Aniline Hydroxylation Research Articles

400 DOES DRUG ADDICTION DUPING PREGNANCY ZNHANCE DRUG BIOTRANSFORMATION BY THE PLACENTA?

The placenta is not merely a passive barrier to drugs and foreign chemicals, but may participate in xenobiotic biotransformation, as well. It is not known whether chronic addiction during pregnancy enhances this placental function. We therefore studied placental homogenates from 5 normal control and 7 drug dependent mothers and compared their oxidoreductase and transferase activities (μmoles/mg protein/h). RESULTS: (1) Reductase activity (measured by neoprontosil reduction) was higher in the soluble than microsomal fractions, due mostly to catalysis by NADPH. No significant difference was seen in the reductase activity of control (0.72 ± 0.06) vs addict (0.74 ± 0.16) in the supernate. (2) Transferee activity (by bilirubin conjugation) was minimal in both soluble and microsomal fractions. No significant difference was noted in the microsomal transferase activity of control (0.11 ± 0.06) vs addict (0.12 ± 0.05) homogenates. (3) Mixed function oxidoreductase activity (by aniline p-hydroxylation) appeared higher in the soluble (0.64 ± 0.24) than the microsomal (0.21 ± 0.14) fractions but due solely to hemoglobin in the soluble fraction which nonenzymatically catalyses aniline hydroxylation (49.4 × 10−4umoles/mg Hgb). No enzymatic oxidoreductase activity was noted in the control or addict homogenates. CONCLUSION: Little to no xenobiotic oxidoreductase and transferase activity was noted in both control and addict placental homogenates. Apparent activity was principally due to nonenzymatic catalysis of substrates by NADPH or Hgb. Thus, despite chronic exposure to drugs, the placenta of drug addicted mothers does not show any enhanced xenobiotic biotransformation activity and therefore offers no adaptive protection to the fetus.

Toxicity of carbon disulfide in developing rats: LD50 values and effects on the hepatic mixed-function oxidase enzyme system

The 24-hr LD50 values of carbon disulfide (CS 2) were estimated in 1-, 5-, 10-, 20-, 30-, and 40-day-old rats. CS 2 was least toxic to 20-day-old rats (LD50 1545 mg/kg, ip) and most toxic to 1-day-old rats (LD50 583 mg/kg, ip). Twenty-four hours after administration of CS 2 (375 mg/kg, ip) to 1-, 5-, 10-, 20-, 30-, and 40-day-old rats, significant inhibition of cytochrome P-450 and aniline hydroxylation was observed in rats of all ages studied except the 1-day-old rats. Following incubation of hepatic microsomes isolated from 1-, 5-, 10-, 20-, 30-, and 40-day-old rats with CS 2, decreases in activity of the hepatic mixed-function oxidase enzyme system and/or concentration of cytochrome P-450 were observed when an NADPH-generating system was present during incubation. When hepatic microsomes isolated from rats of all ages studied were incubated with C 35S 2, 35S was covalently bound to microsomal protein in the presence of an NADPH-generating system. Also, more 35S than 14C was covalently bound to microsomal membranes after incubation of microsomes isolated from rats of all ages studied with C 35S 2 or 14CS 2 in the presence of an NADPH-generating system. The results of this research demonstrated the LD50 of CS 2, the effects of CS 2 on the hepatic mixed-function oxidase enzyme system, and that the conversion of CS 2 to a covalently binding sulfer-containing biotransformation product varied with age in developing rats.

The Effect of Galactosamine on Rat Liver Cytochrome P-450 Activities

The effect of galactosamine on hepatic drug metabolizing activities was examined in rats. In the microsomal fraction, the contents of cytochrome P-450 (P-450) and cytochrome b5 (b5) and the activity of NADPH-cytochrome c reductase (reductase) were examined for 7 days after galactosamine administration. In addition, substrate metabolizing activities in damaged microsomes were examined using four substrates: aminopyrine, aniline, benzo(a)pyrene (B(a)P) and 7-ethoxycoumarine (7-EC). The contents of P-450 and b5 and the activity of reductase showed a minimal value after 3 days of galactosamine administration and then gradually increased, reaching to the control level after 7 days. All four substrate metabolizing activities showed a similar response as the content of P-450, but the decrement among the four activities was not uniform. The activities of B(a)P hydroxylation and 7-EC deethylation were more impared than those of aminopyrine demethylation and aniline hydroxylation. This nonuniformity was clear on the activity based on P-450. This result suggested that galactosamine disturbed the population of multiple P-450 subtypes, and each P-450 subtype was damaged to the various extent by galactosamine administration.

In vivo effect of ozone inhalation on xenobiotic metabolism of lung and liver of rats

Male Jcl:Wistar rats were exposed to 0.4 and 0.8 ppm O3 daily for 7 h for 14 d to examine the effect of O3 on xenobiotic metabolism of lung and liver microsomes. An exposure to 0.4 ppm O3 did not affect the microsomal xenobiotic metabolism of either lung or liver. On the other hand, 0.8 ppm O3 increased significantly the NADPH-cytochrome P-450 reductase activity and the cytochrome P-450 content of lung microsomes. The activities of lung benzo[a]pyrene hydroxylase and 7-ethoxycoumarin O-deethylase also increased significantly on d 7, and remained at a higher level by the d 14. These results show that exposures to 0.8 ppm O3 induce the xenobiotic metabolizing systems in the lung. In the liver, after the first day of exposure to 0.8 ppm O3, a significant reduction occurred in all components of the electron-transport systems examined as well as in the microsomal protein. A significant decrease was also observed in benzo[a]pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and aniline hydroxylation. The decreased activities recovered in the following period of exposure. In contrast, the p-nitroanisole N-demethylase activity was not altered during the 14-d exposures. These results suggest that some isozymes of the hepatic cytochrome P-450 are sensitive to O3 inhalation but other(s) are resistant.

Synthesis and cytochrome P-450-like reactivity of polypeptide-bound porphinatoiron(III)

Polypeptide-bound porphinatoiron(III) was synthesized. This polymer could catalyze the hydroxylation of aniline with H 2O 2 more effectively, and catalyze the monooxygenase-type oxidation of olefins more selectively in the porphinatoiron (III)-O 2-NaBH 4-Me 4NOH system 2) than non-bound porphinatoiron.

Induction of rabbit hepatic microsomal cytochrome P-450 by imidazole: Enhanced metabolic activity and altered substrate specificity

Pretreatment of rabbits with imidazole resulted in a twofold increase in hepatic microsomal cytochrome P-450 content, with the apparent induction of two or more distinct forms of the cytochrome [ K. K. Hajek and R. F. Novak (1982) Biochem. Biophys. Res. Commun. 108, 664–672 ]. The metabolic properties of imidazole-induced microsomes have been compared to those of uninduced, phenobarbital- and β-naphthoflavone-induced preparations. Metabolic activity was enhanced as a consequence of increased P-450 content and as a result of the presence of different forms of the cytochrome. When rates were expressed per nanomole P-450 the following were observed: (a) p-nitroanisole O-demethylation was comparable in all preparations; (b) N,N-dimethylaniline N-demethylation was comparable in imidazole- and β-naphtho-flavone-induced, and uninduced microsomes; (c) polycyclic aromatic hydrocarbon hydroxylase activity was approximately twofold greater in imidazole-induced relative to phenobarbital-induced microsomes, but was only one-half that of β-naphthoflavone-induced microsomes; and (d) metabolism of N,N-dimethylnitrosamine was enhanced fivefold, alcohol oxidation increased three- to fivefold, and aniline hydroxylation was threefold greater in imidazole-induced microsomes compared to phenobarbital- or β-naphthoflavone-induced preparations. Eadie-Scatchard analysis yielded a single K m value for dimethylnitrosamine N-demethylase activity in imidazole-induced microsomes; in contrast, both high- and low- K m values were obtained for phenobarbital- or β-naphthoflavone-induced microsomal preparations. Dimethylnitrosamine N-demethylase activity was P-450 dependent; neither flavin monooxygenase nor monoamine oxidase appeared to contribute significantly to dimethylnitrosamine metabolism. Dimethyl sulfoxide was a competitive inhibitor of dimethylnitrosamine N-demethylase activity in imidazole-, phenobarbital-, and β-naphthoflavone-induced microsomes. Dimethyl sulfoxide competitively inhibited ethanol oxidation in imidazole-induced microsomes; it was a noncompetitive inhibitor of ethanol oxidation in phenobarbitalor β-naphthoflavone-induced microsomes.

The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of nadph-cytochrome P-450 reductase

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-448 1 and MC P-448 2) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-448 1 and MC P-448 2 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-448 1 or MC P448 2. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-448 1 or MC P-448 2 was added to the system containing PB P-450 and limited amount of the reductase. The apparent K m of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-448 1 or MC P-448 2. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.

Selective induction of cytochrome b 5 and NADH cytochrome b 5 reductase by propylthiouracil

Both the cytochrome b 5 level and NADH cytochrome b 5 reductase activity in rat liver microsomes were increased 2-fold by repeated i.p. administration of 1.5 mmol/kg propylthiouracil (PTU) for 2 weeks, but neither the cytochrome P-450 level nor NADPH cytochrome P-450 reductase activity were affected by the treatment. Liver microsomes from PTU-treated rats showed a significant decrease in aminopyrine N-demethylation, but not in benzphetamine N-demethylation, aniline hydroxylation or 7-ethoxycoumarin O-deethylation. A single administration of the compound had no effect on any components of the system. In vitro , drug hydroxylation activities were not affected by PTU up to 1.0 mM. From the above evidence, repeated administration of PTU selectively induced cytochrome b 5 and NADH cytochrome b 5 reductase in rat liver microsomes.

Monooxygenase activity of human hemoglobin: role of quaternary structure in the preponderant activity of the beta subunits within the tetramer.

Catalysis of para hydroxylation of aniline was measured for human ferrihemoglobin and various derivatives in a reconstituted system consisting of the appropriate hemoprotein (at 4 microM heme), reduced nicotinamide adenine dinucleotide phosphate (NADPH), cytochrome P-450 reductase, and aniline under atmospheric O2. The isolated subunits of hemoglobin (alpha 3+ and beta 3+4) were prepared by treatment with p-(hydroxymercuri)benzoate. Semihemoglobin (alpha heme2 beta 02) was prepared from ferrihemoglobin and apohemoglobin. Converse valency hybrids alpha 3+2(beta 2+-CO)2 and (alpha 2+-CO)2 beta 3+2 were prepared from appropriately ligated alpha and beta subunits. After chromatography, the hemoglobin derivatives were characterized by visible and 1H NMR spectroscopy and electrophoresis. At the same concentration of aniline, the alpha and beta subunits were much less active than the normal tetramer. alpha-Semihemoglobin and the alpha 3+2(beta 2+-CO)2 hybrid also displayed lower hydroxylase activity. The (alpha 2+-CO)2 beta 3+2 hybrid was about as active as normal alpha 3+2 beta 3+2. This result suggests that the activity of tetrameric hemoglobin primarily involves the beta subunits. Also transfer of the beta subunits from the beta 4 molecular environment to the alpha 2 beta 2 state enhances their monooxygenase activity approximately 15-fold. The hemoglobin derivatives were differently susceptible to substrate inhibition, the beta 4 species being most sensitive. Estimates of Vmax from the linear portions of the corresponding Lineweaver-Burk plots showed agreement within a factor of 2.5 for all of the hemoglobin derivatives, suggesting that the intrinsic O2-activating capacities of the derivatives are similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrocatalytic hydroxylation of aniline on metal oxides

Studies of Mn, Cr, Cu, La, Sb, V, Co and W oxides as catalysts for the electrocatalytic oxidation of aniline to p-aminophenol have revealed the electrocatalytic activity of WO3. A mathematical model for the kinetics of analine oxidation on WO3 is suggested that agrees fairly well with the experimental data.

Aniline hydroxylation and 7-ethoxycoumarin O-deethylation activities in solubilized and partially resolved liver cytochromes P-450 from ethanol-fed rats.

Chronic ethanol administration in female rats enhances the apparent molar activity of liver microsomes for aniline hydroxylation and 7-ethoxycoumarin O-deethylation. Microsomal cytochromes P-450 from ethanol-fed and control rats have been solubilized and partially resolved in six fractions by anion-exchange chromatography. Induction of aniline hydroxylase activity by ethanol was associated with marked increases in the turnover numbers of the more basic cytochrome P-450 containing fractions in a reconstituted aniline hydroxylation system. Cytochrome P-450, exhibiting by far the highest 7-ethoxycoumarin O-deethylase activity, was eluted in a relatively acidic fraction; its turnover number with 7-ethoxycoumarin after ethanol consumption, however, did not differ significantly from that of the corresponding fraction from control microsomes. These observations suggest that induction of liver microsomal mixed function oxidases by ethanol may reflect the contribution of more than one cytochrome P-450 isozyme.

Gingiva as a site for xenobiotic metabolism in rats

NADPH-dependent N-demethylation of aminopyrine and tripelennamine and hydroxylation of aniline, phenytoin and tripelennamine occurred in rat gingival homogenates. The metabolic profile in gingiva appeared to be similar to that in liver, the major organ in the body for the metabolism of these xenobiotics.

Direct conversion of anilines into aminophenols

Hydroxylation of anilines by hydrogen peroxide in SbF 5-HF yields the three possible aminophenols, the meta isomer being the major product. The reaction implies attack of protonated hydrogen peroxide H 3O 2 + on the N-protonated substrate.

Mouse liver testosterone 15 alpha-hydroxylase (cytochrome P-450(15) alpha). Purification, regioselectivity, stereospecificity, and sex-dependent expression.

Testosterone 15 alpha-hydroxylase (cytochrome P-450(15) alpha) was purified from female 129/J mouse liver microsomes based on its specific activities in the eluates from the columns of octylamino-Sepharose 4B, hydroxylapatite, DEAE-Bio-Gel A, and CM52 chromatography. The 15 alpha-hydroxylation activity was five times higher in female than in male 129/J mouse liver microsomes. The specific cytochrome P-450 content of purified P-450(15) alpha fraction was 14.5 nmol/mg of protein. The Soret peak of the reduced cytochrome P-450-CO complex was 451 nm. The apparent subunit molecular weight of P-450(15) alpha was 48,000, and the protein appeared as only one major band on sodium dodecyl sulfate-polyacrylamide gels. The specific activity of testosterone 15 alpha-hydroxylation reconstituted with the purified P-450(15) alpha was 94 nmol/min/nmol of cytochrome P-450 and 1349 nmol/min/mg of protein, and these were about 65- and 1000-fold higher, respectively, than the activity of solubilized microsomes. The purified P-450(15) alpha exhibited high regioselectivity and stereospecificity for testosterone hydroxylation. More than 95% of the testosterone metabolites formed by the purified P-450(15) alpha was 15 alpha-hydroxytestosterone. Virtually 100% of mouse liver microsomal testosterone 15 alpha-hydroxylation activity can be accounted for by the purified P-450(15) alpha. The P-450(15) alpha fraction was able to catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-de-ethylation, aniline 4-hydroxylation, benzo(alpha)pyrene 3-hydroxylation, acetanilide 4-hydroxylation, and lauric acid (11 + 12)-hydroxylation at various turnover rates, indicating broad substrate specificity of the P-450(15) alpha for the oxidations of xenobiotics. This is in sharp contrast to high regioselectivity and stereospecificity for testosterone hydroxylation.

Effect of successive administration of zopiclone, a new class of minor tranquillizer, on mouse liver microsomal mono-oxygenase system.

Oral administration of zopiclone to mice (10 mg/kg daily) for 14 days had no significant effect on body weight, liver weight, hepatic microsomal protein or cytochrome P-450 contents but produced significant increases in the activities of NADPH-cytochrome c reductase, NADH-ferricyanide reductase, aniline hydroxylation and aminopyrine N-demethylation.

Effect of acetone on aniline hydroxylation by a reconstituted system

Acetone stimulated NADPH-dependent aniline hydroxylation catalysed by cytochrome P-450 purified from phenobarbital-treated rats. No activation by acetone occurred in a reconstituted system containing cytochrome P-448 purified from 3-methylcholanthrene-treated rats. Cumene hydroperoxide-supported aniline hydroxylation catalysed by cytochrome P-450 was not increased by the addition of acetone at the concentration which stimulated NADPH-dependent aniline hydroxylation in the reconstituted system. The NADPH-dependent cytochrome P-450 reduction was stimulated by acetone in the presence or absence of aniline. On the other hand, acetone did not exhibit any stimulatory effect on NADPH-dependent reduction of cytochrome P-448. The stimulatory effect of acetone on aniline hydroxylation was decreased with increasing pH of the reaction media. Furthermore, in the system containing cytochrome P-450, the ratio of product to hydrogen peroxide formation was virtually unchanged by the addition of acetone.

Acute ethanol intoxication decreases subsequent food intake and changes hepatic microsomal enzyme activities similarly to fasting.

The effect of intragastric ethanol, 7.8 g/kg, on the microsomal enzyme activities NADPH cytochrome c reductase, aminopyrine N-demethylase and aniline hydroxylase, were investigated in two sizes (150 g and 260 g) of mature male rats having free access to food and water for the 23 hours following ethanol administration. Controls received intragastric water and were given free access to food and water (fed controls) or only water (fasted controls). The ethanol group had a significantly lower food intake than the fed control group, and food intake in the large rats was more decreased by ethanol than that of the small rats, corresponding to slower recovery from intoxication. Ethanol and fasting increased aniline hydroxylation and decreased acetone enhancement of aniline hydroxylation to the same degree, whereas only fasting caused a significant decrease in aminopyrine N-demethylation. Some of the effects of acute ethanol intoxication on hepatic microsomal enzyme activities appear to be mediated via decreased food intake.

Selenium and drug metabolism—I: Multiple modulations of mouse liver enzymes

Male albino mice were raised on diets containing less than 10 ppb selenium (Se−) or supplemented with 0.5 ppm selenium (Se+) for 6 months. In the (Se−) group total liver selenium was less than 10% of the control, liver selenium-dependent glutathione peroxidase (GSH-Px) less than 2%. The specific activities of catalase and Superoxide dismutase showed essentially no differences between the dietary groups. Several phase I-related specific enzyme activities were measured in liver microsomes. No significant differences between the two animal groups were found for cytochrome P-450 and b 5 content, NADH-cytochrome b 5 reductase, as well as for aniline hydroxylation and aminopyrine dealkylation rates. In (Se−) microsomes, NADPH-cytochrome P-450 reductase activity was about half that found in (Se+) microsomes. An increase in microsomes from (Se−) mice was found for 7-ethoxycoumarine deethylation rate (460%), cytochrome P-450 hydroperoxidase activity (170%), and heme oxygenase (276%). The N-oxidation rate of the flavin-containing monooxygenase decreased by 35%, the N-demethylation rate by 50% in (Se−) animals. Stopped-flow measurements of the reduction rates of microsomal pigments did not support evidence for limitations in microsomal electron supply during selenium deficiency. Among the phase II reactions examined, sulfotransferase activity towards 4-nitrophenol was 47% of the controls in Se-deficient liver cytosols while UDP-glucuronyl transferase activity towards this substrate increased to 215%. Glutathione- S-transferase activity was much higher in (Se−) livers than in (Se+): 310% with 1,2-dichloro-4-nitrobenzene, 255% with 1-chloro-2,4-dinitrobenzene and 120% with ethacrynic acid as substrate. The data indicate that in addition to GSH-Px many other enzyme activities in mouse liver are affected by prolonged dietary selenium deficiency. These effects might be useful in assessing the severity of selenium deficiency. A microsomal selenium-dependent metabolic modulator is discussed as a possible mechanism.

A high spin form of cytochrome P-448 highly purified from PCB-treated rats—II: Characteristic requirement of cytochrome b5 for maximum activity

A high spin form of cytochrome P-448 (PCB P-448-H), highly purified from microsomes of PCB-treated rats, catalyzed oxidations of several compounds and required cytochrome b5 for its full activities in all oxidations examined. PCB P-448-H catalyzed the hydroxylation of aniline and O-dealkylations of p-alkoxy derivatives of aniline and nitrobenzene and 7-alkoxy derivatives of coumarin. Among the activities measured, hydroxylation of aniline and O-dealkylation of p-alkoxy derivatives of aniline were catalyzed by PCB P-448-H more efficiently than by PCB P-448-L, which was a low spin form of cytochrome P-448 purified from liver microsomes of PCB-treated rats. In all reactions, PCB P-448-H required cytochrome b5 for maximum activity. Slight requirements were also seen with PCB P-448-L but varied equivocally depending on the substrates. Cytochrome b5 showed its maximum effects on p-propoxyaniline O-depropylation activity at a molar ratio of cytochrome b5 to PCB P-448-H of 1:2. The enhancement by cytochrome b5 was more pronounced when lower concentrations of either the substrate or NADPH-cytochrome P-450 reductase were added to the reconstituted system. Based on these results, we confirm that PCB P-448-H is a unique form of cytochrome P-448 with respect to the requirements for cytochrome b5 and is a good probe to study the mechanisms involved in the enhancement of drug oxidations by cytochrome b5.

Monooxygenase activities of dioxygenases. Benzphetamine demethylation and aniline hydroxylation reactions catalyzed by indoleamine 2,3-dioxygenase.

Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.