Klebsiella aerogenes exacerbates colon tumorigenesis in the AOM/DSS-induced C57BL/6J mouse.

Klebsiella aerogenes (K. aerogenes, KA) is a gram-negative opportunistic pathogen from the Klebsiella species and the Enterobacteriaceae family. However, the impact of K. aerogenes on colorectal cancer (CRC) remains uncertain. A colitis-associated tumorigenesis animal model was established by administering azoxymethane (AOM) and dextran sulfate sodium (DSS) to C57BL/6J mice. The concentration of K. aerogenes gavage in mice was 109 cfu. The study measured the following parameters: tumor formation (number and size), intestinal permeability (MUC2, ZO-1, and Occludin), colonic inflammation (TNF-α, IL-1ß, IL-6, and IL-10), proliferation and the fluctuation of the intestinal flora. Under the AOM/DSS-treated setting, K. aerogenes colonization worsened colitis by exacerbating intestinal inflammatory reaction and destroying the mucosal barrier. The intervention markedly augmented the quantity and dimensions of neoplasm in the AOM/DSS mice, stimulated cellular growth, and impeded cellular programmed cell death. In addition, K. aerogenes exacerbated the imbalance of the intestinal microbiota by elevating the abundance of Pseudomonas, Erysipelatoclostridium, Turicibacter, Rikenella, and Muribaculum and leading to a reduction in the abundance of Odoribacter, Alloprevotella, Roseburia, and Lachnospiraceae_NK4A136_group. The presence of K. aerogenes in AOM/DSS-treated mice promoted tumorigenesis, worsened intestinal inflammation, disrupted the intestinal barrier, and caused disturbance to the gut microbiota.

Panaxynol alleviates colorectal cancer in a murine model via suppressing macrophages and inflammation.

Currently available colorectal cancer (CRC) therapies have limited efficacy and severe adverse effects that may be overcome with the alternative use of natural compounds. We previously reported that panaxynol (PA), a bioactive component in American ginseng, possesses anticancer properties in vitro and suppresses murine colitis through its proapoptotic and anti-inflammatory properties. Because colitis is a predisposing factor of CRC and inflammation is a major driver of CRC, we sought to evaluate the therapeutic potential of PA in CRC. Azoxymethane-dextran sodium sulfate (AOM/DSS) mice (C57BL/6) were administered 2.5 mg/kg PA or vehicle 3 times/wk via oral gavage over 12 wk. PA improved clinical symptoms (P ≤ 0.05) and reduced tumorigenesis (P ≤ 0.05). This improvement may be reflective of PA's restorative effect on intestinal barrier function; PA upregulated the expression of essential tight junction and mucin genes (P ≤ 0.05) and increased the abundance of mucin-producing goblet cells (P ≤ 0.05). Given that macrophages play a substantial role in the pathogenesis of CRC and that we previously demonstrated that PA targets macrophages in colitis, we next assessed macrophages. We show that PA reduces the relative abundance of colonic macrophages within the lamina propria (P ≤ 0.05), and this was consistent with a reduction in the expression of important markers of macrophages and inflammation (P ≤ 0.05). We further confirmed PA's inhibitory effects on macrophages in vitro under CRC conditions (P ≤ 0.05). These results suggest that PA is a promising therapeutic compound to treat CRC and improve clinical symptoms given its ability to inhibit macrophages and modulate the inflammatory environment in the colon.NEW & NOTEWORTHY We report that panaxynol (PA) reduces colorectal cancer (CRC) by improving the colonic and tumor environment. Specifically, we demonstrate that PA improves crypt morphology, upregulates crucial tight junction and mucin genes, and promotes the abundance of mucin-producing goblet cells. Furthermore, PA reduces macrophages and associated inflammation, important drivers of CRC, in the colonic environment. This present study provides novel insights into the potential of PA as a therapeutic agent to ameliorate CRC tumorigenesis.

Unexpected Liver and Kidney Pathology in C57BL/6J Mice Fed a High-fat Diet and Given Azoxymethane to Induce Colon Cancer.

Multiple animal models have been developed to investigate the pathogenesis of colorectal cancer and to evaluate potential treatments. One model system uses azoxymethane, a metabolite of cycasin, alone and in conjunction with dextran sodium sulfate to induce colon cancer in rodents. Azoxymethane is metabolized by hepatic P450 enzymes and can also be eliminated through the kidneys. In this study, C57BL/6J mice were fed either standard or high-fat diet and then all mice received azoxymethane at 10 mg/kg body weight twice a week for 6 wk. Shortly after the end of treatment, high mortality occurred in mice in the high-fat diet group. Postmortem examination revealed hepatic and renal pathology in mice on both diets. Histologic changes in liver included hepatocytomegaly with nuclear pleomorphism and bile duct hyperplasia accompanied by mixed inflammatory-cell infiltrates. Changes in the kidneys ranged from basophilia of tubular epithelium to tubular atrophy. The results indicate that further optimization of this model is needed when feeding a high-fat diet and giving multiple azoxymethane doses to induce colon cancer in C57BL/6J mice.

Zinc dyshomeostasis in azoxymethane-induced colonic precancerous and cancerous lesions in Fischer rats.

Zinc is an essential micronutrient involved in various biological processes. It is also argued that tumors need zinc for maintenance and proliferation and tumor cell apoptosis. Zinc homeostasis is regulated by the gastrointestinal tract and involves interplay of host, dietary, environmental and social factors such as alcohol consumption. The DNA alkylation agent azoxymethane (AOM), which is primarily activated in the liver, induces a high incidence of initiation and promotion steps of precancerous lesions in the colon of rats. The altered expression of hepatic zinc transporters by AOM may lead to zinc dyshomeostasis in liver. Decreased serum zinc concentration, despite increased liver zinc also indicates altered liver zinc mobilization and failure to regulate zinc homeostasis. During the transformation from normal colonic mucosa to colonic epithelial hyperplasia and aberrant crypt formation, a reduction in zinc concentration is observed. It will be interesting to study further if the same trend continues throughout tumor progression towards adenocarcinomas. Lowered local zinc concentrations in the colon epithelium may not just reflect a bystander effect, but may induce cell proliferation and compromise DNA integrity due to impairment of zinc-containing proteins. In congruence with the tissue zinc concentrations, metallothionein levels were found to be less induced in AOM -administered colon compared to normal healthy colon. Lowered tissue zinc levels in small and large intestine were also associated with increased expression of mRNA and protein ZnT1. In this regard, the mode of zinc responsiveness to ZnT1 mimics that of metallothionein, albeit at a lower level for ZnT1.

Prevention of tumorigenesis in mice by exercise is dependent on strain background and timing relative to carcinogen exposure.

Among cancer diagnoses, colorectal cancer (CRC) is prevalent, with a lifetime risk of developing CRC being approximately 5%. Population variation surrounding the mean risk of developing CRCs has been associated with both inter-individual differences in genomic architecture and environmental exposures. Decreased risk of CRC has been associated with physical activity, but protective responses are variable. Here, we utilized a series of experiments to examine the effects of genetic background (strain), voluntary exercise (wheel running), and their interaction on azoxymethane (AOM)-induced intestinal tumor number and size in mice. Additionally, we investigated how the timing of exercise relative to AOM exposure, and amount of exercise, affected tumor number and size. Our results indicated that voluntary exercise significantly reduced tumor number in a strain dependent manner. Additionally, among strains where exercise reduced tumor number (A/J, CC0001/Unc) the timing of voluntary exercise relative to AOM exposure was crucial. Voluntary exercise prior to or during AOM treatment resulted in a significant reduction in tumor number, but exercise following AOM exposure had no effect. The results indicate that voluntary exercise should be used as a preventative measure to reduce risk for environmentally induced CRC with the realization that the extent of protection may depend on genetic background.

KAG-308, a newly-identified EP4-selective agonist shows efficacy for treating ulcerative colitis and can bring about lower risk of colorectal carcinogenesis by oral administration.

Agonists for EP4 receptor, a prostaglandin E2 receptor subtype, appear to be a promising therapeutic strategy for ulcerative colitis (UC) due to their anti-inflammatory and epithelial regeneration activities. However, the clinical development of orally-available EP4 agonists for mild to moderate UC has not yet been reported. Furthermore, the possibility of an increased risk of colitis-associated cancer (CAC) through direct proliferative effects on epithelial cells via EP4 signaling has not been ruled out. Recently, we identified KAG-308 as an orally-available EP4-selective agonist. Here, we investigated the pharmacological and pharmacokinetic profiles of KAG-308. Then, we compared KAG-308 and sulfasalazine (SASP) for their abilities to prevent colitis and promote mucosal healing in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Finally, the effect of KAG-308 treatment on CAC was evaluated in an azoxymethane (AOM)/DSS-induced CAC mouse model. KAG-308 selectively activated EP4 and potently inhibited tumor necrosis factor-α production in peripheral whole blood and T cells. Oral administration of KAG-308, which showed relatively high bioavailability, suppressed the onset of DSS-induced colitis and promoted histological mucosal healing, while SASP did not. KAG-308 also prevented colorectal carcinogenesis by inhibiting colitis development and consequently decreasing mortality in a CAC model, whereas SASP had marginal effects. In contrast, MF-482, an EP4 antagonist, increased mortality. These results indicated that orally-administered KAG-308 suppressed colitis development and promoted mucosal healing. Moreover, it exhibited preventive effects on colorectal carcinogenesis, and thus may be a new therapeutic strategy for the management of UC that confers a reduced risk of colorectal carcinogenesis.

Dietary linoleic acid and glucose enhances azoxymethane-induced colon cancer and metastases via the expression of high-mobility group box 1.

OBJECTIVE: High-mobility group box 1 (HMGB1) was closely associated with progression and metastasis of colorectal cancer. METHODS: We examined the significance of HMGB1 in causing colon carcinogenesis induced by azoxymethane (AOM) injection in Fischer 344 rats fed on a control diet (group C), a 15% linoleic acid (LA) diet (group L), a control diet with 10% glucose drink (group G), and a 15% LA diet with a 10% glucose drink (group L+G). RESULTS: Group L+G showed the highest body weight and calorie intake. Serum and mucosal HMGB1 levels were temporally increased in all groups, while the highest levels were observed in group L+G. Mucosal HMGB1 levels were correlated with cancer multiplicity and nodal metastases. In the AOM-injected rats fed the 15% LA diet with 10% glucose drink, administration of HMGB1 antibody suppressed serum HMGB1 concentration and cancer multiplicity. CONCLUSION: These data suggest that dietary LA and glucose provided the synergistic effect on AOM-induced rat colon cancer through HMGB1 induction.

Epimorphin deletion protects mice from inflammation-induced colon carcinogenesis and alters stem cell niche myofibroblast secretion.

Epithelial-mesenchymal interactions regulate normal gut epithelial homeostasis and have a putative role in inflammatory bowel disease and colon cancer pathogenesis. Epimorphin is a mesenchymal and myofibroblast protein with antiproliferative, promorphogenic effects in intestinal epithelium. We previously showed that deletion of epimorphin partially protects mice from acute colitis, associated with an increase in crypt cell proliferation. Here we explored the potential therapeutic utility of modulating epimorphin expression by examining the effects of epimorphin deletion on chronic inflammation-associated colon carcinogenesis using the azoxymethane/dextran sodium sulfate (AOM/DSS) model. We found that mice in which epimorphin expression was absent had a marked reduction in incidence and extent of colonic dysplasia. Furthermore, epimorphin deletion in myofibroblasts altered the morphology and growth of cocultured epithelial cells. Loss of epimorphin affected secretion of soluble mesenchymal regulators of the stem cell niche such as Chordin. Importantly, IL-6 secretion from LPS-treated epimorphin-deficient myofibroblasts was completely inhibited, and stromal IL-6 expression was reduced in vivo. Taken together, these data show that epimorphin deletion inhibits chronic inflammation-associated colon carcinogenesis in mice, likely as a result of increased epithelial repair, decreased myofibroblast IL-6 secretion, and diminished IL-6-induced inflammation. Furthermore, we believe that modulation of epimorphin expression may have therapeutic benefits in appropriate clinical settings.

Mitogenic effects of both amidated and glycine-extended gastrin-releasing peptide in defunctioned and azoxymethane-treated rat colon in vivo.

Although there is abundant evidence that gastrin-releasing peptide acts as a mitogen in various carcinoma cell lines, the effect of administration of gastrin-releasing peptide on the colorectal mucosa in vivo has not been reported. The aims of this study were to determine whether continuous infusion of gastrin-releasing peptide stimulated proliferation or accelerated carcinogenesis in the rat gastrointestinal tract and other organs. The possible requirement for C-terminal amidation for mitogenic activity in vivo was also investigated. Proliferation was measured in the colon by metaphase index and by immunostaining for the proliferation marker Ki-67, and in other tissues by immunostaining alone. Acceleration of colorectal carcinogenesis was assessed by counting aberrant crypt foci after treatment with the carcinogen azoxymethane. Defunctioning of the rectum reduced both the proliferative index and the crypt height of the rectal mucosa of untreated rats. Treatment with amidated or glycine-extended gastrin-releasing peptide for 4 weeks using implanted mini-osmotic pumps resulted in a two- to three-fold increase in proliferation, and an increase in crypt height, in the defunctioned rectal mucosa (p<0.001), with smaller but significant increases in the caecum and distal colon. No changes in proliferation were detected in lung, pancreas or gastric mucosa. The numbers of aberrant crypt foci in the mid-colon, distal colon and rectum following treatment with azoxymethane were also significantly increased by infusion with amidated or glycine-extended gastrin-releasing peptide. We conclude that administration of gastrin-releasing peptide to mature rats stimulates proliferation and accelerates carcinogenesis in the colorectal mucosa, and that C-terminal amidation is not essential for either effect. Gastrin-releasing peptides could thus potentially act as promoters of colorectal carcinogenesis.

Tissue-specific attenuation of endogenous DNA I-compounds in rats by carcinogen azoxymethane: possible role of dietary fish oil in colon cancer prevention.

I-compounds are bulky covalent DNA modifications that are derived from metabolic intermediates of nutrients. Some I-compounds may play protective roles against cancer, aging, and degenerative diseases. Many carcinogens and tumor promoters significantly reduce I-compound levels gradually during carcinogenesis. Colon cancer is the second leading cause of cancer death in the United States, whereas cancer of the small intestine is relatively rare. Here we have studied levels of I-compounds in DNA of colon and duodenum of male Sprague-Dawley rats treated with azoxymethane. The effects of dietary lipids (fish oil or corn oil) on colon and duodenal DNA I-compounds were also investigated. Rats fed a diet containing fish oil or corn oil were treated with 15 mg/kg azoxymethane. Animals were terminated 0, 6, 9, 12, or 24 hours after injection. I-compound levels were analyzed by the nuclease P1-enhanced (32)P-postlabeling assay. Rats treated with azoxymethane displayed lower levels of I-compounds in colon DNA compared with control groups (0 hour). However, I-compound levels in duodenal DNA were not diminished after azoxymethane treatment. Animals fed a fish oil diet showed higher levels of I-compounds in colonic DNA compared with corn oil groups (mean adduct levels for fish and corn oil groups were 13.35 and 10.69 in 10(9) nucleotides, respectively, P = 0.034). Taken together, these results support claims that fish oil, which contains a high level of omega-3 polyunsaturated fatty acids, may have potent chemopreventive effects on carcinogen-induced colon cancer. The fact that duodenal I-compounds were not diminished by azoxymethane treatment may have been due to the existence of tissue-specific factors protecting against carcinogenesis. In conclusion, our observations show that endogenous DNA adducts may serve not only as sensitive biomarkers in carcinogenesis and cancer prevention studies, but are also helpful to further our understanding of the chemopreventive properties of omega-3 fatty acids and mechanisms of carcinogenesis.

Experimental evidences on the potential of prebiotic fructans to reduce the risk of colon cancer.

Inulin is extracted from the chicory root. It is a set of fructans with its monomers linked by means of beta(2-1) bonds. This linkage cannot be hydrolysed by either pancreatic or by brush border digestive enzymes in the upper intestinal tract of humans. As such the carbohydrates arrive in the colon, where they are fermented by bifidobacteria and other lactic acid producing bacteria, thus enhancing their relative populations in the gut. Recent research in experimental animal models revealed that inulin has significant anticarcinogenic properties. It acts chemopreventively by reducing the incidence of azoxymethane (AOM) - induced aberrant crypt foci and tumours in the colon. These effects may be due to the stimulation of bifidobacteria, which themselves have been shown to act as antigenotoxic in the colon and to reduce AOM-induced tumours. Also fermentation products, including the short-chain fatty acid butyrate, could contribute to the protective effects. In this case a mechanism may be the induction of apoptosis of already transformed cells. The experimental evidence from animal studies and from studies elucidating potential mechanisms strongly supports the possibility that inulin will contribute to reducing risks for colon cancer in humans. In order to obtain more insight into this possibility, human dietary intervention studies relating biomarkers of reduced risk to inulin consumption are needed.

Effect of tea on the formation of DNA adducts by azoxymethane.

1. The effect of black tea on the conversion of azoxymethane (AOM) to DNA reactive metabolites was studied in four groups of the male F344 rat. Each received 1.25% solutions of tea for 2 or 6 weeks, and simultaneous controls drank water. All rats were injected s.c. twice with 15 mg/kg AOM after the first or fifth week respectively, on tea or water, and again 1 week later. Groups were killed 6 h after the last dose, or 18 h later. The liver and colon were rapidly removed and rinsed with buffer solution, pH 7.0. DNA was isolated from these tissues, and DNA methylation was examined by the typical fluorescence of 06-methylated and N-7-methylated products, separated by HPLC. 2. Two or 6 weeks of tea intake failed to affect significantly the formation of alkylated DNA from liver and colon compared with controls drinking water. Only in the group of rats on tea for 6 weeks, and killed 6 h after the last dose of AOM, was the O6-methyldG and 7-methyldG decreased in DNA obtained from colon. 3. Thus, solutions of tea affected the formation of alkylated products in DNA of the colon of rats given AOM only at one time point, but did not do so under most other experimental conditions. The underlying mechanism is based on our previous finding that tea does not affect cytochrome P4502E1 that our group established to be the enzyme metabolizing AOM.

Mechanism of benzylselenocyanate inhibition of azoxymethane-induced colon carcinogenesis in F344 rats.

Benzylselenocyanate (BSC), a novel organoselenium compound, has been found to inhibit azoxymethane (AOM)-induced colon carcinogenesis in rats during initiation. To investigate its mechanism of action, we examined the effects of BSC feeding on the following parameters: (a) metabolism of [14C]AOM to 14CO2 in vivo; (b) metabolic activation of AOM to MAM and of MAM to formic acid and methanol by rat liver microsomes in vitro; and (c) AOM-induced DNA methylation in rat livers and colons. Five-week-old male F344 rats were fed modified (23% corn oil) AIN-76A diets containing 0 (control), 25, or 50 ppm of BSC or benzylthiocyanate (BTC), a sulfur analogue of BSC which does not inhibit the colon carcinogenicity of AOM. After 3 weeks, rats were either sacrificed for the isolation of liver microsomes or were given 15 mg/kg of [14C]AOM s.c. to determine the rate of carcinogen metabolism in vivo. No difference in [14C]AOM metabolism was found between rats fed the BTC diets and those fed the control diet. In contrast, the rate of [14C]AOM metabolism, as determined by exhaled radioactivity, was 2-3 times higher in rats fed the BSC diets. While liver microsomes from rats fed the BTC diets metabolized AOM and MAM at rates not significantly different from those obtained with control liver microsomes, the metabolic activation of AOM as well as of MAM was stimulated severalfold when assayed with liver microsomes from rats fed the BSC diets. An increase in total liver cytochrome P-450 was also observed in the BSC-fed rats. Following the administration of 15 mg/kg AOM, significantly less O6-methylguanine and 7-methylguanine was present in the colon DNA from rats consuming the BSC diets than in rats fed the BTC or control diets. The body weight gains of rats fed the 25- and 50-ppm BSC-containing diets for 3 weeks were less (27 and 43%, respectively) than those of rats fed either the control or BTC-containing diets. These results indicate that dietary BSC significantly induces the hydroxylation of AOM and the oxidation of MAM in rat liver. An increase in the rates of AOM and MAM metabolism in the liver due to enzyme induction by BSC will result in decreased delivery of MAM to the colon via the bloodstream. This will be reflected in decreased DNA alkylation, as observed, and is likely to be a major factor in the inhibition of AOM-induced colon carcinogenesis by BSC.

Inhibition by dietary oltipraz of experimental intestinal carcinogenesis induced by azoxymethane in male F344 rats.

Epidemiological studies suggest that consumption of cruciferous vegetables rich in dithiolethiones is associated with a reduction in the incidence of cancer in man. The effect of two dose levels of dietary oltipraz [5-(2-pyrazinyl)-4-methyl-1, 2- dithiole-3-thione], a substituted dithiolethione, on azoxymethane (AOM)-induced intestinal carcinogenesis and on serum levels was studied in male F344 rats. The maximum tolerated dose (MTD) of oltipraz was determined in male F344 rats and found to be 500 p.p.m. Oltipraz at levels of 200 p.p.m. (40% MTD) and 400 p.p.m. (80% MTD) diet was tested as inhibitor of intestinal carcinogenesis. At 5 weeks of age, animals were fed the modified AIN-76A (control) diet and experimental diets containing oltipraz. At 7 weeks of age, all animals except the vehicle-treated animals were administered s.c. injection of AOM (15 mg/kg body wt/week for 2 weeks). Animals intended for vehicle treatment were administered s.c. with an equal volume of normal saline. Fifty-two weeks later, all animals were killed and colon and small intestinal tumor incidences and multiplicity were compared among the dietary groups. The results indicate that feeding of 200 and 400 p.p.m. of oltipraz significantly inhibited the incidence of adenocarcinomas in colon and small intestine and multiplicity of colon adenomas and small intestinal adenocarcinomas. Animals fed 400 p.p.m. oltipraz showed increased levels of oltipraz in the serum as compared to those fed 200 p.p.m. oltipraz. The results of this study indicate that dietary oltipraz inhibits intestinal carcinogenesis.

Metabolism of azoxymethane, methylazoxymethanol and N-nitrosodimethylamine by cytochrome P450IIE1.

The metabolism of azoxymethane (AOM), methylazoxymethanol (MAM) and N-nitrosodimethylamine (NDMA) by liver microsomes from acetone-induced rats as well as by a reconstituted system containing purified cytochrome P450IIE1 was examined. The products consisted of MAM from AOM; methanol and formic acid from MAM; and methylamine, formaldehyde, methanol, methylphosphate and formic acid from NDMA. Compared to liver microsomes from untreated rats, the metabolic activity of acetone-induced microsomes was approximately 4 times higher for all three carcinogens. Using the reconstituted system, the enzyme activities (nmol substrate metabolized/nmol P450/min) for AOM, MAM and NDMA were 2.88 +/- 1.14, 2.87 +/- 0.59 and 9.47 +/- 2.24 respectively. Incubations carried out in the presence of a monoclonal antibody to cytochrome P450IIE1 resulted in a 85-90% inhibition of all three reactions in this system. These results provide conclusive evidence that AOM, MAM and NDMA are metabolized by the same form of rat liver cytochrome P450. In addition, the stoichiometry of NDMA products formed in these reactions indicates that denitrosation, a presumed detoxication process, and alpha-hydroxylation, an activation reaction, are also catalyzed by the same cytochrome P450 isozyme.

Inhibition by dietary ethanol of experimental colonic carcinogenesis induced by high-dose azoxymethane in F344 rats.

Epidemiological studies have shown an association between consumption of alcoholic beverages and increased occurrence of large bowel carcinoma, but studies in experimental models of colonic carcinogenesis have produced conflicting results. We assessed the effects of chronic dietary ethanol consumption during the preinduction and induction phase (period of acclimatization and carcinogen administration) in a high-dose azoxymethane-treated rat model (14 mg/kg/wk for 10 wk). Ten-wk-old male Fischer 344 rats were given 33% of calories as ethanol or no ethanol (controls). Pair-feeding with Lieber-DeCarli-type liquid diets provided comparable total carbohydrates, proteins, fats, and calories. After 3 wk of dietary acclimatization, injections of azoxymethane (AOM) were given s.c. to all rats in Wk 1 to 10. At necropsy in Wk 25, dramatic suppression of gastrointestinal tumorigenesis was evident in the ethanol-fed group: the prevalence of colonic tumors was 5% as compared with 91% in controls; and the prevalence of small bowel tumors was 0% versus 74% (P less than 0.0001). In an analogous study of [14C]AOM metabolism, exhaled 14CO2 was decreased in the ethanol-fed rats, indicating suppression of AOM metabolism. Similarly, in the ethanol-fed rats the levels of the DNA adducts O6-methylguanine and 7-methylguanine 24 h after AOM injection were reduced in the colonic mucosa to 14 +/- 7% and 61 +/- 11% of controls and in the liver to 80 +/- 9% and 86 +/- 6 of controls. By contrast, rats changed from the ethanol diet to no-ethanol diet for 12 h prior to the dose of [14C]AOM metabolized the carcinogen at a faster rate than controls, indicating loss of suppression with cessation of ethanol intake along with induction of metabolizing enzymes; DNA adduct levels were reduced in the colonic mucosa to 90 +/- 13% and 76 +/- 9% of controls and in the liver to 81 +/- 6% and 85 +/- 3% of controls. Our findings indicate that dietary ethanol during the preinduction and induction phase of the AOM model dramatically inhibits tumorigenesis, even with high dosage of carcinogen, and suggest that: (a) inhibition of tumorigenesis may result from suppression of metabolic activation of AOM and the consequent reduced formation of DNA adducts during the induction (initiation) phase of the model; (b) these anti-initiation effects of ethanol are unrelated to the epidemiological association between consumption of alcoholic beverages and large bowel cancer; and (c) mechanisms of action of agents found to modulate carcinogenesis in experimental models should be determined before the results can be generalized to human beings.

Effects of timing and quantity of chronic dietary ethanol consumption on azoxymethane-induced colonic carcinogenesis and azoxymethane metabolism in Fischer 344 rats.

Epidemiological studies have shown an association between consumption of alcoholic beverages and carcinoma of the large bowel, but studies in experimental models of colonic carcinogenesis have yielded conflicting results. We assessed the effects on azoxymethane-induced colonic carcinogenesis of both timing of chronic dietary ethanol consumption relative to carcinogen administration and quantity of ethanol consumption. Ten-week-old male Fischer 344 rats were given 11%, 22%, or 33% of calories as reagent ethanol or no ethanol by pair feeding with Lieber-DeCarli-type liquid diets providing comparable total carbohydrates, proteins, fats, and calories. Ten weekly s.c. injections of the bowel carcinogen azoxymethane (AOM), 7 mg/kg, were given to all rats in weeks 1-10. Three experimental groups were given their respective ethanol diet during acclimatization and AOM administration (preinduction and induction phases) and then were given the no-ethanol diet from week 11 until sacrifice in week 26 (postinduction phase). Three other groups received the no-ethanol diet during acclimatization and AOM administration and then were changed to their respective ethanol diet until sacrifice. The control AOM group received the no-ethanol diet throughout the study. Suppression of colonic tumorigenesis occurred in the groups with high levels of chronic dietary ethanol consumption during acclimatization and AOM administration: in the 33% and 22% diet groups, the prevalence of colonic tumors was 3% and 20% as compared with 50% in control (P less than 0.001 and P less than 0.02, respectively). Tumorigenesis in the left colon was more affected than in the right colon, as tumor prevalence in the left colon was decreased in both the 33% and 22% diet groups (0% in both versus 24% in control, P less than 0.005), whereas prevalence in the right colon was decreased only in the 33% diet group (3% versus 38%, P less than 0.001). By contrast, prevalence of colonic tumors in the 11% diet group was not significantly different from control. Chronic dietary ethanol consumption after AOM administration had no effect on tumor outcome, regardless of quantity of consumption. In an analogous study of [14C]AOM metabolism in rats fed the 33% diet during acclimatization and AOM administration, 14CO2 was exhaled at a slower rate than in rats fed no-ethanol diet (P = 0.05), indicating suppression of AOM metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhancement of rat liver microsomal metabolism of azoxymethane to methylazoxymethanol by chronic ethanol administration: similarity to the microsomal metabolism of N-nitrosodimethylamine.

We compared the metabolism of azoxymethane (AOM) and of N-nitrosodimethylamine (NDMA) by liver microsomes obtained from male F344 rats pair-fed for 3 weeks either a control liquid diet or an isocaloric liquid diet containing ethanol at a concentration of 6.6% by volume. High-performance liquid chromatographic analysis of the products of the microsomal metabolism of AOM showed that methylazoxymethanol was the only primary metabolite. While the formation of small (less than 4% of methylazoxymethanol) quantities of methanol and formaldehyde could also be detected in this reaction, these products could be accounted for almost entirely by the spontaneous decomposition of methylazoxymethanol. With NDMA as the substrate in the incubation system, the formation of methylamine, formaldehyde, methanol, and an additional, as yet unidentified metabolite was detected. Liver microsomes obtained from rats fed the ethanol-containing diet up to the time of sacrifice were 12-18 times more active in the metabolism of both AOM and NDMA than liver microsomes obtained from rats fed the control, ethanol-free diet for the same period. When rats fed the ethanol diet for 20.5 days were fed the control diet for 0.5 days and then sacrificed, only a 2- to 3-fold increase in the metabolism of both AOM and NDMA by liver microsomes was observed, indicating that cessation of ethanol intake results in a rapid decrease of the ethanol-induced metabolic enzymes. Hepatocytes isolated from ethanol-fed rats showed a significantly enhanced sensitivity to AOM- as well as to NDMA-induced unscheduled DNA synthesis, indicating that the increased rate of microsomal metabolism induced by ethanol is associated with enhanced carcinogen activation in vitro. The metabolism of AOM and NDMA by liver microsomes was inhibited to similar extents by carbon monoxide, pyrazole, sodium azide, aminoacetonitrile, imidazole, and ethanol. In addition, both ethanol and NDMA were found to inhibit competitively the microsomal metabolism of AOM. These results suggest that AOM and NDMA are metabolized by very similar, indeed perhaps the same rat liver microsomal enzyme(s).

Lack of genetic and in vitro metabolic activity of potently carcinogenic azoxyalkanes.

4 carcinogenic azoxyalkanes (azoxymethane, azoxymethane and the 2 mixed methyl-ethyl compounds) were examined for activity in the Salmonella histidine reversion assay and in a lambda-lacZ prophage induction assay. Because azoxyalkanes are isomeric with nitrosodialkylamines, and might be expected to generate the same active intermediates, their biological activity was investigated under conditions which would allow direct comparison with these well-studied carcinogens. However, none of the azoxyalkanes, which are liver carcinogens, showed significant activity in either microbial assay in the presence of liver S9. In addition, metabolism studies with liver microsomes or hepatocytes indicated that the compounds were metabolized only to a small extent, if at all, under the conditions examined. This inactivity of the azoxyalkanes contrasts with the considerable activity in these assays - and the substantial metabolism - of the isomeric nitrosodialkylamines, also liver carcinogens. These results suggest that the carcinogenic action of azoxyalkanes proceeds through alternative metabolic pathways that are not adequately modeled by the assays and in vitro conditions used here.

The metabolism and cellular interactions of some aliphatic nitrogenous carcinogens.

The alkylation of nucleic acids of the liver of rats and Syrian hamsters was measured in relation to carcinogenesis by a number of nitrosamines and azoxyalkanes, most of which induce tumors of the liver in both species following chronic treatment. Two compounds, nitroso-2,6-dimethylmorpholine and nitrosobis(2-hydroxypropyl)amine were not liver carcinogens in rats, but did induce liver tumors in hamsters; there was much less alkylation by these compounds in the rat than in the hamster. In both rats and hamsters, azoxymethane produced a greater extent of alkylation, both at N-7 and O-6 guanine, than did nitrosodimethylamine, although the former is no more potent than the latter as a carcinogen in either species. Both methyl groups of azoxymethane gave rise to N-7 methylation. Nitrosobis(2-oxopropyl)amine (BOP) and nitroso(2-hydroxypropyl) (2-oxopropyl)amine (HPOP) produced considerable methylation of liver nucleic acids in both species, comparable with that by nitrosodimethylamine, and they induce liver tumors in both rats and hamsters. However, in male rats the extent of alkylation by BOP was much smaller than in females and no O-6-methylation was detected in the former; this correlates with the failure of BOP to induce liver tumors in male rats by gavage, whereas liver tumors are induced in females.