Effects of cytochrome b5 on aniline hydroxylation catalyzed by a reconstituted system containing acetone or 2,2'-bipyridine

Cytochrome b5 did not exert any effect on NADPH-dependent aniline hydroxylation in the absence of acetone or 2,2'-bipyridine, whereas cytochrome b5 exhibited a stimulatory effect on the reaction in the presence of acetone or 2,2'-bipyridine. In addition, cytochrome b5 did not have any significant effect on the cumene hydroperoxide-dependent reaction in the presence of acetone or 2,2'-bipyridine.     

Multiple drug metabolism: p-nitroanisole reversal of acetone enhanced aniline hydroxylation

The unique effect of acetone on the p-hydroxylation of aniline was evaluated in microsomes prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated animals. The existence of multiple forms of the hydroxylase was evaluated as an explanation of the acetone enhancement. Simultaneous metabolism of aniline in the presence of either p-nitroanisole (pNA) or ethylmorphine (EM) was evaluated to probe the participation of different mixed function oxidase systems. Aniline inhibited both N- and O-demethylation, while pNA and EM both inhibited p-hydroxylation of aniline. Acetone decreased the individual demethylation reactions, but enhanced aniline hydroxylation. In multiple drug reactions, acetone decreased N-demethylation and proportionately increased aniline p-hydroxylation. On the other hand, p-nitroanisole blocked the acetone enhancement of aniline metabolism. Kinetic evaluation of the acetone and p-nitroanisole effects on aniline metabolism indicated that each agent increased the apparent K_m′ by 4- to 5-fold for aniline in the hydroxylation reaction, but only acetone increased the V_max′. From the Eadie-Scatchard analysis of the rates of aniline hydroxylation, acetone appeared to produce a biphasic increase in the hydroxylation above 0.75 mM aniline, even in the presence of pNA. Thus, multiple forms of the aniline p-hydroxylase are indicated by their altered activities in the presence of other drugs, and acetone seemed to specifically alter a species having a higher K_m′ for aniline.     

Species differences in the hydroxylation of aniline and N-ethylaniline by liver microsomes

Summary The velocity of the N- and p-hydroxylation of aniline and N-ethylaniline by NADPH-dependent hydroxylases in guinea pig liver microsomes was found to be as high as in rabbit liver microsomes. Microsomes prepared from cat livers were less active.The effect of 2,4-dichlorophenol, p-chloromercuribenzoate, semicarbazide, and 8-hydroxyquinoline on the microsomal hydroxylations was observed to be not uniform in the microsomes of the species studied.Carbon monoxide inhibited the p-hydroxylation of N-ethylaniline by the microsomes prepared from the livers of rabbits, guinea pigs, dogs, and rats. The N-hydroxylation of N-ethylaniline by microsomes from rat's liver was inhibited like the p-hydroxylation by carbon monoxide. The N-hydroxylation by dog and guinea pig microsomes was scarcely, if at all, inhibited by the same carbon monoxide pressure. These enzymes showed a lower affinity for oxygen than the N-hydroxylating enzyme in rat microsomes and the p-hydroxylating enzymes.With 1 Figure in the TextResults of this study were presented at a meeting of the Deutsche Pharmakologische Gesellschaft in Mainz on April 26, 1965.     

Stimulation of the mouse hepatic microsomal aniline p-hydroxylases by acetone and polyamines

The effects of acetone +/- spermine on the high (AH-I) and low (AH-II) affinity forms of aniline hydroxylases in the mouse hepatic microsomes were investigated under in vitro conditions. The addition of either acetone or spermine alone stimulated both AH-I and AH-II activities at low concentration while some decline in stimulation was noted at higher concentrations. In the presence of both the modifiers the observed monoxygenation rates were greater than those produced by any one enhancer alone for AH-I and more than additive for AH-II. The results suggest that the enhancement of aniline p-hydroxylation by the acetone and spermine in the mouse hepatic microsomes involves at least two separate and possibly interdependent mechanisms.     

The effect of carbon monoxide on the hydroxylation of aniline and N-ethylaniline by microsomal enzymes

Summary Lidoflazine, a new and very longacting coronary vasodilator was given to 18 dogs with a chronically developing stenosis and occlusion of the circumflex coronary artery for 4 and 6 weeks respectively. 18 other dogs with chronically developing stenoses leading to occlusion of the circumflex artery were used as controls. Lidoflazine in a dosis of 20 mg/kg/day increased the speed of development of the collateral circulation and prohibited symptoms of permanent cardiac hypoxia. Permanent myocardial hypoxia, caused by a higher degree of physical activity in a group of freely exercising dogs produced the same favorable effects on the canine coronary collateral circulation.With 6 Figures in the Text     

CYP2E1 hydroxylation of aniline involves negative cooperativity

CYP2E1 plays a role in the metabolic activation and elimination of aniline, yet there are conflicting reports on its mechanism of action, and hence relevance, in aniline metabolism. Based on our work with similar compounds, we hypothesized that aniline binds two CYP2E1 sites during metabolism resulting in cooperative reaction kinetics and tested this hypothesis through rigorous in vitro studies. The kinetic profile for recombinant CYP2E1 demonstrated significant negative cooperativity based on a fit of data to the Hill equation (n = 0.56). Mechanistically, the data were best explained through a two-binding site cooperative model in which aniline binds with high affinity (K_s = 30 μM) followed by a second weaker binding event (K_ss = 1100 uM) resulting in a threefold increase in the oxidation rate. Binding sites for aniline were confirmed by inhibition studies with 4-methylpyrazole. Inhibitor phenotyping experiments with human liver microsomes validated the central role for CYP2E1 in aniline hydroxylation and indicated minor roles for CYP2A6 and CYP2C9. Importantly, inhibition of minor metabolic pathways resulted in a kinetic profile for microsomal CYP2E1 that replicated the preferred mechanism and parameters observed with the recombinant enzyme. Scaled modeling of in vitro CYP2E1 metabolism of aniline to in vivo clearance, especially at low aniline levels, led to significant deviations from the traditional model based on non-cooperative, Michaelis–Menten kinetics. These findings provide a critical mechanistic perspective on the potential importance of CYP2E1 in the metabolic activation and elimination of aniline as well as the first experimental evidence of a negatively cooperative metabolic reaction catalyzed by CYP2E1.     

Stimulation of tolbutamide hydroxylation by acetone and acetonitrile in human liver microsomes and in a cytochrome P-450 2C9-reconstituted system.

Organic solvents are often used to solubilize lipophilic new chemical entities before their addition to in vitro test systems such as microsomal stability or cytochrome P-450 (CYP) inhibition. However, the effect of these organic solvents on the test systems is not usually characterized. This study was initiated to evaluate the effect of acetonitrile and acetone, in addition to other organic solvents, on the tolbutamide hydroxylation activity of CYP2C9 in both human liver microsomes and a CYP2C9-reconstituted system. Both acetonitrile and acetone significantly stimulated the NADPH-dependent tolbutamide hydroxylation by nearly 2- to 3-fold in human liver microsomes and CYP2C9-reconstituted system when incubated at 2 and 4% final solvent concentrations. When cumene hydroperoxide was used instead of NADPH, both acetone and acetonitrile significantly inhibited tolbutamide hydroxylation. This NADPH-dependent stimulatory effect was further evaluated by examining the effect of a series of other organic solvents with different carbon chain lengths and various functional groups, including hydroxyl, ketone, and aldehyde. Unlike acetone, two other ketone-containing solvents, methyl ethyl ketone (2-butanone) and diethyl ketone (3-pentanone) failed to significantly enhance tolbutamide hydroxylation. Other solvents tested, including methanol, ethanol, propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, acetaldehyde, and dimethyl sulfoxide significantly inhibited NADPH-dependent tolbutamide hydroxylation. Overall, the stimulatory effect of both acetonitrile and acetone on tolbutamide hydroxylation was found to be primarily due to a consistent increase in V(max), whereas K(m) was unchanged in both human liver microsomes and the reconstituted CYP2C9 system. These data suggest that acetone and acetonitrile stimulate NADPH-mediated tolbutamide hydroxylation via the CYP reductase and not by modifying the affinity of tolbutamide for the CYP2C9 enzyme.     

Preparation of a high-active microsomal monooxygenase system reconstituted by means of self-assembly

The present work is devoted to study of the role of glycerol in preservation of the lipoprotein nature of cytochrome P-450 during the stages of sodium cholate-caused solubilization of microsomal fraction and subsequent self-assembly of a monooxygenase system after prolonged dialysis. The system thus reconstituted is characterized by the highest catalytic activity of cytochrome P-450 compared to other reconstituted liver microsomal hydroxylating systems presently described in literature.     

Effect of diphenylhydantoin on hepatic drug hydroxylation

Summary Two experiments were done on eight males who were to receive diphenylhydantoin (DPH) for epilepsy: the metabolic disposition of phenazone (antipyrine, 14 mg/kg p.o.) was examined before and after 2.5–6 months of treatment with DPH (300 mg/day). The average half-life of phenazone fell from 10.0 h in the first study to 6.1 h after DPH treatment; and the urinary excretion of unchanged phenazone decreased within 24 h from 28.5 mg (2.8% of the dose) to 18.1 mg (1.8% of the dose). Similar results were obtained when data were calculated on the basis of the output of creatinine. The amount of 4-OH-phenazone, the principal hydroxylated metabolite excreted in the urine in the 24 h after dosing, increased from 185 mg (18% of the dose) to 300 mg (29.5% of the dose) during treatment with DPH. In an additional epileptic patient, who suffered from Icterus juvenilis Meulengracht, the half-life of phenazone decreased from 20.7 h to 6.6 h after 22 days treatment with DPH (300 mg/day); the serum bilirubin concentration fell from 2.6 mg% to 1.0 mg% during the same period. The results are considered to show increased activity of hepatic microsomal drug hydroxylating systems, which can be attributed to treatment with DPH for several weeks.     

A comparative study of two procedures used in the determination of hepatic microsomal aniline hydroxylation

The hepatic microsomal parahydroxylation of aniline is measured by the determination of its product p-aminophenol (pAP). The pAP formed in incubation mixtures is assayed either after ether extraction or after trichloroacetic acid (TCA) precipitation. The TCA-precipitation method is simpler and less time-consuming than the ether-extraction method, but it has been found that the apparent recovery of pAP was lower in TCA supernatants than with the ether-extraction method. In the present work, it was found that the recovery of pAP by the TCA-precipitation method depended on the nature of NADPH-generating system used in the incubation mixture. When “soluble fraction” was used as a source of glucose-6-phosphate dehydrogenase for the reduction of NADP to NADPH, the recovery of pAP using the TCA-precipitation method was less than that with the ether-extraction method. But when “soluble fraction” was replaced by yeast glucose-6-phosphate dehydrogenase or by chemically prepared NADPH, the recovery of pAP was approximately equal with both the methods. Lowered recovery of pAP when added to the soluble fraction or its dialyzate might be due to the presence of sulfhydryl reacting groups. The addition of mercuric chloride to the soluble fraction dialyzate resulted in almost “full” recovery of pAP by the TCA-precipitation method, i.e., the apparent loss of pAP was prevented.