CASP-8; Assay ID Rn00574069 m1; Applied Biosystems) were amplified using TaqMan Universal Master Mix (PN 4304437, Applied Biosystems). Total RNA input was normalized based on cycle threshold (Ct) values for the glyceraldehyde 3-phosphate dehydrogene housekeeping gene (GAPD; 4352338E; Applied Biosystems, Foster City, CA, USA). The fold change was calculated using the 2−Ct method. Further information is given in Supplementary Material (section III). 2.5. Biochemical analysis Cholesterol (mg/dl), triglyceride (TG; mg/dl), aspartate aminotransferase (AST; U/l), alanine aminotransferase (ALT; U/l), malondialdehyde (MDA; nmol/g), and reduced glutathione (GSH; nmol/g) levels were biochemically analyzed in serum samples according to our description in Supplementary Material (section IV). 2.6. Computerized tomography (CT) CT exam was performed according to our previous report (Kannen et al., 2012) to determine the densities of hepatic fat (HF), intra-abdominal fat (IAF) and visceral adipose tissue (VAT). Further information is given in Supplementary Material (section V). 2.7. Detection of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) by high-performance liquid chromatography (HPLC) The colon 5-HT and 5-HIAA levels (ng/mg) were analyzed by HPLC as we described previously (Kannen et al., 2011a, 2012). Further information is given in Supplementary Material (section VI).
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Fig. 1. Influence of food deprivation on body weight in rats. (A) Food intake development during the 8-week experiment (3 measurements per week). (B) Body weight development during the 8-week experiment (# P < 0.05, SD/C vs. SD/D; b P < 0.05, SD/C vs. DR/C; *P < 0.05, SD/D vs. DR/D). (C) Body weight gain during the 8-week experiment (**P < 0.01 vs. SD/C; ***P < 0.001 vs. SD/D).
2.8. Statistical analysis Data were analyzed using GraphPad Prism 5 (Graph Pad Software Inc., San Diego, CA, USA). Two-way ANOVA test (Bonferroni post hoc test) was used to separately analyze carcinogen-treated (D groups) and non-carcinogen-treated (C groups) rats. The relationship between the following parameters was determined using linear regression analysis: hepatic MDA levels and VAT density (i); MDA levels in liver and colon tissues (ii); and colonic 5-HT and MDA levels (iii). Comparisons of values with different units (i and iii) were performed based on fold change calculations between SD and DR, always noting the difference between the C and D groups. The Mann–Whitney test was used to analyze dysplasia, since such lesions were only detected in carcinogen-treated rats. P < 0.05 was considered to be statistically significant. All values are reported as mean ± standard error of the mean (SEM).
3. Results 3.1. Effects of food deprivation on colonic dysplasia Food intake was similar for the two SD groups and for the two DR groups (Fig. 1A). Considering a calorie value of 3.3 kcal/g for the chow (Moura et al., 2012), the feeding efficiency (mg/kcal) was calculated by the formula (BWG/TCC [total calories consumed]) (Neyrinck et al., 2012), and found almost 1.3-fold reduced in the food deprived rats exposed to carcinogen (SD/D, 7 ± 0.7 vs. DR/D, 5.5 ± 1.6; P > 0.05). Average body weight increase slowed down significantly from the fifth week until the eighth week in non-carcinogen-treated and carcinogen-treated rats in those that were subjected to the food deprivation (Fig. 1B). Surprisingly, carcinogen-treated rats receiving standard diet showed significantly higher body weights than non-carcinogen-treated rats during the two last weeks of the eight-week experiment (Fig. 1B). BWG was significantly decreased in both groups subjected to the food restriction regimen (Fig. 1C). Dysplastic preneoplastic lesions are observed at the initial stage of colon carcinogenesis (Fig. 2A). Since our aims and protocols were
designed to study preneoplastic lesions, no tumors were found or could be expected in the colon tissues at the end of the experimental period. We found that food deprivation significantly increased the total number of dysplastic lesions in the carcinogen-treated group (Fig. 2B). Although food deprivation reduced cryptal size in the non-carcinogen treated group (total cell number per crypt; SD/C, 92.2 ± 11.8 vs. DR/C, 71.6 ± 7.1; SD/D, 106.5 ± 11.5 vs. DR/D, 106.6 ± 4.2; P < 0.05; Fig. 2C), it resulted in increased cell proliferation in the same group, while the proliferation remained unchanged in carcinogen-treated rats under this feeding regimen (Fig. 2D). Food deprivation downregulated expression of the genes encoding CASP-8 and CASP-3 in carcinogen-treated rats 20% and 37%, respectively (Fig. 2E and F). Hence, food deprivation seemed to enhance the formation of carcinogen-induced preneoplastic lesions in colon tissue. 3.2. Effects of food deprivation on body weight, serum parameters and fat tissue Biochemical analysis and CT exam were performed to understand the results above. Although serum cholesterol levels remained unchanged at the end of the experiment (Fig. 3A), serum TG levels were significantly reduced in both food deprived groups (Fig. 3B). Analysis of transaminase levels (ALT and AST) showed that hepatic function was unaffected at the end of the experimental period (Fig. 3C and D). Analysis of fat density by CT exam (Fig. 3E) revealed that the hepatic adipose tissue density remained unchanged in all groups (SD/C, 74 ± 3.2 vs. DR/C, 75 ± 4.5; SD/D, 73 ± 10 vs. DR/D, 78.5 ± 2.9; P > 0.05). Although abdominal adipose tissue also remained equal among groups (Fig. 3F), visceral adipose tissue density was significantly decreased in both food deprived groups in comparison with their respective control groups (Fig. 3G). Therefore, the lower
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Fig. 2. Histopathological analysis and gene expression in colon tissue of rats exposed to food deprivation. (A) Representative image from a DMH-exposed animal under standard diet shows a colon preneoplastic area (sectioned red line), partial loss of cell polarity (sectioned yellow lines), large lymphocytic infiltrate (sectioned white line), enhanced number of microvessels (white arrows), compressed cryptal luminal opening (red arrow), enlarged luminal opening (green arrow), among other pathological features as reduced number of goblet cells, and increase number of mitosis figures and apoptotic corpuscles. Pictures was taken at 200× magnification, scale bars represent 20 m. (B) Quantification of dysplastic lesions in rats colons, expressed as the number of lesions/m2 (*P < 0.05 vs. SD/D). (C) Colon tissue sections from (1) SD/C, (2) DR/C, (3) SD/D, and (4) DR/D groups stained with anti-PCNA antibody. Pictures were taken at 200× magnification, scale bars represent 20 m. (D) Labeling index for staining with anti-PCNA antibody (**P < 0.01 vs. SD/C). (E) Gene expression for CASP-8, and CASP-3 (F; P > 0.05) in colon tissue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
body weight in the food deprived groups was independent of hepatic malfunction and carcinogen activity, but was associated with reduced serum TG levels and VAT density. 3.3. Effects of food deprivation on oxidant and antioxidant patterns in the liver and colon To investigate the effects of the food deprivation on peroxidative stress, hepatic and colon tissue samples were subjected to further biochemical analysis. Hepatic MDA levels were significantly increased in food deprived control and carcinogen-treated rats (Fig. 4A), while GSH levels were significantly decreased in
these same groups (Fig. 4B). Regression analysis revealed that the reduction of visceral adipose tissue was related to high hepatic MDA levels among non-carcinogen-treated and carcinogen-treated rats subjected to the food deprivation regimen (DR/C, r2 = 0.9042, P = 0.013; DR/D, r2 = 0.9942, P = 0.0029). In colon tissue, food deprivation also increased MDA significantly in both non-carcinogen-treated and carcinogen-treated rats, respectively (Fig. 4C). Colonic GSH levels were significantly decreased in these two groups (Fig. 4D). Pearson correlation analysis showed that high hepatic MDA levels were associated with its colonic enhancement in food deprived rats exposed to carcinogen (SD/D, r2 = 0.7219, P = 0.0684; DR/D, r2 = 0.8094, P = 0.0376).
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Fig. 3. Effects of food deprivation on biochemical serum patterns and adipose tissue in rats. (A) Biochemical analysis of serum cholesterol (P > 0.05), (B) triglycerides (TG; **P < 0.01 vs. SD groups), (C) alanine transaminase (ALT; P > 0.05), and (D) aspartate transaminases (AST; P > 0.05). (E) Representative images from CT exams of rats in the SD/D (1, 3, and 5) and DR/D (2, 4, and 6) groups, diaphragmatic area cardiac apex (E1 and E2), hepatic area (E3 and E4; green sectioned lines), and abdominal (red sectioned lines) and visceral areas (E5 and E6; yellow sectioned lines). (F) CT exam revealed the fat density in abdominal (P > 0.05), and (G) visceral areas (***P < 0.001 vs. SD/C; **P < 0.01 vs. SD/D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
Our data suggest that food deprivation interferes with hepatic and colonic peroxidative processes. 3.4. Effects of food deprivation on the colonic serotonergic system The serotonergic system plays a major role in regulating functions related to feeding in organs that range from the brain to the colon. Hence, we investigated whether food deprivation affected serotonergic system-related events in colon tissue. In
carcinogen-treated rats, food deprivation significantly decreased 5-HT and 5-HIAA (5-HT-metabolite) levels, while in control group rats only 5-HIAA was reduced (Fig. 5A and B). In food deprived rats exposed to carcinogen, low 5-HT colonic levels were associated with high colonic MDA levels by linear regression analysis (DR/D, r2 = 0.9686, P = 0.0156). This regimen also decreased SERT mRNA levels 85% in carcinogen-treated rats; in control rats, SERT expression was not affected by food deprivation (Fig. 5C). Expression of two 5-HT-related receptors was downregulated in
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Fig. 4. Influence of food deprivation on biochemical parameters in rats. (A) Biochemical analysis of liver tissue was performed to determine the MDA (***P < 0.001 vs. SD groups), and (B) GSH levels (**P < 0.01 vs. SD groups). (C) Biochemical analysis of colon tissue was performed to determine the MDA (***P < 0.001 vs. SD/C; **P < 0.01 vs. SD/D), and (D) GSH levels (*P < 0.05 vs. SD/C; ***P < 0.001 vs. SD/D).
carcinogen-treated rats that were subjected to food deprivation: 56% for serotonin receptor 5-HT1A (Fig. 5D); and, 77% for serotonin receptor 5-HT2C (Fig. 5E). In control rats, the expression of the 5-HT related receptors was not affected by food deprivation. Therefore, food deprivation might deregulate colonic serotonergic system-related activities in a carcinogen-dependent manner.
4. Discussion Our previous studies argued that initial steps in colon carcinogenesis were increased by suppression of the serotonergic system (Kannen et al., 2011a, 2012). Actually, we reported that the activity of a high-fat diet which enhanced the formation of preneoplastic lesions was related to the suppression of the serotonergic system in colon tissue (Kannen et al., 2012). Further, we have shown that inhibiting the serotonin reuptake in colon tissue, which enhances serotonin levels without hormonal treatment, reduced the number of dysplastic lesions in colon tissue of carcinogen-exposed rats (Kannen et al., 2011a). Here, we hypothesize that food deprivation might enhance the number of preneoplastic lesions in colon tissue by promoting liver and colon lipid peroxidation-related processes that suppress the colon serotonergic system. In this context should be considered that a food restriction altered carcinogen-induced DNA methylation in liver tissue (Sohn and Fiala, 1995) and induced a negative feedback in 5-HT control reducing the brain tissue of 5HT levels (Haleem, 2009). DMH-induced preneoplastic lesions were mainly related to increased oxidant and lipid peroxidation events when antioxidant capacity became impaired (Baskar et al., 2012; Bird, 1995; Bird and Good, 2000; Dadkhah et al., 2011; Devasena et al., 2002; Sangeetha et al., 2012). 5-HT exhibited antioxidant potential (Herraiz and Galisteo, 2004; Marshall et al., 1996; Miura et al., 1996; Noda et al., 1999), preneoplastic and tumor cell populations were differentially sensitive to amine hormone activities during colon carcinogenesis (Kannen et al., 2011a; Tutton and Barkla,
1987), and DMH enhanced ROS production while disrupting the serotonergic system (Arutjunyan et al., 2001; Kannen et al., 2011a). Calorie and food restrictive regimens are currently often assumed to reduce the risk of developing various kinds of malignancy. Some elegant reports have argued that such regimens lowered the incidence of cancer only if applied early or through the entire life of the organism (Birt et al., 1991; Mattison et al., 2012). Colman et al. reported that a calorie restriction regimen reduced the incidence of intestinal adenocarcinoma by 50% in nonhuman primates. It should be remembered that this was a life-term experiment (Colman et al., 2009). Moreover, calorie restrictive regimens have reduced calorie amounts by altering carbohydrate or fat sources, while vitamin and mineral content are increased through supplementation to compensate for the calorie decrease (Colman et al., 2009; Lasko and Bird, 1995; Mattison et al., 2012; Raju and Bird, 2003). On the other hand, nutritional deficiencies were suggested to enhance cancer, because experimental animal studies found that certain nutrient deficiencies increased the chemically induced carcinogenesis (Yang and Newmark, 1987). Much less clear are the influences of food deprivation phases later in life and of severe food restriction situations imposing malnutrion on cancer risk. Tomita has shown that calorie restriction reduced colon preneoplastic lesions in F344 rats (Tomita, 2012). The author did not reproduce the previous description for calorie restriction in murine models (Lasko and Bird, 1995; Raju and Bird, 2003), but instead used a food deprivation strategy similar to ours. However, food deprivation was started a week before the first carcinogen treatment, whereas we started both conditions at same time. This means that our study represented an acute situation of carcinogenic exposure plus food deprivation. Lasko and Bird observed that calorie restriction counteracted the pro-carcinogenic effects of a high-fat diet, but did not reduce preneoplastic lesions in comparison with lean animal under standard diet (Lasko and Bird, 1995). This supports our previous findings that a high-fat diet enhanced initial carcinogenic steps (Kannen et al.,
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Fig. 5. Effects of food deprivation on colon serotonergic system in rats. (A) HPLC analysis of 5-HT (**P < 0.01 vs. SD/D), and (B) 5-HT metabolite 5HIAA levels (*P < 0.05 vs. SD/C and SD/D). (C) Gene expression performed by qRT-PCR analysis of the serotonin transporter (SERT; P > 0.05), (D) serotonin 5HT1A (P > 0.05), (E) and 5HT2C receptors (P > 0.05).
2012), whereas our current findings surprisingly showed that food deprivation also increased preneoplasia. Birt et al. determined that calorie restriction (without malnutrition) significantly decreased the incidence of skin carcinoma, whereas food restriction (40% less calories and other nutritional components, similar to our conditions) did not; thus, mice subjected to food restriction had larger tumors than those under calorie restriction after 24 weeks. Neither caloric nor food restriction reduced early preneoplastic lesions (Birt et al., 1991). In another study, a fasting/refeeding regimen increased colonic preneoplastic lesions, mainly due to disturbance of the proliferative and apoptosis rates, driving mutated colon cells in carcinogen-treated animals toward the S-phase of the cell-cycle (Premoselli et al., 1996, 1998). Regarding findings in humans, Elias et al. reported that food restriction imposed on the Dutch female population during World War II increased breast cancer risk and incidence (Elias et al., 2004, 2005, 2007). Food deprivation increased preneoplastic lesions among carcinogen-exposed rats, while it did not enhance proliferation, but reduced apoptosis-related genes in colon tissue. This dietary
regimen further impaired antioxidant defense and enhanced lipid peroxidation. This might mean that the colon tissue was unable to keep up its normal self-defense against carcinogenic insult, allowing the manifestation of early-carcinogenic mutations. It should be remembered that serum TG levels are an important and circulating source of energy (Parks, 2002) that require hepatic lipid peroxidation to generate adenosine-5 -triphosphate (Mitra et al., 2008; Rolo et al., 2012), releasing MDA as the final product of the metabolic reaction (Tsukamoto, 1993). Increasing hepatic lipid metabolism may enhance lipid peroxidation rates to generate energy (ChavezJauregui et al., 2010; Mitra et al., 2008; Parks, 2002; Rolo et al., 2012). An earlier study in rats found that dietary restriction reduced body weight and TG content in visceral adipose tissue (Moura et al., 2012). Another report showed that the decrease in energy during fasting depleted fatty acids in the adipose tissue of rats by promoting the release of fatty acids from adipocytes and by mobilizing triglycerides in retroperitoneal adipose tissue (Raclot and Groscolas, 1995). In addition, the carcinogen DMH increased lipid peroxidation (Sangeetha et al., 2012), which impaired hepatic and
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colonic antioxidant capacity while promoting lipid peroxidation (Baskar et al., 2012; Sangeetha et al., 2012). Therefore, DMH promoted colon preneoplastic lesions, and thus tumors, by increasing hepatic and colonic oxidative status. This implies that there is a positive feedback mechanism between liver and colon that promotes malignancy (Baskar et al., 2012; Bird, 1995; Bird and Good, 2000; Dadkhah et al., 2011; Devasena et al., 2002; Sangeetha et al., 2012). It seems reasonable to suggest that the antioxidant capacity, which is already impaired under food deprivation, is unable to counteract DMH activity in liver tissue. In this situation, DMH may exert its lipid peroxidation activity fully, supporting the development of preneoplastic lesions. Previous reports indicate possible connections between the effects of food deprivation on lipid peroxidation and on the serotonergic system in colon tissue. Actually, calorie restriction is known to alter hormonal parameters, circulating growth factors, and receptor activities (Lasko and Bird, 1995). It is already known that anion and hydroxyl radicals degrade serotonergic terminals (Yang et al., 1997), and 5-HT synthesis is dependent on the balance between oxidant and antioxidant levels in the brain tissue (Trabace and Kendrick, 2000). It was reported that 5-HT has antioxidant properties that enable it to either scavenge nitric oxide or hypochlorous acid and peroxyl radicals by donating protons to them (Herraiz and Galisteo, 2004; Noda et al., 1999). 5-HT thus prevents phospholipid and lipid peroxidation at cell membrane surfaces (Marshall et al., 1996; Miura et al., 1996). Although the relationships between carcinogen exposure, lipid peroxidation, and the colonic serotonergic system are not well elucidated, we previously demonstrated that high 5-HT levels were a marker of the reaction of the colon to DMH treatment early in colon carcinogenesis, and that lowering 5-HT levels promoted preneoplastic lesions (Kannen et al., 2011a, 2012). Thus, DMH is likely to upregulate the ROS production, disturbing the neural and colonic serotonergic systems (Arutjunyan et al., 2001; Kannen et al., 2011a). Hence, we suggest that our current and previous findings, as well as the literature data, shed light on another major role that the serotoninergic system may play besides being involved in the feeding behavior and its control: the modulation of cancer risk by dietary factors. To summarize, our findings suggest that food deprivation enhanced initial tumor formation steps in the colon by suppressing the colonic serotonergic system through enhanced lipid-peroxidative processes in liver and colon tissues. Additionally, food deprivation may have further supported carcinogen-induced formation of preneoplastic lesions by compromising the physiological antioxidant defense situation. Because of its potential implications for (extreme) fasting regimens in humans, more studies are warranted to fully clarify our current observations. Funding This work was supported by the following agencies: Deutscher Akademischer Austausch Dienst (DAAD), Coordenac¸ão de Aperfeic¸oamento de Pessoa de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPQ), and Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP). Ethical approval The experimental protocol for this multidisciplinary and multilaboratory project, which was coordinated by Prof. Dr. Sergio B. Garcia, was approved by the Animal Care and Use Committee at the at the Medical School of Ribeirao Preto, University of Sao Paulo. This project involved 180 rats that were divided into 18 experimental groups according to treatments; of these 18 groups, results from 4
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