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 dodecyi 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 fact that nutriture affects drug metabolism and drug action in laboratory animals is undisputable. Activation or detoxification of drugs and potential carcinogens can also be modified by diet. The quantity and quality of dietary fat affects lipid composition and physical characteristics of biological membranes and enzymatic activity of several components of the drug metabolizing enzyme system. These changes have been associated with alterations in the physiological response to drugs and to the resulting mutagenicity and carcinogenicity of procarcinogens. It is suggested by these data that dietary fat, by altering fatty acid composition of biological membranes, alters the physical and biochemical characteristics of these membranes, thereby directly affecting drug entrance into the membrane; the stability of the membrane; the potential for lipid peroxidation; and the activity of the phospholipid dependent enzymes associated with these membranes. The fact that these membrane associated changes can occur rapidly and that brief periods of fatty acid deprivation can profoundly affect the inducibility of these enzymes by xenobiotics suggests that the potential for drug-nutrient interactions exists in the absence of frank nutrient deficiency states.

Administration of a single dose of the potent interferon inducer poly rI:rC to Swiss Webster mice depressed hepatic cytochrome P-450 to 75% of control, ethylmorphine N-demethylase to 56% of control and DMN N-demethylases I and II to about 80% of control. Although each enzyme responded in a unique manner, maximum depression occurred at 24 hours after poly rI:rC administration and the concurrent administration of inhibitors of protein synthesis (actinomycin D or cycloheximide) prevented this depression. These data suggest that poly rI:rC effects on the mixed function oxidases are not species specific although depression follows a time course shorter than that reported in the rat (maximum depression at 40 hours after poly rI:rC administration) and that depression occurs through the stimulation of a protein responsible for degrading cytochrome P-450.

Partially purified fractions of cytochrome P-450 were prepared from hepatic microsomes recovered from male rats 12 h after administration of either saline or polyriboinosinic:polyribocytidylic acid (poly I:C). Poly I:C reduced the microsomal concentration of cytochrome P-450 by 19% and decreased the maximal binding spectrum (delta Amax) resulting from addition of the type-II substrate 2,4-dichloro-6-phenylphenoxyethylamine to one fraction (B2) while increasing the affinity of that fraction for this substrate. Poly I:C also reduced the microsomal hydroxylation of benzo(a)pyrene and the N-demethylation of benzphetamine by the other fraction (B1). Since 14C-leucine incorporation into cytochrome P-450 was increased in poly I:C-treated rats, it is suggested that poly I:C depresses hepatic mixed-function oxidase activity by increasing the rate of degradation of specific cytochrome P-450s.

Hepatic microsomes from male Holtzman albino rats fed a synthetic fat-free diet for 21 days had significantly less cytochrome P-450 and exhibited less binding capacity (delta Amax/mg protein) for aniline and octylamine than microsomes from similar rats fed a diet containing 10% corn oil. Treatment with 3-methylcholanthrene (3-MC) increased the concentrations of cytochrome P-450 (as measured by CO binding spectra) to nearly equal levels in both dietary groups, but the binding of aniline and octylamine to microsomes of rats fed the fat diet exceeded the increase in cytochrome P-450 concentration. Nuclear envelope concentrations of cytochrome P-450 were unaffected by diet. The administration of 3-MC to rats fed a fat-free diet failed to induce nuclear envelope P-450; however, in rats fed the corn oil diet, 3-MC increased this CO binding pigment over twofold. The affinity of nuclear envelope P-450 towards type II substrates was at least equal to that of microsomes, except in control rats fed the fat-free diet. In general, 3-MC pretreatment increased the binding affinity of nuclear envelop and microsomes toward aniline, while increasing affinity for SKF 525-A binding only to nuclear envelope. Molecular weight species in the region known to contain the cytochrome P-450 were quantified by fluorescence gel electrophoresis. Molecular weight species of 48,000 and 53,000 in the nuclear envelope had their counterparts in the microsomal preparation, but a 50,000 dalton component of nuclear envelope was not detected in microsomes. 3-Methylcholanthrene increased only a species with molecular weight 45,500 in the microsomal and nuclear envelope preparations. Rats fed the diet containing corn oil had microsomes with increased capacity for binding CO, but this was not accompanied by increased cytochrome P-450 protein concentration, as measured by quantitative fluorescence gel electrophoresis.

The interferon inducing agents, polyriboinosinic: polyribocytidylic acid and tilorone, and Freund's complete adjuvant cause a marked depression of several components of the hepatic mixed-function oxidase system. Separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitation by fluorescence gel scanning of different molecular weight species of cytochrome P-450 indicate that the depressant effect of these agents on the apoprotein moieties of cytochrome P-450 is of a specific nature.

Rats were fed diets resembling a normal human diet, except that, in a complete factorial fashion, safflower oil and/or mineral oil were substituted for part of the fat, while fructose, lactose and/or cellulose were substituted for carbohydrate, and zein was substituted for milk protein. Food intake and weight gain were not influenced by cellulose and mineral oil, but zein decreased both substantially. Plasma cholesterol was unchanged by safflower oil or mineral oil but was decreased by fructose. Plasma glucose was reduced as the starch-to-sugar ratio increased. The rate of aniline metabolism was increased by lactose and/or zein, but the rate of ethylmorphine metabolism was decreased by safflower oil or mineral oil.

Ingestion of high levels of thiamin significantly decreased the activity of cytochrome P-450, NADPH cytochrome c reductase, and the metabolism of aniline and ethylmorphine. Apparent VmaxS for ethylmorphine N-demethylase and aniline hydroxylase were decreased by high levels of riboflavin even though NADPH cytochrome c reductase was elevated. High levels of dietary pyridoxine significantly decreased only the Vmax for aniline hydroxylase. Generally, norethindrone produces either no change or slight depression of cytochrome P-450 regardless or diet, whereas the administration of norethindrone produced no change or an increase in activity of c reductase and ethylmorphine N-demethylase. Norethindrone induces aniline hydroxylase in animals fed all diets except those deficient in thiamin and riboflavin. The activities of the four parameters of the drug metabolizing system measured in these studies as well as the effects of norethindrone are clearly affected by the dietary status of the animal.

Hepatic microsomes from female rats fed a thiamin deficient diet for three weeks had approximately three times the capacity to metabolize mestranol as microsomes from similar rats fed a diet rich in thiamin. The incremental addition of thiamin to the diet depressed mestranol O-demethylation, NADPH cytochrome c reductase, and cytochrome P-450 content in a dose related manner up to 2 μg thiamin per gram of feed. Pair-feeding experiments indicate that thiamin ingestion is responsible for the depression of mestranol O-demethylation and NADPH cytochrome c reductase activity while carbohydrate ingestion is responsible for the decrease in cytochrome P-450. The absorbance spectra generated by the binding of ethylisocyanide to microsomes yield data which suggest that there are no qualitative alterations in cytochrome P-450 due to diet.

Hepatic drug metabolism in the chicken was investigated. White leghorn chickens were administered 20 mg of 3-methylcholanthrene (3MC) per kg 72 and 48 hr before killing. Levels of hepatic cytochrome P-450 were increased approximately 4-fold. In vitro ethylmorphine N-demethylase (ND) activity was enhanced approximately 1.7-fold, aniline hydroxylase (AH) was increased 2.5-fold, aryl hydrocarbon hydroxylase was increased 20-fold, and NADPH-cytochrome c reductase was unchanged. The Vmax was increased for both ND and AH activities, but the KM for demethylation was depressed whereas that for hydroxylation of aniline was increased. The metabolism of hexobarbital in vivo was not enhanced by 3MC treatment. In brief, the distinctive features of the hepatic mono-oxygenase system of the 3MC-treated chicken were: (a) enhanced ethylmorphine N-demethylase activity, (b) a shift in the Soret peak in the CO-difference spectrum of reduced cytochrome P-450 from the control value of 452 nm to 449 nm, and (c) proliferation and pronounced vesiculation of the hepatic endoplasmic reticulum as revealed by electron-microscopic examination.

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.

The interactions of drug-metabolizing enzyme inducers, inhibitors, and substrates with washed liver microsomes and a solubilized cytochrome P-450 were investigated using spectral shift techniques. Results indicate that the microsomal enzyme inducer, phenobarbital, binds to microsomes and to soluble cytochrome P-450, is readily displaced by hexobarbital (type I substrate) or aniline (type II substrate), and does not greatly inhibit the interaction of substrates with cytochrome P-450. On the other hand, enzyme inhibitors such as SKF 525-A and 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) bind to microsomes and to soluble cytochrome P-450, are not readily displaced by substrates, and greatly inhibit the interactions of substrates with cytochrome P-450. Barbital, which is not metabolized appreciably, does not bind to microsomes or to soluble cytochrome P-450, nor does it affect the binding of other substances. Aniline and DPEA added to solubilized cytochrome P-450 produced a modified type II spectral shift. The Ks for aniline binding was much higher than that observed with washed microsomes, whereas the Ks for DPEA was identical in both systems. Nicotinamide (a type II compound). SKF 525-A, and hexobarbital (type I compounds) produced typical difference spectra with soluble cytochrome P-450. Puromycin, a potent type II binder to microsomal cytochrome P-450, failed to produce a spectral shift when added to soluble cytochrome P-450. These results suggest that type I and type II binding sites are inter-related, that drug binding affects both sites, and that the extent of mutual displacement between two drugs is governed by their dissociation constants regardless of the type of difference spectrum produced.

In the present study, we have examined the effect of dietary fat on paracetamol-induced liver injury in an in vitro rat liver slice model. Rats were fed, for 7–10 days, diets containing either butter or polyunsaturated vegetable margarine, two fat sources commonly consumed in the human diet. Liver slices were then exposed to paracetamol for 2 hr and further incubated for 4 hr without paracetamol. Cell damage in the slices was quantified at 6 hr by measuring leakage of lactate dehydrogenase, increase in water content and potassium loss. Covalent binding of radioactive paracetamol to liver and the membrane fatty acid composition of the liver were also measured. Liver slices from rats fed butter diets were significantly more sensitive to the toxic effects of paracetamol than those from margarine fed rats. The membrane lipid composition of the livers also reflected the differing fatty acid content of the two diets.

The administration of small amounts of thiamin (0.3 μgday or more, i.p., for 21 days) depressed musomal cytochrome P-450 content and the Vmax of aniline hydroxylase when compared to values obtained from rats fed a thiamin-deficient diet (approximately 0.1 μg of thiamine/day in basal diet). The concurrent administration of neopyrithiamin (50 μgday, i.p.) eliminated the depressant effect of 10.0 μig of thiamin/ day, but was without significant effect in rats receiving more than 100 μg of thiamin/ day. In contrast, 100 μg of oxythiamin/day had no thiamin-opposing effect on cytochrome P-450 content and only partially counteracted the effects of 1 μg of thiamin/day on aniline hydroxylase activity. These treatments were without significant effect on the Km for this reaction. Using ethyl isocyanide as the ligand, there appears to be a qualitative change induced in the cytochrome P-450 from thiamin-deficient rats. The absorption peak height ratios indicate that cytochrome P1-450 is increased in a manner analogous to that produced by the administration of 3-methylcholanthrene. This was supported by the fact that aniline binding, as evidenced by increased ΔAmax, is enchanced in musomes from thiamin-deficient animals, whereas the hexobarbital spectral shift was unaltered.

One hundred and forty rats were treated with ovex (p-chlorophenyl p-chlorobenzenesulfonate) 100 mg/kg orally, paired with an equal number of controls, grouped (20 treated and 20 control) and designated as 12, 24, 48, 72, 96, 240, and 360 hr. Ten treated and ten control were given a lethal dose of parathion (100 mg/kg, oral) at the designated times after ovex and the length of survival recorded. Similar groups were killed at the designated times and the liver wt/body wt ratio, and α-naphthyl acetate hydrolyzing activity of 9000X g liver supernate determined. Significant increases in liver wt/body wt ratio and α-naphthyl acetate hydrolyzing activity were observed. When the above increases in liver size and enzyme activity were combined and expressed as a percentage, peak activity was seen at 72 hr (181%) and was present up to 240 hr (129%). Ovex (100 mg/kg oral) pretreatment appeared to provide more protection against parathion than paraoxon toxicity. It increased the rate of parathion metabolism by whole liver homogenates but had only a marginal effect on the rate of metabolism of paraoxon (p < 0.10). Ovex treatment reduced the liver organophosphate content of parathion but not paraoxon-exposed rats. An effect of ovex on plasma and brain organophosphate concentrations was not demonstrated.

The effects of ascorbic acid (vitamin C) deficiency on components of drug-hydroxylating systems in guinea pig liver were investigated. Although the liver weight-body weight ratio was increased, the concentration of microsomal protein was markedly less in ascorbic acid-deficient guinea pigs. This was reflected in a decrease in aniline and hexobarbital hydroxylation reactions when calculated on a unit of liver weight; however, when analyzed per unit of microsomal protein, ethylmorphine demethylase activity was unaffected. The Km's for these substrates, as well as the ethyl isocyanide difference spectra, were unchanged, indicating that no qualitative changes had occurred in the enzymes responsible for their metabolism or in the cytochrome P-450. Aniline metabolism per unit protein was depressed by ascorbic acid deficiency, as was the content of cytochromes P-450 and b5. The return of function by a single injection of ascorbic acid given 1-24hr. prior to decapitation was not frequently observed. Induction with sodium phenobarbital was not blocked by this dietary deficiency state.