Adelbert E. Wade

Dietary fatty acid-induced alterations of hepatic microsomal drug metabolism☆

Abstract Male and female weanling, Sprague-Dawley rats were fed synthetic fat-free (FF) or corn oil-containing diets for 3 weeks. The apparent K m and V max for the metabolism of ethylmorphine and hexobarbital were lower in washed hepatic microsomes from male rats fed a FF diet than in rats fed diets containing 3 or 10 per cent corn oil. The apparent V max for aniline hydroxylase was also depressed by feeding a FF diet to male rats, although aniline K m was not altered. Feeding a FF diet to female rats decreased V max for both hexobarbital oxidase and aniline hydroxylase, but the apparent K m for the substrates was not changed. Gas-liquid Chromatographie analysis of fatty acids derived from microsomal membranes revealed marked alterations in the relative content of fatty acids in response to FF feeding. Content of cytochrome P-450 was lower in both male and female rats fed a FF diet. Associated with the decreased cytochrome P-450 content were decreases in the ability of microsomes to bind aniline and hexobarbital. A qualitative change produced in cytochrome P-450 was indicated by a decrease in the ratio of ethyl isocyanide peak heights (455:430 nm) in microsomes from FF-fed rats.

Eicosanoid synthesis in 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in Sprague-Dawley rats fed primrose oil, menhaden oil or corn oil diet

The comparative effects of high-fat diets (20%, w/w) on eicosanoid synthesis during mammary tumor promotion in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rats were studied using diets containing 20% primrose oil (PO), 20% menhaden oil (MO) or 20% corn oil (CO). Sprague-Dawley rats fed the PO or MO diet had 21% or 24% fewer adenocarcinomas, respectively, than rats fed the CO diet. Histologically (i.e., mitotic figures, inflammatory cell infiltration and necrosis), the CO-fed rats exhibited the highest frequency of changes within tumors. Plasma fatty acid composition was significantly altered by diet, reflecting the composition of the oils which were being fed. Only the plasma of PO-fed rats contained detectable levels of gamma-linolenic acid (GLA). Arachidonic acid (AA) levels were significantly higher (p<0.05) in PO-fed than in CO- or MO-fed rats. MO-fed rats had significantly higher levels of plasma palmitic acid, while palmitoleic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were detected only in MO-fed rats. As expected, linoleic acid (LA) and AA levels were lower (p<0.05) in the MO-fed rats than in PO- or CO-fed groups. The plasma of the CO-fed rats contained significantly higher levels of oleic acid. Eicosanoid synthesis in mammary carcinomas of rats fed the 20%-fat diets was 2–10 times higher than in mammary fat pads of control rats. The synthesis of PGE1 and LTB4 was significantly (p<0.05) higher in PO-fed rats than in CO-fed or MO-fed rats, although PGE values were significantly (p<0.05) higher in CO-fed rats than in Mo or PO groups. The synthesis of eicosanoids in both mammary fat pads and mammary carcinomas of MO-fed rats was lower (p<0.05) than in tissues of rats fed either CO or PO diets due to less AA precursor being fed and/or to competition between n−6 and n−3 fatty acids for cyclooxygenase and lipoxygenase. The ratios of monoenoic to dienoic eicosanoids in both mammary fat pads and mammary carcinomas were higher in the PO group than in the MO or CO groups. These results suggest that inclusion of GLA (PO feeding) or EPA and DHA (MO feeding) in the diet may decrease malignancy by altering eicosanoid profiles.

Influence of tamoxifen and its N-desmethyl and 4-hydroxy metabolites on rat liver microsomal enzymes

Tamoxifen (Nolvadex; TAM) and its major metabolites, N-desmethyl- (DMT) and 4-hydroxy-tamoxifen (HT), were shown to be potent inhibitors of hepatic cytochrome P-450-dependent mixed function oxidations. From in vitro experiments, all three were found to be potent inhibitors of oxidation of Type-I substrates (ethylmorphine and aminopyrine) and less potent, non-competitive inhibitors of Type-II substrates (aniline and dimethylnitrosamine). TAM, DMT and HT were of essentially equal potency and had a much more pronounced effect on Type-I substrates than on Type-II compounds studied. Their action appears to parallel SKF-525A in type and potency of inhibition produced. Spectral binding studies suggest that TAM and its metabolites exert their effects by occupying the Type-I binding site of cytochrome P-450 and thus limiting the accessibility of other substrates to the active site of the enzyme. TAM (and its metabolites) also inhibits its own metabolism, altering the distribution and elimination half-lives of tamoxifen-derived species. In addition, tamoxifen metabolism was found to be sensitive to the presence of other drugs. These results raise concern regarding the role that continued administration of tamoxifen plays in changing its own disposition as well as in the detoxification of drugs administered with it.

Temperature compensation in the hepatic mixed-function oxidase system of bluegill

Bluegill (Lepomis macrochirus R.) were acclimated to 12, 22 or 32 degrees C for 5 or 14 days. Liver weight to body weight ratio and the rate of metabolism of benzo[alpha]pyrene by liver microsomes varied inversely with the acclimation temperature of the fish. Concentration of microsomal cytochrome P-450, as determined by CO-difference binding spectra, was not significantly affected by acclimation temperature. There were no qualitative or quantitative differences in the electrophoretic patterns of proteins with molecular weights similar to those reported for cytochrome P-450. There were no shifts in the temperature optima of the microsomal benzo[alpha]pyrene hydroxylase activity.

Effect of different levels of corn oil in the diet upon the rate of hexobarbital, heptachlor and aniline metabolism in the liver of the male white rat

Abstract At low dietary intakes of corn oil there is a decreased ability of the liver, in an in vitro system, to metabolize drugs. Half of this decrease in rate of aniline, hexobarbital and heptachlor occurs at corn oil intakes equivalent to linoleate intakes of 0.1, 1 and 1% of calories, respectively. High corn oil intakes are equally deleterious (half decrease at intakes equivalent to 9% linoleate) in the case of hexobarbital. Biochemical results were confirmed by sleeping time measurements. Results are interpreted as confirming the linoleate requirement of the male rat as being about 2% of calories, and it is suggested that the optimal linoleate intake is about 3% of calories.

Dietary fat—A requirement for induction of mixed-function oxidase activities in starved-refed rats☆

Male rats were starved 0-48 hr, and then refed diets containing 0% (F.F.) to 20% corn oil (C.O.) lab chow or 20% coconut oil (C.C.O.) for 1-4 days. Some received phenobarbital sodium (80 mg/kg, i.p. daily) for 1-3 days prior to decapitation. Five cytochrome P-450-dependent indicators were assayed as measures of altered hepatic microsomal function: ethylmorphine N-demethylase (EMDM), N-nitrosodimethylamine (DMN)-N demethylase, aniline hydroxylase (AH), benzo[a]pyrene hydroxylase (AHH) and CO-difference spectra (P-450). Increasing dietary corn oil (0, 0.5, 10, 20%) in control rats resulted in a progressive increase in the activities of these five enzymes. Dietary fat influenced phenobarbital (Pb) inducibility of all mixed-function oxidase (MFO) enzymes measured except AHH. Pb induced the remaining enzymes only 11-22% in animals fed fat-free diet as compared to 119-246% in animals fed coconut oil and corn oil. Rats fed fat-free diet for 21 days without prior food deprivation and administered Pb had 79% more EMDM, 34% more AH and 120% more P-450 than non-induced controls, whereas rats fed 20% corn oil diet had 227% more EMDM, 143% more AH and 128% more P-450. A requirement of dietary fat for induction of MFO by Pb was demonstrated by these starvation-refeeding experiments. Coupled with data recovered from the 21-day studies, these experiments suggest that a compensatory mechanism may be operative during chronic feeding of the fat-free diet to partially return inducibility to the drug-metabolizing system.

Effect of dietary fat on the induction of hepatic microsomal cytochrome P450 isozymes by phenobarbital

Dietary polyunsaturated fatty acid is needed for optimal induction of cytochrome P450. In this study we quantitated cytochrome P450 hemoproteins in male Sprague-Dawley rats that were starved for 36 hr and then refed a fat-free diet (FF) or a diet containing 20% corn oil for 4 days. Some received phenobarbital (Pb) sodium (80 mg/kg, i.p., daily) for 3 days prior to decapitation. Microsomal cytochrome P450 levels were measured by carbon monoxide binding spectra, and the P450 isozymes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were quantitated by gel scanner. Cytochrome P450 PB-B was quantitated by a Western blot technique. Rats fed FF diet and administered Pb had only 21% more microsomal P450 than non-induced controls, whereas rats fed 20% corn oil diet had 59% more P450 and Pb-treated rats fed 20% corn oil diet had 181% more P450 than FF controls. Analysis of gels showed 32, 59 and 124% more P450 protein, respectively, in FF Pb, corn oil control or corn oil Pb groups than in FF controls. Cytochrome P450 PB-B was not detected in non-induced groups, but quantitation by Western blot yielded 0.32 and 0.70 nmol/mg protein, respectively, in FF Pb and corn oil Pb groups. Our findings suggest that deprivation of dietary fat reduces the total amount of cytochrome P450 hemoprotein and its inducibility by Pb through decreased P450 hemoprotein synthesis. The limiting factor(s) restricting synthesis of new cytochrome P450 hemoproteins in rats refed a diet devoid of fat may be the inability to respond to the inducer (Pb) or the paucity of utilizable fatty acids needed for synthesis of the phospholipid matrix of the endoplasmic reticulum necessary for the support and proper juxtapositioning of these protein molecules.

The effects of temperature on the cytochrome P-450 system of thermally acclimated bluegill

The effects of temperature acclimation at 10, 20 and 30 degrees C on the concentration and activity of the mixed function oxidase system in bluegill are as follows. Liver weight/body weight varied inversely with temperature. Significant (P less than 0.05) differences in concentration of cytochrome P-450 of hepatic microsomes were seen and varied inversely with temperature. Benzo(a)pyrene hydroxylase activity tested in vitro at incubation temperatures of 10, 20 and 30 degrees C showed significant differences in Km, but no differences in Vmax. SDS polyacrylamide gel electrophoresis revealed some quantitative differences in cytochrome P-450 isozymes between groups.

Nutritional factors affecting drug-metabolizing enzymes of the rat.

Abstract Feeding diets rich in thiamin depresses aniline hydroxylase, cytochrome P-450 and cytochrome b5 within 9–14 days. Pair-feeding experiments suggest that the depression of aniline hydroxylase, cytochrome c reductase and ethylmorphine demethylase is due to the thiamin; however, the depression of cytochrome P-450 and b5 may be due primarily to the increased amount of carbohydrate ingested by rats fed the enriched diet. When starch was substituted for sucrose, cytochrome P-450 was not lowered by high thiamin ingestion, although cytochrome b5 and NADPH cytochrome c reductase were depressed similarly to that of rats fed high thiamin levels in a sucrose-based diet. Although aniline hydroxylase and ethylmorphine demethylase activities were significantly depressed by both high thiamin diets, this effect was more pronounced in rats fed the sucrose-based diet.

Relationship of Dietary Essential Fatty Acid Consumption to Hepatic Drug Hydroxylation

Maximal aniline hydroxylase and hexobarbital oxidase activities were evident in rats fed between 1 and 10 µl arachidonate/day whereas cytochrome P-450 was at a maximum between 10 and 30 µ\l &