Microsomal Aniline Hydroxylase Activity Research Articles

The effects of ethanol and of a mixture of ethanol and higher-alcohols on the activity of microsomal aniline hydroxylase in the rat liver.

The effects of ethanol and of a mixture of ethanol and higher-alcohols on the activity of microsomal aniline hydroxylase in the rat liver.

RNA expression profiling reveals alterations in lipid metabolism-related molecules in response to antipsychotic drug treatment: Insights into the pathophysiology of psychiatric disorders

The effect of aging on the inhibition by ethanol of drug metabolism activity was examined in liver microsomes of female Fischer 344 rats aged 4, 14 and 24 months. Inhibition of aniline hydroxylase activity in microsomes from 4-month-old females occurred at low concentrations of ethanol (0.1 mM) and was predominentaly competitive. Aging was associated with a significat increase in apparent Km for aniline in the absence of ethanol (24 ± 2, 20 ± 2 and 32 ± 1μM in microsomes from 4-, 14- and 24-month-old rats, respectively) and a change from competitive to non-competitive inhibition by ethanol. Inhbition of benphetamine N-demethylase activity occurred only at high concentrations of ethanol (100 mM) and was non-competitive in nature. There was no significant effects of aging on the kinetics of the reaction or the type of inhibition produced by ethanol. Microsomal ethanol oxidation rates were measured in liver microsomes of 4-, 15- and 25-month-old Fischer 344 rats of both sexes. Ethanol oxidation in males was greater than in females and was decreased significantly in old age. Ethanol oxidation in female rats was unaffected by aging. The results suggest that significant changes in drug/ethanol interactions can occur as a consequence of aging.

Modulation of microsomal cytochrome P450 by Scutellariae Radix and Gentianae scabrae Radix in rat liver.

The present study has determined the effects of Scutellariae Radix (Huangqin) and Gentianae scabrae Radix (Longdan) on liver microsomal cytochrome P450 (P450)-dependent mono-oxygenases using rats pretreated with crude extracts of medicinal herbs. Scutellariae Radix resulted in a 53% decrease of pentoxyresorufin O-dealkylase activity in liver microsomes. In contrast, Gentianae scabrae Radix caused a 50% increase of benzo(a)pyrene hydroxylase activity. Immunoblotting analysis of liver microsomes revealed that Scutellariae Radix induced and suppressed the levels of P450 1A and 2B proteins, respectively. Scutellariae and Gentianae scabrae Radixes had no effects on microsomal aniline hydroxylase activity and P450 2E1 protein.

Enhancement of hepatic microsomal esterase activity following soman pretreatment in guinea pigs

Soman (pinacolyl methylphosphonofluoridate), a highly toxic organophosphate compound, has been found to be a strong inhibitor of hepatic microsomal carboxylesterase in vitro, but an enhancer of carboxylesterase when administered in vivo. In response to this paradoxical observation, the objective of this study was to determine if soman could cause true enhancement of the metabolism of drugs in the guinea pig and, if so, to characterize the enhanced enzyme activity. Following the pretreatment of guinea pigs with 90% ld 50 soman, enhancement of microsomal esterase activity was noted 12 and 24 hr after pretreatment. Using Michaelis-Menten enzyme kinetic studies, enhancement was found to occur with liver carboxylesterase and procaine esterase, but not with aniline hydroxylase. Since the soman-enzyme complex was known to undergo aging with the release of pinacolyl alcohol and the subsequent formation of pinacolone, the effects of these metabolites on the activity of liver microsomal enzymes in vitro were explored. Pinacolone and pinacolyl alcohol produced enzyme enhancement in vitro in a manner similar to that produced by soman pretreatment. These effects were compared with those made by acetone in the same incubations, since the enhancing influence of acetone has already been well documented. Similarity was found between the in vitro effects of acetone and the effects of pinacolone and pinacolyl alcohol. Lastly, the in vivo effects of pinacolone on the activities of the same liver microsomal enzymes were studied following pretreatment of the guinea pigs with 90% LD Lo (lowest published lethal dose) pinacolone. Pretreating guinea pigs with pinacolone prior to killing them enhanced liver microsomal carboxylesterase and procaine esterase activities, but had no effect on microsomal aniline hydroxylase activity. This pattern of enzyme enhancement was similar to that observed after soman pretreatment. Therefore, soman was found to enhance hepatic microsomal esterase activity in the guinea pig in a manner similar to that seen with its metabolites, as well as acetone. This information may give insight into how the efficacy and toxicity of therapeutic drugs, other xenobiotics, and endogenous materials may be altered in individuals who survive an exposure to soman.

Drug and xenobiotic metabolising enzymes in camel liver: Multiple forms and species specific expression

1. Previous studies have demonstrated the presence of phase I mixed-function oxidases (cytochrome P 450-dependent) and phase II conjugation (glutathione S-transferase) enzymes in camel liver. This study represents further characterisation of these drug metabolising enzyme systems in camel liver by comparing their catalytic and immunochemical properties with enzymes of rat and mouse liver. 2. Using the specific P 450 substrate aniline, the microsomal aniline hydroxylase activity of camel liver was found to be significantly lower than that of rat and mouse. The K m values of the enzyme for aniline was similar in rat and camel liver; however, the V max for camel liver enzyme was 50% of the rat liver enzyme. Aminopyrene N-demethylase activity in camel liver, was lower than that of rat but higher than in mouse. Microsomal NADPH cytochrome C-reductase and NADPH-supported lipid peroxidation activities were similar in all three species. 3. The cytosolic phase II conjugation enzyme glutathione S-transferase and glutathione peroxidase activities in camel liver were markedly lower than those of rat and mouse enzymes. However, GSH concentration was similar in all three species. 4. Immunodot blot and Western blot analysis of liver cytosols, using antibodies to specifie GST isoenzymes, have shown that camel liver like mouse and rat, expresses predominantly the Alpha and Mu classes of GST. GST Pi on the other hand, was abundant in mouse liver and was underexpressed in camel and rat liver. 5. Our results demonstrate that there are multiple forms of phase I (P 450) and phase II (GST) enzymes in camel liver and that they are comparable with the drug metabolising enzymes of rat and mouse. The lower activties of drug metabolising enzymes in camel liver compared with rat and mouse appear to be related to the differential expression of selective P 450 and GST isoenzymes.

Ethanol-inducible microsomal aniline hydroxylase activity and cytochrome P-450 isozymes in adult hen liver

1. Cytochrome P-450 was induced in adult hen liver by administering 15% ethanol in drinking water and compared with other inducers such as phenobarbital and β-naphthonavone. 2. Aniline was the only substrate whose turnover was induced by ethanol treatment when measured in the presence of 100 μM α-naphthoflavone. 3. The inhibitor α-naphthoflavone differentiated aniline and p-nitrophenol hydroxylase activities, while p-hydroxyphenyl imidazole and SKF differentiated p-nitrophenol hydroxylase activity between ethanol- and β-naphthoflavone-induced microsomes. 4. Ethanol treatment also slightly induced some P-450 isozymes related to phenobarbital and β-naphthoflavone inducers.

Stimulatory and inhibitory effects of dimethyl sulfoxide on microsomal aniline hydroxylase activity

Dimethyl sulfoxide (DMSO) at a single dose of 3 ml kg body wt, administered i.p. to male rats, caused a significant increase in the hepatic microsomal aniline hydroxylase activity. However, the level of cytochrome P-450, the activities of NADPH-cytochrome c reductase, benzphetamine N-demethylase and aminopyrine N-demethylase were unchanged at 24 h post-treatment. DMSO interacted with control rat liver microsomes in vitro and produced a type II spectral change (peak at 420 nm and trough at 392 nm). On the other hand, liver microsomes from DMSO-treated rats gave qualitatively similar spectra, but with a higher magnitude of binding. Liver microsomes from DMSO-treated rats showed a 3.4-fold increase in V max for aniline hydroxylase, while K m was found to be the same when compared with control rat liver microsomes. In vitro addition of 6 mM DMSO to microsomal incubations from control and DMSO-treated rats caused a 9-fold and a 25-fold increase in K m, respectively, while V max values for aniline hydroxylase were unchanged. When DMSO (6 mM) was incubated with rat liver microsomes in the presence of NADPH, there was formation of formaldehyde. The results suggest an interaction of DMSO with microsomal cytochrome P-450.

Effects of ethanol on microsomal drug metabolism in aging female rats. II. Inhibition

The effect of aging on the inhibition by ethanol of drug metabolism activity was examined in liver microsomes of female Fischer 344 rats aged 4, 14 and 24 months. Inhibition of aniline hydroxylase activity in microsomes from 4-month-old females occurred at low concentrations of ethanol (0.1 mM) and was predominentaly competitive. Aging was associated with a significat increase in apparent K m for aniline in the absence of ethanol (24 ± 2, 20 ± 2 and 32 ± 1 μM in microsomes from 4-, 14- and 24-month-old rats, respectively) and a change from competitive to non-competitive inhibition by ethanol. Inhbition of benphetamine N-demethylase activity occurred only at high concentrations of ethanol (100 mM) and was non-competitive in nature. There was no significant effects of aging on the kinetics of the reaction or the type of inhibition produced by ethanol. Microsomal ethanol oxidation rates were measured in liver microsomes of 4-, 15- and 25-month-old Fischer 344 rats of both sexes. Ethanol oxidation in males was greater than in females and was decreased significantly in old age. Ethanol oxidation in female rats was unaffected by aging. The results suggest that significant changes in drug/ethanol interactions can occur as a consequence of aging.

Effects of Methionine Deficiency and Ethanol Ingestion on Acetaminophen Metabolism in Mice

The interaction of the effect of dietary methionine on the avallability of hepatic glutathione (GSH) and the effect of chronic ethanol (EtOH) consumption on the activity of the hepatic oxidation system was studied in relation to acetaminophen (ACAP) metabolism in mice. Adult male Swiss-Webster mice were pair-fed for 4 wk an EtOH-containing liquid diet that provided 50 or 100% of the methionine requirement in a 2 × 2 factorial design. Hepatic microsomal protein, relative liver weight and microsomal aniline hydroxylase activity were higher in EtOH-fed groups than in non-EtOH-fed groups. After an ACAP dose of 300 mg/kg body wt i.p., serum inorganic sulfate, endogenous hepatic methionine and GSH concentrations were lower, whereas uridine diphosphoglucuronosyltransferase activity was not changed compared to controls. GSH levels were lowered to a greater extent in the methionine-deficient groups than in methionine-sufficient groups. Incorporation of [35S]methionine into hepatic proteins was lower in all treatment groups after ACAP administration than in controls. The distribution of ACAP into the urinary sulfate conjugates was lower in methionine-deficient than in methionine-sufficient groups, and the percentage of sulfate and mercapturic acid conjugates formed as determined by high-performance liquid chromatographic analysis was higher in mice fed EtOH than in controls. Methionine deficiency compromises the normal pathways of ACAP disposition in the mouse, and chronic EtOH ingestion may potentiate this effect by increasing the amount of activated ACAP formed.

Ethanol-induced increase in NADH-dependent monooxygenase activities of hepatic microsomal cytochrome P-450

NADH-dependent hepatic microsomal monooxygenase activities were measured in the presence and absence of NADPH in material from adult male rats given ethanol in a liquid diet. Controls were given a liquid control diet (control group; lipid as substitute for ethanol) or rat chow (untreated group). Ethanol feeding elevated microsomal aniline hydroxylase activity and did not change ethylmorphine N-demethylase activity. NADH supported 21–24% of the NADPH-driven aniline hydroxylase activity in ethanol, control and untreated microsomes, but only about 6% of ethylmorphine N-demethylase activity. In the presence of NADPH, NADH gave 13–14% increase in aniline hydroxylase activity in microsomes from control and untreated rats, but only 3% in ethanol microsomes. In contrast, the presence of NADPH increased many times the effect of NADH on ethylmorphine N-demethylation with no striking difference between the groups. In another series of experiments, demethylation of 4-nitroanisole was elevated after ethanol feeding (4-fold with NADPH; 5-fold with NADH) and phenobarbital treatment (8-fold with NADPH, 2-fold with NADH). In the ethanol-induced activity, NADH and NADPH were less than additive. In the control and untreated and the phenobarbital-induced activities, NADH and NADPH were additive or possibly synergistic in driving the activity. Both ethanol and phenobarbital elevated cytochrome P-450; ethanol also elevated cytochrome b 5 measured as NADH-reducible cytochrome. NADPH caused oxidation of some cytochrome b 5 when added to NADH-reduced microsomes from ethanol-fed rats, but enhanced reduction of cytochrome b 5 when added to NADH-reduced microsomes from control or untreated rats or rats pretreated to induce either microsomal desaturases (fasted rats refed lipid-free high-carbohydrate diet) or monooxygenases (phenobarbital-treated rats). The finding suggests a tight connection between the ethanol-induced cytochrome P-450 and cytochrome b 5 and that ethanol-induced and phenobarbital-induced cytochrome P-450 differ with respect to communication with the NADH-dependent electron transport system which is usually of minor importance in delivering electrons to cytochrome P-450. These findings may be of some importance in the development of metabolic tolerance to ethanol in the liver.

Changes in the rat hepatic mixed function oxidase system associated with chronic ethanol vapor inhalation

Chronic ethanol vapor inhalation by rats increased hepatic microsomal aniline hydroxylase activity, increasing the turnover number and decreasing the K m. Activity of ethanol-induced microsomes toward other substrates was also examined. The increase in aniline hydroxylase activity as a result of ethanol treatment is attributed to an increase in a form of cytochrome P-450 with a high specific activity toward aniline. Since the ethanol effect on aniline hydroxylation had disappeared 24 hr after treatment was discontinued, a high rate of turnover of this enzyme was deduced. Dimethylsulfoxide (56 mM) produced a reverse type I spectral change in ethanol-induced, but not in control, microsomes. This was interpreted as being due to a change in the spin state of the cytochrome P-450 in these microsomes. Acetone added to the incubation produced an increased rate of aniline hydroxylation by microsomes from control and ethanol-induced rats. The difference between the rate of aniline hydroxylation by control microsomes and the rate by ethanol-induced microsomes was, however, abolished at higher acetone concentrations.

Sub-acute and sub-chronic toxicity of mono-2-ethylhexyl phthalate in the rat.

Groups of six male Sprague-Dawley rats were administered single oral doses of mono-2-ethylhexyl phthalate (MEHP) at 50, 100 or 200 mg/kg or di-2-ethylhexyl phthalate (DEHP) at 2,000 mg/kg in 2% gum acacia and were observed clinically for seven days. Body weight and food consumption were not affected by treatment. The liver weight increased in the groups receiving 100 mg or 200 mg mono-2-ethylhexyl phthalate and 2,000 mg diethylhexyl phthalate. Hepatic microsomal aniline hydroxylase activity was not altered by treatment. In a subsequent 28-day experiment, groups of ten weanling Sprague Dawley rats were fed diets containing 0, 25, 100, 400, 1,600 or 6,400 ppm of mono-2-ethylhexyl phthalate. Decreased growth rate occurred in the group receiving the highest dose level. Increased heart and liver weights were observed in animals from the 1,600 and 6,400 ppm groups. Minor alterations in serum biochemical values included decreased SDH and calcium levels, and elevated alkaline phosphatase activity in some treated groups. A mild reduction in red cells saturation and hematocrit was noted in some groups. In 3-month and 6-month feeding studies, groups of ten male and female weanling Sprague-Dawley rats were fed diets containing 1, 5, 25, 125 or 625 ppm of mono-2-ethylhexyl phthalate. Growth rate and food consumption were not affected at any dose level or time interval. Relative organ weights of rats of both sexes were not altered in the 3-month period, but the liver weights of female rats in the 6-month experiment were increased. Changes in clinical chemistry and hematological values were mild. These included lower LDH, SGOT, hemoglobin, and hematocrit values in male rats at the 3-month period and reduced potassium content at the 6-month period. Histological changes were mild in both male and female rats at both time intervals. Treatment-related lesions were found in the liver, heart, and adrenals. Alteration in the liver consisted of midzonal and periportal eosinophilic cytoplasmic inclusions and vacuolations with isolated binucleated and necrotic hepatocytes. There was a mild enlargement of myocardial nuclei and segmental deregistration of myocardial striations in test animals. The adrenal glands exhibited vacuolation of the zona fasciculata which was less severe in the 6-month period than the 3-month counterpart. In conclusion, mono-2-ethylhexyl phthalate is similar to its parent compound diethylhexyl phthalate in that it possesses a low order of oral toxicity in rats.

The interaction of the antineoplastic drug thalicarpine with aniline hydroxylase and microsomal cytochrome of rat liver.

Abstract1. The new antineoplastic agent thalicarpine inhibits aniline hydroxylase activity of rat liver microsomes.2. Pre-treatment with thalicarpine (75mg/kgx3) does not affect hepatic microsomal aniline hydroxylase activity, microsomal cytochrome P-450, cytochrome b5, NADPH cytochrome c reductase, RNA or nuclear DNA.3. Thalicarpine pre-treatment prevents the methylcholanthrene-induced increase in aniline hydroxylase, microsomal RNA and nuclear DNA, but does not affect the induction of cytochromes P-450 and b5.

Electron Transport Components of Hepatic Microsomes: SOLUBILIZATION, RESOLUTION, AND RECOMBINATION TO RECONSTITUTE ANILINE HYDROXYLASE ACTIVITY

Abstract Components of the hepatic microsomal P-450 hemoprotein electron transfer system (TPNH-cytochrome c reductase and P-450 hemoprotein) and the microsomal cytochrome b5 electron transfer system (DPNH-cytochrome b5 reductase and cytochrome b5) were solubilized using Triton N-101 and glycerol, and separated using DEAE-cellulose chromatography. Microsomal aniline hydroxylase activity was reconstituted when the TPNH-cytochrome c reductase fractions and the P-450 hemoprotein fraction were combined. Cross-activities of the TPNH reductases and P-450 hemoproteins were obtained with preparations from phenobarbital- and 3-methylcholanthrene-treated rats. The reconstituted aniline hydroxylase system resembled the native system with respect to turnover number, reducibility of the P-450 hemoprotein component with TPNH, and inhibition with carbon monoxide, p-chloromercuribenzoate, and vitamin K3, but differed in its ability to react with type I substrates such as ethylmorphine and aminopyrine.

The influence of in vivo administration of dimethyl sulfoxide on the in vitro metabolism of some monocyclic aromatic chemicals by hepatic microsomal preparations

The stimulation of hepatic microsomal aniline hydroxylation caused by treating rats with dimethyl sulfoxide (DMSO) was studied further. Stimulation of aniline p-hydroxylation was not abolished by the concurrent administration of DMSO and actinomycin D or cycloheximide. Concurrent administration of DMSO and phenobarbital did not cause any increase in the in vitro aniline hydroxylase activity above that found with DMSO treatment alone; however, concurrent administration of DMSO and 3-methylcholanthrene resulted in a decrease in microsomal aniline hydroxylase activity compared with rats given DMSO alone. Stimulation of hepatic microsomal aniline hydroxylase, associated with DMSO administration, was demonstrated in male, female, and newborn rats, male mice, and male rabbits. DMSO treatment of rats stimulated the hepatic microsomal metabolism of acetanilide, and p-nitroanisole, but did not stimulate microsomal zoxazolamine hydroxylation, and depressed the microsomal demethylation of dimethylaniline.