A 90-day oral toxicity study of purple corn color, a natural food colorant, in F344 rats.

A subchronic oral toxicity study of purple corn color (PCC), a natural food colorant, was performed with groups of 10 male and 10 female F344 rats fed the agent at dietary levels of 0%, 0.5%, 1.5% and 5.0% for 90 days. No mortalities occurred during the treatment period. No treatment-related changes in the body weight, food and water consumption, ophthalmology, hematology, organ weight data and histopathology were observed. Regarding general conditions and gross pathology, staining of fur and black feces were noted in rats of the 1.5% and 5.0% diet groups. Moreover, brown urine and black material in the stomach, small and large intestine were evident in rats receiving 5.0%. These changes were considered due to the anthocyanin content. On clinical chemistry analysis, total cholesterol, phospholipid and triglyceride were significantly lowered in both sexes of the 5.0% group, but these were not considered to be toxicologically significant. Thus, the No-observed-adverse-effect-level (NOAEL) was judged to be 5.0% in diet for both sexes (male: 3542 mg/kg/day, female: 3849 mg/kg/day) for PCC under the present experimental conditions.     

Chemopreventive efficacy of gallic acid,an antioxidant and anticarcinogenic polyphenol,against 1,2-dimethyl hydrazine induced rat colon carcinogenesis

Colon cancer is a major cause of morbidity and mortality in developed and developing countries and its etiology is known to be a combination of hereditary, environmental, dietary factors and lack of physical activity. Chemoprevention offers a novel approach to control the incidence of colon cancer. Gallic acid (GA) is a polyphenol widely present in tea and other plants which is popularly used in the traditional medicine of China. The present study was to evaluate the efficacy of GA supplementation on tissue lipid peroxidation and antioxidant defense system in 1,2-dimethyhydrazine (DMH) induced colon carcinogenesis in male Wistar rats. The rats were assorted into six groups, viz., group1 control rats received modified pellet diet; group 2 rats received GA (50 mg/kg body weight) orally along with modified pellet diet; group 3 rats received DMH (20 mg/kg body weight) subcutaneously once a week for the first 15 weeks; groups 4, 5 and 6 rats received GA along with DMH during the initiation, post- initiation stages and the entire period of study respectively. All the rats were sacrificed at the end of 30 weeks and the tissues were evaluated biochemically. We observed decreased lipid peroxidation (LPO) products such as thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH) and conjugated dienes (CD) and diminished levels of antioxidants such as superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione reductase (GR) and glutathione peroxidase (GPx) in the tissues of DMH treated rats, which were elevated significantly on GA supplementation. Moreover, enhanced activity of ascorbic acid and α-tocopherol levels were also observed in DMH alone treated rats which were significantly reduced on GA supplementation. Our results suggest that GA could exert a significant chemopreventive effect on DMH induced colon carcinogenesis.     

Reduction of 1,2-Dimethylhydrazine-induced Colorectal Proliferative Lesions in Mice by Aloe arborescens var. natalensis (Kidachi Aloe)

We examined the modifying effects of freeze-dried whole-leaf Aloe arborescens Miller var. natalensis Berger (Japanese name, Kidachi aloe; designated as ‘ALOE’) on 1,2-dimethylhydrazine (DMH)-induced colorectal tumorigenesis in mice. Female ICR mice (7-weeks old) were given a basal diet or a diet containing 1, 0.5 or 0.1% ALOE for 32 weeks. One week later, all mice were injected i.p. with DMH (20?mg/kg, once weekly for 10 weeks) or vehicle (1?mM?EDTA solution, pH 6.5). At 32 weeks, animals were killed by exsanguination, and the colorectums were processed for histological examination. The administration of ALOE (1, 0.5 or 0.1% in diet) did not induce diarrhea or reduction of body weight. In mice given DMH and 1% ALOE (Group 2), the incidence and multiplicity of colorectal adenomatous hyperplasias were significantly decreased as compared with mice given DMH alone (Group 1) (both p < 0.05), whereas the incidence and multiplicity of tumors (adenoma and adenocarcinoma) in Group 2 tended to be lower than those in Group 1. In addition, the incidence and multiplicity of the colorectal proliferative lesions (the total of adenomatous hyperplasias, adenomas and adenocarcinomas in mouse colorectum) in Group 2 were significantly decreased as compared with Group 1 (both p < 0.01). No colorectal proliferative lesions were found in animals that did not receive DMH. These results indicated that ALOE reduces the incidence and multiplicity of DMH-induced colorectal proliferative lesions, especially adenomatous hyperplasia, in mice.     

Influence of dietary components associated with high or low risk of colon cancer on apoptosis in the rat colon.

Although there is much epidemiological evidence for an interaction between diet and colorectal cancer risk, the mechanisms by which diet might protect against colorectal cancer are still unclear. Here we report the significant up-regulation of carcinogen-induced apoptosis in the colon of rats fed a diet containing low-risk factors for colon cancer, namely low fat content, high calcium and high non-digestible carbohydrate. The dose-dependent induction of apoptosis in colonic crypts by the carcinogen 1,2-dimethylhydrazine (DMH) was significantly greater in rats receiving the low-risk compared with a high-risk (high fat, low calcium, low non-digestible carbohydrate) diet (P<0.001). There were also significant interactions of colon region with DMH dose and region by diet, with the greatest increases in apoptosis occurring in the mid and distal regions of the colon compared with the proximal region. Since we have previously shown the low-risk diet to be non-toxic, these new results suggest a diet-induced up-regulation of apoptosis, which may represent a mechanism of protection against the early stages of carcinogenesis in the colon.     

Phenolics: blocking agents for heterocyclic amine-induced carcinogenesis.

Chemopreventive effects of synthetic and naturally occurring antioxidants on heterocyclic amine (HCA)-induced rat carcinogenesis and mechanisms of inhibition were assessed. In a medium-term liver bioassay, combined treatment with 0.03% 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and synthetic antioxidants such as 1-O-hexyl-2,3,5-trimethylhydroquinone (HTHQ), butylated hydroxyanisole (BHA), butylated hydroxutoluene (BHT), tert-butylhydroquinone (TBHQ) or propyl gallate, each at a dose of 0.25%, inhibited development of preneoplastic glutathione S-transferase placental form (GST-P) positive foci as compared with MeIQx alone, after initiation with diethylnitrosamine (DEN). Of these antioxidants, HTHQ showed the greatest activity. 8-Hydroxydeoxyguanosine (8-OHdG), a marker for DNA damage induced by active oxygen species, and malonedialdehyde and 4-hydroxynonenal levels were not largely influenced by the treatment with MeIQx or antioxidants, either alone or in combination. In the same medium-term liver bioassay, effects of some naturally occurring antioxidants, such as green tea catechins (GTC), hesperidin, chlorogenic acid, quercetin, rutin, curcumin, daidzin, ferulic acid and genistein were also examined. Of these antioxidants, only GTC tended to inhibit GST-P positive foci development, while quercetin, rutin, curcumin, daidzin, ferulic acid and genistein all exerted significant enhancing effects. Examination of HTHQ influence in a medium term liver bioassay with HCA Glu-P-1, in which the experimental period was extended for up to 26 weeks, also demonstrated a significant decrease in the incidence of liver tumours to 40% in the group treated with 0.5% HTHQ and 0.03% 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) as compared with the Glu-P-1 alone value of 89%. Effects of HTHQ on colon carcinogenesis induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were evaluated in a two-stage colon carcinogenesis model using 1,2-dimethylhydrazine (DMH) as an initiator. At week 36, the multiplicity of colon tumours induced by 0.02% PhIP after DMH initiation (9.1+/-6.2/rat) was dose-dependently decreased by the combined treatment with 0.5% HTHQ (3.6+/-1.8, P < 0.001) and 0.125% HTHQ (6.2+/-3.2, not significant). Similarly, the incidence of mammary carcinomas in female F344 rats induced by oral administration of 0.02% PhIP (40%) for 52 weeks was significantly decreased by simultaneous treatment with 0.5% HTHQ (5%). Alpha-tocopherol and chlorophyllin only reduced the multiplicity of carcinomas. Analysis of the influence of HTHQ on metabolic activation of Glu-P-1 or PhIP after incubation with rat S9 mixture and NADPH by HPLC, revealed that each major metabolite was strongly reduced by the addition of HTHQ. Immunohistochemically detected PhIP-DNA adduct positive nuclei in the colon induced by continuous oral treatment with 0.02% PhIP for 2 weeks decreased by the combined treatment with 0.5 or 0.125% HTHQ. These results indicate that synthetic antioxidant HTHQ is a very strong chemopreventor of heterocyclic amine (HCA)-induced carcinogenesis and that depressed metabolic activation rather than antioxidant activity is responsible for the observed effect.     

Dose-response,sex difference,and the effect of bran in dimethylhydrazine-induced intestinal tumorigenesis in rats

Dose-response and sex difference were examined in rats to the experimental colonic carcinogen dimethylhydrazine (DMH). Ten male and ten female weanling Sprague-Dawley rats were used in each group: Group 1-control, Group II-20% bran in the diet, Group III-15 mg/kg DMH given as 10 weekly oral doses, Group IV-bran and 15 mg/kg DMH, Group V-30 mg/kg DMH given as 10 weekly oral doses, and Group VI-bran and 30 mg/kg DMH. Male rats in Groups III and V had 2.1 and 6.4 colonic tumors/rat, respectively, and at the high dose of DMH, there was a 100% incidence of rats with colonic tumors. The addition of bran to the diet reduced the formation of colonic tumors. Female rats in Groups III and V had 0.2 and 0.6 colonic tumors/rat respectively, but only a 30% incidence of colonic tumors at the high dose. Dietary bran had no effect on the production of intestinal tumors in females. Thus, in this model system, the effects of a dose-response and sex difference are important considerations in experimental design.     

Efficacy of the potential chemopreventive agent, hesperetin (citrus flavanone), on 1,2-dimethylhydrazine induced colon carcinogenesis

Our current study is an effort to identify a potent chemopreventive agent against colon cancer. Here we have investigated the efficacy of hesperetin on tissue lipid peroxidation, antioxidant defense system and colonic histoarchitecture in male Wistar rats in colon carcinogenesis. Rats in groups 3, 4, 5 and 6 were treated with DMH (20 mg kg body weight s.c.) once a week for 15 weeks. Group 1 rats received modified pellet diet and served as control; group 2 received modified pellet diet along with hesperetin (20 mg/kg body weight, p.o., every day); and hesperetin was given to the rats as in-group 2 during the initiation, post-initiation and entire period stages of colon carcinogenesis. Lipid peroxidation was studied by measuring the formation of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH) and conjugated dienes (CD), and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), reduced glutathione (GSH), in the liver and colonic tissues of DMH administered rats. (1) Decreased levels of lipid peroxidation in the colonic tissues; (2) decreased activities of antioxidant enzymes SOD, CAT, GPX, GR and GSH levels in the tissues on DMH treatment. Hesperetin supplementation during the initiation, post-initiation and entire period stages of carcinogenesis significantly reversed these activities. These results indicate that hesperetin may be a potential chemopreventive agent against DMH-induced colon cancer.     

Dietary Chlorella protects against heterocyclic amine-induced aberrant gene expression in the rat colon by increasing fecal excretion of unmetabolized PhIP

The food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines formed by cooking meat and fish at high temperature. PhIP induces colorectal adenoma risk in male rats when administered orally. This study used male Fisher 344 rats to investigate the impact of dietary Chlorella on PhIP metabolism and aberrant colonic gene expression following short-term PhIP treatment. High-performance liquid chromatography analysis revealed that fecal excretion of unmetabolized PhIP was significantly increased in rats whose diets were supplemented with Chlorella compared to rats in a PhIP-only group (P < 0.001). Quantitative realtime PCR confirmed that the increase in beta-catenin and cyclin D1 mRNA in the colon induced by PhIP was ameliorated in rats pre-fed with Chlorella (P = 0.052 for beta-catenin; P = 0.005 for cyclin D1). The increase in DNA shearing that is a hallmark of caspase-8-mediated apoptosis by PhIP was also significantly diminished in the colons of rats pre-fed Chlorella (P = 0.012). These results suggested that administering dietary Chlorella with a Western-style diet concomitantly or immediately before mutagen exposure might be beneficial in blocking the absorption of food mutagens such as PhIP.     

Modification of Gastrointestinal Tumor Development in Rats by Dietary Butylated Hydroxytoluene

Modification of Gastrointestinal Tumor Development in Rats byDietary Butylated Hydroxytoluene. Lindenschmidt, R. C., TRYKA,A. F., AND WITSCHI, H. (1987). Fundam. Appl. Toxicol. 8, 474–481.Male Fischer 344 rats were given two or four injections of 1,2-dimethylhydra-zine(DMH), 40 mg/kg sc, and then fed a diet containing 0.5% butylatedhydroxytoluene (BHT). Five months later, the animals treatedwith two doses of DMH had a significantly higher incidence ofcolon tumors than the animals fed a BHT-free control diet. Inanimals treated with four injections of DMH, the increase incolon tumor incidence was statistically not significant, butBHT appeared to produce a shift in tumor distribution. In asecond experiment, Fischer 344 rats were treated with 2 x 40mg/kg of DMH and fed a diet of 0.5 or 0.1% BHT for 6 months;these animals had a significantly increased incidence of smallintestinal tumors (duodenum, jejunum, and ileum) compared withanimals fed the control diet. In rats treated with DMH and givena diet of 0.5% butylated hydroxyanisole (BHA), overall incidenceof gastrointestinal tract tumors was higher than in controlanimals, although the difference was statistically not significant.Administration of N-nitroso-N-methylurea (NMU; 90 mg/kg givenorally) produced stomach and colon tumors; 0.5% BHT in the dietdid not modulate tumor incidence. It is concluded that dietaryBHT may enhance development of gastrointestinal tumors producedby DMH, but not by NMU, provided exposure to BHT occurs afterexposure to the carcinogen.     

Influence of a fat-rich diet,folic acid supplementation and a human-relevant concentration of 2-amino-1-methyl-6-phenylimidazo[4,5-<Emphasis Type="Italic">b</Emphasis>]pyridine on the induction of preneoplastic lesions in the rat colon

In the present study, the effect of three controversially discussed risk factors for colorectal cancer, a fat-rich diet (16% raw fat content), dietary folic acid supplementation (50 mg folic acid/kg lab chow) and a human-relevant concentration (0.1 ppm) of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), either alone or in combination, on the induction of aberrant crypt foci (ACF) in the colon of male Fischer 344 rats was analyzed. The mean number of ACF per rat in the case of the four groups fed a fat-rich diet tended to be higher than that of the four groups being fed a standard diet. However, the increase in the mean number of ACF per rat only reached statistical significance in the case of the rats receiving a fat-rich lab chow supplemented with 50 mg/kg folic acid. Moreover, a concentration of 0.1 ppm PhIP per se, either in the standard or in the fat-rich lab chow, did not lead to an increase in the mean number of ACF per rat. In conclusion, the present study provides additional evidence for a colon cancer promoting effect of folic acid supplementation when rodents are fed the compound in supraphysiological concentrations.     

Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP-DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats.

The chemopreventive properties of dietary indole-3-carbinol (I3C) were evaluated by assessing its effect on DNA adduct formation and metabolism of the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and the induction of cytochromes P450 1A1 and -1A2 in female F344 rats. In experiment 1, animals on I3C diets (0, 0.02% or 0.1%, w/w) were treated by gavage with 1mg/kg/day of PhIP for 23 days. On days 2, 9, 16 and 23, their 24-hr urine was collected and unmetabolized PhIP was measured by GC/MS. On day 24, the animals were sacrificed, and DNA from pancreas, spleen, white blood cells (WBCs), lung, colon, kidney, mammary epithelial cells, caecum, heart, small intestine, liver and stomach was isolated for determination of PhIP-DNA adduct levels by (32)P-postlabelling assays. Except in the mammary gland, I3C diets significantly inhibited PhIP-DNA adduct formation in WBCs and in all organs, ranging from 34.7 to 67.7% with the 0.02% I3C diet to 68.4 to 95.3% with the 0.1% I3C diet. I3C diets also significantly decreased the concentration of urinary unmetabolized PhIP to 29.5-38.4% (0.02% I3C) and 12.8-17.8% (0.1% I3C) of values obtained with the I3C-free diet. In experiment 2, animals were either treated by intubation of I3C at 100 or 200mg/kg for 2 consecutive days or given an I3C-containing diet (0.02% or 0.1%, w/w) for 2 weeks. The expression and activity of cytochromes P450 1A1 and -1A2 were studied by Northern blots, Western blots, and in vitro enzyme determinations. Both the expression and activity of these cytochromes were induced by all of the I3C treatments. It is concluded that, in the female F344 rat, dietary I3C inhibits PhIP-DNA adduct formation and accelerates PhIP metabolism, probably through induction of cytochromes P450 1A1 and -1A2. The chemopreventive properties of I3C in PhIP-induced carcinogenesis are probably mediated through enhancement of PhIP detoxification pathways.     

A 13-week subchronic toxicity study of paprika color in F344 rats.

The fruit of the paprika (Capsicum annuum) has been widely used in various countries as a spice and food-coloring additive. As a part of the safety assessment of paprika color (Paprika oleoresin), a 13-week subchronic toxicity study was performed in F344 rats. To establish a no-observed-adverse-effect level (NOAEL) for application in subsequent long-term studies, rats were fed powder diet containing paprika color at dose levels of 0 (basal diet), 0.62, 1.25, 2.5 and 5% (maximum) for 13 weeks. During the experiment, there were no remarkable changes in general appearance and no deaths occurred in any experimental group. Although serum total cholesterol was dose-dependently increased in both sexes, no related histopathological changes were observed in the liver. Slight inflammatory cell infiltration in the myocardium and vacuolation of hepatocytes were noted in both control and paprika color-treated animals, but there were no clear differences between groups. In conclusion, paprika color even at 5% in the diet (0.67 g/rat/day or 2948.4 mg/kg bw/day for male rats and 0.43 g/rat/day or 3197.4 mg/kg bw/day for female rats) did not cause remarkable adverse effects in F344 rats. Thus, the NOAEL and the maximum dose level for carcinogenicity testing of paprika color were concluded to be 5% in the diet.     

Safety assessment of dietary administered paprika color in combined chronic toxicity and carcinogenicity studies using F344 rats

Combined chronic toxicity and carcinogenicity studies of paprika color, used as a food additive in various countries, were performed in male and female F344 rats. Dietary concentrations of 0%, 0.62%, 1.25%, 2.5% and 5% were applied in a 52-week toxicity study and 0%, 2.5% and 5% in a 104-week carcinogenicity study. Treatment with paprika color caused a significant increase in incidence of hepatocellular vacuolation in 5% males, but no toxicological effects were found with reference to survival rates, body weights, hematological or serum biochemical parameters and organ weights at any dose level in either sex in the chronic toxicity study. Also, paprika color did not induce specific tumors nor did it exert significant influence on the development of spontaneous tumors in any of the organs examined in the carcinogenicity study. In conclusion, based on slight histopathological changes observed in 5% male livers, the no-observed-effect level (NOEL) was estimated to be 2.5% in the diet (1,253 mg/kg bw/day) and the no-observed-adverse-effect level (NOAEL) was determined to be 5% in the diet (2,388 mg/kg bw/day) for male rats, and for females, the NOEL was concluded to be 5% in the diet (2,826 mg/kg bw/day). Additionally, paprika color was not carcinogenic to male and female F344 rats under the present experimental conditions.     

Co-carcinogenic effect of carbon black ingestion with dietary fat on the development of colon tumors in rats

Previous studies in our laboratory on the co-carcinogenic effect of the ingestion of an industrial carbon black (CB) on chemically induced colon cancer in rats and mice demonstrated no differences in tumor incidences attributable to CB feeding. The present study examined the effect of CB ingestion within the context of a high fat diet, formulated to simulate the typical diet of western industrialized nations. Corn oil was added to ground commercial chow at 20% by weight and CB added at 2.05 g/kg diet and fed for 52 weeks to female Sprague-Dawley rats. Colon tumors were induced with 16 weekly injections of 1,2-dimethylhydrazine (DMH, 10 mg/kg body weight). Tumor incidences in DMH-treated rats ingesting CB were significantly (P less than 0.05) higher than in those with no CB added to the diet (76% vs. 60%). The survival of CB + DMH treated animals (64%) was also lower than that of animals treated with DMH and not ingesting CB (80%). These findings may implicate CB ingestion as a co-carcinogen for industrial workers when acting in synergism with high fat diets and other unknown colon carcinogens.     

Lack of chemoprevention of dietary Agaricus blazei against rat colonic aberrant crypt foci

The mushroom Agaricus blazei (Ab) has been widely used in folk medicine to treat various diseases including cancer. No information is available on its possible protective effects on the development of colon cancer. The potential blocking effect of Ab intake on the initiation stage of colon carcinogenesis was investigated in a short-term (4-week) bioassay using aberrant crypt foci (ACF) as biomarker. Male Wistar rats were given four subcutaneous injections of the carcinogen 1,2-dimethylhydrazine (DMH, 40 mg/kg bw, twice a week), during 2 weeks to induce ACF. The diet containing Ab at 5% was given 2 weeks before and during carcinogen treatment to investigate the potential beneficial effects of this edible mushroom on DMH-induced ACF. All groups were killed at the end of the fourth week. The colons were analyzed for ACF formation in 1% methylene blue whole-mount preparations and for cell proliferation in histological sections immunohistochemically stained for the proliferating cell nuclear antigen (PCNA). All DMH-treated rats developed ACF mainly in the middle and distal colon. Agaricus blazei intake at 5% did not alter the number of ACF induced by DMH or the PCNA indices in the colonic mucosa. Thus, the results of the present study did not confirm a chemopreventive activity of Ab on the initiation stage of rat colon carcinogenesis.     

Açai (Euterpe oleracea Mart.) feeding attenuates dimethylhydrazine-induced rat colon carcinogenesis

This study investigated the protective effect of spray-dried açaí powder (AP) intake on colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male Wistar rats. After 4 weeks of DMH administrations, the groups were fed with standard diet, a diet containing 2.5% or 5.0% AP or a diet containing 0.2% N-acetylcysteine (NAC) for 10 weeks, using aberrant crypt foci (ACF) as the endpoint. Additionally, two groups were fed with standard diet or a diet containing 5.0% AP for 20 weeks, using colon tumors as the endpoint. In ACF assay, a reduction in the number of aberrant crypts (ACs) and ACF (1–3 AC) were observed in the groups fed with 5.0% AP (37% AC and 47% ACF inhibition, p = 0.036) and 0.2% NAC (39% AC and 41% ACF inhibition, p = 0.042). In tumor assay, a reduction in the number of invasive tumors (p < 0.005) and tumor multiplicity (p = 0.001) was observed in the group fed with 5.0% AP. Also, a reduction in tumor Ki-67 cell proliferation (p = 0.003) and net growth index (p = 0.001) was observed in the group fed with 5.0% AP. Therefore the findings of this study indicate that AP feeding may reduce the development of chemically-induced rat colon carcinogenesis.     

Modifying effects of chitin, chitosan and their related compounds on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in a rat medium-term hepatocarcinogenesis model, and their post-initiation effects in a female rat 2-stage multi-organ carcinogenesis model

The modifying effects of chitin, chitosan, chitin-oligo sugar, chitosan-oligo sugar and chlorophyllin-chitosan on 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated in a rat medium-term hepatocarcinogenesis model. Male F344 rats were injected with diethylnitrosamine (DEN) and starting 2 weeks later, received 0.03% MeIQx alone, MeIQx plus each chemical (0.4%), or each chemical alone (0.1%) in diet for 6 weeks. Three weeks after the DEN injection, animals were subjected to 2/3 partial hepatectomy. The numbers and areas of glutathione S-transferase placental form (GST-P) positive foci given MeIQx plus test chemicals were similar to the MeIQx alone values. In a second experiment, post-initiation effects of chitin and chitosan on major organs were examined in female F344 rats after initiation with 1,2-dimethylhydrazine (DMH), 7,12-dimethylbenz[a]anthracene (DMBA) and 2,2′-dihydroxy-di-n-propylnitrosamine (DHPN). In rats fed a diet containing 1.0% chitin for 36 weeks, the development of palpable mammary tumors tended to be delayed and the final incidence and multiplicity of adenocarcinomas, were significantly lowered. However, in the colon, the number of aberrant crypt foci (ACF) in the chitin and chitosan groups was significantly increased. These results indicate that chitin, chitosan and related compounds do not exert unequivocal chemopreventive effects on heterocyclic amine-induced hepatocarcinogenesis, and that effects in other organs may be tissue specific with possible inhibitory action in the mammary gland being offset by promotion of colon lesion development.     

One-year chronic toxicity of madder color in F344 rats – Induction of preneoplastic/neoplastic lesions in the kidney and liver

To evaluate chronic toxicity of madder color (MC), a natural food colorant extracted from the roots of Rubia tinctorum L., F344 rats were fed diet containing 0%, 0.2%, 1.0% or 5.0% MC for 53 weeks. Hematological changes including anemia and serum biochemical alterations indicating hepatotoxicity were demonstrated at 5.0% in both sexes. Relative weights of the liver were significantly increased from 1.0% in both sexes, and those of the kidney were significantly increased from 1.0% in males and from 0.2% in females. Histopathologically, atypical renal tubule hyperplasias were increased at 1.0% or higher in both sexes in association with increase of cell proliferative activity in the tubules. A renal cell adenoma was observed in a male rat receiving 5.0% MC. In addition, glutathione S-transferase placental form-positive liver cell foci were significantly increased at 5.0% in both sexes. These results indicate that MC has chronic toxicity targeting kidney, liver and blood cells. Moreover, the results strongly suggest that MC may have the carcinogenic potential in the kidney and the liver.     

Polymeric black tea polyphenols inhibit 1,2-dimethylhydrazine induced colorectal carcinogenesis by inhibiting cell proliferation via Wnt/beta-catenin pathway

Tea polyphenols like epigallocatechin gallate and theaflavins are established chemopreventive agents for colorectal carcinogenesis. However, studies on evaluating similar chemopreventive properties of thearubigins or polymeric black tea polyphenols (PBPs), the most abundant polyphenols in black tea, are limited. Hence, in the present study we aim to investigate chemopreventive effects along with probable mechanisms of action of PBP extract employing 1,2-dimethylhydrazine (DMH)-induced colorectal carcinogenesis in Sprague-Dawley rats as experimental model. The present study suggests that PBPs, like other tea polyphenols, also inhibit DMH-induced colorectal tumorigenesis by decreasing tumor volume and multiplicity. This study also shows that although the pretreatment with PBP extract could induce detoxifying enzymes in hepatic and colorectal tissue, it did not show any additional chemopreventive effects when compared to treatments with PBP extract after initiation with DMH. Mechanistically, PBP extract may inhibit colorectal carcinogenesis by decreasing DMH-induced cell proliferation via Wnt/beta-catenin pathway. Treatments with PBP extract showed decreased levels of COX-2, c-MYC and cyclin D1 proteins which aid cell proliferation probably by regulating beta-catenin by maintaining expression of APC and decreasing inactivation of GSK3beta. DMH-induced activation of MAP kinases such as ERK and JNK was also found to be inhibited by treatments with PBP extract. In conclusion, the protective effects of PBP extract could be attributed to inhibition of DMH-induced cellular proliferation probably through beta-catenin regulation.     

Effects of lycopene,synbiotic and their association on early biomarkers of rat colon carcinogenesis

This study evaluated whether a synergy exists for the combined treatment with lycopene and synbiotic on early biomarkers of colon carcinogenesis. Male Wistar rats received a diet containing 300 mg/kg of lycopene and/or synbiotic (Bifidobacterium lactisplus oligofructose/inulin) or their combination 2 weeks before and during carcinogen treatment with 1,2-dimethylhydrazine (DMH). Twenty-four hours after the last DMH application, the colons were processed for immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), p53 protein, hematoxylin–eosin staining for apoptosis analysis and genotoxicity of fecal water by comet assay. Eight weeks after the last DMH application, the colons were analyzed for development of classical aberrant crypt foci (ACF) and mucin-negative ACF. Treatment with lycopene, synbiotic or their combination significantly increased apoptosis, reduced the PCNA and p53 labeling indexes and the development of classical ACF and mucin-negative ACF. Furthermore, a lower genotoxicity of fecal water was also detected in the groups treated with the chemopreventive agents. An additive/synergistic effect of the combined treatment with lycopene/synbiotic was observed only for the fecal water genotoxicity and mucin-negative ACF parameters. These results indicate that an additive/synergistic of the combination of chemopreventive agents on the initiation phase of colon carcinogenesis can be detected using selective early biomarkers.