Evaluation of microleakage in posterior nanocomposite restorations with adhesive liners.

AIMS AND OBJECTIVES: To compare the microleakage in class II nanocomposite restorations without liner, with resin-modified glass ionomer liner and flowable composite liner. MATERIALS AND METHODS: Thirty-six sound premolars extracted for orthodontic reasons were selected and randomly assigned into three groups of 12 teeth each (Group I, II and III). Class II cavities of specified dimensions were prepared with margins located in the enamel. Cavities in group I were lined with resin modified glass ionomer (GC Fuji II LC-Improved), group II were lined with flowable composite (Filtex Z350 Flowable Restorative) and no liner was placed for cavities in group III. All the teeth were restored with nanocomposite (Z 350 Universal Restorative). The teeth were immersed in 0.5% methylene blue dye, sectioned mesiodistally and observed under stereomicroscope. RESULTS: Group III showed maximum leakage compared to group I and II which was statistically significant. Microleakage was lesser in group lined with resin-modified glass ionomer as compared to flowable composite group but not statistically significant. CONCLUSIONS: Placement of liner beneath nanocomposite restoration results in significant reduction in microleakage. Both resin-modified and flowable composite liners under nanocomposite restorations result in comparable reduction of microleakage.

Chemoprevention of colon cancer by a glutathione conjugate of 1,4-phenylenebis(methylene)selenocyanate, a novel organoselenium compound with low toxicity.

We have consistently shown that several synthetic Organoselenium compounds are superior cancer chemopreventive agents and less toxic than selenite or certain naturally occurring selenoamino acids. 1,4-Phenylenebis(methylene)selenocyanate (p-XSC) is the lead Organoselenium compound in that it has been shown to be the most effective and the least toxic agent in several experimental cancer models. It is not known whether p-XSC or one of its metabolites is responsible for its chemopreventive efficacy. As an initial step, we synthesized one of its putative metabolites, i.e., the glutathione conjugate of p-XSC (p-XSe-SG), and determined its stability in the pH range from 2 to 8 and in the diet under normal feeding conditions. We also assessed its maximum tolerated dose and examined its chemopreventive efficacy against azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. p-XSe-SG proved to be very stable over the pH range tested. The maximum tolerated dose of p-XSe-SG determined in a 6-week subchronic toxicity study was found to be >210 ppm (>40 ppm selenium) when the compound was added to AIN-76A high-fat diet. To assess the efficacy of this agent in the postinitiation period of colon carcinogenesis, male F344 rats 6 weeks of age were fed the high-fat diet, and at beginning of weeks 7 and 8, all of the rats intended for carcinogen treatment were given AOM at a dose of 15 mg/kg body weight by s.c. injection. Two days after the carcinogen treatment, the groups of rats consuming the high-fat control diet began their respective high-fat experimental diet regimens with 0, 56, or 84 ppm p-XSe-SG (0.1, 10, and 15 ppm of selenium) supplementation. All animals continued on their respective diets for 38 weeks after the AOM-treatment and were then killed. Colon tumors were evaluated histologically using routine procedures and were also analyzed for cyclooxygenase (COX)-1 and COX-2 expression and enzymatic activities. The results indicate that p-XSeSG administered during the post-initiation stage significantly inhibited both the incidence (P < 0.05-0.01) and the multiplicity (P < 0.05-0.005) of AOM-induced colon adenocarcinomas. This agent also greatly suppressed the multiplicity (P < 0.01-0.001) of AOM-induced exophytic adenocarcinomas in a dose-dependent manner. Feeding of 56 or 84 ppm p-XSe-SG in the diet significantly suppressed total COX activity (P < 0.02 to -0.01) and COX-2 specific activity (P < 0.005-0.0005) but had minimal effect on the protein expression levels of COX-1 and COX-2. These results suggest that the newly developed synthetic Organoselenium compound, p-XSe-SG, is stable in the diet and at wide pH ranges, inhibits colon carcinogenesis when administered during the postinitiation stage, and inhibits COX activity. Compared with previous efficacy studies and considering the toxicity associated with selenium, p-XSe-SG seems to be the least toxic Organoselenium chemopreventive agent thus far tested in the experimental colon carcinogenesis. Studies are in progress to delineate whether p-XSe-SG is also effective when administered during the progression stage of colon carcinogenesis.

Preventive potential of wheat bran fractions against experimental colon carcinogenesis: implications for human colon cancer prevention.

Epidemiological studies suggest an inverse relationship between the intake of dietary fiber, particularly fiber from cereal grains, and colon cancer risk. Animal model assays have demonstrated that the protective effects of dietary fiber on colon cancer development depend on the nature and source of the fiber. Wheat bran (WB) appears to inhibit colon tumorigenesis more consistently than do oat bran or corn bran. This study was designed to determine whether specific WB fractions such as WB fiber, WB lipids, or phytic acid differentially affect colon carcinogenesis in a well-established colon cancer model. In addition, the modulating effect of specific fractions of WB on the activities of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-1 and COX-2 enzymes were assessed in colon tumors as those have been shown to play a role in tumor progression. At 5 weeks of age, groups of male F344 rats were assigned to one of six diets: a high-fat diet containing 10% WB (control diet) and experimental high-fat diets containing 10% dephytinized WB (WB-P), 10% defatted WB (WB-F), 10% dephytinized and defatted WB (WB-PF), 10% WB-PF fortified with 2% bran oil and/or with 0.4% phytate. At 7 weeks of age, all eats except those in the vehicle-treated groups were given two weekly s.c. injections of azoxymethane (AOM) at a dose rate of 15 mg/kg body weight/week. They continued to receive their respective diets until 50 weeks after carcinogen treatment and were then killed. Colon tumors were analyzed for iNOS, COX-1, and COX-2 expression and enzymatic activities. Colon tumors were evaluated histopathologically and classified as adenomas and adenocarcinomas. We found that removal of phytic acid (WB-P) or lipids (WB-F) from WB had no significant effect on colon tumor incidence (% animals with tumors) or multiplicity (tumors/ animal), whereas removal of both phytate and lipids from WB (WB-PF) significantly increased colon tumor multiplicity and volume. Interestingly, WB-PF fortified with excess bran oil or with bran oil plus phytate significantly inhibited colon tumor incidence, multiplicity, and volume; but supplementation of WB-PF with phytate alone had no significant effect on colon tumorigenesis in rats suggesting that lipid fraction of WB possesses tumor-inhibitory properties. Moreover, feeding WB-PF diet significantly increased iNOS, total COX and COX-2 enzyme activities, and iNOS protein expression in colon tumors as compared with wheat bran control diet. Feeding the WB-PF that was fortified with excess bran oil alone or with bran oil plus phytate significantly suppressed the activities of iNOS and COX-2 as well as the expression of iNOS and COX-2 in colon tumors compared with that in rats fed the WB diet or WB-PF diet. The study demonstrates for the first time that the lipid fraction of wheat bran has strong colon tumor inhibitor properties. The exact mechanism(s) by which the lipid fraction of WB inhibits colon carcinogenesis in addition to alteration of iNOS and COX activities remains to be elucidated. Additional studies are warranted to identify biologically active constituents of lipid fraction of WB and their relative role in colon tumor inhibition.

Chemoprevention of familial adenomatous polyposis development in the APC(min) mouse model by 1,4-phenylene bis(methylene)selenocyanate.

Epidemiological and experimental studies have suggested that dietary supplementation with selenium can inhibit the development of cancers at several organ sites. We have consistently shown that 1, 4-phenylene bis(methylene) selenocyanate (p-XSC) is a highly effective cancer chemopreventive agent against the development of chemically induced cancers in several laboratory animal species. This is the first report describing the preventive effects of p-XSC in an animal model of familial adenomatous polyposis (FAP) containing a germline mutation of the APC gene. Six-week old male (heterozygous) C57BL/6J-APC(min) or wild-type mice were fed high fat diets containing 0, 10 or 20 p.p.m. p-XSC. After 80 days, the mice were killed and their intestines were excised and evaluated for polyps. Multiple samples were also harvested from normal appearing small intestine and colon for molecular analysis. Both the mucosa and polyps from the intestine and colon were assayed for beta-catenin, cyclooxygenase (COX)-2 expression and COX isoform activities. Administration of p-XSC in the diet significantly decreased the rate of formation of small intestinal tumors (P < 0. 0001) and colon tumors (P < 0.002) in APC(min) mice. p-XSC produced a dose-dependent inhibition of tumors in both small intestine (P < 0. 0001) and colon (P < 0.035). Mice fed 20 p.p.m. p-XSC had significantly lower levels of beta-catenin expression and COX-2 activity in polyps. These observations demonstrate for the first time that the synthetic organoselenium compound p-XSC possesses antitumor activity against genetically predisposed neoplastic lesions, such as FAP. While the exact mechanism(s) for this antitumor activity of p-XSC remains to be elucidated, it appears that modulation of beta-catenin expression and COX-2 activity is associated with inhibition of intestinal polyps.

Mechanisms in the chemoprevention of colon cancer: modulation of protein kinase C, tyrosine protein kinase and diacylglycerol kinase activities by 1,4-phenylenebis-(methylene)selenocyanate and impact of low-fat diet.

Epidemiological and experimental studies suggest an inverse relationship between the intake of dietary selenium and/or low fat-intake and colon cancer risk. Efficacy studies in rodents suggest that the organoselenium compound 1, 4-phenylenebis(methylene)selenocyanate (p-XSC), is a more effective and less toxic chemopreventive agent than other organic or inorganic selenium compounds such as selenomethionine and Na2SeO3. The efficacy of p-XSC against colon cancer is significantly augmented by a low-fat diet. To explore the mechanisms by which this combined inhibiting effect against colon carcinogenesis comes about, we have investigated protein kinase C (PKC), tyrosine protein kinase (TPK), diacylglycerol kinase (DGK) activities and 8-isoprostane levels in colonic mucosa and tumor tissues in an azoxymethane (AOM)-induced rat colon cancer model. Weanling male F344 rats were fed the semipurified AIN-76A diet until seven weeks of age. Then various experimental groups were fed the low- or high-fat diets containing 0 or 20 ppm p-XSC (10 ppm as selenium). At seven weeks of age, groups of rats were injected s.c. with azoxymethane (AOM; 15 mg/kg body wt., once weekly for 2 weeks) and continued on their respective experimental diets until 38 weeks after the second AOM treatment. They were then sacrificed and colonic mucosal and tumor samples were evaluated for PKC, TPK, DGK and 8-isoprostane levels. Administration of p-XSC along with a low-fat diet significantly inhibited Ca+2-dependent and -independent PKC (P<0.05-0.01) activities in colonic mucosa and tumors. Administration of p-XSC either low-fat or high-fat diet significantly suppressed both colonic mucosal and tumor TPK activity (P<0.05-0.01). Suppression of TPK activity was more pronounced in rats maintained on a low-fat diet containing p-XSC. In contrast, rats receiving p-XSC with either low- or high fat diet showed significantly increased DGK activity (P<0.01-0.0001). Rats fed low-fat or high-fat plus p-XSC had lower-levels of 8-isoprostane in the colonic tumors than animals who had been given low- or high-fat diets without the organoselenium compound. Interestingly, 8-isoprostane levels were lower in the colon tumors of the rats fed the low-fat diet than those fed the high-fat diet. Our findings suggest that p-XSC induced down-regulation of PKC and TPK activities and up-regulation of DGK activity. These events may in part be responsible for the chemopreventive activity against colon carcinogenesis. Further, this study implies that p-XSC with a low-fat dietary regimen will augment regulation of PKC, TPK and DGK activities in the colon.

Prevention of colonic preneoplastic lesions by the probiotic Lactobacillus acidophilus NCFMTM in F344 rats.

The experiments described here were aimed at developing novel probiotic strains that may aid in the reduction of colon cancer risk. We assessed the potential anticancer properties of Lactobacillus acidophilus NCFMTM in male F344 rats using inhibition of the formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy. At 6 weeks of age, groups of rats were fed the experimental diets containing 0, 2% or 4% lyophilized cultures of L. acidophilus NCFMTM. At 7 weeks of age, all animals in each dietary group, except the vehicle-treated rats, were s.c. injected with AOM (15 mg/kg body weight) once weekly for two weeks. The vehicle-treated groups were given s.c. injections of normal saline. All rats were necropsied 10 weeks after the last AOM injection and ACF in formalin-fixed, methylene blue-stained colonic tissues were counted under the light microscope. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity. Diet supplementation with the probiotic strain NCFMTM significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P<0.01 - 0.001). NCFMTM inhibited AOM-induced colonic ACF formation in a dose-dependent manner (P<0.01). A significant dose-dependent reduction of cecal beta-glucuronidase activities was observed in the rats fed 2% (P<0.04) and 4% (P<0.0001) NCFMTM. These results suggest that Lactobacillus acidophilus NCFMTM may potentially prevent colon cancer development. Further studies are warranted to determine the full potential of this probiotic strain in preclinical efficacy studies.

Prevention of colonic aberrant crypt foci and modulation of large bowel microbial activity by dietary coffee fiber, inulin and pectin.

The present experiments were aimed at developing novel dietary fibers to aid in reduction of colon cancer risk. We assessed the effects of coffee (non-fiber fraction), coffee fiber (arabino-galactose polymer) and inulin (oligo-fructose) in male F344 rats using formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in the colon as the measure of preventive efficacy (or lack of such). At 5 weeks of age, groups of rats were fed the AIN-76A (control) and experimental diets that contained 1% coffee, 10% coffee fiber, 10% inulin, 10% pectin (positive control for fiber) or 200 p.p.m. piroxicam (a known ACF inhibitor). At 7 weeks of age, all animals were s.c injected with AOM (15 mg/kg body wt) once weekly for 2 weeks. All rats were killed 8 weeks after the last AOM injection and ACF were counted. The contents of the cecum were analyzed for bacterial beta-glucuronidase activity and short-chain fatty acids (SCFAs). Dietary administration of coffee fiber significantly suppressed AOM-induction of colonic ACF, in terms of total number, as well as crypt multiplicity and number of ACF/cm2 colon (P < 0.01-0.001). Inulin diet had no significant effect on total ACF, but had reduced the number of ACF/cm2 (P < 0.05). Whereas coffee had no effect on ACF formation, 10% pectin diet and 200 p.p.m. piroxicam significantly suppressed colonic ACF (P < 0.001) as had been expected. A significant reduction of cecal beta-glucuronidase activity was observed in the rats fed coffee, coffee fiber and pectin diets. Further, coffee fiber, inulin and pectin increased cecal SCFA levels 3- to 5-fold. These results suggest that coffee fiber can prevent colon cancer risk. Further studies are warranted to determine the full potential of this fiber in pre-clinical efficacy studies.

Enhancement of experimental colon cancer by genistein.

Several phytochemicals and micronutrients that are present in fruits and vegetables are known to exert cancer chemopreventive effects in several organs, including the colon. Among them, the soybean isoflavonoid genistein received much attention due to its potential anticarcinogenic, antiproliferative effects and its potential role in several signal transduction pathways. The present study was designed to investigate the effect of genistein on azoxymethane (AOM)-induced colon carcinogenesis and to study its modulatory role on the levels of activity of 8-isoprostane, cyclooxygenase (COX), and 15-hydroxyprostaglandin F2alpha dehydrogenase (15-PGDH) in the colonic mucosa and colon tumors of male F344 rats. At 5 weeks of age, groups of male F344 rats were fed control (AIN-76A) diet or a diet containing 250 ppm genistein. Beginning 2 weeks later, all animals except those in the vehicle-treated groups were given weekly s.c. injections of AOM (15 mg/kg body weight) for 2 successive weeks. All rats were continued on their respective dietary regimen for 52 weeks after AOM treatment and were then sacrificed. Colon tumors were evaluated histopathologically. Colonic mucosae and tumors were analyzed for COX, 15-PGDH, and 8-isoprostane levels. Administration of genistein significantly increased noninvasive and total adenocarcinoma multiplicity (P < 0.01) in the colon, compared to the control diet, but it had no effect on the colon adenocarcinoma incidence nor on the multiplicity of invasive adenocarcinoma (P > 0.05). Also, genistein significantly inhibited the 15-PGDH activity (>35%) and levels of 8-iosoprostane (50%) in colonic mucosa and in tumors. In contrast, genistein had no significant effect on the COX synthetic activity, as measured by the rate of formation of prostaglandins and thromboxane B2 from [14C]arachidonic acid. The results of this investigation emphasize that the biological effects of genistein may be organ specific, inhibiting cancer development in some sites yet showing no effect or an enhancing effect on the tumorigenesis at other sites, such as the colon. The inhibition of 8-isoprostane levels by genistein indicates its possible antioxidant potential, which is independent of the observed colon tumor enhancement, yet this agent may also possess several biological effects that overshadow its antioxidant potential. The exact mechanism(s) of colon tumor enhancement by genistein remain to be elucidated; it is likely that its colon tumor-enhancing effects may, at least in part, be related to inhibition of prostaglandin catabolic enzyme activities.

Wheat bran and psyllium diets: effects on N-methylnitrosourea-induced mammary tumorigenesis in F344 rats.

BACKGROUND: Experimental and epidemiologic evidence suggests that increased dietary fiber is associated with decreased breast cancer risk. Little is known about the role played by different types of fiber and, particularly, mixtures of soluble and insoluble fibers similar to those consumed by human populations in reducing breast cancer risk. High intake of fiber may suppress bacterial hydrolysis of biliary estrogen conjugates to free (absorbable) estrogens in the colon and thus may decrease the availability of circulating estrogens necessary for the development and growth of breast cancers. PURPOSE: The purpose of this study was to evaluate the effect of wheat bran (an insoluble fiber) and psyllium (a soluble fiber) alone and in combination on overall estrogen status, on fecal bacterial beta-D-glucuronidase (a key diet-responsive estrogen-deconjugating enzyme) activity, and on the induction of mammary tumors in rats treated with N-methylnitrosourea (MNU). METHODS: One hundred fifty virgin female F344 rats were fed the NIH-07 diet from 28 days of age until 50 days of age; they were then given a single dose (40 mg/kg of body weight) of MNU by tail vein injection. Three days later, they were randomly assigned to one of five experimental dietary groups (30 animals per group). Soft, white wheat bran (45% dietary fiber content) and psyllium (80% dietary fiber content) were added to a modified (high-fat) American Institute of Nutrition (AIN)-76A diet at the following percents, respectively: 12% + 0% (group 1), 8% + 2% (group 2), 6% + 3% (group 3), 4% + 4% (group 4), and 0% + 6% (group 5). Blood, urine, and feces were collected and analyzed by radioimmunoassay techniques for estrogens. Cecal contents were analyzed for bacterial beta-D-glucuronidase activity. After 19 weeks on the experimental diets, the rats were killed, and mammary tumors were counted and classified by histologic type. Cumulative tumor incidence was evaluated by the Kaplan-Meier life-table method and the logrank test. Tumor number was evaluated by the chi-squared test of association, and tumor multiplicity was evaluated by the Mantel-Haenszel chi-squared test. All statistical tests were two-tailed. RESULTS: As the level of psyllium relative to that of wheat bran increased, the total tumor number and multiplicity of mammary adenocarcinomas in rats decreased as a statistically significant linear trend across groups 1-5 (P < .05). Compared with the group given wheat bran alone, the group given the 1:1 (wheat bran:psyllium) combination had maximum protection against mammary tumorigenesis, while the groups given the 4:1 or 2:1 (wheat bran:psyllium) combination or psyllium alone had intermediate protection. No statistically significant differences in circulating estrogens or urinary estrogen excretion patterns were observed among the five experimental groups. Fecal estrogen excretion, however, decreased with increasing levels of psyllium (P < .01), and cecal beta-D-glucuronidase activity exhibited a decreasing trend with respect to the increasing psyllium content of the diet across groups 1-5 (P < .01). CONCLUSIONS: The addition of a 4%:4% mixture of an insoluble (wheat bran) fiber and a soluble (psyllium) fiber to a high-fat diet provided the maximum tumor-inhibiting effects in this mammary tumor model. Although increasing levels of dietary psyllium were associated with decreased cecal bacterial beta-D-glucuronidase activity, these changes were not reflected in decreased circulating levels of tumor-promoting estrogens. Therefore, the mechanism(s) by which mixtures of soluble and insoluble dietary fibers protect against mammary tumorigenesis remains to be clarified.

Effect of amount and types of dietary fat on intestinal bacterial 7 alpha-dehydroxylase and phosphatidylinositol-specific phospholipase C and colonic mucosal diacylglycerol kinase and PKC activities during stages of colon tumor promotion.

It is evident from many studies that the effect of dietary fat on colon tumor promotion depends not only on the amount of fat but especially on fatty acid composition. Animal model studies have shown that diets which are high in omega-6 fatty acids increase colon tumor promotion, whereas diets rich in omega-3 fatty acids have no such enhancing effect. The mechanisms by which the high fat content of the diet promotes colon carcinogenesis may include the production of secondary bile acids in the colon and the modulation of colonic luminal bacterial 7 alpha-dehydroxylase that is involved in generating secondary bile acids, phosphatidylinositol-specific phospholipase C (PI-PLC), and mucosal PI-PLC, as well as diacylglycerol (DAG) kinase and protein kinase C (PKC). In the present study, we investigated the effect of high-fat diets that are rich in omega-3 and omega-6 fatty acids on cecal bacterial 7 alpha-dehydroxylase and PI-PLC, fecal secondary bile acids, and colonic mucosal DAG kinase and PKC activities during different stages of colon carcinogenesis in male F344 rats. At 5 weeks of age, groups of animals were fed a low-fat diet containing 5% corn oil (LFCO). Beginning at 7 weeks of age, all animals, except those intended as vehicle controls, received azoxymethane (AOM) s.c. once weekly for 2 weeks at a dose rate of 15 mg/kg body weight. Vehicle-treated groups received s.c. injections of normal saline. One day after the second AOM or saline treatment, the experimental groups of animals were transferred to a high-fat diet containing 23.5% corn oil (HFCO) or 20.5% fish oil + 3% corn oil (HFFO). One group continued on the LFCO diet. Animals were sacrificed at weeks 1, 12, and 36 after the AOM or saline treatment. Colonic mucosa were harvested at weeks 1, 12, or 36, and the colonic tumor tissues were examined for PKC and DAG kinase activities. Contents of the cecum were analyzed for bacterial 7 alpha-dehydroxylase and PI-PLC activities. Stool samples collected at week 12 were analyzed for bile acids. High corn oil content of the diet significantly increased the cecal bacterial 7 alpha-dehydroxylase and PI-PLC activities as compared to the diets with high fish oil or low corn oil content. Animals fed the HFCO diet excreted higher levels of secondary bile acids, such as deoxycholic acid and lithocholic acid, than those fed the LFCO or HFFO diets. Carcinogen treatment significantly enhanced the activities of DAG kinase and total membrane PKC activities in colonic mucosa compared to saline treatment in all dietary groups. Animals treated with saline or AOM and fed HFCO showed increased levels of DAG kinase and membrane PKC activities in the colonic mucosa when compared to LFCO and HFFO groups. DAG kinase and membrane PKC activities were higher in colon tumors than in the surrounding colonic mucosa, and also increased levels of these enzyme activities were found in the HFCO diet group. These results indicate that the modifying effect of dietary fat on colonic bacterial enzymes, secondary bile acids, colonic mucosal and tumor DAG kinase, and PKC that may play a role in colon carcinogenesis depends on the types and amount of fat given. The colon tumor-enhancing effect of a HFCO diet in contrast to the high dietary fish oil may be, in part, explained on the basis of its modulating effect on these bacterial and colonic mucosal enzymes and colonic secondary bile acids relevant to colon tumor promotion.

Modulating effect of amount and types of dietary fat on colonic mucosal phospholipase A2, phosphatidylinositol-specific phospholipase C activities, and cyclooxygenase metabolite formation during different stages of colon tumor promotion in male F344 rats.

Epidemiological and laboratory animal model studies suggest that the effect of dietary fat in colon carcinogenesis depends not only on the amount but on its fatty acid composition. Animal model studies demonstrated that high dietary corn oil or safflower oil rich in omega-6 fatty acids increased the colon tumor promotion, whereas diets containing fish oil high in omega-3 fatty acids had no such enhancing effect. One of the mechanisms by which high dietary fat enhances colon carcinogenesis may be through the modulation of colonic mucosal phospholipase A2 (PLA2) and phosphatidylinositol-specific phospholipase C (PI-PLC), which are dominant pathways for arachidonic acid release and formation of eicosanoids. PI-PLC is also responsible for diacylglycerol formation and protein kinase C-dependent signal transduction and cell proliferation. In the present study, we investigated the modulating effect of high fat diets rich in omega-3 and omega-6 fatty acids on colonic mucosal PLA2, PI-PLC activities, and eicosanoid (prostaglandins and thromboxane B2) formation from arachidonic acid via cyclooxygenase (COX) during different stages of azoxymethane (AOM)-induced colon carcinogenesis in male F344 rats. At 5 weeks of age, groups of animals were fed the low-fat diet containing 5% corn oil. Beginning at 7 weeks of age, all animals except those intended for vehicle treatment received AOM s.c. once weekly for 2 weeks at a dose rate of 15 mg/kg body weight. Vehicle-treated groups received an equal volume of normal saline. One day after the second AOM or vehicle treatment, groups of animals were transferred to experimental diets containing 23.5% corn oil and 20.5% fish oil + 3% corn oil, whereas one group continued on the low-fat diet containing 5% corn oil. Groups of animals were then sacrificed at weeks 1, 12, and 36 after the second AOM-or saline-treatment. Colonic mucosa harvested at weeks 1, 12, and 36 and colonic tumors obtained at week 36 were analyzed for PLA2, PI-PLC, and eicosanoid formation from arachidonic acid by the action of COX. The results demonstrate that colon carcinogen treatment increases the activities of colonic mucosal PLA2 and PI-PLC and the formation of prostaglandins and thromboxane A2 from arachidonic acid through COX throughout the study period compared to saline-treated animals fed similar diets. The activities of PLA2, PI-PLC, and COX were significantly higher in colon tumors compared to colonic mucosa. These results also demonstrate that a high-fat diet containing corn oil increases colonic mucosal and tumor PLA2 and PI-PLC and the formation of prostaglandins and thromboxane B2 by the action of COX as compared to low dietary corn oil or a diet high in fish oil. The results of our study offer one of the mechanisms by which the amount and types of dietary fat modulate colon carcinogenesis.

Enhancement of experimental colon carcinogenesis by dietary 6-phenylhexyl isothiocyanate.

Naturally occurring and related synthetic isothiocyanates are known to exert chemopreventive effects in several organs in rodent models. The present study was designed to investigate the efficacy of 6-phenylhexyl isothiocyanate (PHITC), a potent chemopreventive agent in the lung tumor model in strain A mice, on azoxymethane-induced colon tumorigenesis. Another aim was to study the modulating effect of PHITC on colonic mucosal and tumor phospholipase A2 (PLA2), phosphatidylinositol-specific phospholipase C (PI-PLC), lipoxygenase (LOX), and cyclooxygenase (COX) activities. At 5 weeks of age, groups of male F344 rats were fed control diet or diets containing 320 or 640 ppm of PHITC representing 40 and 80% maximum tolerated dose levels, respectively. At 7 weeks of age, all animals except those in the vehicle-treated groups were given two weekly s.c. injections of azoxymethane at a dose rate of 15 mg/kg body weight/week. All animals continued on their respective dietary regimen for 52 weeks after the carcinogen treatment; then the study was terminated. Colonic mucosa and tumors were analyzed for PLA2, PI-PLC, prostaglandin (PG) E2, COX, and LOX activities. Intestinal tumors were evaluated histopathologically and classified as invasive or noninvasive adenocarcinomas. Intestinal tumor incidence (percentage of animals with tumors) and tumor multiplicity (tumors/animal; tumors/tumor-bearing animal) were compared among the dietary groups. At the 640-ppm dose level, dietary PHITC significantly increased the incidence of intestinal (small intestine plus colon) adenocarcinomas (P < 0.05) as well as the multiplicities of invasive and noninvasive adenocarcinomas of the colon (P < 0.05 to 0.01). At the 320-ppm dose level, PHITC increased the multiplicity (tumors/animal) of noninvasive adenocarcinomas and total (invasive plus noninvasive) adenocarcinomas of the colon (P < 0.05). Dietary PHITC also increased the colon tumor volume (2- to 4.3-fold) in a dose-dependent manner. Moreover, PHITC significantly enhanced the activities of PLA2 (50-100%) and levels of PGE2 (2-fold) in the colonic mucosa and in tumors, but it had no significant effect (P > 0.05) on PI-PLC activity. The formation of COX metabolites, particularly PGE2, PGF2 alpha, PGD2, 6-keto PGF1 alpha, and thromboxane B2, as well as LOX metabolites such as 8(S)-, 12(S)- and 15 (S)-hydroxyeicosatetraenoic acids, were significantly increased in the colonic mucosa and tumors of animals that were fed 640 ppm of PHITC. Although the exact mechanism by which PHITC promotes colon tumorigenesis remains to be elucidated, it is likely that the tumor-promoting effects of PHITC may, at least in part, be related to increased eicosanoid metabolism in the colon.

Chemoprevention of colon carcinogenesis by phenylethyl-3-methylcaffeate.

Previous studies from this laboratory have established that caffeic acid esters present in propolis, a natural resin produced by honey bees, are potent inhibitors of human colon adenocarcinoma cell growth, carcinogen-induced biochemical changes, and preneoplastic lesions in the rat colon. The present study was designed to investigate the chemopreventive action of dietary phenylethyl-3-methylcaffeate (PEMC) on azoxymethane-induced colon carcinogenesis and to examine the modulating effect of PEMC on phosphatidylinositol-specific phospholipase C (PI-PLC), phospholipase A2, lipoxygenase (LOX), and cyclooxygenase activities in the colonic mucosa and tumor tissues in male F344 rats. At 5 weeks of age, groups of rats were fed the control (modified AIN-76A) diet, or a diet containing 750 ppm of PEMC. At 7 weeks of age, all animals except those in the vehicle (normal saline)-treated groups were given 2 weekly s.c. injections of azoxymethane at a dose rate of 15 mg/kg body weight/week. All groups were maintained on their respective dietary regimen until the termination of the experiment 52 weeks after the carcinogen treatment. Colonic tumors were evaluated histopathologically. Both colonic mucosa and tumors were analyzed for PI-PLC, phospholipase A2, cyclooxygenase, and LOX activities. The results indicate that dietary administration of PEMC significantly inhibited the incidence and multiplicity of invasive, noninvasive, and total (invasive plus noninvasive) adenocarcinomas of the colon (P < 0.05-0.004). Dietary PEMC also suppressed the colon tumor volume by 43% compared to the control diet. Animals fed the PEMC diet showed significantly decreased activities of colonic mucosal and tumor PI-PLC (about 50%), but PEMC diet had no effect on phospholipase A2. The production of 5(S)-, 8(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids via the LOX pathway from arachidonic acid was reduced in colonic mucosa and tumors (30-60%) of animals fed the PEMC diet as compared to control diet. PEMC had no effect on the formation of colonic mucosal cyclooxygenase metabolites but inhibited the formation in colonic tumors by 15-30%. The precise mechanism by which PEMC inhibits colon tumorigenesis remains to be elucidated. It is likely that the chemopreventive action may be related, at least in part, to the modulation of PI-PLC-dependent signal transduction and LOX-mediated arachidonic acid metabolism.

Chemoprevention of colon carcinogenesis by sulindac, a nonsteroidal anti-inflammatory agent.

Epidemiological and laboratory animal model studies have suggested that nonsteroidal anti-inflammatory drugs reduce the risk of development of colon cancer. The present study was designed to investigate the chemopreventive action of 160 and 320 ppm (equivalent to 40 and 80% maximum tolerated doses) sulindac, a nonsteroidal anti-inflammatory drug, fed during initiation and postinitiation stages and 320 ppm sulindac fed during promotion/progression stages of azoxymethane-induced colon carcinogenesis in male F344 rats. Also investigated was the modulating effect of this agent on the colonic mucosal and tumor phospholipase A2, phosphatidylinositol-specific phospholipase C, lipoxygenase, and cyclooxygenase activities. At 5 weeks of age, groups of male F344 rats were fed control diet or diets containing 160 and 320 ppm of sulindac. At 7 weeks of age, all animals except those in the vehicle-treated groups were given two weekly s.c. injections of azoxymethane at a dose rate of 15 mg/kg body weight/week. Animals intended for tumor promotion/progression study were administered 320 ppm of sulindac in diet starting at 14 weeks after a second azoxymethane treatment. All animals continued on their respective dietary regimen until the termination of the experiment at 52 weeks after the carcinogen treatment. Colonic tumors were evaluated histopathologically. Colonic mucosa and tumors were analyzed for phospholipase A2, phosphatidylinositol-specific phospholipase C, prostaglandin E2, cyclooxygenase, and lipoxygenase activities. The levels of sulindac and its metabolites in stomach, cecal, and fecal contents and in serum were analyzed. The results indicate that dietary sulindac at 160 and 320 ppm levels inhibited the incidence of invasive and noninvasive adenocarcinomas of the colon (P < 0.01-0.001) as well as their multiplicity (P < 0.01-0.0001) in a dose-dependent manner. Also, feeding sulindac during promotion/progression stages significantly suppressed the incidence (P < 0.0001) and multiplicity (P < 0.0001) of colonic adenocarcinomas. Dietary sulindac also suppressed the colon tumor volume by > 52-62% compared to the control diet. Dietary sulindac significantly decreased the activities of phosphatidylinositol-specific phospholipase C (32-51%) and levels of prostaglandin E2 (> 40%) in the colonic mucosa and tumors, but it had no significant (P > 0.05) effect on phospholipase A2 activity. The formation of cyclooxygenase metabolites, particularly prostaglandin E2, prostaglandin F2 alpha, prostaglandin D2, 6-ketoprostaglandin F1 alpha, and thromboxane B2, and lipoxygenase metabolites such as 8(S)- and 12(S)-hydroxyeicosatetraenoic acids were significantly reduced in colonic mucosa and tumors of animals fed 320 ppm sulindac.(ABSTRACT TRUNCATED AT 400 WORDS)

Chemoprevention of colon carcinogenesis by dietary curcumin, a naturally occurring plant phenolic compound.

Human epidemiological and laboratory animal model studies have suggested that nonsteroidal antiinflammatory drugs reduce the risk of development of colon cancer and that the inhibition of colon carcinogenesis is mediated through the alteration in cyclooxygenase metabolism of arachidonic acid. Curcumin, which is a naturally occurring compound, is present in turmeric, possesses both antiinflammatory and antioxidant properties, and has been tested for its chemopreventive properties in skin and forestomach carcinogenesis. The present study was designed to investigate the chemopreventive action of dietary curcumin on azoxymethane-induced colon carcinogenesis and also the modulating effect of this agent on the colonic mucosal and tumor phospholipase A2, phospholipase C gamma 1, lipoxygenase, and cyclooxygenase activities in male F344 rats. At 5 weeks of age, groups of animals were fed the control (modified AIN-76A) diet or a diet containing 2000 ppm of curcumin. At 7 weeks of age, all animals, except those in the vehicle (normal saline)-treated groups, were given two weekly s.c. injections of azoxymethane at a dose rate of 15 mg/kg body weight. All groups were continued on their respective dietary regimen until the termination of the experiment at 52 weeks after the carcinogen treatment. Colonic tumors were evaluated histopathologically. Colonic mucosa and tumors were analyzed for phospholipase A2, phospholipase C gamma 1, ex vivo prostaglandin (PG) E2, cyclooxygenase, and lipoxygenase activities. The results indicate that dietary administration of curcumin significantly inhibited incidence of colon adenocarcinomas (P < 0.004) and the multiplicity of invasive (P < 0.015), noninvasive (P < 0.01), and total (invasive plus noninvasive) adenocarcinomas (P < 0.001). Dietary curcumin also significantly suppressed the colon tumor volume by > 57% compared to the control diet. Animals fed the curcumin diet showed decreased activities of colonic mucosal and tumor phospholipase A2 (50%) and phospholipase C gamma 1 (40%) and levels of PGE2 (> 38%). The formation of prostaglandins such as PGE2, PGF2 alpha, PGD2, 6-keto PGF1 alpha, and thromboxane B2 through the cyclooxygenase system and production of 5(S)-, 8(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids via the lipoxygenase pathway from arachidonic acid were reduced in colonic mucosa and tumors of animals fed the curcumin diet as compared to control diet. Although the precise mechanism by which curcumin inhibits colon tumorigenesis remains to be elucidated, it is likely that the chemopreventive action, at least in part, may be related to the modulation of arachidonic acid metabolism.

Evaluation of organoselenium compounds for potential chemopreventive properties in colon carcinogenesis.

As a part of a program aimed to develop less toxic and more effective chemopreventive organoselenium compounds than inorganic selenium, we have evaluated benzyl selenocyanate (BSC) and its o-, m-, p-nitro and -methoxy isomers, o-, m-, and p-isomers of phenylenebis(methylene)selenocyanate (XSC), dibenzyl diselenide (DDS), and 2,2'-diselenobis[((N,N-dimethylamino)methyl)- benzene]bis(hydrochloride salt) (DSBDB) for their potential colon tumor inhibitory properties using azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF), a preneoplastic lesion, in male F344 rats prior to preclinical efficacy study. In the first experiment, the effect of these agents administered during initiation and postinitiation periods of carcinogenesis was investigated. Male F344 rats were fed diets containing 8 ppm Na2SeO3 or 10 ppm of each BSC and its analogues, DDS and DSBDB or 20 ppm of each XSC analogue, two weeks prior to AOM (15 mg/kg body wt., once weekly for two weeks, s.c.) administration and during and until 8 weeks after AOM treatment. Formalin-fixed and methylene blue stained colons were scored for AOM-induced ACF using the light microscope. Taking body weight gains and multiplicity of 4 or more AC/focus, the inhibitory effects of Na2SeO3, o-, m- and p-methoxy-BSC, p-XSC and DDS were much greater than those of the other selenium compounds. In the second study, the effects of these agents when administered during the initiation or postinitiation periods were investigated. The results indicated that o-, m-, and p-methoxy-BSC, DDS and p-XSC significantly inhibited crypt multiplicity during the initiation period whereas o-, and p-methoxy-BSC, p-XSC and DDS suppressed crypt multiplicity during the postinitiation period. It is concluded that o-, and p-methoxy-BSC, p-XSC and DDS possess potential chemopreventive properties in colon cancer. Further studies are warranted to evaluated these agents for chemopreventive properties in preclinical efficacy studies.

Biochemical epidemiology of colon cancer: effect of types of dietary fiber on colonic diacylglycerols in women.

BACKGROUND/AIMS: In view of the potential significance of dietary fat and fiber in colon cancer and the possible indirect involvement of diacylglycerols (DAGs) in the pathogenesis of colon cancer, the effect of types of dietary fiber on fecal DAG in premenopausal women was investigated. METHODS: Forty-eight women consuming a typical western diet provided two 24-hour stool specimens and two sets of preintervention 4-day food records. They were randomly assigned to one of the fiber groups, namely, a wheat, oat, or corn bran supplement. They consumed their control diet plus 13-15 g of dietary fiber from each source for 8 weeks. At the end of the fiber period, each subject provided two 24-hour stool specimens and 4-day food records. Stool samples collected during the two periods were analyzed for total fat and DAG fatty acids. RESULTS: All sources of dietary fiber increased the amount of fecal fat excreted. Dietary wheat bran decreased the concentrations of total DAG and DAG containing lauric acid, myristic acid, palmitic acid, stearic acid, and linoleic acid, whereas oat bran increased the DAG composed of oleic acid and linoleic acid compared with the control diet. Corn bran decreased the DAG containing stearic acid. CONCLUSIONS: These results show that the modifying effect of dietary fiber on DAG depends on the type of fiber consumed.

Molecular markers as intermediate end-points in chemoprevention of colon-cancer - modulation of ras activation by sulindac and phenylhexylisothiocyanate during colon carcinogenesis.

Recent evidence suggests that activation of ras proto-oncogenes and inactivation of suppressor genes induce malignant phenotype in colonic cells. Thus the identification of clonal population of cells expressing activated ras may lead to a valuable intermediate biomarker to detect premalignant lesions amenable to chemoprevention. Previously, we demonstrated that sulindac inhibited the carcinogen-induced colon tumor development whereas phenylhexylisothiocyanate (PHITC) promoted the tumor outcome. The present study was conducted to investigate the effect of sulindac and PHITC on azoxymethane (AOM)induced activation of ras proto-oncogenes in order to explore the plausibility of using ras as an intermediate biomarker in chemoprevention of colon cancer. Male F344 rats were fed the AIN-76A diet containing 0, 320 ppm sulindac or 640 ppm PHITC and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt/week for 2 weeks. Vehicle control groups received s.c. equal volume of normal saline. Animals were sacrificed 52 weeks after AOM or saline treatment and their colonic mucosa and tumors were analyzed for mutations in codon 12 and 13 of K-ras and the expression of ras p21. As an alternative non-invasive approach, we developed a simple and sensitive one-step mutant-enriched PCR method to detect these genetic lesions in stools collected at 16, and again at 24 weeks after AOM treatment. AOM-induced G to A transitions were observed at the second nucleotide of 12th codon of K-ras substituting amino acid asp with wild-type gly. Sulindac not only suppressed the selective amplification of initiated cells possessing AOM-induced mutated K-ras codon 12, but significantly inhibited the AOM-induced expression of total and mutant ras-p21. PHITC did not exert any inhibitory effect on AOM-induced ras activation. Results indicated a strong correlation between ras activation and tumor outcome. Data suggest that ras activation may be a useful intermediate molecular marker in chemoprevention of colon cancer.