Inhibition of intestinal carcinogenesis in rats: effect of difluoromethylornithine with piroxicam or fish oil.

An inhibitor of ornithine decarboxylase, difluoromethylornithine (DFMO), and two inhibitors of prostaglandin biosynthesis, piroxicam and menhaden fish oil, were examined for their effect on intestinal tumorigenesis in male Sprague-Dawley rats fed a 5% fat semisynthetic diet. Each agent was given individually in one of two doses as follows: DFMO, 0.05% and 0.1% in the drinking water; piroxicam, 65 mg/kg diet and 130 mg/kg diet; and menhaden fish oil, 1.25% and 2.50% of the diet. Additional animal groups were given combinations of the lower dose of DFMO and the lower dose of either piroxicam or fish oil. Intestinal tumors were induced by sc injections of azoxymethane (AOM; CAS: 25843-45-2) at 8 mg/kg (body wt) weekly for 8 weeks. Test diets were started 1 week prior to the first dose of AOM, and the rats were sacrificed 26 weeks later. Rats that received either dose of DFMO or the high dose of piroxicam developed significantly fewer intestinal tumors compared to controls. The low dose of piroxicam and the fish oil given at either dose level had no effect. The combination of the low dose of DFMO and the low dose of piroxicam reduced tumor formation more than either dose of DFMO alone, whereas the low dose of DFMO and fish oil together was no more effective than either dose of DFMO alone. These results show that a combination of a small amount of DFMO and piroxicam, each acting through a different mechanism, exerts an additive inhibitory effect on intestinal tumor formation in rats.

Effect of dietary fiber on azoxymethane-induced intestinal carcinogenesis in rats.

The effect of alfalfa, bran, and cellulose on intestinal tumor formation and fecal billary steroid levels was studied in male Sprague-Dawley rats given injections of azoxymethane (AOM). Animals received weekly injections of 8 mg AOM/kg and were fed diets containing 10% fiber (wt/wt) and 35% beef fat or 20 or 30% fiber and about 6% beef fat. Control animals in each instance were fed fiber-free diets. The addition of 10% fiber to the high-fat diet did not significantly reduce the intestinal tumor frequency (average No. of tumors/rat). However, addition of 20 or 30% fiber to the 6% fat diet significantly reduced the intestinal tumor frequency. The concentration of fecal biliary steroids (mg/g dry feces) was significantly lowered in the groups with reduced tumor frequencies, whereas the total excretion of fecal biliary steroids (mg/day) did not show a similar correlation. These observations suggest that intestinal tumor frequency can be reduced by increased dietary fiber only when fat intake is not at a high level. The effect of fiber may be due to dilution of promoters and/or carcinogens in the intestinal tract.

Combined inhibitors of carcinogenesis: effect on azoxymethane-induced intestinal cancer in rats.

Outbred male Sprague-Dawley CD rats were fed a complete semisynthetic diet and were given supplemental low doses (2 ppm) of selenium as H2SeO3 in their drinking water or 50 mg 13-cis-retinoic acid (13-cis-RA) and 2 g beta-sitosterol/kg diet either singly, in combinations of two, or in combinations of all three. Intestinal tumors were induced with eight weekly sc injections of 8 mg azoxymethane (AOM)/kg body weight, and inhibition of tumor formation was determined by tumor counts after 26 weeks. Noncarcinogen controls for each dietary group received eight injections of sterile water. Tumor inhibition was statistically significant in 2 groups of animals: Dietary control animals had a tumor frequency of 5.07 tumors/rat, rats receiving selenium- plus 13-cis-RA supplementation had a tumor frequency of 3.77, and those being given the combination of all three inhibitors had 2.75 tumors/rat. Analysis of fecal steroids from 3 AOM groups (dietary controls, the beta-sitosterol plus 13-cis-RA-supplemented group, and the group receiving all three additives) after 4 months of supplementation showed that the addition of beta-sitosterol to the diet had no effect on acidic or neutral steroids, regardless of the observed difference in tumor frequency. These results suggest that subpharmacologic doses of inhibitors, particularly those that inhibit the process by different mechanisms, while ineffective alone, may provide significant inhibition of tumorigenesis when used in combination.

Structural requirements for stimulation of colonic cell proliferation by oxidized fatty acids.

The primary autoxidation products of polyunsaturated fatty acids are known to stimulate DNA synthesis and induce ornithine decarboxylase activity in colonic mucosa. In the present study we have determined the structural features of the oxidized fatty acids necessary for the stimulation of these two components of mitogenesis. Compounds were instilled intrarectally in either aqueous or mineral oil vehicles and 3 h later (ornithine decarboxylase activity) or 12 h later (tritiated thymidine incorporation), the animals were killed and the colonic mucosa harvested for measurement of the two parameters of cell proliferation. Hydroperoxy and hydroxy fatty acids derived from oleate and stearate were studied. Ricinoleic acid and the alpha,beta-unsaturated ketone derived from oleic acid were also investigated. The minimal requirement for stimulation of cell proliferation is the presence of an oxidized functionally adjacent to a carbon-carbon double bond. All active compounds studied were roughly equipotent, which suggests a common mediator may be involved. These results imply that, in addition to biliary steroids, the autoxidation products of unsaturated fatty acids may play a role in the enhancement of tumorigenesis by high levels of dietary fat. Furthermore, the data suggest a possible mechanism of action for the active compounds.

Effect of inhibitors of tumorigenesis on the formation of O6-methylguanine in the colon of 1,2-dimethylhydrazine-treated rats.

The level of O6-methylguanine (O6MeGua) in the colonic DNA of rats treated with 1,2-dimethylhydrazine was determined. The effect of various tumorigenesis inhibitors on the formation of this modified base was also studied. Rats were given a single s.c. injection of 1,2-[14C]dimethylhydrazine. Six hr later, they were killed, and colonic DNA was extracted and analyzed by high-pressure liquid chromatography. The inhibitors tested were disulfiram (DSF), pyrazole, sodium selenite, butylated hydroxyanisole, butylated hydroxytoluene, potassium ascorbate, and 13-cis-retinoic acid. The level of O6MeGua in control rats was 29.9 [(O6MeGua X 10(6)/guanine)]. When rats were fed 0.25% (w/w) DSF, this value was reduced to 10.2, and at 0.5% DSF there was no detectable O6MeGua formed. Injection of pyrazole (40 mg/kg i.p.) 2 hr prior to 1,2-dimethylhydrazine treatment reduced the O6MeGua level to 2.4. All the other tumorigenesis inhibitors had no effect on either O6MeGua levels or the cpm/mg DNA in treated rats. With O6MeGua as a measure of the extent of initiation, these results confirm that DSF and pyrazole inhibit the initiation phase of carcinogenesis. This is to be expected as both have been shown to block the metabolism of azoxymethane, which is a crucial metabolite in the activation of 1,2-dimethylhydrazine. The other substances, all known tumorigenesis inhibitors, may act on the promotional phase of carcinogenesis and are worthy of further study for the role in cancer prevention.

Ornithine decarboxylase activity in the rat and human colon.

We have investigated the effect of age, a high-fat diet, sodium deoxycholate, and the ornithine analogue alpha-difluoromethylornithine on ornithine decarboxylase (ODC) activity in the rat colon. The relative levels of ODC activity were also determined in normal mucosa and tumor tissue from rat and human colon. The colonic ODC activity induced by intrarectal instillation of sodium deoxycholate in male Sprague-Dawley rats was highest in young animals, and it decreased with increasing age. A high level of dietary fat caused both an increased in basal colonic ODC activity and enhanced ODC induction by deoxycholate. alpha-Difluoromethylornithine given in drinking water inhibited, in a dose-dependent fashion, deoxycholate-induced ODC activity. The frequency of azoxymethane-induced intestinal tumors was also significantly reduced by alpha-difluoromethylornithine. Since colonic ODC activity is increased in carcinogenesis by known promoting agents and decreased by tumor inhibitors, this short-term assay may provide a useful system for identifying colon tumor promoters and inhibitors. The ODC activity in colon tumors of Sprague-Dawley rats was found to be significantly higher than in normal-appearing mucosa in the same animals. Similarly, ODC activity in human colon cancer was found to be higher than that of the normal-appearing mucosa in the same specimen. These results strengthen the utilization of the rat model for studies, the results of which may apply to the human situation.

Promotion of azoxymethane-induced intestinal cancer by high-fat diet in rats.

Promotional properties of a high-fat diet in intestinal cancer were studied by feeding a 30% beef fat diet to 8 groups of rats (25 rats/group) for time periods varying from 1 to 21 weeks after 8 weekly s.c. injections of azoxymethane (AOM) (8 mg/ kg). Two other groups were fed the high-fat diet, one for 8 weeks prior to and the other during AOM injections. A 5% fat diet was fed to rats when not on the 30% fat diet and to a control group of 25 animals. High fat diet increased intestinal tumor frequency up to 2-fold when given for at least 4 weeks after but not during or prior to AOM injections; this increase occurred even after a prolonged interval (10 weeks) between the last AOM injection and the high-fat diet. In general, tumor frequency increased according to the length of time animals were fed the high-fat diet after AOM. Therefore, the high-fat diet in this model exhibited most of the properties of promoters developed from murine skin cancer, thus adding support to the concept that excess dietary fat acts at the promotional phase of carcinogenesis.

In vivo stimulation of DNA synthesis and induction of ornithine decarboxylase in rat colon by fatty acid hydroperoxides, autoxidation products of unsaturated fatty acids.

The effect of intrarectal instillation of hydroperoxy and hydroxy fatty acids on colonic DNA synthesis and ornithine decarboxylase activity in male Sprague-Dawley rats was examined. A mixture of hydroperoxy-arachidonic acid isomers was prepared by methylene blue-sensitized photooxygenation. Pure 13-hydroperoxy-9,11-octadecadienoic acid was prepared by the action of soybean lipoxygenase on linoleic acid. Sodium borohydride reduction yielded the respective hydroxy fatty acids. Twelve hr after instillation of solutions of either hydroperoxy or hydroxy fatty acids, at concentrations up to 10 mM, DNA synthesis was increased in a dose-dependent fashion up to 240% above control values. The induction of ornithine decarboxylase occurred over a similar concentration range 3 hr after instillation of oxidized linoleic acid. In this case, the hydroxy acids (49-fold increase at 10 mM), were more stimulatory than the hydroperoxy derivatives (23-fold at 10 mM). Highly purified linoleic and arachidonic acids did not stimulate either activity at concentrations up to 50 mM. These data indicate that autoxidation products of unsaturated fatty acids, likely components of high-fat diets, can evoke proliferative responses in colonic mucosa. These responses may be relevant to the promotional effect of high dietary fat on colon carcinogenesis.

Energy-dependent export of the 13-oxooctadecadienoic acid-glutathione conjugate from HT-29 cells and plasma membrane vesicles.

Numerous studies have identified members of the multidrug resistance protein (MRP) family of ABC transporters as ATP-dependent GS-X pumps responsible for export of various xenobiotic conjugates, and the few known glutathione conjugates of endogenous metabolites. In the present study we have investigated the possibility that the glutathione conjugate of 13-oxooctadecadienoic acid (13-OXO-SG), is exported from HT-29 cells by one of these GS-X pumps. The precursor 13-oxooctadecadienoic acid (13-OXO) is a metabolic oxidation product of linoleic acid. The transport of 13-OXO-SG is compared to that of the glutathione conjugate of chlorodinitrobenzene (DNP-SG). The results show that the efflux of 13-OXO-SG is ATP-dependent. In cultured HT-29 cells as well as in inside-out vesicles prepared from these cells, significant inhibition of conjugate export is achieved by the energy disrupters, beta,gamma-methylene ATP, sodium vanadate, and 2-deoxyglucose. Significant inhibition of the vesicle-mediated transport is also observed in the presence of genistein and verapamil. In inside-out vesicles, the transport of both conjugates exhibits saturation with an apparent K(m) of 325.5 microM and a V(max) of 0.0669 nmol/mg protein per min for 13-OXO-SG and a K(m) of 169 microM and a V(max) of 0.496 nmol/mg protein per min for DNP-SG. Furthermore, co-inhibition is observed when both conjugates are present simultaneously which is consistent with the involvement of common pumps. The data in this report demonstrate the involvement of an ATP-dependent pump in the metabolic disposition of endogenously derived metabolites of linoleic acid.

Specific protein targets of 13-oxooctadecadienoic acid (13-OXO) and export of the 13-OXO-glutathione conjugate in HT-29 cells.

The linoleic acid metabolite, 13-oxooctadecadienoic acid (13-OXO), is reactive with cellular thiols. In the present report, incubations of HT-29 or CaCo-2 homogenates with 13-OXO and GSH indicate that HT-29 cell homogenates produce a 13-OXO-GSH conjugate. The conjugate formed was likely of enzymatic origin as chiral-phase HPLC showed the major product consisted of only one of two possible diastereomers. The glutathione transferase activity (GST), using chlorodinitrobenzene, was found to be 126 nmol/mg/min in HT-29 cells and 21 nmol/mg/min in CaCo-2 cells. These levels of activity are consistent with the relative ability of the two cell lines to conjugate GSH to 13-OXO. Incubation of intact HT-29 cells with either 13-OXO, or the metabolic precursor 13-hydroxyoctadecadienoic acid (13-HODE), showed detectable 13-OXO-GSH conjugate in the media, but none in the cells. The stereochemistry of the extracellular conjugate suggested an enzymatic origin. In additional experiments, the labeling of cellular protein by 13-HODE was much more specific than the labeling of protein by 13-OXO suggesting that in situ generation of 13-OXO from 13-HODE confers selectivity on the reactions between cellular thiols and 13-OXO. These results demonstrate that in HT-29 cells, 13-HODE is converted to 13-OXO which then either reacts with cellular protein or is conjugated to GSH by GST. The 13-OXO-GSH conjugate is then exported from the cell.

Metabolism of oxidized linoleic acid: characterization of 13-hydroxyoctadecadienoic acid dehydrogenase activity from rat colonic tissue.

An oxidized derivative of linoleic acid, 13-hydroxyoctadecadienoic acid (13-HODE), is dehydrogenated by an NAD+ dependent dehydrogenase present in rat colon mucosa. The product of the reaction is the 2,4-dienone, 13-oxooctadecadienoic acid. Enzyme activity was determined by HPLC analysis of incubation mixtures as well as by measuring the increase in absorbance at 285 nm, which represents formation of the 2,4-dienone chromophore. Characteristics of the reaction with respect to protein concentration, time of incubation and substrate dependence were investigated. Several inhibitors of known dehydrogenases had no effect on the 13-HODE dehydrogenase. These include, ethanol, indomethacin, 6-methyl-17-hydroxyprogesterone acetate, 4-(diethylamino)-benzaldehyde, and aspirin. The enzyme was mildly inhibited by pyrazole, 4-methylpyrazole and ibuprofen. Disulfiram was found to be a potent inhibitor of enzyme activity with an IC50 of 200 microM. Inhibitor specificity, and other characteristics of the reaction suggest the enzyme is neither alcohol dehydrogenase, diol dehydrogenase, nor a prostaglandin dehydrogenase. It is possible this enzyme plays an important role in the response of the colonic mucosa to the mitogenic effect of oxidized fatty acids.

Fatty acids and colon cancer in experimental models.

Experimental models have several advantages in the study of colon cancer. They can be used to tightly control diet, examine putative intermediate markers, test hypotheses about mechanisms of carcinogenesis, and quantify development of tumors in a short time. Dietary issues that have been studied in animal models but are unresolved include the concept of the effects of total fat compared with energy intake, composition of the basal diet, linoleic acid requirements, and interactions of fat with other nutrients. Intermediate markers that have been probed in animal or in vitro studies include cytokinetics, aberrant crypt foci, eicosanoids and hydroxyoctadecadienoic acids, ornithine decarboxylase, tyrosine kinase, protein kinase C, and gene expression. Colon cancer is studied in animals primarily with use of chemicals that are relatively specific inducers of these tumors, but transplantable models and transgenic animals are also used. Total dietary fat is generally thought to affect colon tumorigenesis, but there does not appear to be any specific fatty acid that promotes the development of colon cancer. Several studies indicate that n-3 fatty acids from marine sources alter a variety of biological intermediates and inhibit colonic tumorigenesis; this is probably mediated via the eicosanoid pathway. Although there are undoubtedly multiple cellular changes elicited by certain fatty acids, our current knowledge of this area suggests that specific fatty acid metabolites or their targets are the likely mediators in this sequence.

Decreased levels of 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase in neoplastic tissue of human colon biopsies.

Recent studies have identified a role for the oxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (13-HODE) in cell proliferation. The enzyme 13-HODE dehydrogenase catalyzes the conversion of 13-HODE to 13-oxooctadecadienoic acid. This enzyme has been shown to correlate with the degree of differentiation of intestinal cells in both in vitro and in vivo models. Higher enzyme levels are found in more differentiated cell types. The present study was done to determine if enzyme levels of 13-HODE dehydrogenase are predictive of the differentiation status of biopsies from human colonic mucosa. Twenty-eight patients who underwent diagnostic colonoscopy (10 patients with adenocarcinoma and 18 with adenomatous polyps) had biopsies taken from both normal rectal mucosa and neoplastic mucosa. The determination of 13-HODE dehydrogenase activity was conducted by high-performance liquid chromatography analysis of all biopsy samples. Sixteen of the 18 patients with polyps had lower 13-HODE dehydrogenase activity in the adenoma than in the uninvolved rectal mucosa (P = 0.001). The colon adenocarcinomas also had less 13-HODE dehydrogenase activity in the cancer biopsy tissue than in uninvolved rectal mucosa (P = 0.041) These data are consistent with a role for 13-HODE dehydrogenase in intestinal cell differentiation. Understanding the precise role of this enzymatic reaction could be important potentially in the therapy and biology of colon cancer. In addition, measurements of 13-HODE dehydrogenase may be a useful parameter by which to ascertain the differentiation status of intestinal cells in vitro.

Effect of alkyl sulfides on diazomethane-induced methylation of DNA in vitro.

Organosulfur compounds found in Allium species such as garlic and onions inhibit carcinogenesis induced by alkylating agents. One potential mechanism for this activity involves scavenging of ultimate carcinogenic species by the nucleophilic sulfur atom. Carcinogenic methylating agents such as methyl-nitrosourea produce, among others, O6-methylguanine and N7-methylguanine in DNA both in vivo and in vitro. In the present study we have determined the effect of several alkyl sulfides on the formation of O6-methylguanine and N7-methylguanine in DNA reacted with the methylating agent diazomethane in vitro. Dipropyl sulfide and diallyl sulfide affect guanine methylation by increasing the O6/N7 ratio without drastic alterations in the total amount of adduct formed. Three similar compounds--diallyl disulfide, allyl methyl sulfide and diallyl ether--had no appreciable effect on the amount of alkylation at either position. These data suggest that scavenging of diazomethane-like ultimate carcinogens does not play a major role in the inhibition of carcinogenesis by organic sulfides.

Importance of the duration of inhibition on intestinal carcinogenesis by difluoromethylornithine in rats.

The effect of the duration and sequence of inhibition of intestinal tumor formation in rats was studied to determine whether part time inhibition has any value. Four groups of male Sprague-Dawley rats were given 8 weekly s.c. injections of azoxymethane (AOM) 8 mg/rat. Three groups were given the inhibitor, difluoromethylornithine (DFMO) in the drinking water; one for the entire 26 weeks of the study, one for the first 13 weeks only, and one for the last 13 weeks. A control group was not given the inhibitor. While the continuous treatment group developed the least number of tumors per rat (1.5 vs. 5 for controls), still both groups given the inhibitor for just 13 weeks also developed fewer tumors than controls 5 vs. 3.2 (early treatment) and 5 vs. 2.8 (late treatment). These results show that part time inhibition, including its late application, does reduce intestinal tumor formation in rats.

Regulation of the induction of ornithine decarboxylase in short-term rat colon organ culture by dexamethasone and 13-hydroxyoctadecadienoic acid (13-HODE).

The activity of rat colon mucosal ornithine decarboxylase (ODC) was found to dramatically increase within three hours after placement of mucosal explants under organ culture conditions. Increases of 224-fold above the initial levels were observed 24 hours after establishment of cultures. During the next 72 hours, activity gradually declined although it never reached in vivo levels. Inclusion of either dexamethasone (DEX) or 13-hydroxyoctadecadienoic acid (13-HODE) in the media suppressed the early induction of ODC activity but did not abolish the increases. The effect of these compounds was reversible. Within 24 hours after removal of either dexamethasone or 13-HODE the ODC activity increased to the level found in untreated control cultures. These data suggest that glucocorticoids and 13-HODE may play a role in the regulation of colonic cellular proliferative activities in intact animals. The findings with 13-HODE add to the growing list of examples of regulation of biological activity by oxidized derivatives of linoleic acid.

Formation of adducts between 13-oxooctadecadienoic acid (13-OXO) and protein-derived thiols, in vivo and in vitro.

Linoleic acid is metabolized by numerous tissues to oxidized derivatives possessing biological activity. In the current experiments, we have investigated the reaction of 13-oxooctadecadienoic acid (13-OXO) and the metabolic precursor 13-hydroxyoctadecadienoic acid (13-HODE) with cellular macromolecules and model cellular nucleophiles. Colonic mucosal explants from Sprague-Dawley rats were incubated in the presence of [1-14C]-13-OXO or [1-14C]-13-HODE. The binding of radiolabel to the protein and nucleic acid fractions was analyzed by isopycnic centrifugation in Cs2SO4. Cellular homogenates incubated with either 13-OXO or 13-HODE resulted in the binding of radiolabel to cellular protein. No significant amounts of reaction with cellular RNA or DNA were observed. To assess possible modes of reaction with cellular constituents, the oxidized fatty acids were incubated in vitro with oxygen, sulfur, or nitrogen, nucleophiles including, serine, cysteine, glutathione, methionine, lysine, adenosine, and guanosine. Under physiologic conditions, in the absence of cellular homogenates, only 13-OXO was reactive. In addition, only the sulfur-containing compounds cysteine and glutathione showed significant rates of reaction. Furthermore, treatment of colonic homogenates with N-ethlymaleimide reduced the binding of [1-14C]-13-OXO to cellular protein. These data support the suggestion that 13-HODE requires metabolic activation, by dehydrogenation to 13-OXO, prior to binding to cellular protein and that protein-derived thiol groups are involved in the binding reactions.

Copper-containing IUDs are not associated with an increase of malondialdehyde levels in cervical mucus.

The malondialdehyde (MDA) content of cervical mucus from 23 healthy adult females was measured using an ion-pairing HPLC method capable of detecting 10 pmol MDA. Ten of the women were wearing copper IUDs, four were wearing plastic IUDs, and nine controls were not wearing an IUD. Cervical mucus was sampled during the follicular, periovulatory, luteal, and menstrual phases. The study was designed to determine if there is a relationship between MDA formation and the use of a copper IUD. A total of 79 samples were analyzed. Only 16 of the samples had sufficient MDA for reliable quantitation with the level ranging from 0.1 nmol/g to 2.32 nmol/g. In 19 of the samples, trace levels (less than 0.06 nmol/g) were detected but could not be reliably quantitated. In the remaining 44 samples, no MDA was detectable. There was no correlation between the presence of copper- or non-copper-containing IUDs and the level of MDA. These results are contrary to a previously published report that used a less specific method for MDA analysis.

Modulation of prostaglandin H synthase activity by conjugated linoleic acid (CLA) and specific CLA isomers.

Conjugated linoleic acid (CLA) has been shown to inhibit tumorigenesis in animal models and is cytostatic to numerous cell lines in vitro. However, the mechanism of action is unknown. In the current study, we determined the effects of CLA and specific isomers of CLA on the rate of oxygenation of arachidonic acid by prostaglandin H synthase (PGHS) in ram seminal vesicle microsomes. The enzyme was incubated with 0.1 to 100 microM CLA or specific isomers of CLA for 2 min prior to the addition of 44 to 176 microM arachidonate. The isomers tested were 9(E),11(E) CLA; 9(Z),11(E) CLA; 9(Z),11(Z) CLA, and 10(E),12(2) CLA. For a positive inhibitor control, flurbiprofen was used at 0.75 to 2.50 microM. Enzyme activity was assessed by measuring the rate of oxygen consumption. Inclusion of CLA or specific isomers of CLA in the incubation mixtures inhibits PGHS. The efficacy differs for each isomer, with the 9(Z),11 (E) CLA isomer being the most effective and the 9(Z),11 (Z) CLA isomer being the least effective inhibitor among the four CLA isomers tested. The Ki values obtained by Dixon replots range from 18.7 microM for the most effective isomer, 9(Z),11 (E) CLA, to 105.3 microM for the least effective isomer, 9(2),11(2) CLA. The Ki value for flurbiprofen with ram seminal vesicle microsomes was 0.33 microM. As the concentration of arachidonate was increased, the CLA-dependent inhibition of PGHS decreased, suggesting competitive inhibition. The results of this study demonstrate the potential of CLA and specific isomers of CLA to modulate prostaglandin biosynthesis.

The essential fatty acid requirement for azoxymethane-induced intestinal carcinogenesis in rats.

The essential fatty acid requirement for the development of intestinal carcinogenesis was determined and compared to the overall essential fatty acid status of the animals as measured by the triene/tetraene ratio in the plasma, liver and colon. To induce tumors, male Sprague-Dawley rats were given two weekly injections (20 mg/kg body wt) of azoxymethane. Two weeks after the last injection, the rats were divided into groups of 25 and given one of six diets containing various levels of essential fatty acids (as linoleate). The diets contained 5% total fat and were prepared by mixing safflower oil (high essential fatty acids, beef fat (low essential fatty acids), and medium chain triglyceride oil (no essential fatty acids). One group of rats was fed a 20% beef fat diet. The range of essential fatty acids was from less than 0.03% to 1.28% (w/w). Twenty-six weeks after the first azoxymethane injection, the animals were killed and intestinal tumor incidence and multiplicity were determined. Samples of plasma, liver and colon were also taken for measurement of the triene/tetraene ratio by gas chromatography. Large bowel tumor incidence showed a dependence on the essential fatty acid content of the diet. The results were as follows: (percent essential fatty acids: percent tumor incidence) Group A (1.28: 72.4), Group B (0.60: 73.3), Group C (0.11: 55.2), Group D (0.08: 39.3), Group E (less than 0.03: 37.9) and Group F, which was fed 20% beef fat, (0.34: 88.5).(ABSTRACT TRUNCATED AT 250 WORDS)