Aniline Hydroxylation Research Articles

Differential Induction of Peroxygenase-Dependent Microsomal Aniline Hydroxylase by Chronic Ethanol Ingestion

The liver microsomal-mediated hydroxylation of aniline, which is selectively induced by chronic (EtOH) ingestion, has been studied as a function of NADPH plus dioxygen (O2)- or hydroperoxide-dependent reactions. Consistent with the well-documented induction of aniline hydroxylase following chronic ethanol -ingestion, the results showed selectivity towards aniline hydroxylase by the NADPH plus O2- and tert-butyl hydroperoxide (t-BuOOH)-dependent reactions with microsomes from EtOH-fed rats. On the other hand, the cumene hydroperoxide (CumOOH)-dependent aniline hydroxylase activity was not discriminated between microsomes from EtOH- and pair-fed rats. In parallel experiments with positive controls, CumOOH did show selectivity for phenobarbital (PB)-induced microsomal aniline hydroxylase compared to chow-fed rats. The Kcat/KM values, which indicate the efficiency of enzyme catalysis, for NADPH plus O2-, t-BuOOH, and CumOOH-dependent aniline hydroxylase from EtOH-fed rats were 102, 37, and 5 and from pair-fed rats were 68, 4, and 4 (nmol p-aminophenol/min/nmol cytochrome P-450)/mM aniline, respectively. The relative Kcat/KM ratio for EtOH-fed to that of pair-fed microsomal aniline hydroxylase from NADPH plus O2-, t-BuOOH-, and CumOOH-dependent reactions were 1.5, 7.4, and 1.2, respectively. The present preliminary studies indicate that the catalytic efficiency of EtOH-induced aniline hydroxylase is significantly greater for the t-BuOOH-dependent reaction.

Inhibition of hepatic microsomal cytochrome P450 by cannabidiol in adult male rats.

The mechanism of inhibitory effect of cannabidiol (CBD) on the hepatic drug-metabolizing enzyme system was studied in adult male rats in vivo. Time course studies revealed that microsomal d-benzphetamine N-demethylation and testosterone 2 alpha-, 16 alpha- and 17-oxidation were markedly suppressed 6 to 48 h after the single administration of CBD (10 mg/kg, intraperitoneally). Decreases in activities of aniline hydroxylation and p-nitroanisole O-demethylation and in content of total cytochrome P450 were intermittent and moderate. On the other hand, no change was observed in reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase activity or cytochrome b5 content in the hepatic microsomes of the CBD-treated rats. Western blotting analysis showed a marked decrease in the male-specific cytochrome P450 UT-2 in the hepatic microsomes, especially 24 to 48 h after pretreatment with CBD. It is possible that CBD given 6 to 12 h before the sacrifice might interact with cytochrome P450 as a substrate, resulting in inhibition of the drug-metabolizing enzyme activities in the earlier stages. In the later stages from 24 to 48 h after CBD treatment, the reduction in content of the male-specific cytochrome P450 UT-2 may play a major role in the inhibitory effect of CBD on the hepatic drug-metabolizing enzyme system in the adult male rat in vivo.

Effect of aging on in vivo and in vitro ethanol metabolism and its toxicity in F344 rats

To investigate the effect of aging on ethanol metabolism, 24 male and female F344 rats aged 2 and 12 mo that were fed a laboratory diet received ethanol (1.2 and 2.5 g/kg body wt) intraperitoneally. In male rats, in vivo ethanol elimination significantly decreased according to age both at high (436 ± 38 vs. 294 ± 27 mg/kg h; p < 0.01) and low (365 ± 19 vs. 261 ± 8 mg/kg · h; p < 0.01) blood ethanol concentrations. Age did not influence the specific activity of hepatic or gastric alcohol dehydrogenase, whereas the activity was significantly decreased with age in the liver (p < 0.05) and in the stomach (p < 0.001) when related to body weight. In addition, the activity of the hepatic microsomal ethanol oxidizing system decreased significantly according to age (8.7 ± 0.5 vs. 6.00 ± 0.3 nmol/min · mg micr. protein; p < 0.001). To study the response of ethanol-metabolizing enzymes to chronic ethanol ingestion, 2- and 19-mo-old male F344 rats were pair-fed nutritionally adequate liquid diets containing 36% of total calories either as ethanol or isocaloric carbohydrate for 3 wk. In this experiment specific alcohol dehydrogenase activity was not significantly affected by age, whereas the hepatic microsomal function estimated by the determination of cytochrome P450, microsomal ethanol oxidizing system, and aniline hydroxylation as well as hepatic mitochondrial low Km-acetaldehyde dehydrogenase activity was found to be markedly depressed with age (p < 0.01). Chronic ethanol consumption increased microsomal enzyme activities in older rats to levels comparable to those observed in young animals prior to ethanol administration. Chronic ethanol feeding also resulted in an increased hepatic fat accumulation, which was significantly enhanced in older rats. In contrast to male rats, in vivo ethanol metabolism was practically identical for 2- and 12-mo-old female rats. These data demonstrate an enhanced toxicity of alcohol in older compared to younger male but not female rats associated with a delay in alcohol elimination both at high and low ethanol blood concentrations and a decrease in ethanol- and acetaldehyde-mptabolizing enzyme activities.

Differential effects of cyclosporin on hepatic and renal heme, cytochrome P-450 and drug metabolism: Possible role in nephrotoxicity of the drug

Treatment of rats with 25 or 50 mg/kg cyclosporin A for 6 days elicited vastly different responses in hepatic and renal heme and drug metabolism activities. In the liver, cytochrome P-450 concentration was decreased significantly (to 70–75 % of the control). This was accompanied by a marked reduction in benzo[ a]pyrene hydroxylase activity (to 20–28% of the control). Aniline hydroxylation was also decreased, but to a lesser extent (to 77% of the control). In contrast, in the kidney cytochrome P-450 concentration was significantly increased to (145–170% of the control), along with a modest decreasc in benzo[ a]pyrene hydroxylation activity. In this organ, the concentration of porphyrins was severely decreased (to 30% of the control). Also, the activities of δ-aminolevulinate (ALA) synthetase and ALA dehydratase, as well as that of heme oxygenase, were inhibited. It is suggested that in the kidney the inhibition of degradation, rather than an enhanced rate of synthesis of the heme molecule, contributes to the observed increase in cytochrome P-450 concentration. In the liver, the decrease in the cytochrome concentration could not be explained in terms of an alteration in the rate of heme biosynthesis or degradation. Therefore, the observed decrease in cytochrome P-450 concentration could reflect the direct inactivation of the hemoprotein or regulation of apoprotein production by cyclosporin and/or its metabolites(s). The possible relevance of the observations to cyclosporin nephrotoxicity is discussed.

Comparison of Effects of Long‐Term Ethanol Consumption on the Heart and Liver of the Rat

Alterations in heart and liver metabolism were determined periodically in Sprague-Dawley rats pair-fed a liquid diet (ethanol, 36% of calories) for times as long as 1 year. In liver mitochondria the rate of ATP synthesis was lowered significantly after ethanol administration for 1 month and longer feeding periods. In liver microsomes from ethanol-fed animals, ethanol oxidation and aniline hydroxylation increased 1.5- and 3.5-fold, respectively, after 1 month and remained elevated at the longer feeding intervals. Electron microscopic analyses of heart left ventricles revealed no alterations from ethanol consumption for 1 month. Alterations including disrupted mitochondrial cristae, dilatation of sarcoplasmic reticulum, and widening of the intercalated discs were observed after 6.5-month feeding periods. Myocardial concentrations of creatine, creatine phosphate, ATP, ADP, and Pi remained constant even after ethanol consumption for 9 months. After a 12-month feeding period slight changes in cardiac mitochondrial energy-linked properties were observed which were not as pronounced as those occurring in liver mitochondria. The activity and oligomycin sensitivity of the ATPase were not altered in cardiac mitochondria, whereas in liver preparations significant alterations in these properties of the ATPase were apparent after ethanol consumption for 1 month and the longer feeding periods. These observations suggest that the liver responds more quickly and dramatically to chronic ethanol consumption than does the heart.

Ozagrel hydrochloride monohydrate, a thromboxane synthase inhibitor, and its metabolites as inhibitors of hepatic microsomal drug metabolism.

The change in the hepatic oxidative drug-metabolizing capacity in humans treated with ozagrel hydrochloride monohydrate (OZA), an imidazole derivative and a new thromboxane A2 synthase inhibitor, was studied and the inhibitory potencies of the metabolites of OZA (M-1 and M-2) on the mouse hepatic microsomal monooxygenase system in vitro were compared with that of OZA. In vitro, M-1 and M-2, which are the beta-oxidized form and the reduced form of OZA, respectively, inhibited aminopyrine N-demethylation, aniline hydroxylation and testosterone hydroxylations in mouse hepatic microsomes and produced type II difference spectra in the same manner as OZA. The kinetic data indicated that the inhibitory potencies and the affinities of these compounds for cytochrome P-450 were decreased in the order of M-2 greater than OZA greater than M-1. The ratio of 6 beta-hydroxycortisol (6 beta-OHF) to cortisol (F) in urine, used as an indicator of oxidative drug-metabolizing capacity in humans, did not change significantly during oral treatment with 400 mg/d of OZA, while the ratio decreased to 80-85% of the original level during treatment with 800 mg/d of OZA. Although the participation of the metabolites of OZA in the reduction of drug-metabolizing capacity in vivo is not yet clear, the results suggest that hepatic oxidative drug-metabolizing enzyme activities in humans are inhibited by treatment with a relatively high dose of OZA.

N-demethylation reactions in intact erythrocytes and erythrocyte supernatant.

While it is generally accepted that the liver is the primary organ of xenobiotic metabolism, it is also known that other tissues can contribute both to the activating and inactivating reactions. Recently mutagenicity tests using erythrocytes indicated that the latter can mediate in metabolic activation of premutagens (Norppa et al 1984). However, biochemical evidence is still scarce although hemoglobin can catalyze the hydroxylation of aniline (Mieyal et al 1976) and red blood cells seem to have intracellular enzymic systems similar to those of the microsome with regard to the NADH- and NADPH-dependant reductases and cytochrome b5 (Kuma et al 1976). This paper describes the N-demethylation of methylaniline in rat erythrocytes in different media and the effects of potential inhibitors in the rate of metabolite formation.KeywordsDiethylene Triamine Pentaacetic AcidPotassium CyanideGlucose SystemNADPH Generate SystemIntact ErythrocyteThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effects of mutagenic and non-mutagenic aniline derivatives on rat liver drug-metabolizing enzymes.

1. The effect of 4,4'-methylene bis(2-chloroaniline) (MOCA), 4,4'-methylene dianiline (MDA) and 4,4'-sulphonyldianiline (Dapsone) in vivo on xenobiotic biotransformation in male rat liver was studied. 2. Treatment with MOCA or MDA but not Dapsone caused a dose-dependent increase in ethoxyresorufin O-deethylase activity and a concomitant decrease in aldrin epoxidase activity in male rats. 3. Treatment with MOCA or MDA resulted in dose-dependent increases in ethoxycoumarin O-deethylation and epoxide hydrolation, while only MOCA induced cytosolic glutathione S-transferase activity. 4. Treatment with Dapsone resulted in no changes in xenobiotic biotransformation except for the induction of aniline hydroxylation. 5. The results are consistent with the contention that there is a relationship between carcinogenic chemicals and particular alterations in the activities of biotransformation enzymes.

A novel form of cytochrome P-450 in Beagle dogs: P-450-D3 is a low spin form of cytochrome P-450 but with catalytic and structural properties similar to P-450d

A form of cytochrome P-450, P-450-D3, cross reactive with antibodies to rat P-450d was purified from liver microsomes of polychlorinated biphenyl (PCB)-treated female Beagle dogs to an electrophoretic homogeneity. Judging from the result of sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of P-450-D3 was estimated to be 54,000. The oxidized form of P-450-D3 showed a peak at 416 nm indicating that the cytochrome is mostly in a low spin state. The carbon monoxide bound reduced form of P-450-D3 showed a peak at 448 nm. In a reconstituted system, P-450-D3 catalyzed drug oxidations including benzphetamine and aminopyrine N-demethylations, 7-ethoxycoumarin and p-propoxyaniline O-dealkylations, and aniline and benzo( a)pyrene hydroxylations. The rate of aniline hydroxylation catalyzed by P-450-D3 was similar to that catalyzed by P-450c which is a low spin form of cytochrome P-450 purified from liver microsomes of PCB-treated rats, whereas the catalytic activities of P-450-D3 for 7-ethoxycoumarin O-deethylation and benzo( a)pyrene hydroxylation were considerably lower than those of P-450c. The amino terminal portion of P-450-D3 was found to be highly similar to those of P-450d, human P 3-450 and P 3-450 when four amino acid deletions were tentatively inserted between fifth and sixth amino acids from the N-terminal, but not that of P-450c which is a low spin form of cytochrome P-448 purified from rat liver microsomes. These results indicate that Beagle dogs possess a low spin form of cytochrome P-450 with spectral properties similar to P-450c but with catalytic and structural properties similar to P-450d.

Hydroxylation of aniline mediated by heme-bound oxy-radicals in a heme peptide model system

Hydroxylation of aniline mediated by heme-bound oxy-radicals in a heme peptide model system

The effect of di-isopropyl 1,3 dithiol-2-ylidenemalonate (Malotilate) on the hepatic changes induced by ethanol administration in the rat

The sulphur-containing drug, di-isopropyl-1,3-dithiol-2-ylidenemalonate (Malotilate) protects against the increase in hepatic triglyceride concentration after acute ethanol administration (either 6 g/kg p.o. or 2 g/kg i.p.)inrats. The compound had no influence on the increased hepatic NADH: NAD ratio (measured as the lactate : pyruvate and 3-hydroxybutyrate : acetoacetate ratios) after acute ethanol dosing (2 g/kg i.p.), but was found to lower hepatic acetaldehyde concentrations and prevent some of the disturbances in lipid metabolism observed in liver slices from ethanol-treated animals (e.g. decreased oxidation of [1- 14C]palmitate to 14CO 2) after this ethanol dose. The drug did not inhibit ethanol metabolism in this acute experiment. Administration of Malotilate to Wistar rats (100 mg/kg/day orally) during chronic feeding of ethanol as 36% of the total calorie intake in a liquid diet, resulted in a lower intake of the alcohol-containing diet by ethanol-fed animals and reduced body weight gain in rats which received the drug, without blood ethanol levels or the ethanol intake (expressed in g/kg body weight/day) being affected. In ethanol-fed animals, Malotilate prevented the production of fatty liver and the adaptive increase in the ethanol elimination rate (EER) normally seen in ethanol-fed animals, although the drug actually caused a slight increase in EER in glucose pair-fed controls. Malotilate did not significantly decrease the degree of induction of microsomal cytochrome P-450 by ethanol, but the increase in aniline hydroxylation was much less marked in animals receiving ethanol and Malotilate, suggesting that the activity of the inducible microsomal ethanol oxidising system (MEOS) may be reduced by the compound. Determination of hepatic acetaldehyde concentrations during ethanol feeding, and during an acute ethanol challenge test following long-term ethanol treatment showed that the compound significantly lowered the level of this ethanol metabolite in the liver under both circumstances. This reduction of hepatic acetaldehyde concentrations, probably resulting in part from the reduced EER as well as increased low- K m aldehyde dehydrogenase activities and glutathione contents seen in the livers of Malotilate-treated rats, are possible mechanisms by which the drug protects against triglyceride accumulation after ethanol administration.

Identification of P-450 ALC in microsomes from alcohol dehydrogenase-deficient deermice: Contribution to ethanol elimination in vivo

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450 ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450 ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH −) and -positive (ADH +) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH − than from naive ADH + animals. Ethanol treatment induced the protein about 3-fold in the ADH + strain and about 4-fold in the ADH − strain. The antibody to rabbit P-450 ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450 ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450 ALC homolog, increased about 3-fold upon ethanol treatment in the ADH + strain and about 4-fold in the ADH − strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH + strain and 1.9-fold and 3.3-fold, respectively, in the ADH − strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450 ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450 ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH + and ADH − strains, respectively. After chronic ethanol treatment, P-450 ALC could account maximally for 8% of the total ethanol elimination in the ADH + strain and 22% in the ADH − strain. Further, cytochrome P-450 ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450 ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant. Peroxisomal β-oxidation capacity was increased 40% over control values by ethanol treatment, consistent with the hypothesis that the increase in ethanol elimination in the ADH-negative deermouse is mediated predominantly via catalase-H 2O 2.

Inhibition of hepatic microsomal drug metabolism by the steroid hydroxylase inhibitor SU-10'603

SU-10'603 is a pyridine derivative that has been widely used as a steroid 17-hydroxylase inhibitor. Studies were done to compare the effects of SU-10'603 with those of the structurally related compound, metyrapone, on hepatic microsomal drug metabolism in vitro in rats and guinea pigs. In rat liver microsomes, SU-10'603 produced a concentration-dependent (0.01 to 1.0 mM) inhibition of ethylmorphine demethylation, aniline hydroxylation, and benzo[a]pyrene hydroxylation. A concentration of 0.1 to 0.2mM decreased the metabolism of all three substrates by approximately 50%. SU-10'603 was a more potent inhibitor of ethylmorphine metabolism than metyrapone, and its relative potency was even greater with respect to aniline and benzo[a]pyrene metabolism. Similar results were obtained with guinea pig liver microsomes. SU-10'603 and metyrapone produced type II spectral changes in hepatic microsomes, but the apparent affinity of SU-10'603 for cytochrome(s) P-450 was greater than that of metyrapone. Both compounds inhibited the binding of type I substrates to microsomal cyto-chromes P-450; SU-10'603 was the more potent inhibitor. The results indicate that SU-10'603 is a potent inhibitor of hepatic microsomal monooxygenases whose mechanism of action is similar to that of metyrapone.

Biochemical and immunochemical evidence for the induction of an ethanol-inducible cytochrome p-450 isozyme in male syrian golden hamsters

The effects of ethanol and of phenobarbital pretreatment on hamster microsomal metabolism of aniline and p-nitrophenol have been investigated. Hydroxylation of both compounds was increased over 2-fold by ethanol pretreatment, whereas phenobarbital pretreatment had little effect on either activity. Ethanol pretreatment had no effect on the specific content of total cytochrome P-450, while phenobarbital pretreatment increased the specific content 1.6-fold. Comparison of the specific activities for aniline hydroxylation and p-nitrophenol hydroxylation of individual microsomal samples from control, ethanol-pretreated and phenobarbital-pretreated animals showed a high degree of correlation r 2 = 0.98) consistent with the involvement of the same site for catalysis of these two compounds. Antibody to rabbit ethanol-inducible cytochrome P-450 (isozyme 3a) inhibited over 80% of the aniline (high affinity) and p-nitrophenol hydroxylase activities of microsomes from ethanol-treated hamsters. A comparison of the antibody-inhibitable rates for both hydroxylase activities with microsomes from untreated, ethanol- or phenobarbital-pretreated hamsters suggested that an isozyme homologous to rabbit isozyme 3a (hamster cytochrome P-450alc) was induced in hamsters about 3.5-fold by ethanol and was unaffected by phenobarbital. The induction of hamster cytochrome P-450alc was confirmed by immunoblot analysis of hamster microsomes. A single protein with a molecular weight of approximately 54,000 was recognized by antibody to the rabbit isozyme. Quantification of the immunoblots demonstrated that the hamster isozyme was increased about 3-fold, in good agreement with the induction determined by a comparison of the antibody-inhibitable rates. The results indicated that, although there was no change in the total spectrally observable cytochrome P-450, there was a marked change in the distribution of the isozymes of cytochrome P-450, with an increase in the alcohol-inducible form after 28-day ethanol consumption by chow-fed hamsters. This isozyme can be readily monitored by either high-affinity aniline or p-nitrophenol hydroxylation or by Western immunoblot analysis and appears to be the ethanol-inducible form of cytochrome P-450 in hamsters.

The possible role of the ethanol-inducible isozyme of cytochrome P450 in the metabolism and distribution of carbon disulfide

Acute treatment with ethanol and other alcohols has been shown to potentiate the hepatotoxicity of certain xenobiotics, in part via induction of the mixed-function oxidase (MFO) system. Carbon disulfide (CS 2)-induced hepatotoxicity and inhibition of the MFO system have been shown to be a consequence of MFO metabolism. In the present study, the ability of several different alcohols to induce the hepatic MFO metabolism of CS 2 and the effects of this induction on CS 2 distribution and hepatotoxicity were examined in rats. Eighteen hours after alcohol administration ( 1 2 LD50 dose, po), CS 2 microsomal MFO metabolism was significantly enhanced, in order of descending potency, by isopropanol, methanol, and ethanol pretreatments, but not by isobutanol pretreatment. The degree of enhancement of CS 2 metabolism by different alcohols paralleled the enhancement of nitroanisole O-demethylation and aniline hydroxylation, MFO activities associated with the ethanol-inducible isozyme of cytochrome P 450. CS 2 (1 mg/kg, ip, 3 hr) inhibited only the cytochrome P 450-mediated activities enhanced by alcohol pretreatment. These results suggest that CS 2 metabolism is catalyzed by the ethanol-inducible isozyme. Alcohol-induced rats had significantly more 14CS 2-derived radioactivity in the liver than control and isobutanol-pretreated rats 3 hr after dosing (1 mg/kg, ip). However, only methanol pretreatment resulted in an increased retention of 14CS 2-derived radioactivity in plasma, brain, and kidney. Unlike other alcohol pretreatments, methanol decreased the total 14C expired during the 3-hr period after CS 2 dosing and caused a significant (twofold) increase in plasma glutamic-pyruvic transaminase, measured 24 hr after CS 2 exposure (625 mg/kg). These data indicate that alcohol induction of MFO-dependent CS 2 metabolism per se is not sufficient to result in CS 2-induced hepatic damage although it does lead to loss of specific cytochrome P 450 function.

Effects of chronic acetone administration on ethanol-inducible monooxygenase activities in the rat

Liver microsomal monooxygenase activities known to be ethanol-inducible were determined in female Sprague-Dawley rats after 2-week treatment with 1% v/v) acetone. Daily acetone intake was in the order of 1.2 g/kg. The final body weight, liver weight and microsomal protein content of acetone-treated rats were identical to those of untreated controls. Microsomal NADPH-cytochrome c reductase activity was also unaffected, while cytochrome P-450 content was only increased 12–18%. Ethanol-inducible p-nitrophenol hydroxylation, aniline hydroxylation and 7-ethoxycoumarin Odeethylation activities were enhanced 5.3-, 4.4- and 2.6-fold, respectively, by chronic acetone treatment. The sex-dependent inducing effect of ethanol on benzphetamine N-demethylation activity in female rats was not observed however, after acetone. Addition of acetone in vitro had a stimulatory effect on aniline hydroxylation by microsomes from control and acetone-induced rats. Acetone, however, was found to be a competitive inhibitor of p-nitrophenol hydroxylation activity (apparent K i = 1.8 mM), an observation suggesting that p-nitrophenol is a more selective substrate than aniline for rat liver ethanol- and acetoneinducible cytochrome P-450j. Interruption of the chronic acetone treatment for 24 hr resulted in the almost complete disappearance of its inducing affects, this treatment apparently reproducing only the rapidly reversible preferential inducing effects of chronic ethanol administration. This experimental model of induction by acetone in the rat, when compared to chronic ethanol administration, would thus permit a more selective look at the consequences of these common inducing effects in particular, with respect to drug metabolism and toxicity in vivo, and this, in the absence of the hepatotoxic effects of ethanol itself.

Einfluß von Azoxybenzen auf mikrosomale Leberenzyme der Ratte

Oral administrations of azoxybenzene (0, 25, 50 and 100 mg/kg b.w.) over a period up to 7 days caused a time and dose dependent decrease in the amount of cytochrome P-450 as well as in the ability of aminopyrine-N-demethylation and aniline hydroxylation in the hepatic microsomes of male and female rats. In both sexes there were retardations in the body weight gain and increases in the relative weight of the liver. The amount of the microsomal cytochrome b5 was increased.

Time Course Study of Liver Lipid, Mixed-Function Oxidase, and Estradiol Metabolism in Laying Hens Fed Different Diets

The purpose of this investigation was to determine if hepatic mixed-function oxidase (MFO) activity, plasma estradiol, and estradiol metabolism changed in concert with liver lipid content when laying hens were fed two diets of different composition. White Leghorn laying hens were fed either a corn-soybean meal (CS) diet or CS plus fish meal, alfalfa meal, and torula yeast (FAY) for 24 days. Hens were sampled at the start of the experiment and at 4, 24, 10, 17, and 24 days after the dietary changes. By Day 17, liver weight per unit of body weight and liver lipid content were significantly lower for hens fed the FAY diet compared to those fed the CS diet. Plasma estradiol concentration was significantly higher for hens fed the FAY diet at 4 days, but a reversal occurred at later times and estradiol concentration tended to be higher for birds fed the CS diet for the remainder of the experiment. An in vitro estimate of the estradiol metabolism in the liver showed that by Day 17 the quantity of radioactive estradiol converted to water soluble metabolites was significantly increased in samples from hens fed the FAY diet. Feeding the FAY diet also significantly increased hepatic aminopyrine demethylation and aniline hydroxylation at Day 24.

Identification of a human liver cytochrome P-450 exhibiting catalytic and immunochemical similarities to cytochrome P-450 3a of rabbit liver

Immunoblot analysis of liver microsomes from nine patients demonstrated that each contained a cytochrome P-450 that reacted with an antibody directed against the ethanol-inducible rabbit liver cytochrome, P-450 3a. Two of the liver specimens exhibited high concentrations of the immunoreactive protein, high rates of aniline hydroxylation and N-nitrosodimethylamine demethylation, and extensive inhibition of activity in the presence of antibody to P-450 3a. One other liver specimen exhibited a very low rate of aniline hydroxylation with significantly less antibody inhibition. The variability witnessed was independent of the alcohol history of the individual patients, suggesting that the human cytochrome may be under some other environmental, dietary or genetic regulation. Its inducibility by ethanol was not directly studied in this investigation. However, we conclude that there is a cytochrome P-450 present in human liver which is immunochemically and catalytically similar to the ethanol-inducible P-450 of rabbit liver.

Significance of cytochrome P-450 (P-450 HFLa) of human fetal livers in the steroid and drug oxidations

The purpose of this study was to clarify the pharmacological and physiological significance of P-450 HFLa. Thus, correlations between cytochrome P-450 (P-450 HFLa) level and different monooxygenase activities were investigated in liver homogenates from human fetuses. Poor correlation was seen between P-450 HFLa level and the activity of benzphetamine N-demethylation or aniline hydroxylation. In contrast, the content of P-450 HFLa was highly correlated with the activity of benzo( a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation or testosterone 6β-hydroxylation. In microsomes from human adult livers, a moderate relationship was also observed between testosterone 6β-hydroxylation and P-450 HFLa level. Furthermore, antibodies to P-450 HFLa inhibited testosterone 6β-hydroxylase activity in fetal and adult livers to similar extents. We conclude that P-450 HFLa is a form of cytochrome P-450 which catalyzes testosterone 6β-hydroxylation and limited drug oxidations in human fetal and adult livers.