Relationship between alveolar PO2 and the rate of p-nitroanisole O-demethylation by the cytochrome P-450 pathway in isolated rabbit lungs.

The relationship between alveolar PO2 and the rate of O-demethylation of p-nitroanisole, a model substrate for cytochrome P-450 -linked mixed-function oxidation, was evaluated in the isolated rabbit lung perfused with Krebs-Ringer bicarbonate buffer. The appearance of the product, p-nitrophenol, in the pulmonary perfusate was measured spectrophotometrically, The PO2 of the ventilating gas was varied with an accurate gas mixing pump and measured with an electrochemical O2 analyzer. In control lungs ventilated with 5% CO2 in air, the rate of p-nitrophenol production was approximately equal to 3.1 +/- 0.04 (mean +/- SE; n = 9) mumol/h per g dry wt. p-Nitrophenol production was unaltered when O2 in the ventilating gas was decreased to 1%, but it was depressed reversibly when alveolar O2 WAS 0.1% OR LESS AND WAS ABOLISHED DURING VENTILATION WITH 0.005% O2. The rate of the reaction was inhibited by 50% when alveolar PO2 was 0.3 mm Hg representing and intracellular [O2] OF approximately equal to muM. In the presence of metyrapone (0.1--1 mM), an inhibitor of cytochrome P-450-dependent reactions, p-nitrophenol production was 0.07--0.17 mumol/h per g dry wt. Ventilation of lungs with varying CO concentration in 20% O2 resulted in 50% inhibition of p-nitrophenol production when CO concentration was 10% (CO/O2 = 0.5). These results indicated that O-demethylation of p-nitroanisole by the lung is a cytochrome P-450-dependent reaction and that its rate is not affected until alveolar PO2 is less than 1 mm Hg.

Effect of 2-aminonaphthalene on glutathione content and cytochrome P-450 p-nitroanisole O-demethylation activity in mouse liver.

Four days after single i.p. administrations of 2-aminonaphthalene to C3H mice, the liver content of reduced glutathione and the liver microsomal cytochrome P-450 O-demethylation activity were decreased. No changes were observed in oxidized glutathione.

The effects of a marginally lipotrope-deficient diet on the hepatic levels of S-adenosylmethionine and on the urinary metabolites of 2-acetylaminofluorene in rats.

Hepatic levels of S-adenosylmethionine (AdoMet), of glutathione, and of the microsomal enzymes p-nitroanisole demethylase and benzo(a)pyrene hydroxylase were measured in male and female rats fed a diet marginally deficient in choline and methionine and void of folic acid (lipotrope deficient) or an adequate diet for 0 to 14 weeks with and without added 2-acetylaminofluorene (AAF). The urinary metabolites of AAF were determined throughout the experimental period. After 2 to 4 weeks of dietary administration, the hepatic AdoMet levels were 43% lower in male rats fed the lipotrope-deficient diet than in male rats fed the lipotrope-adequate diet; no differences were found in hepatic AdoMet of females fed the lipotrope-deficient or lipotrope-adequate diets for 2 to 14 weeks. Administration of AAF to lipotrope-deficient female rats for 2 weeks led to a transient decrease in hepatic levels of AdoMet. The administration of AAF for 2 to 14 weeks did not significantly affect hepatic AdoMet in female rats fed the lipotrope-adequate diet or in male rats fed either diet. Female rats fed the lipotrope-deficient diet and treated with AAF excreted decreased proportions of N-hydroxy-2-acetylaminofluorene and increased proportions of 5-hydroxy-2-acetylaminofluorene in their urine. However, the urine of lipotrope-deficient male rats treated with AAF contained increased proportions of N-hydroxy-2-acetylaminofluorene and decreased levels of 5-hydroxy-2-acetylaminofluorene. The urinary excretion of 7-hydroxy-2-acetylaminofluorene by male and female lipotrope-deficient rats treated with AAF was generally similar to that in lipotrope-adequate rats. The lipotrope-deficient diet did not appear to alter the hepatic levels of glutathione, p-nitroanisole demethylase, or benzo(a)pyrene hydroxylase activity was lower in the livers of lipotrope-deficient male rats treated with AAF for 8 to 14 weeks than in the livers of lipotrope-deficient rats not receiving the carcinogen. The altered metabolism of AAF correlated well with the previously reported effects of a marginal lipotrope deficiency on AAF carcinogenesis.

A comparative study on the influence of cysteamine and metyrapone on mixed-function oxygenase activities in variously pretreated liver microsomes from rats and mice.

It has been found that metyrapone can inhibit both type I and type II mixed-function oxygenase reactions, while cysteamine inhibits only type I activity in this mammalian system. Following pretreatment with phenobarbital and 3-methylcholanthrene the half-maximal inhibiting concentrations for the O-demethylation of paranitranisol are increased for cysteamine and decreased for metyrapone. Both cysteamine and metyrapone give type II binding spectra with oxidized cytochrome P-450. The negative and positive peaks are at 393 and 426 nm respectively for metyrapone, and 410 and 434 nm for cysteamine. Cysteamine showed no binding comparable to that of metyrapone for reduced cytochrome P-450. Metyrapone showed little or no inhibition of the NADH cytochrome-c reductase (EC 1.6.1.1) or NADPH (EC 1.6.2.3) cytochrome-c reductase while cysteamine had a more or less strong inhibiting effect depending on the pretreatment of animals. Neither the binding to P-450 heme nor the inhibition of NADH and NADPH cytochrome-c reductase correlates well with cysteamine inhibition of total activity. It is therefore suggested that cysteamine reacts with an intermediate electron carrier of non-heme iron or glycoprotein character thus inhibiting mixed-function oxygenase activity.

Dietary cholesterol caused modification in the structure and function of rat hepatic microsomes, studied by fluorescent probes.

A 4% cholesterol diet fed to rats for four weeks was found to increase the phospholipid and cholesterol contents and the activities of drug metabolizing enzymes in rat liver microsomes. Microsomes from rats on a high cholesterol diet were able to enhance the fluorescence of membrane bound 1-anilinonaphthalene 8-sulphonate (1,8-ANS) and ethidium bromide more than microsomes from rats on a standard diet. In the case of 1,8-ANS, the enhanced fluorescence was found to be due to the increased affinity of the molecules for microsomes. In the case of ethidium bromide the fluorescence increased partly because of the larger amount of binding sites and partly because of the enhanced quantum yield of the molecules. P-nitrophenol was found to compete with 1,8-ANS for the same binding sites in microsomes. On the other hand, 1,8-ANS lowered the rate of drug metabolism when present in the incubation mixture. In vitro treatments of microsomes with trypsin, phospholipase A or digitonin altered the binding properties of 1,8-ANS and ethidium bromide to microsomes. It is concluede that the binding sites of 1,8-ANS in microsomes are important for the activity of drug-metabolizing enzymes. The mechanisms of dietary cholesterol in enhancing the drug metabolism and the role of microsomal phospholipids in regulating the activity of drug-metabolizing enzymes are discussed.

Food restriction and stimulation of monooxygenation of p-nitroanisole in perfused rat liver.

This study assessed the effect of food restriction on the metabolism of model monooxygenase substrates in the perfused rat liver. Female Sprague-Dawley rats has access ad lib. to a Purina 5001 nonpurified diet (control) or were given 65% of the intake of controls for 3 weeks. Livers were perfused with oxygenated Krebs-Henseleit buffer using a non-recirculating system, and the rates of monooxygenation of p-nitroanisole and 7-ethoxycoumarin were measured. The results indicate that food restriction stimulated p-nitroanisole O-demethylation from 2.9 +/- 0.2 to 4.6 +/- 0.5 mumol/(g.hr) when saturating concentrations of p-nitroanisole were infused. Concomitantly, the ratio of beta-hydroxybutyrate to acetoacetate (B/A) and the rates of ketogenesis (B + A) were increased significantly by food restriction. Further, p-nitroanisole (200 mumol/L) increased hepatic malate concentration nearly 3-fold in liver extracts from food-restricted rats. However, infusion of either a low concentration of p-nitroanisole (50 mumol/L) or 7-ethoxycoumarin (200 mumol/L) did not alter these parameters. On the other hand, food restriction did not alter rates of monooxygenation in isolated microsomes supplemented with excess NADPH. Taken together, these data support the hypothesis that high concentrations of p-nitroanisole increased monooxygenation in food-restricted rats by stimulating fatty acid oxidation, which elevates the mitochondrial NADH/NAD+ ratio. This, in turn, increases the availability of reducing equivalents in the form of NADPH by a malate-pyruvate exchange system, leading to increased drug metabolism.

Regulation of NADPH-dependent mixed-function oxidation in perfused livers. Comparative studies with sorbitol and ethanol.

Sorbitol and ethanol were shown to have opposite effects on p-nitroanisole O-demethylation in perfused livers from fasted, phenobarbital-treated rats. Sorbitol (2 mM) stimulated drug metabolism by 50% while ethanol (20 mM) caused 80% inhibition. Both sorbitol and ethanol infusion decreased the NAD+/NADH ratio and increased fluorescence of pyridine nucleotides monitored from the liver surface; however, the NADP+/NADPH ratio was decreased by sorbitol but tended to be increased by ethanol. Stimulation of drug metabolism by sorbitol was abolished by pretreatment of fasted rats with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt, but was not affected by aminooxyacetate, a transaminase inhibitor. These results indicate that sorbitol stimulated p-nitroanisole metabolism by providing NADPH via the pentose phosphate shunt. Ethanol and sorbitol caused changes in intracellular concentrations of NADPH in livers from fasted rats which correlated directly with changes in hepatic levels of citrate and aspartate. Furthermore, aspartate infusion reduced the inhibition of p-nitroanisole O-demethylation by ethanol. This inhibition was also reversed partially by sorbitol in livers from 6-aminonicotinamide-treated rats. It is concluded that ethanol inhibits mixed-function oxidation primarily by decreasing the concentrations of citric acid cycle intermediates which leads to depletion of cytosolic NADPH.

p-Nitroanisole O-demethylation in perfused hamster liver. High rates of pentose cycle-independent mixed-function oxidation.

Rates of p-nitroanisole O-demethylation in perfused livers from Syrian golden hamsters were three to four times greater than comparable rates measured in preparations from Sprague-Dawley rats. Hamsters also had greater microsomal p-nitroanisole O-demethylase activity and cytochrome P-450 contents than rats. In general, phenobarbital caused similar increases in these properties in both species. Fasting of hamsters for 24 hr increased p-nitroanisole O-demethylase activity in microsomes but did not affect rates in perfused livers. Rates were also unaffected in the perfused liver by pretreatment with 6-aminonicotinamide, an inhibitor of the pentose phosphate shunt. Hamster livers had low activities of pentose cycle enzymes but high activities of malic enzyme and isocitrate dehydrogenase compared to rats. In hamster livers, maximal rates of p-nitroanisole O-demethylation were not maintained but declined steadily over 40 min with prolonged p-nitroanisole infusion. The decreased rates of mixed-function oxidation in the non-recirculating perfusion system could not be explained by diminished tissue viability or degradation of cytochrome P-450 but were likely due to a decline in the formation of reduced cofactor. Hepatic concentrations of alpha-ketoglutarate and malate increased during p-nitroanisole infusion. Furthermore, rates of p-nitroanisole O-demethylation were inhibited by ethanol and aminooxyacetate, agents which inhibit the generation and/or movement of mitochondrial reducing equivalents into the cytosol. The infusion of pyruvate stimulated p-nitroanisole O-demethylation in perfused livers from fasted hamsters. This effect was maximal with 0.1 mM pyruvate, did not require gluconeogenesis, and was insensitive to 6-aminonicotinamide treatment. Thus, stimulation of p-nitroanisole metabolism by pyruvate in hamster livers is likely related to the mitochondrial oxidation of pyruvate, rather than to increased NADPH generation via the pentose phosphate cycle. These data indicate that mitochondrial sources of NADPH supply reducing equivalents for mixed-function oxidation in hamster liver.

Effect of repeated administration of 11-hydroxy-delta 8-tetrahydrocannabinol, an active metabolite of delta 8-tetrahydrocannabinol, on the hepatic microsomal drug-metabolizing enzyme system of mice.

The effects of delta 8-tetrahydrocannabinol (delta 8-THC) and its major and active metabolite, 11-hydroxy-delta 8-tetrahydrocannabinol (11-OH-delta 8-THC), on the hepatic microsomal drug-metabolizing enzyme system were studied in mice. The repeated administration of 11-OH-delta 8-THC (5 mg/kg/day, i.v.) for 3 or 7 days increased significantly the activities of aniline hydroxylase and p-nitroanisole O-demethylase. By the same treatment, cytochrome P-450 content (3 days) or NADPH-cytochrome c reductase activity (7 days) was also increased significantly. The treatment with delta 8-THC for 7 days (5 mg/kg/day, i.v.) significantly increased aniline hydroxylase only. 11-OH-delta 8-THC increased the Vmax, but not the Km, values for both drug-metabolizing enzymes, whereas delta 8-THC decreases significantly the Km value (270 microM) for p-nitroanisole O-demethylase as compared with the control (398 microM). Repeated administration of these cannabinoids for 7 days also increased the metabolism of delta 8-THC by hepatic microsomes; this was attributed to an enhanced formation of 11-OH-delta 8-THC. In contrast, microsomal formation of 7 alpha-OH-delta 8-THC was decreased significantly by treatment with delta 8-THC. 11-OH-delta 8-THC, but not delta 8-THC, treatment increased the metabolism of 11-OH-delta 8-THC by hepatic microsomes. These findings indicate that delta 8-THC and 11-OH-delta 8-THC treatment can induce hepatic microsomal drug-metabolizing enzymes and affect differently the catalytic properties of the enzymes.

Interaction and electron transfer between cytochrome b5 and cytochrome P-450 in the reconstituted p-nitroanisole O-demethylase system.

The interaction and electron transfer between cytochrome b5 and cytochrome P-450B1 were investigated using the reconstituted p-nitroanisole O-demethylase system. Apocytochrome b5 was prepared from detergent-solubilized cytochrome b5 by the acid-butanone method. The apocytochrome b5 thus obtained has been substituted with several metalloporphyrin derivatives. The reconstituted system containing cytochrome b5 substituted with heme derivatives such as proto-, meso-, and deuteroheme exhibited demethylation activity at the maximum turnover rates of 94, 58, 30%, respectively, compared to that containing the native cytochrome b5, while neither apocytochrome b5 nor cobaltic protoporphyrin-cytochrome b5 displayed the activity. Kinetic analysis showed the formation of a 1:1 complex between cytochrome P-450B1 and each of these substituted cytochrome b5's, except for cobaltic protoporphyrin-cytochrome b5; the affinities differed with the cytochrome b5 species used. The synergistic effect with the addition of the NADH-linked electron transport system was more remarkable at the lower reduction levels of cytochrome b5 in the steady state. Interaction between the components involved in NADH- and NADPH-linked electron transport systems was modulated by the existence of Triton X-100. The optimal concentration in the reconstituted system for the demethylation was observed at around 0.03% of Triton X-100, where the reduction rates for cytochrome b5 and cytochrome P-450B1 by the respective reductases were maximal. These results indicate that the two electron transport systems are closely coupled and exhibit the demethylase activity.

Effect of trichlorophenols on xenobiotic metabolism in the rat.

Unlike halogenated benzenes, trichlorophenols did not induce xenobiotic metabolism in the rat. 2,3,5-, 2,3,6-, 2,4,5-, and 2,4,6-Trichlorophenol at doses as high as 400 mg/kg p.o. daily for 14 days did not alter EPN detoxification. Only 2,4,5-trichlorophenol at the highest dose decreased microsomal NADPH-cytochrome c reductase activity and cytochrome P-450 content. In vitro, all 4 isomers inhibited EPN detoxification and the demethylation of p-nitroanisole. UDP-glucuronyltransferase was not altered in vivo and was only slightly inhibited in vitro by 2,3,5- and 2,4,5-trichlorophenol. The compounds were not hepatotoxic as assessed by measurement of hepatic glucose-6-phosphatase and serum sorbitol dehydrogenase.

Liver injury by the false morel poison gyromitrin.

After oral application of the mushroom poison gyromitrin a time and dose dependent decrease of cytochrome P-450 was found in rat liver microsomes. The maximal decrease to about 50-60% of the control (after 200 mg/kg, 80% of LD50) was observed 8-12h after application, a normalization after 48 h. The inhibition of cytochrome P-450 mediated metabolism of aminopyrine and p-nitroanisole corresponds to the decrease of cytochrome P-450. The specific activity of cytochrome P-450 remains unchanged while that of cytochrome P-448 is decreased as shown by means of the metabolism of ethoxycoumarin or ethoxyresorufin. Comparable results were obtained after application of N-methyl-N-formylhydrazine (MFH) which is formed from gyromitrin rapidly by hydrolysis. An attack on the endoplasmatic membrane with a stimulation of lipid peroxidation is discussed.

Potentiation of carbon tetrachloride hepatotoxicity in rats by pretreatment with polychlorinated biphenyls.

Pretreatment of male rats with Aroclor 1254 at a dose of 25 mg/kg i.p. for 6 days resulted in potentiation of the hepatotoxicity of inhaled carbon tetrachloride (CCl4) as evidenced by a decrease in liver glucose-6-phosphatase and elevations of serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), isocitrate dehydrogenase, and sorbitol dehydrogenase. Aroclor 1254 alone did not demonstrate hepatotoxicity. Aroclor 1254 administration resulted in large increases in cytochrome c reductase, cytochrome P-450 (448) AND P-Nitroanisole demethylation. Subsequent exposure to CCl4 vapor resulted in over 70% decreases in the latter two parameters. The potentiation was dose-dependent with a dose of 5 mg/kg or higher being effective. Aroclor 1260 administration gave results similar to those of Aroclor 1254, but Aroclor 1221 enhanced CCl4 toxicity to a lesser extent.

On the inhibition of microsomal drug metabolism by polychlorinated biphenyls (PCB) and related phenolic compounds.

The in vitro metabolism of p-nitroanisole, aminopyrine, and aniline by rat liver microsomal monoxygenases were studied in the presence of different polychlorinated biphenyl (PCB) mixtures and some related hydroxybiphenyls. The tested PCB mixtures contained preferably dichloro- (di-CB), tetrachloro- (tetra-CB), or hexachlorobiphenyls (hexa-CB). All PCB were competitive inhibitors of only aminopyrine demethylation by normal microsomes (Ki 22-39 micron). In microsomes of PCB-pretreated rats the aminopyrine demethylation was inhibited noncompetitively by di-CB and hexa-CB whereas tetra-CB remained a competitive inhibitor (Ki 12 micron). Moreover, after PCB pretreatment all PCB were competitive inhibitors of p-nitroanisole demethylation. 2-OH-biphenyl and 4-OH-biphenyl caused competitive inhibition of aminopyrine demethylation and aniline hydroxylation but failed to inhibit p-nitroanisole metabolism by normal microsomes. Chlorinated 4-hydroxybiphenyls inhibited competitively the metabolism of both type I and type II substrates. However, after PCB pretreatment all phenolic compounds caused uncompetitive inhibition of aniline hydroxylation.

Modifications of drug metabolism by disulfiram and diethyldithiocarbamate. I. Mixed-function oxygenase.

Disulfiram and diethyldithiocarbamate were administered to rats for 4 days alone (300 mg/kg, daily, per os) or in combination with phenobarbital (80 mg/kg, daily, i.p.), in order to observe the effects of these compounds on the microsomal membrane components and on the mixed-function oxygenase system. Both disulfiram and diethyldithiocarbamate increased the liver to body weight ratio, and the total hepatic protein content. Disulfiram significantly increased also the microsomal protein and phospholipid contents. Diethyldithiocarbamate and disulfiram partially prevented the increase of microsomal protein and phospholipid contents caused by phenobarbital. Disulfiram and diethyldithiocarbamate decreased the amount of cytochrome P-450 and P-420, and the activity of p-nitroanisole O-demethylase. These changes were more pronounced after diethyldithiocarbamate than after disulfiram treatment. On the contrary, the activity of NADPH-cytochrome c reductase was enhanced only by disulfiram. The induction by phenobarbital of cytochrome P-450 and p-nitrosanisole O-demethylase was partially prevented on concomitant treatment with disulfiram and diethyldithiocarbamate. These compounds. however, had an additive effect with phenobarbital in enhancing the microsomal NADPH-cytochrome c reductase activity.

Comparative study of hepatic microsomal and cytosolic enzyme activities in three murine strains.

A comparative study on hepatic microsomal p-nitroanisole-O-demethylase, aminopyrine-N-demethylase, NADPH-cytochrome c reductase and cytosolic glutathione (GSH) S-transferase was performed in both sexes of C3Hf, C57BL/6J and DBA/2 mice. In control and phenobarbital (PB)-induced mice similarities in hepatic microsomal activities were found with slight variations among strains and sexes. The Ah locus nonresponsive DBA/2 mice showed uninduced microsomal activities after beta-naphthoflavone (BNF), as expected; the other two strains were induced by BNF to a similar extent. Differences in cytosolic GSH S-transferase were detected among strains and sexes; C57BL/6J mice showed the highest basal and induced levels. The males of all the three strains examined were found to have higher GSH S-transferase activity than females.

Stability of microsomal mono-oxygenase during incubations for the liver microsomal assay with S9 fractions of mouse liver under various inductions.

Aminopyrine-N-demethylase and p-nitroanisole-O-demethylase activities were determined in incubation mixtures for the liver microsomal assay at time zero and after 1 h of incubation in the conditions for the mutagenic assay. The experiments were performed with the S9 liver fraction of mice in the basal state and induced with sodium phenobarbital, beta-naphthoflavone or both. Lipid peroxidation was also determined. The experiments were repeated with female mice and also in the presence of styrene, as an example of a xenobiotic substance. The activity of pNAD was much more stable than that of APD in all the conditions tested. The pattern of stability, however, was similar for the two activities: more stable than controls with S9 fractions from beta-NF-induced mice, less stable than controls in PB-induced mice, intermediate between controls and PB-induced mice in those with combined induction by PB + beta NF. Styrene 50 mM in the incubation mixtures led to a marked inactivation of enzymic activity, similar in all cases and reaching about 90% in 1 h. S9 fractions from female mice gave enzymes slightly more stable in almost all cases. Lipid peroxidation was appreciably more elevated in basal than in induced animals. It was concluded that, for a mutagenesis test on an unknown xenobiotic, S9 fractions from mice following PB and beta-NF induction are to be preferred from the point of view of activation.

Dietary fats and properties of endoplasmic reticulum: II. Dietary lipid induced changes in activities of drug metabolizing enzymes in liver and duodenum of rat.

Rats were fed cholesterol, cacao butter, or olive oil diets to determine the effect of dietary lipids on the rate of drug biotransformation in the liver and duodenum. The cholesterol rich diet maintained the hepatic aryl hydrocarbon hydroxylase activity at the same level as did the standard diet. Rats fed olive oil and cacao butter diets showed lower hepatic aryl hydrocarbon hydrorylase activity. The p-nitroanisole O-demethylase activity was doubled in hepatic microsomes of rats fed the high cholesterol diet when compared to rats fed the standard diet. The hepatic uridine diphosphate glucuronosyltransferase activity showed different patterns depending on the in vitro treatment of the microsomal membranes. If the enzyme activity was assayed from the native, untreated microsomes, an increase in the measurable uridine diphosphate glucuronosyl transferase activity was found in rats having cholesterol rich diet. After the in vitro activation of membrane-bound uridine diphosphate glucuronosyltransferase by trypsin, the increase in measurable activity was 10 fold in the group fed the standard diet, 6 fold in group fed cholesterol, 4 fold in group fed cacao butter, and 3 fold in group fed olive oil. Trypsin digestion of microsomes increased the measurable uridine diphosphate glucuronosyltransferase activity less in rats fed diets rich in neutral fats than those fed the standard diet. In the duodenal mucosa, lipid diets decreased the activities of drug hydroxylation and glucuronidation.

Mixed function oxidation and intermediary metabolism: metabolic interdependencies in the liver.

Both hepatic lipogenesis from glucose and gluconeogenesis from lactate are inhibited by substrates for mixed function oxidation such as aminopyrine. These effects are inducible with phenobarbital pretreatment and absent with a demethylated product of aminopyrine metabolism, aminophenylpyrazolone. The data indicate that an interaction between biosynthesis and drug metabolism occurs in the intact liver cell. In the case of lipogenesis, direct competition for cytosolic NADPH occurs, whereas in the case of gluconeogenesisi, enhanced drug metabolism diverts key intermediates, e.g., malate from glucose synthesis toward NADPH generation. Furthermore, rates of mixed function oxidation were modified following perturbations of intermediary metabolism, e.g., fasting, addition of substrates for glucose synthesis, inhibition of energy metabolism, etc. The data support the hypothesis that in the presence of sufficiently high concentrations of substrate and oxygen, the rate-limiting step for mixed function oxidation in the intact cell is supply of NADPH.

Effect of dietary vitamin E (alpha-tocopherol) on aflatoxin B metabolism.

Intraperitoneal injection of rats with 2 mg/kg ring labelled 14C AFB1 (spec. act. 110 mCi/mM/nmole) showed a higher level of radioactivity in the urine of test animals on diets containing 600 mg/kg vit. E 24 h after pretreatment. Analysis of the urine by chloroform extraction, thin layer chromatography and liquid scintillation counting of the various fractions showed less aflatoxin M1 (AFM1) and less unmetabolized AFB1 in test samples than in controls. Incubation of ring labelled 14C AFB1 with hepatic 10,000 g supernatant fractions, however, showed increased metabolism of AFB1 by fractions from test animals as compared with the controls. Rate of disappearance of 14C AFB1 and the consequent formation of AFM1 was greater in the test fractions than in the controls. At 30 days all test animals showed higher levels of serum vitamin E than the controls. Hepatic aniline hydroxylase and ethyl morphine N-demethylase activities of the liver fractions and blood glutathione reductase activity were greater in the tests. P-nitroanisole-O-demethylase activity was reduced while hepatic and serum reduced glutathione levels remained basically unaltered.