Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--I. Factors that influence NADPH kinetic estimations during mixed function oxidase reactions.

The localisation of significant amounts of nucleotide pyrophosphatase activity in the rat hepatic microsomal fraction results in erroneous values of apparent K_m (NADPH) for aminopyrine-Ndemethylase. This was demonstrated by the inclusion of 20 mM pyrophosphate, which inhibits nucleotide pyrophosphatase activity and reduces the apparent K_m (NADPH) value from 27.9 μM to 7.92 μM. The apparent K_m (NADPH) values determined in the presence of three Type I substrates were not statistically different from each other, but the value in the presence of aniline was lower. The kinetic constants of NADPH for NADPH cytochrome P450-reductase in the presence of either aminopyrine, ethylmorphine or aniline, are also reported.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--IV. Inhibition of aminopyrine and ethylmorphine-N-demethylases by 2.6-dihydroxy-acetophenone.

Pyridine nucleotide kinetics for both aminopyrine and ethylmorphine-N-demethylases were evaluated in the presence of 2.6-dihydroxyacetophenone. (DiHAP) which inhibits certain microsomal mixed function oxidase reactions. The nature of the inhibition in the presence of varying NADPH concentrations was shown to be slope-linear, intercept-linear non-competitive. DiHAP was shown to combine preferentially with the ferric cytochrome P₄₅₀-substrate complex in the presence of aminopyrine, but had a greater affinity for the ferrous cytochrome P₄₅₀-substrate complex when ethylmorphine was present.There were changes in the values of K_ii and K_is without changes in the type of inhibition of aminopyrine-N-demethylase in the presence of both NADH and DiHAP, whereas with ethylmorphine-N-demethylase, the nature of the inhibition kinetics changed to slope-hyperbolic, intercept-hyperbolic non-competitive inhibition in the presence of both NADH and DiHAP.These results are consistent with the proposal that NADH interacts with the ferric cytochrome P₄₅₀-substrate complex prior to the reduction of this complex by an electron from NADPH.     

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism--III. The influence of the 1,4,5,6-tetrahydronicotinamide analogue of NADP on the NADPH kinetics of aminopyrine-N-demethylation.

1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (NADH₃), a structural analogue of NADH, was unable to support the demethylation of aminopyrine or the reduction of the cytochrome P450-aminopyrine complex. However, the combination of NADH₃ with NADPH stimulated the NADPH dependent reduction of the cytochrome P450-aminopyrine complex. There was no significant alteration in the apparent K_m (NADPH) value, but there was an 80 per cent increase in apparent V of NADPH for NADPH-cytochrome P450-reductase (plus aminopyrine) when the kinetic constants were determined in the presence of 100 μM NADH₃. The inclusion of NADH₃ in the medium for aminopyrine demethylation also resulted in a significant increase in apparent V compared to the value obtained in the absence of NADH₃. The results suggest that the structure of NADH, as well as its capacity to donate the electron, is responsible for the NADH mediated increase in aminopyrine metabolism.     

New heterocyclic modifiers of oxidative drug metabolism--II. Steric factors in the interaction of isomeric 2-(naphthyl)methylbenzimidazoles with rat hepatic microsomal cytochrome P-450 and monooxygenase activities

The inhibitory potency of the two isomeric 2-(naphthyl)methylbenzimidazoles towards three monooxygenase activities (aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase and aniline p-hydroxylase) was assessed in hepatic microsomal fractions from untreated, phenobarbitone-induced and beta-naphthoflavone-induced rats. The isomers were essentially equipotent with each other as inhibitors of the phenobarbitone-induced monooxygenases (the ratio of the I50s of the isomers was about 1.0 in each case) but differences between the isomers were noted in the inhibition potencies against three monooxygenase activities from beta-naphthoflavone-induced liver. The isomer 2-(1'-naphthyl)methylbenzimidazole was approximately twice as potent as the 2'-naphthyl isomer against 7-ethoxyresorufin O-deethylase activity, whereas the opposite was observed with respect to 7-ethoxycoumarin O-deethylase inhibition; aniline p-hydroxylase was poorly inhibited by both isomers. The binding affinity and extent of binding, assessed from double-reciprocal plots of spectral binding studies, of the 1'-isomer was much greater than that of the 2'-isomer in beta-naphthoflavone-induced microsomes. Inhibition data in untreated hepatic microsomes were more complex and the finding of principal interest was that the 1'-isomer was poorly inhibitory towards aniline p-hydroxylase activity whereas the 2'-isomer enhanced this activity. These studies suggest that the steric conformations of the isomeric naphthylmethylbenzimidazoles at the cytochrome P-450 active centre determines the extent to which the inhibitors modulate a specific monooxygenase activity, and that multiple binding sites with the capacity to interact to different extents with benzimidazole derivatives are present in P-450 in beta-naphthoflavone-induced hepatic microsomes. The apparent importance of steric conformation as a determinant of inhibition and enhancement of aniline p-hydroxylase in untreated microsomal fractions may well reflect specific interactions with multiple binding sites.     

Impairment of hepatic microsomal drug metabolism in the rat during daily disulfiram administration.

Male rats were given 0.1 or 0.4 g/kg of disulfiram (DS) daily by gavage for up to 12 days, in order to study the effects of chronic DS administration on hepatic microsoma] drug metabolism. Administration of 0.4 g/kg of DS resulted in significant impairment of aniline (ANL) hydroxylase after 1 day. but 2 days of DS treatment were required for significant inhibition of ethylmorphine (EtM) metabolism and depression of cytochrome P-450 levels. At this time, maximum impairment of ANL and EtM metabolism and maximum reduction of cytochrome P-450 levels were seen. Continued administration of DS for 10 additional days produced no further change in these parameters. ANL hydroxylase was also significantly reduced in treated animals throughout a 12-day period during which 0.1 g/kg of DS was given. EtM N-demethylase activity and cytochrome P-450 levels were also reduced in animals so treated, but not until DS had been given for at least 5 days. However, by the end of the 12-day experimental period. EtM metabolism and cytochrome P-450 levels had returned almost to control levels and only ANL hydroxylase was significantly different from control activity. Daily DS administration (0.4 g/kg produced small but significant increases in microsomal cytochrome b₅ levels and in NADPH-cytochrome c reductase activity, whereas NADPH oxidase and NADPH-cytochrome P-450 reductase activities were significantly lower in treated rats. In addition to these effects in vivo, DS competitively inhibited EtM N-demethylase in vitro and bound to cytochrome P-450, producing a type I difference spectrum, thus providing additional mechanisms to account for impairment in vivo of drug metabolism by DS.     

Pyridine nucleotide flux and glutathione oxidation in the cultured rat conceptus.

It is proposed that protection of the developing embryo from chemical and environmental insults that produces oxidative stress requires a proper glutathione (GSH) and pyridine nucleotide status in both the embryo and extra-embryonic membranes. Modulation of pyridine nucleotide flux [NAD(H) and NAD(P)H] in the visceral yolk sac (VYS) by the thiol oxidants diamide and tert-butyl hydroperoxide (tBH) was studied in real time using microfiberoptic sensors in GD 10 rat conceptuses. Consecutive 5-min exposures to 125- and 250-microM diamide resulted in a fluorescence decrease of 14 and 32 Arbitrary Fluorescence Units (AFU). An additional consecutive exposure to 500-microM diamide caused an attenuated decrease followed by a rebound increase of 22 AFU. Consecutive 5-min exposures to tBH at 250 and 500 microM produced fluorescence decreases similar to that of 500 microM diamide, but the decreases were attenuated at 1000 microM. However, there was variability in the rebound increase. A 5-min exposure to tBH (500 microM) alone caused a fluorescence decrease of 14 AFU followed by a rebound increase of 8 AFU. The rate of fluorescence decrease was attenuated by 50% with pretreatment with the glutathione reductase (GSSG-Rd) inhibitor, BCNU (1,3, bis(2 chloroethyl)-1-nitrosourea), indicating that the decrease in surface fluorescence was probably attributable to a decrease in NADPH. Decreases in fluorescence, observed from the surface of the VYS, correlated with decreases in GSH/GSSG ratios in the embryos and the VYS. After exposure to tBH, GSH levels in conceptuses decreased at the end of 5 and 15 min, with a corresponding increase in oxidized glutathione (GSSG) at the end of 3, 5, and 15 min. Our results demonstrate that the increased production of GSSG on exposure to thiol oxidants correlates with a decrease in the reduced pyridine nucleotide, implying the presence of an active GSSG-Rd pathway in the conceptus during organogenesis, and implicating an important role of the pyridine nucleotides in the restoration of GSH homeostasis in the developing rat conceptus during organogenesis.     

Selenium and drug metabolism--II. Independence of glutathione peroxidase and reversibility of hepatic enzyme modulations in deficient mice

Male mice were fed a diet containing less than 0.01 ppm selenium (Se-) for 6 months. A control group received the same diet containing 0.5 ppm selenium (Se+). In the livers of the Se- animals a drastic decrease in glutathione peroxidase (GSH-Px) activity was observed. It reached undetectable levels after 17 days of the Se- diet. At that time, GSH-transferase activity began to increase significantly, followed by changes in many other enzyme activities. After the 60th day, these enzyme modulations had reached a plateau with the following percentage changes compared to controls: GSH-transferases: 320% (1,2-dichloro-4-nitrobenzene), 218% (1-chloro-2,4-dinitrobenzene); glutathione reductase: 160%; ethoxycoumarin deethylase: 330%; cytochrome P-450-hydroperoxidase: 230%; heme oxygenase: 240%; UDP-glucuronyltransferase: 200%; GSH-thioltransferase: 64%; sulphotransferase: 62%; NADPH-cytochrome-P-450-reductase: 65%; flavin-containing mono-oxygenase: 57%. No significant changes were observed for GSH-transferase activity assayed with ethacrynic acid or for microsomal H2O2 formation and aniline hydroxylase activity. In single-pulse repletion experiments by injection of 250 micrograms selenium/kg body wt, different individual time constants for the recovery process of the enzymatic perturbations were observed. The half-times for the recovery ranged from 5.7 hr for the microsomal NADPH-cytochrome-P-450 reductase to over 29 hr for GSH-Px up to 44 hr for part of the GSH-transferase activity. 250 micrograms selenium/kg body wt were needed to restore 50% of GSH-Px activity in the long-term Se- mice compared to Se+ controls. All other enzymatic changes in the Se- mice needed a dose of 7 micrograms selenium/kg body wt for 50% restorage . The results demonstrate that processes other than those related to GSH-Px take place in a later phase of selenium deficiency in mouse liver with a chronologically common beginning. The different repletion and depletion kinetics as well as the different need of these processes for the trace element are discussed with respect to the existence of two separate selenium pools.     

Evidence for the inductive effects of hexafluorobenzene on hepatic microsomal enzymes in the male rat

Acute administration of hexafluorobenzene (HFB) (1200 mg/kg body wt i.p.), induced significant increases in male rat hepatic microsomal protein content (39% increase), microsomal cytochrome P-450 content (150% increase) and microsomal glucose-6-phosphatase activity (25% increase). The assays were carried out 24 h after treatment. Hexafluorobenzene also significantly shortened (by 22%) hexobarbitone-induced sleeping time.     

Guinea pig and rat hepatic microsomal metabolism of monocrotaline.

The comparative metabolism of the pyrrolizidine alkaloid, [14C]monocrotaline, was studied using rat and guinea pig hepatic microsomes. Metabolites were quantified to the nanomole level using HPLC and radiometric detection. Triorthocresylphosphate and carbon monoxide were used to assess the involvement of carboxylesterases and cytochrome P-450 in the hepatic microsomal metabolism of monocrotaline, respectively. Esterase hydrolysis accounted for 92% of the metabolism in the guinea pig; the rat displayed no esterase activity. This result may explain the guinea pig's resistance to pyrrolizidine alkaloid toxicity. Dehydropyrrole was found to be the major pyrrolic metabolite in the guinea pig, although colorimetric analysis indicated multiple pyrrolic moieties in the rat microsomal incubations.     

Propylene glycol as a drug solvent in the study of hepatic microsomal enzyme metabolism in the rat

Propylene glycol administered ip to rats at a dose of 4 ml/kg twice a day for 3 days caused a significant elevation of the in vitro hepatic microsomal metabolism of aniline and p-nitroanisole, but at the same time caused a significant decrease in aminopyrine demethylation with no significant change in p-nitrobenzoic acid metabolism. There was no change in cytochrome P-450 concentrations with this treatment, but the response, which was dose-dependent, could not be repeated by the in vitro addition of propylene glycol to incubating systems. In vivo inhibition of drug metabolism was demonstrated by increased hexobarbital sleeping times and zoxazolamine paralysis times after propylene glycol treatment. When administered concurrently with 75 mg of phenobarbital/kg for 3 days. an additive response was obtained with aniline and p-nitroanisole metabolism. Phenobarbital appeared to abolish the depressant effects observed in aminopyrine metabolism and the slight changes observed in p-nitrobenzoic acid reduction. Kinetic studies with microsomes from treated rats showed a reduced K_m and V_max for aminopyrine demethylation, while for aniline hydroxylation there was an increase in V_max but an unchanged K_m value.     

Effect of acute and repeated chlordimeform treatment on rat hepatic microsomal drug metabolizing enzymes

The effect of chlordimeform (CDM) treatment on the hepatic microsomal drug metabolizing enzymes was examined in male and female rats following either acute or repeated treatment. After acute administration of chlordimeform (100 mg/kg, i.p., 1 hour before killing) differential effects were observed in various parameters of the hepatic microsomal mixed function oxidase system with significant decreases in ethylmorphine metabolism, cytochrome P-450 content, NADPH cytochrome c reductase, and in the spectral binding of hexobarbital and aniline while no changes were found in the metabolism of aniline or p-nitroanisole. Durations of zoxazolamine-induced paralysis and pentobarbital-induced hypnosis were increased significantly after acute CDM administration. Following repeated administration of CDM (75 mg/kg, i.p., for 4 days) to adult male rats, a decrease was observed in zoxazolamine-induced paralysis time while pentobarbital-induced hypnosis was not altered. Metabolism studies using isolated hepatic microsomal fractions showed a decreased rate of biotransformation of ethylmorphine and aniline while the activity of p-nitroanisole O-demethylase was not changed. No differences were found in cytochrome P-450 levels whereas microsomal spectral binding of hexobarbital was reduced while that of aniline was not affected. Following acute or repeated administration of CDM to adult female rats, decreases in the hepatic microsomal metabolism of aniline, but not ethylmorphine or p-nitroanisole, were observed. Addition of CDM to microsomal suspensions yielded a Type I binding curve.     

Biochemical basis for impaired drug metabolism in tumor-bearing rats: Evidence for altered regulation of hepatic microsomal hemeprotein synthesis

The pharmacologie effects of many drugs are enhanced in animals bearing tumors. This apparently stems from a decrease in the microsomal metabolism of these compounds in liver cells, owing to a decrease in either the activities of certain enzymes or in the content of cytochrome P-450 in microsomes, or both. Since impaired drug metabolism may have a direct bearing on the outcome of chemotherapy, a study of the biochemical basis for this alteration was begun. In female Sprague-Dawley rats bearing Walker 256 solid tumors i.m., pentobarbital metabolism was impaired as judged from prolonged sleeping-times. This effect was accompanied by a decrease in the microsomal content of cytochrome P-450. Analysis of hepatic microsomes by sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that a microsomal protein of ~53,000 daltons was diminished in livers of tumor-bearing animals, when compared to normal controls, as determined by staining with Coomassie Blue. When rats were injected with [³H]leucine, although the dpm/100 μg of microsomal protein were the same for both normal and tumor-bearing rats, ~40% less ³H was associated with the microsomal proteins in the cytochrome P-450 region (~43,000 to ~58,000 daltons) of tumor-bearing animals than with those of normal controls. This was attributed to a decrease in the rate of synthesis of these, but not of other, microsomal proteins. Either staining of microsomal proteins with 3,3',5,5'-tetramethyl-benzidine, or labelling of them with δ-[¹⁴C]aminolevulinic acid, revealed that the overall content of microsomal hemeproteins of tumor-bearing rats was reduced considerably, when compared to normal controls (~70% as judged by the incorporation of ¹⁴C). These observations prompted investigations of the apparent rate-limiting synthetic and degradative enzymes of heme. We found that in the livers of 7-day tumor-bearing rats, the activity of δ-aminolevulinic acid synthetase was only 16% of control activity; conversely, the activity of hepatic microsomal heme oxygenase in the tumor-bearing rats was nearly eight-times greater than that of the normal animals. Together, these data indicate that perturbations in heme- and hemeprotein-synthesis cause the reduced content of cytochrome P-450 seen in tumor-bearing rats, and they provide a partial explanation for diminished drug metabolism by hepatic microsomes in the presence of a growing, transplantable, non-hepatic tumor.     

In vivo and in vitro effects of thiuram disulfides and dithiocarbamates on hepatic microsomal drug metabolism in the rat.

The effects of tetramethylthiuram disulfide (TMTDS) and dimethyldithiocarbamate (DMDTC) on hepatic microsomal drug metabolism were studied after in vivo administration to male rats (1 g/kg, po) and after in vitro addition of the compounds to control microsomal suspensions. Results were compared to the effects of the known inhibitor of drug metabolism, disulfiram (DS, tetraethylthiuram disulfide), its reduced metabolite diethyldithiocarbamate (DDTC), and a common metabolite of all four compounds, carbon disulfide. Twenty-four hours after administration of the disulfides (TMTDS and DS) impairment of microsomal aniline hydroxylase and carboxylesterase activities was observed, while cytochrome P-450 and ethylmorphine N-demethylase activity were unchanged. The reduced thiols (DMDTC and DDTC) caused significant decreases in microsomal cytochrome P-450 and impaired all three microsomal enzymes. In vitro addition of all four compounds to control microsomes at a final concentration of 1 mm impaired aniline hydroxylase and carboxylesterase activity. However, only in vitro addition of TMTDS and DS significantly decreased ethylmorphine N-demethylase activity. This effect may be due to the fact that TMTDS and DS bind to cytochrome P-450 producing a type I spectral change and may, therefore, compete with the type I compound, ethylmorphine, for binding sites on cytochrome P-450. Impairment of aniline hydroxylase activity is the most sensitive indicator of an inhibitory effect of all four compounds on microsomal drug metabolism; this action is not dependent on decreases in cytochrome P-450. In vivo impairment of ethylmorphine N-demethylation by DMDTC and DDTC is related to decreases in microsomal cytochrome P-450 produced by these compounds, which may be due, in part, to their decomposition to CS₂ in the gut. The data indicate that industrial or agricultural exposure to compounds such as TMTDS and DMDTC may impair hepatic metabolism, and thereby enhance pharmacological activity of drugs taken by exposed individuals.     

Effect of acetone administered in vivo upon hepatic microsomal drug metabolizing activity in the rat

The effects of acetone administered to female rats in vivo, upon the metabolism of drugs by the hepatic microsomal subcellular fraction have been studied. There is a rapid increase, maximal at 0·5 hr in aniline p-hydroxylation of 69 per cent, and an inhibition of aminopyrine N-demethylation of 61 per cent. Thereafter levels return to control values. There is a slower increase in aniline p-hydroxylation of 168 per cent, maximal at 48 hr. whilst aminopyrine N-demethylation is unaltered. Cycloheximide has no effect upon the changes in activity after 0·5 hr but blocks the increase in aniline p-hydroxylation 48 hr after the administration of acetone. It is suggested that acetone may contribute to the changes in drug metabolizing activity found in diabetic animals.