Aniline Hydroxylation Research Articles

Role of NADPH and the NADPH-dependent methemoglobin reductase in the hydroxylase activity of human erythrocytes

Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal, J. Biol. Chem. 254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations ( K m NADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP +, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation.

Substrate specificity of age-related changes in the inducibility of hepatic microsomal monooxygenases in middle-aged rats

Hepatic microsomal monooxygenase induction was investigated in young-adult and middle-aged male Fischer 344 rats. Monooxygenase components and drug metabolism activities were determined in liver microsomes prepared from rats treated with phenobarbital (PB), β-naphthoflavone (BNF) or methyltestosterone (MT) and compared with values from untreated rats. PB and BNF effects on cytochrome P-450 concentration and cytochrome c reductase activity were similar in young-adult and middle-aged animals. However, the extent of cytochrome P-450 induction by MT was less in the older animals. The age-related changes in induction of drug metabolism activities differed with different substrates for the monooxygenase system. In contrast to the inducibility of benzphetamine N-demethylation and aniline hydroxylation, which was dimished in the older rats, the inducibility of nitroanisole O-demethylation was enhanced. The results imply that qualitative changes in the microsomal enzyme system occurred as the animals progresses from young to middle adulthood.

Kinetics and mechanism of aniline hydroxylation by cumene hydroperoxide in the presence of catalase

In a phosphate buffer with pH=7.4 at 20°C the kinetics of aniline hydroxylation to p-aminophenol by the catalase-cumene hydroperoxide system has been studied. The reaction mechanism of this system is discussed.

Preservation of glucuronidation in carbon tetrachloride-induced acute liver injury in the rat

The influence of carbon tetrachloride on several routes of drug metabolism has been investigated in the rat in vivo, in isolated perfused livers, and in hepatic microsomal preparations to evaluate the hypothesis that hepatic glucuronyl transferase activity is preserved when mixed-function oxidase activity is impaired by this hepatotoxin. Impaired oxidative metabolism after acute administration of carbon tetrachloride was confirmed in vivo by a 65 per cent reduction in the rate of elimination of 14CO 2 in breath after [ 14C]aminopyrine administration and a 58 per cent reduction in the clearance of lorazepam in the whole animal. In the isolated perfused livers of control rats, glucuronide metabolism of lorazepam accounted for 26 per cent of its overall elimination; thus, by inference, oxidative metabolism accounted for the remainder. Carbon tetrachloride pretreatment resulted in a 63 per cent reduction in total lorazepam clearance. In microsomal preparations, cytochrome P-450 concentration, cytochrome P-450 reductase activity, and activities of o-demethylation of p-nitroanisole and hydroxylation of aniline were reduced by 63, 32, 85 and 95 per cent, respectively, after exposure to carbon tetrachloride. In contrast, in the carbon tetrachloride-damage isolated perfused liver the proportion of lorazepam conjugated to lorazepam glucuronide increased from 26 to 43 per cent of the dose administered. Furthermore, in vitro microsomal glucuronidation of o-aminophenol was not decreased by carbon tetrachloride pretreatment, while glucuronidation of p-nitrophenol actually increased significantly by 49 per cent. Solubilization of microsomes with Triton X-100 resulted in a 10.4-fold increase in glucuronyl transferase activity in control microsomes, but in only a 5.7-fold increase in microsomes from carbon tetrachloride-treated rats. This suggests that carbon tetrachloride enhanced basal glucuronidating activity but decreased the total amount of enzymes present. These overall results suggest that microsomal glucuronidation activity is spared in an experimental liver injury in which oxidative metabolism is impaired.

1H nuclear relaxation study of the aniline-binding site in human hemoglobin

Hemoglobin-catalyzed hydroxylation of aniline may be taken as a model for similar reactions catalyzed by cytochrome P-450. Using ultraviolet-difference spectroscopy and 1H nuclear relaxation techniques, the binding of aniline to hemoglobin was examined. From the magnitude of paramagnetic effects of ferric iron on aniline protons, using the correlation time determined from the magnetic field dependence of water proton relaxation rates, aniline was found to bind to methemoglobins such that the aromatic protons are 8.5 ± 0.7 A, away from the high-spin Fe3+. A mode of binding is proposed where the aniline molecule is hydrogen bonded to the distal histidine of hemoglobin.

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.

Structural and electronic factors influencing the inhibition of aniline hydroxylation by alcohols and their binding to cytochrome P-450

A series of 22 homologous and isomeric alcohols have been investigated by quantum mechanical calculations by modified neglect of diatomic overlap (MNDO) and multiple regression analysis by quantitative structure-activity relationships (QSAR) with the goal of gaining some insight into the mechanism of inhibition of aniline hydroxylation and the mode of binding to cytochrome P-450 of these alcohols. The biological data used are the IC 50 values of the alcohols (concentration to give a 50% inhibition of microsomal aniline para-hydroxylation) reported by Cohen and Mannering [11]. The lipophilic-steric (log P, number of carbon atoms in the main chain, lateral bulk) parameters appearing in the regression equations point to a bilinear increase in activity with increasing log P or number of carbon atoms and to an optimal activity for alcohols displaying minimal lateral bulk. This is interpreted in terms of the alcohols binding to a hydrophobic cleft. In addition, electronic parameters obtained by quantum calculations and appearing in the regression equations (energy of the frontier orbitals) indicate charge transfer processes between the ferric ion and the alcohols to be a component of their inhibitory activity. In particular, the inhibitory capacity is found to increase with both nucleophilicity and electrophilicity of the alcohols. Based on these results, a mechanism is proposed in order to explain the inhibitory action of alcohols (Scheme I). The latter are suggested to bind as ligands to the fraction of the cytochrome P-450-aniline complex existing in the high spin form. This ligand binding of an alcohol is competitive with the first reduction of the high spin cytochrome P-450-aniline complex initiating biotransformation.

Decline in hepatic microsomal monooxygenase components in middle-aged Fischer 344 rats

Monooxygenase components and drug metabolism activities were determined in liver microsomes from young-adult (3–5 months) and middle-aged (14 months) male Fischer 344 rats. Several components of the monooxygenase system were decreased in middle-aged rats including total cytochrome P-450, cytchtomre b 5, NADPH-cytochrome, reductase activity and phospholipids. However, the reduction of cytochrome P-450 by NADPH, thought to be rate-limiting for monooxygenase activity, was not decreased. Drug metabolism activities in microsomes from middle-aged rats were increased (nitroanisole O-demethylation), decreased (aniline hydroxylation) or unchanged (benzphetamine N-demethylation). Aging decreased the microsomal content of phospholipids and changed the relative percentages of several microsomal fatty acids. The substrate selectivity of the age-related changes in drug metabolism activities may be related to changes in the fatty acid composition of microsomal phospholipids.

Induction par le lindane des monoxygenases microsomales du foie chez le rat: Effets d'un jeûne de 72 heures

Young male rats were divided into a control group and a group treated with lindane by addition of 60 ppm to the semi-synthetic diet for 4 wk. All the animals were then killed, half of each group after an 18-hr fast and the rest after a prolonged 72-hr fast. Ingestion of lindane accelerated the biotransformation of aminopyrine and of aniline by the liver microsomes and increased the P-450 content and the proportion of linoleic acid in the phosphatidylcholine. Prolonged fasting increased aniline hydroxylation and depressed aminopyrine demethylation. In lindane-treated rats, the fasting partly counteracted the induction of aminopyrine N-demethylase and of P-450 and completely suppressed the characteristic effect of the pesticide on the linoleic acid content of the phosphoglyceride. It is suggested that lindane treatment leads to the induction of a particular form of P-450 sensitive to starvation and bound in the membrane to a phosphatidylcholine rich in linoleic acid.

Inhibition of the microsomal oxidation of ethanol and 1-butanol by the free-radical, spin-trapping agent 5,5-dimethyl-1-pyrroline-1-oxide

The free-radical, spin-trapping agent, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO), inhibited the microsomal oxidation of ethanol and 1-butanol as well as the metabolism of 2-keto-4-thiomethylbutyrate. DMPO also blocked the stimulation of ethanol oxidation caused by the addition of iron-EDTA to microsomes. Previous electron spin resonance studies with DMPO by others had shown that the free radical produced by rat liver microsomes and augmented by iron-EDTA was the hydroxyl radical. In the current study, DMPO had no effect on microsomal oxygen consumption with NADPH as substrate, nor on NADPH-cytochrome c reductase activity, nor on the demethylation of aminopyrine or the hydroxylation of aniline. Therefore, a general toxic action of DMPO on microsomes is excluded. DMPO also failed to inhibit the catalase-dependent pathway of ethanol oxidation by microsomes. Experiments with Chelex 100 resin and the chelating agent, diethylenetri-aminepentaacetic acid, rule out any major role for contaminating iron in the reagents. These results tend to identify the free radical responsible for microsomal metabolism of ethanol, 1-butanol, and 2-keto-4-thiomethylbutyrate as the radical trapped by DMPO, namely the hydroxyl radical.

Differential monooxygenase-like activity of fetal and adult erythrocytes

The monooxygenase-like activity of human erythrocytes was measured by monitoring the rate of para-hydroxylation of aniline. Erythrocytes from umbilical cord blood samples were found to be 3–5 times more active than erythrocytes from adult peripheral venous blood samples. This result may be attributed to an intrinsic difference in the reactivity of the particular form of hemoglobin which predominates in each of these erythrocyte types. Thus, the fetal hemoglobin isolated and purified from the cord blood displayed 2–6 times more activity than purified adult hemoglobin when each was tested in reconstituted aniline hydroxylation systems containing NADPH.

Split lung perfusion: A new method to test the effects of chemical agents on pulmonary metabolism

An isolated and perfused lung preparation was developed which allows increased flexibility and efficiency in evaluating the effects of either gaseous or circulating chemical agents on pulmonary metabolism. This split lung perfusion (SLP) combines the advantages of isolated whole lung perfusion studies with the advantages derived from the use of paired organs. The right and left lungs are perfused and ventilated simultaneously and independently. Using aniline hydroxylation and p-nitroanisole- O-demethylation as parameters of tissue mixed-function oxidase metabolism, we found that enzyme activities were the same in the right and left lung. When the left lung was ventilated with NO 2 (20 ppm) and the right lung was ventilated with air (internal control), aniline hydroxylase activity was significantly inhibited about 20%. Using statistical tests to analyze the effects of acute exposure to ventilated NO 2 on pulmonary aniline hydroxylation, it was found that the coefficient of variation was reduced five-fold using the SLP, as compared with data obtained using the whole lung perfusion technique. It is concluded that the SLP allows for more efficient testing of the effects of chemical agents on pulmonary metabolism by reducing experimental variability and requiring few animals.

Inhibition of rabbit liver microsomal oxidative metabolism and substrate binding by tamoxifen and the geometric isomers of clomiphene

The inhibitory effects of the triarylethylene antiestrogens tamoxifen (TAM) and enclomiphene (EC), and of the estrogenic geometric isomer of the latter compound (zuclomiphene, ZC) on the binding and metabolism of standard substrates in the presence of rabbit liver cytochrome P-450 have been studied. In the presence of microsomes at pH 7.4, the triarylethylenes interfered with the binding of β-diethylaminoethyl diphenylpropylacetate (SKF 525-A) and with the N-demethylation of ethylmorphine. Their inhibitory effects were less pronounced on the binding and metabolism (hydroxylation) of aniline. Kinetic studies showed that EC and TAM interfered with SKF 525-A binding by a mixed mechanism. These results suggest that the triarylethylenes affect preferentially the binding of ‘Type I’ substrates with cytochrome P-450.

Preservation of rat hepatic microsomal enzyme activities: Effect of low temperature and freeze-drying

About 30–70% of microsomal hydroxylation of aniline, 0-demethylation of 4-NO 2-anisole, N-demethylation of aminopyrine, were lost when whole organs were frozen and kept at low temperatures (0°, −20°, −196°C). When 9000 × g or 105000 x g fractions were prepared from fresh liver and subsequently frozen to different temperatures, there was little or no such loss of activity. The kinetics of the decrease in microsomal enzyme activities was followed during storage of frozen or freeze-dried microsomes at various temperatures. N-demethylation of aminopyrine appeared to be the most sensitive marker of microsome denaturation.

Inhibition of In Vitro Cytochrome P-450-Catalyzed Reactions by Substituted Pyridines

A series of substituted pyridines was investigated as inhibitors of cytochrome P-450-catalyzed reactions. The relative potencies for the in vitro inhibition of aminopyrine demethylation and aniline hydroxylation are reported for a series of 2-, 3-, and 4-substituted pyridines.

Enhancement by cyanide of aniline p-hydroxylation activity in rat liver microsomes

Aniline p-hydroxylation activity of rat liver microsomes was found to be enhanced, rather than inhibited, at aniline concentrations higher than about 3 mM. The cyanide-induced enhancement increased as the oxygen tension was increased. The activation by cyanide was, however, significantly diminished with liver microsomes from phenobarbital- and 3-methylcholanthrene-pretreated rats. The enhancement was also decreased when liver microsomes were fortified with NADPH-cytochrome P-450 reductase. Aniline hydroxylation by reconstituted systems consisting of partially purified preparations of several species of cytochrome P-450 and the reductase was inhibited by cyanide, though the degree of inhibition was dependent on the species of cytochrome P-450 used for reconstitution. In several respects, the cyanide-induced enhancement of aniline hydroxylation is different from the enhancement caused by acetone and 2,2'-bipyridine, but is similar to the activation by ethylisocyanide.

Effect of chronic heat exposure on in-vitro drug metabolism in the rat

The in-vitro rate of drug metabolizing enzymes was investigated in chronically heat exposed male rats. Exposure to 35±2°C for 30 days resulted in significant reduction in the rates of both hepatic N-demethylation of parachloro-N-methylaniline and aniline hydroxylation. Substantial elevation of rectal temperature and marked reduction in body and liver weights was observed in the same rats. The significance of these results is discussed.

Characterization of aniline hydroxylation by a cytochrome P-450 model and a possible hydroxylation mechanism.

Aniline hydroxylation with a hemin-cysteine system was studied under various conditions as a model for cytochrome P-450 enzymes. The reaction was characterized by using various inhibitors, such as hydroxyl radical scavengers, a singlet oxygen trapping agent, superoxide dismutase and catalase. The system was compared with simple model systems consisting of cupric ion, ascorbic acid and hydrogen peroxide or the Udenfriend system. The hemin-cysteine model system was not inhibited appreciably by any of the inhibitors tested. On the basis of these findings and the fact that the thiolate-heme iron linkage was found to be present in the hemin-cysteine system, a possible mechanism for hydroxylation by the model system is proposed.

Planning of experiments for the testing of chronic toxicity in rats taking into account biorhythms.

During the course of testing chronic toxicity in rats several physiological and biochemical parameters were investigated. Many of the processes studied show a circadian and infradian rhythm, which can clearly be influenced from the second year onwards by age dependent alterations. The daily rhythm of the following processes were investigated: Hexobarbital sleeping time, aminophenazone demethylation, Hb (Hemoglobin), Hc (Hematocrit), GPT (Glutamate-pyruvate-transaminase) and total plasma proteins. The seasonal changes of total plasma proteins, Hexobarbital sleeping time and aniline hydroxylation were also examined. The results obtained indicate, that for comparison and interpretation of data on acute and chronic toxicity the knowledge of daily and seasonal changes is necessary and has to be considered for an exact planning of experiments.

Induction of rat hepatic microsomal enzymes by toxaphene pretreatment.

The effects of pretreatment of rats with toxaphene on hepatic drug metabolizing enzymes and several other parameters of the mixed function oxidase system were investigated. Adult male Sprague-Dawley rats were fed diets containing 0, 50, 100, 150 and 200 ppm of toxaphene for 14 days. The body weight gain was unaltered as well as the food consumption in all the toxaphene fed groups. There was no change in the weights of brain, kidney, heart, and testes but the liver weight was significantly increased. The thymus weight in all the toxaphene fed grups was decreased. Hydroxylation of pentobarbital and aniline was significantly enhanced in rats exposed to toxaphene. Ethylmorphine-N-demethylase activity in the toxaphene treated rats was also elevated. Enhanced hydroxylation of pentobarbital was also evident from the decreased sleeping time following pentobarbital administration. Exposure to toxaphene increased cytochrome P-450, NADPH-cytochrome c-reductase and dehydrogenase in hepatic microsomal fractions. The binding of aniline and hexobarbital to microsomes was also enhanced, suggesting that the intermediate steps in the electron-transfer system were increased. In conclusion, pretreatment of rats with toxaphene for fourteen days resulted in the induction of the hepatic mixed function oxidase system.