Metabolism Of Aniline Research Articles

Induction par le lindane des monoxygenases microsomales du foie chez le rat: Effets d'un regime hypocalcique

Two groups of young male rats were fed either a diet with a normal calcium content (1%) or a low-calcium (0·06%) diet for 6 wk. After the first 2 wk, each group was subdivided equally into a control group and a group treated for the remaining 4 wk with 60 ppm lindane in the diet. The rats were then killed, livers and kidneys were removed and weighed and hepatic microsomal fractions were prepared for protein, phosphatidylcholine and enzyme measurements. Lindane increased the kidney weights and the rates of aminopyrine and aniline metabolism in the liver, and induced the NADPH-dependent microsomal electron-transport system (cytochrome P-450, cytochrome c reductase, phosphatidylcholine). Under the experimental conditions used, the low-calcium diet did not appear to affect the metabolism of exogenous substances. although it lowered the P-450 content of the liver microsomes. The maintenance of the normal level of metabolism may be explained by an increase in electron transfer towards the cytochrome from NADPH with induction of NADPH-cytochrome c reductase and NADPH-oxidase. Lindane treatment prevented the characteristic inducing effect of calcium deficiency on the specific molecular activity of NADPH-cytochrome c reductase [reduction of P-450 (Fe 3+)].

Effects of short-term administration of maleic hydrazide or hydrazine on rat hepatic microsomal enzymes

The herbicide maleic hydrazide (MH), in amounts greatly exceeding its residue level in foods, had no apparent effect on the hepatic microsomal enzyme system of Fischer rats. In vivo, up to 1 g/kg of MH did not affect hexobarbital sleep times (HST) and in vitro, 2 g/kg of the sodium salt of MH altered neither the metabolism of benzo(a)pyrene and aniline nor the activities of cytochrome b 5 and P-450. Hydrazine-SO 4, a frequent contaminant of commercial MH preparations, in moderately high doses prolonged HST and diversely altered metabolism in vitro. The effects of hydrazine impurities, if present, could be significant in toxicity tests where excessive doses of MH are administered.

Estimation of Michaelis--Menten constants in microsomal enzyme systems by the direct linear plot and least squares regression.

1. Data from in vitro liver microsomal metabolism of ethylmorphine aniline and benzpyrene in male rats and male and female quokkas (Setonix Brachyurus) were fitted to the Michaelis--Menten equation by Wilkinson's non-linear regression procedure and Eisenthal and Cornish-Bowden's direct linear plot. 2. Ethylmorphine and aniline metabolism in the male quokka were non-ideal, but in all other instances there were no differences in the performance of the two methods.

Effect of acute and chronic cadmium treatment on hepatic drug metabolism in male rats.

The effect of acute and chronic cadmium administration on hepatic drug metabolism was investigated in the male rat. 3 days after the acute administration of cadmium by either the intraperitoneal (0.84 mg Cd/kg) or the oral (greater than 80 mg Cd/kg) route, there was a significant potentiation in duration of hexobarbital hypnosis and inhibition of hepatic microsomal metabolism of hexobarbital and aniline. Administration of cadmium in the drinking water at levels of 100 or 200 ppm Cd for periods of 2--12 weeks or at levels of 5 or 20 ppm Cd for 50 weeks did not produce alterations in either drug response or hepatitic drug metabolism. Significant levels of metallothionein, a cadmium binding protein, found in the liver of the rats receiving cadmium chronically may offer an explanation for the observed differences in drug metabolism between the acute and chronic administration of cadmium. In additional studies, pretreatment of the rats with subthreshold doses of cadmium (0.21 or 0.42 mg Cd/kg) intraperitoneally produced a tolerance to the alterations in drug metabolism induced by the previous cadmium dose (0.84 mg Cd/kg, i.p.). However, chronic cadmium treatment (5 or 20 ppm Cd for 50 weeks) did not impart any such tolerance to subsequently administered Cd (0.84 mg/kg) by the intraperitoneal route. The hepatic levels of metallothionein induced by the chronic cadmium treatment were only 30--60% of those induced by the subthreshold cadmium and thus may not have bound enough of the large challenge cadmium dose to produce the tolerance phenomenon.

Potentiation of hexobarbital and amphetamine effects in male and female rats physically dependent on morphine

Morphine dependence (following s.c. implantation of 207 mg morphine in pellet form) increases hexobarbital sleeping time in both male and female rats; this is accompanied by an apparent increase in brain sensitivity to hexobarbital. Only male morphine-dependent rats manifested a decrease in in vitro hepatic metabolism of hexobarbital as well as a significantly faster rate of decline of hexobarbital from the brain. In vivo uptake of hexobarbital into the brain revealed no difference between male and female morphine-dependent and sham-implanted animals. The suggested mechanism for the observed changes in male rats is a combination of inhibition of hepatic metabolism and an additive effect of morphine and hexobarbital. In female rats only an additive effect is manifested. In male rats morphine dependence also increases amphetamine-stimulated locomotor activity (open-field), increases amphetamine brain levels, and decreases in vitro metabolism of aniline. Morphine-dependent female rats manifested increases in brain levels of amphetamine but no other changes were observed. The in vivo decline of amphetamine from the brain of either male or female morphine-dependent rats was not significantly different from their respective controls. The suggested mechanism for the changes in male morphine-dependent rats is an increase in the brain uptake of amphetamine.

Evidence that Acetaldehyde Irreversibly Impairs Glycoprotein Metabolism in Liver Slices

When liver slices were incubated for 1 hr in a medium containing either acetaldehyde or ethanol and then switched to a fresh medium free from acetaldehyde but containing the appropriate radioactive precursors and reincubated for 3 additional hr, a decreased incorporation of both [14C]glucosamine and [14C]leucine into hepatocellular and secretory proteins was observed. Further studies were carried out comparing the inhibitory effects of ethanol and acetaldehyde on drug metabolism. Higher concentrations of ethanol were necessary to impair aniline hydroxylation when compared to its effect on glycoprotein metabolism. Acetaldehyde did not affect aniline metabolism, suggesting some specificity for its effect on intracellular metabolic functions. Our results show that the acetaldehyde-induced inhibition of glycoprotein synthesis is irreversible under these short term experimental conditions and is not dependent on the physical presence of acetaldehyde in the incubation medium.

Lack of effect of vasectomy on hepatic mixed function monooxygenase enzymes in the rat

The activities of several of the hepatic mixed function monooxygenase enzymes have been studied in vasectomized and sham-operated rats. The in vitro metabolism of aniline and aminopyrine was slightly but insignificantly depressed in the vasectomized group. Likewise, no significant differences in the hepatic microsomal metabolism of androst-4-en-3,17-dione, or the microsomal content of cytochrome P-450, cytochrome b5, or microsomal protein, were detected between the two groups. In addition, vasectomy appeared to have no effect on the weights of the liver, heart, adrenals, kidneys, or testes.

Interaction of nitromethane with reduced hepatic microsomal cytochrome P-450

Nitromethane interacts with sodium dithionite reduced rabbit liver microsomes to generate a difference spectrum characterized by maxima at 388, 423, 454 and 520 nm and minima at 405, 436, 480 and 550 nm. Spectral binding constants ( K s ) of 0.306 ± 0.082 mM ( A max = 0.032 ± 0.004), 0.178 ± 0.029 mM ( A max = 0.040 ± 0.002), and 1.168 ± 0.250 mM ( A max = 0.035 ± 0.006) were calculated for the 388, 423 and 454 nm peaks respectively. These difference spectra are qualitatively different from those previously reported for aromatic nitro compounds [L. A. Sternson and R. E. Gammans, Drug Metab. Dispos. 3, 266 (1975)]. Interaction of nitromethane with microsomes from rabbits pretreated with phenobarbital produced absorbance maxima and minima within 2 nm of the controls. Interaction of nitromethane with reduced microsomes from 3-methyl-cholanthrene (3-MC)-pretreated animals produced ferrohemochromes in which maximal absorbance changes were shifted to maxima at 384, 422, 451 and 514 nm and minima at 407, 433, 473 and 552nm. Peak-height ratios derived from difference spectra generated by the addition of 1 mM nitromethane to the sample cuvette were considerably different depending on whether the microsomes were obtained from control, phenobarbital- or 3-MC-pretreated rabbits and may indicate that phenobarbital, like 3-MC, induces qualitative changes in cytochrome P-450. Nitromethane apparently competes with carbon monoxide for a common binding site. Addition of nitromethane to CO-saturated microsomes reduced the magnitude of the 450 nm peak with a concomitant increase in the 423 nm peak of nitromethane. Similarly, addition of CO to reduced microsomes containing nitromethane caused reduction of the 423 nm peak of nitromethane with a corresponding increase of the 450 nm peak of CO. Nitromethane does not generate difference spectra with oxidized microsomes nor does it alter the K s or A max of aminopyrine, hexobarbital, aniline or zoxazolamine binding spectra. Nitromethane does inhibit the binding of the type II compound, nicotinamide. Addition of nitromethane to incubation flasks enhanced the metabolism of aniline while tending to inhibit the oxidative demethylation of ethylmorphine.

Content and activities of microsomal electron transport components during the development of dieldrin-induced hypertrophic hypoactive endoplasmic reticulum

The addition of dieldrin (50 ppm) to the diet of male and female rats produced an early induction of drug metabolism accompanied by an increase in the microsomal electron transport components. However, the rates of metabolism of aldrin, ethylmorphine, and aniline in males and of aldrin and aniline metabolism in females declined toward control values during subsequent weeks. These decreases occurred despite continuous exposure to dieldrin and continued induction of cytochrome P-450, NADPH oxidase, and NADPH cytochrome c reductase. In several instances, the metabolism of substrate per unit of cytochrome P-450 was lower in dieldrin-induced microsomes than in microsomes from controls. This condition has previously been termed hypoactive hypertrophic endoplasmic reticulum (HHER). In the male, HHER appeared to be related to alterations in the binding of the Type I substrates ethylmorphine and aldrin to cytochrome P-450. However, decreases in aniline metabolism in both males and females appeared to be unrelated to changes in binding. HHER did not develop in microsomes from female rats with respect to ethylmorphine and aldrin metabolism. Our results suggest that the appearance of HHER may not represent a decompensation phase of dieldrin response, but instead may be due to qualitative changes in the induced cytochrome P-450.

The effects of albumin upon the spectral changes and metabolism by the hepatic microsomal fraction

Bovine serum albumin added to the microsomal fraction produced a reverse type I spectral change, and defatted albumin produced a greater change than untreated albumin. Washing the microsomal fraction several times, although removing the albumin, did not eliminate the spectral change. Organic solvent extracts of the defatted albumin after its interaction with microsomes, as well as fractions of the extract from thin-layer chromatography, were capable of causing a type I spectral change in extracted and unextracted microsomes. The microsomal fraction washed with albumin exhibited a greater spectral change with compounds producing a type I difference spectrum (ethylmorphine and laurate); an apparent decreased type II spectral change was obtained with aniline due to the conversion of an absorption peak of ferric cytochrome P-450 from 390 to 420 nm. The metabolism of ethylmorphine was enhanced and the metabolism of aniline was inhibited after washing with albumin. It is concluded that the effects of albumin are due to the displacement of endogenous compounds bound to the cytochrome P-450.

On the inhibition of microsomal drug metabolism by olychlorinated biphenyls (PCB) and related phenolic compounds

The in vitro metabolism of p-nitroanisole, aminopyrine, and aniline by rat liver microsomal monoxygenases were studied in the presence of different polychlorinated biphenyl (PCB) mixtures and some related hydroxybiphenyls. The tested PCB mixtures contained preferably dichloro- (di-CB), tetrachloro- (tetra-CB), or hexachlorobiphenyls (hexa-CB). All PCB were competitive inhibitors of only aminopyrine demethylation by normal microsomes ( K i 22–39 μM). In microsomes of PCB-pretreated rats the aminopyrine demethylation was inhibited noncompetitively by di-CB and hexa-CB whereas tetra-CB remained a competitive inhibitor ( K i 12 μM). Moreover, after PCB pretreatment all PCB were competitive inhibitors of p-nitroanisole demethylation. 2-OH-biphenyl and 4-OH-biphenyl caused competitive inhibition of aminopyrine demethylation and aniline hydroxylation but failed to inhibit p-nitroanisole metabolism by normal microsomes. Chlorinated 4-hydroxybiphenyls inhibited competitively the metabolism of both type I and type II substrates. However, after PCB pretreatment all phenolic compounds caused uncompetitive inhibition of aniline hydroxylation.

Influence of Norethindrone on Drug-Metabolizing Enzymes of Female Rat Liver in Various B-Vitamin Deficiency States

Ingestion of high levels of thiamin significantly decreased the activity of cytochrome P-450, NADPH cytochrome c reductase, and the metabolism of aniline and ethylmorphine. Apparent VmaxS for ethylmorphine N-demethylase and aniline hydroxylase were decreased by high levels of riboflavin even though NADPH cytochrome c reductase was elevated. High levels of dietary pyridoxine significantly decreased only the Vmax for aniline hydroxylase. Generally, norethindrone produces either no change or slight depression of cytochrome P-450 regardless or diet, whereas the administration of norethindrone produced no change or an increase in activity of c reductase and ethylmorphine N-demethylase. Norethindrone induces aniline hydroxylase in animals fed all diets except those deficient in thiamin and riboflavin. The activities of the four parameters of the drug metabolizing system measured in these studies as well as the effects of norethindrone are clearly affected by the dietary status of the animal.

Nadh synergism of microsomal aniline metabolism in the presence of enhancing agents

NADH, which normally has no effect upon the NADPH-dependent metabolism of aniline by the microsomal fraction, exerted a synergistic effect in the presence of various enhancing agents, including acetone. Acetone enhanced the rate of aniline metabolism but inhibited the NADPH-dependent reduction of cytochrome P-450, showing that this is not the rate-limiting step in the metabolism of aniline. It is suggested, however, that acetone might facilitate the transfer of the second electron to cytochrome P-450 and that this may normally be the rate limiting step in the metabolism of aniline. The increase in metabolism did not, of itself, appear to be responsible for the synergistic effect exhibited by NADH in the presence of enhancing agents.

Disposition kinetics of barbital in hamsters with hereditary heart failure

Within a few minutes after oral administration of sodium barbital, 100 mg/kg, in hamsters with hereditary heart failure, the concentration of the drug was found to be lower in the plasma, kidney, brain and striated muscle of these animals than in the tissue of control hamsters. This could be explained by delayed intestinal absorption, as evidenced by greater amounts of drug recovered from the intestine of cardiomyopathy animals a few minutes after barbital administration. However, 6–24 hours later, the concentration of the drug in these tissues was higher than in those of control hamsters; also, the elimination rate of the drug from these tissues was found to be slower than in control animals. This slower elimination rate of the drug from the tissue of animals in heart failure was responsible for the greater drug bioavailability in the tissues, as reflected by the area under the curve of drug concentration in the tissue vs. time. Fromin vitro studies of aniline and aminopyrine metabolism by liver microsomal enzymes of hamsters in heart failure, it is concluded that impairment of drug metabolism by the liver may be partially responsible for the slower drug elimination from the body of patients in heart failure.

Mechanism of inhibition by carbonyl cyanide m-chlorophenylhydrazone and sodium deoxycholate of cytochrome P-450-catalysed hepatic microsomal drug metabolism.

1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the 'binding protein' for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.

Effect of water and food deprivation on hepatic microsomal metabolism of hexobarbital and aniline.

Water deprivation for 48 hr with its accompanying decrease in food intake significantly lowered the in vitro rate of hexobarbital metabolism by hepatic microsomes isolated from male rats. Pair-fed rats allowed water ad libitum had a significantly lower level of hexobarbital metabolism than those deprived of water. Rats starved for 24 hr with or without water also had levels of hexobarbital metabolism significantly lower than their controls; with those animals allowed water ad libitum, the level was significantly lower than for those deprived of water. In vivo hexobarbital "sleeping time" experiments were in general agreement with these results. The in vitro metabolism of aniline was increased in both male and female rats following 24 hr starvation and in female rats (but not males) the effect was greater when water was allowed than when deprived. The differences between hydrated and dehydrated animals were not attributable to reduction in concentration of microsomal protein or the water content of liver. It is concluded that water consumption accentuates the effect of food deprivation on hepatic microsomal metabolism.

In vitro metabolism of aniline by sheep intestine

1. 1. The major metabolite after incubating aniline with sheep intestine was acetanilide. 2. 2. Other metabolites detected in smaller amounts were 2-aminophenol, 4-aminophenol, 2-acetamidophenol and 4-acetamidophenol. 3. 3. The rumen, abomasum duodenum, jejunum, ileum and colon were all able to acetylate aniline. 4. 4. 4-Aminophenol, 4-aminobenzoic acid, 4-anisidine and 4-nitroaniline were also acetylated.

Inhibition of NADPH oxidation and related drug oxidation in liver microsomes by zinc

Rat liver microsomes were incubated in the presence of zinc and the rate of NADPH oxidation and related metabolism of aniline and ethylmorphine by appropriate oxidases were studied. A competitive mechanism of the inhibition of NADPH oxidation by zinc was found, with V max = 10.3 nmoles NADP/min/mg of protein and K i amounting to 7.22 μM zinc. In microsomes dialyzed against EDTA, addition of Mn 2+ but not of Mg 2+ enhanced the rate of NADPH oxidation. A complex relation of Zn 2+ and Mn 2+ in liver microsomes was found, the data not obeying the rigorous treatment for enzyme kinetics. The activity of aniline hydroxylase and ethylmorphine- N-demethylase was inhibited by zinc; 50 per cent inhibition was reached at 60 and 55 μM Zn 2+ respectively. Another microsomal enzyme, glucose 6-phosphatase, independent of NADPH, was not affected by zinc. The content and spectral characteristics of cytochrome P-450 were not affected by zinc. It is concluded that Zn 2+ inhibits oxidation of NADPH and prevents this pyridine nucleotide from functioning in the microsomal electron transport system. The possibility that Zn 2+ may interfere with other ions or enzymes involved in microsomal electron transport cannot be excluded.

Studies on the in vitro metabolism of dimethylnitrosamine by rat liver

The biotransformation of dimethylnitrosamine (DMN) to formaldehyde, generally attributed to the mediation of a demethylase enzyme associated with the microsomal mixed function oxidase system, has been investigated in rat liver preparations. All of the enzyme activity was found in the postmitochondrial fraction and the microsomes contained approximately 50% of this activity. The restoration of total activity resulting from the addition of the cytosol to the microsomal fraction was found to be due to the presence of diffusible, heat-labile constituents in the cytosol. Enzyme kinetic studies revealed that DMN was metabolized to formaldehyde by either a multistep or a multicomponent process. DMN demethylase was found to be relatively stable to storage in contrast to cytochrome P-450 and a number of mixed function oxidase enzyme activities. In spectral interaction studies DMN was found to form an atypical interaction spectrum with either control, phenobarbitone-pretreated or phospholipid-depleted microsomal preparations. DMN had little effect on the Type II spectral interaction of aniline, but non-competitively inhibited the Type I spectral interactions of benzphetamine and biphenyl. Whilst the mixed function oxidase enzyme inhibitors SKF 525A and metyrapone markedly reduced the metabolism of ethylmorphine and aniline, DMN demethylase was little affected by the former compound and appreciably enhanced by metyrapone. Moreover, DMN demethylase was strongly inhibited by a number of metal chelators. Finally, DMN, in vitro, did not inhibit a number of hepatic mixed function oxidases, but significantly reduced anaerobic nitroreductase activity. The results of these studies reveal important differences between the properties of the enzymatic systems which metabolize DMN and mixed function oxidase substrates, and are consistent with the conclusion that the degradation of DMN to formaldehyde by rat liver preparations is a multicomponent system not rate limiting with respect to cytochrome P-450.

Ethanol—A direct inducer of drug metabolism

The inductive effects of chronic phenobarbital and ethanol ingestion on hepatic microsomal drug metabolism were examined in rats consuming different proportions of dietary fat. Eight groups of rats were fed for 1 week on matched amounts of nutritionally adequate liquid diets providing 2, 5, 10 or 41% of calories as fat (safflower oil), two groups at each level. One group at each level received phenobarbital (80 mg/kg daily by intubation), the other getting water. In a parallel experiment, one group at each fat level received 36% of calories as ethanol, the other as sucrose, and these regimens were continued for 1 month. Dietary fat content per se had no significant effect on body weight, liver weight, microsomal protein, cytochrome P-450 content, or hepatic triglycerides (TG) during the 1-week experiment, but significantly increased all these measures in the 1-month experiment. Both phenobarbital and ethanol treatment increased all these measures except body weight, but phenobarbital had a greater effect on liver weight, microsomal protein and cytochrome P-450, while ethanol had a greater effect on hepatic TG and a significant interaction with dietary fat content in relation to TG levels. In contrast, microsomal metabolism of aminopyrine, aniline and meprobamate in vitro was increased approximately equally by phenobarbital and ethanol, while dietary fat content had no effect alone, and no significant interaction with phenobarbital or ethanol. Ethanol, like phenobarbital, therefore, appears to induce microsomal drug metabolism independently of its effects on TG metabolism.