Renal necrosis,glutathione depletion,and covalent binding after acetaminophen

A dose-dependent, acute renal necrosis occurred in male Fischer rats following a single subcutaneous injection of acetaminophen. Doses of [³H]acetaminophen (750–900 mg/kg) causing renal and hepatic necrosis in rats markedly depleted target organ glutathione and resulted in large amounts of radiolabeled metabolite being bound to renal and hepatic protein. Pretreatment with cobalt chloride, an inhibitor of hepatic and renal drug metabolism, decreased both the irreversible binding of metabolite and the glutathione depletion in target organ tissues while concomitantly protecting against tissue damage. Pretreatment with 3-methylcholanthrene enhanced hepatic necrosis and covalent binding of metabolite to hepatic protein in vivo and to microsomal protein in vitro but had little effect on the corresponding renal parameters. Covalent binding of radiolabeled acetaminophen to rat renal or hepatic microsomes was enzyme dependent and required NADPH and oxygen. Thus, acetaminophen-induced renal and hepatic necrosis apparently result from in situ activation of acetaminophen to a chemically reactive species capable of covalently binding to target organ macromolecules.     

Hepatic microsomal metabolism and covalent binding of 2,4-dinitrotoluene

The effects of 2,4-dinitrotoluene (2,4-DNT) on xenobiotic metabolizing enzymes and the hepatic metabolism and covalent binding of this compound to microsomal proteins in vitro were studied. Male Fischer-344 rats received po doses of DNT daily for 5 days at 14, 35, and 70 mg/kg/day. Hepatic oxygen-insensitive cytosolic azoreductase activity was increased and microsomal nitroreductase was decreased by DNT treatments. A small but significant increase in liver/body weight ratio and in hepatic cytochromes P-450 and b₅ occurred in the absence of changes in microsomal biphenyl hydroxylase or aryl hydrocarbon hydroxylase activities. The patterns of in vitro microsomal metabolism of DNT were dependent on oxygen tension: under aerobic conditions, 2,4-dinitrobenzyl alcohol (DNBAlc) was the major metabolite whereas under anaerobic conditions no DNBAlc was detected; 2-amino-4-nitrotoluene (2A4NT) and 4-amino-2-nitrotoluene (4A2NT) were the major metabolites. Pretreatment of rats with phenobarbital or Aroclor 1254 increased the metabolism of 2,4-DNT to DNBAlc by six- to sevenfold. Metabolism to the alcohol was inhibited by SKF-525A. These data suggested that oxidative metabolism of 2,4-DNT to DNBAlc was mediated by cytochrome P-450-dependent mixed-function oxidases. Covalent binding studies showed that a maximum of only 7 pmol of 2,4-DNT-derived radioactivity was bound per milligram of microsomal protein per hour; this binding was increased to 1.0 nmol bound/mg protein/hr in microsomes from phenobarbital of Aroclor 1254-pretreated rats. It is concluded that 2,4-DNT treatment had little effect on the activity of some hepatic xenobiotic metabolizing enzymes and was readily metabolized by liver preparations in vitro. The pathways of in vitro metabolism were dependent on oxygen tension. This in vitro metabolism produced mostly polar metabolites which did not bind appreciably to microsomal macromolecules.     

In vivo studies on the target tissue metabolism,covalent binding,glutathione depletion,and toxicity of 4-ipomeanol in birds,species deficient in pulmonary enzymes for metabolic activation

Comparative studies on the target organ for covalent binding and toxicity by 4-ipomeanol, a furan derivative metabolically activated by and toxic to the nonciliated bronchiolar epithelial (Clara) cells in mammalian lung, were performed in birds. In birds, whose lungs lack the typical pulmonary ciliated and nonciliated bronchiolar cells and are deficient in enzymes necessary for the metabolic activation of 4-ipomeanol, the target organ for toxicity and covalent binding by 4-ipomeanol was the liver. Tissue damage was not observed in lungs or kidneys of either Japanese quail or roosters at any dose of 4-ipomeanol tested. Likewise, the levels of irreversibly bound 4-ipomeanol metabolites were highest in liver, with much lower levels in lungs, kidneys, and all other organs studied. In addition, toxic doses of 4-ipomeanol markedly depleted hepatic, but not pulmonary or renal glutathione in Japanese quail. The covalent binding of 4-ipomeanol metabolites and concomitant depletion of tissue glutathione in the liver were both time and dose dependent. In quail, over a dose range of 5 to 75 mg/kg, no dose threshold for covalent binding or hepatic damage which depended upon substantial glutathione depletion was observed. Pretreatment of quail with phenobarbital had no effect on liver cytochrome P-450 levels, nor did it have any effect on tissue covalent binding or toxicity by 4-ipomeanol. Although 3-methylcholanthrene pretreatment nearly tripled the microsomal cytochrome P-450 levels in quail liver, it had relatively little or no effect on the hepatic covalent binding or toxicity by 4-ipomeanol. Prior treatment with the drug metabolism inhibitor, piperonyl butoxide, markedly decreased the covalent binding, glutathione depletion, and the toxicity of 4-ipomeanol. Treatment of quail with diethylmaleate produced a dose-dependent depletion of hepatic, renal, and pulmonary glutathione, and it markedly increased hepatic covalent binding by 4-ipomeanol, but had no effect on renal or pulmonary covalent binding. Diethyl maleate markedly enhanced the hepatic necrosis by 4-ipomeanol. The results are consistent with the view that 4-ipomeanol is metabolized preferentially in quail liver to highly reactive metabolites which can be detoxified by glutathione. It appears unlikely however, that differences in detoxification via glutathione are the primary determinants of the remarkable tissue selectivity for covalent binding and toxicity of 4-ipomeanol in different animal species in vivo. The present studies support the view that the tissue selectivity of 4-ipomeanol resides primarily in the ability of the target tissue to rapidly metabolize 4-ipomeanol to its ultimate toxic metabolite(s). These investigations also suggest that the bird may be a potentially useful animal model in studies on the relationship of target organ toxicity to the formation of reactive metabolites by other chemicals requiring metabolic activation.     

Interaction of capsaicinoids with drug-metabolizing systems. Relationship to toxicity

The interaction of capsaicin with microsomal drug-metabolizing systems was assessed to determine the role that bioactivation of capsaicin may play in the induction of hepatotoxicity and neurotoxicity. Capsaicin produced a type I spectral change in rat hepatic microsomes in a high affinity (K_s = 8 μM) concentration-dependent manner and was approximately equipotent with SKF-525A in inhibiting ethylmorphine demethylation. Capsaicin (10 mg/kg, s.c.) inhibited biotransformation in vivo as measured by prolongation of pentobarbital sleep time. Reactive metabolites of capsaicin were studied using [³H]dihydrocapsaicin. [³H]Dihydrocapsaicin bound irreversiblyto hepatic microsomal protein after in vitro incubation or in vitro administration. No binding was observed in spinal cord or brain. Although the bioactivation and subsequent covalent binding of capsaicin equivalents may initiate events associated with the hepatotoxicity of capsaicin, it appears that capsaicin-induced neuropathy does not involve covalent interactions with neuroproteins in spinal cord or brain.     

Hepatic drug metabolism in rats with experimental chronic renal failure

The activities of several hepatic microsomal, mitochondrial, and cytosolic drug-metabolizing enzymes, as well as the components of the cytochrome P-450 system, were determined in vitro for control, sham-operated, and uremic rats. Chronic renal failure (CRF) was produced by a two-stage surgical procedure. In this model, the animals were maintained for 21 days postoperatively before assay. During this time, serum urea nitrogen (SUN) levels rose from control levels of 21 mg/dl to an average of 63 mg/dl. EnzymesassayedincludedmicrosomalN-, O-, and S-demethylases, esterase, and UDP-glucuronyl transferase; monoamine oxidase; and alcohol dehydrogenase. CRF caused decreases of 24–32% in N- and O-demethylase activities, while S-demethylase, esterase, UDP-glucuronyl transferase, and monoamine oxidase activities were not altered significantly. Alcohol dehydrogenase activity was increased 71%. In addition, the functional components of the microsomal mixed-function oxidase system were assayed. CRF caused a 26% decrease in cytochrome P-450 levels, as compared to shamoperated controls, but cytochrome b₅ and NADPH-cytochrome c (P-450) reductase were not altered. CRF caused an increase in hexobarbital sleeping time of more than 7-fold. In each case, alterations in enzyme activities or cytochrome P-450 correlated with the extent of renal failure, as determined by elevated SUN levels.     

In vitro binding of metabolically activated [14C]-ledakrin, or 1-nitro-9-14C-(3'-dimethylamino-N-propylamino) acridine, a new antitumor and DNA cross-linking agent, to macromolecules of subcellular fractions isolated from rat liver and HeLa cells.

A new antitumor drug named Ledakrin or Nitracrine, 1-nitro-9-(3′-dimethylamino-n-ropylamino)acridine, which has been shown to be a latent DNA cross-linking agent in both mammalian and bacterial cells, was investigated to determine whether it irreversibly binds to cellular macromolecules in vitro. Incubation of [¹⁴C]-Ledakrin with subcellular fractions of either rat liver or HeLa cells in the presence of a NADPH-regenerating system led to an irreversible binding of as much as about 30 per cent of the drug radioactivity (up to 57 nmoles/mg protein) with subcellular macromolecules after exhaustive extraction with cold trichloroacetic acid, ethanol and ether. The binding seems to be covalent. The difference between irreversible binding in the presence of intact and heat-inactivated enzymatic subcellular fractions indicates that the metabolites of the drug, rather than Ledakrin itself, are responsible for the irreversible binding with macromolecules in vitro. The dependence of the macromolecule binding of Ledakrin radioactivity with subcellular macromolecules of post-mitochondrial or microsomes on microsomal enzyme, on substrate concentration, oxygen and NADPH, as well as induction of this reaction with phenobarbital or 3-methylcholanthrene rat pretreatment, indicates that the oxidative macromolecule binding of Ledakrin metabolites is catalyzed in vitro by mixed-function oxidases, probably by the unspecific drug metabolizing system involving cytochrome P-450 of liver microsomes. Irreversible binding in vitro was less pronounced under anaerobic conditions than in incubations under air. The reductive irreversible macromolecule binding of Ledakrin metabolites is catalyzed in vitro by unknown rat liver enzymes resistant to allopurinol or dicoumarol inhibition. To account for oxidative binding of Ledakrin through a metabolic activation in vitro, three pathways are considered likely: (1) C-hydroxylation; (2) N-alkylhydroxylamine formation and (3) aromatic N-hydroxylation. The elevated oxidative macromolecule binding of Ledakrin metabolites when an epoxide hydrase was inhibited is evidence for the formation of a reactive acridine epoxide intermediate during the drug binding reaction. The ineffectiveness of SKF 525-A, a specific inhibitor of microsomal C-oxidation, in decreasing the irreversible binding is indirect evidence that besides microsomal C-oxidation other oxidative activations are involved. Arylhydroxylamines formed under air in vitro with all subcellular fractions studied, as determined colorimetrically. An aliphatic N-hydroxylation of the amino group of Ledakrin side chain can be involved in the drug oxidative binding in vitro too. To account for irreversible macromolecule binding of Ledakrin metabolites under highly anaerobic conditions, a N-arylhydroxylamine arising from the nitro group reduction seems to be an intermediate determined colorimetrically. Moreover, the metabolism of the nitro group of Ledakrin to its parent 1-N-hydroxylamine was directly related to the irreversible binding of the drug metabolite(s) with subcellular macromolecules in vitro under nitrogen. Reduced glutathione trapped in vitro reactive electrophilic Ledakrin metabolite(s) formed most probably by establishing a chemically stable thioether bond and thereby protected macromolecules against irreversible binding. Finally, four reactive species are postulated in the irreversible macromolecule binding of Ledakrin metabolites in vitro.     

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.     

Mechanism of chloroform nephrotoxicity: IV. Phenobarbital potentiation of in vitro chloroform metabolism and toxicity in rabbit kidneys

Metabolism of chloroform (CHCl₃) by a cytochrome P-450-dependent process to a reactive metabolite may be required to elicit hepatic and renal toxicities. Specific inducers or inhibitors of cytochrome P-450 have been employed frequently as tools to demonstrate this relationship between metabolism and toxicity in the liver. The experiments reported herein were designed to identify the relationship between metabolism and toxicity of CHCl₃ in the kidney of rabbits, a species in which renal cytochrome P-450 is induced by phenobarbital. Pretreatment with phenobarbital enhanced the toxic response of renal cortical slices to CHCl₃in vitro as indicated by decreased p-aminohippurate and tetraethylammonium accumulation. Phenobarbital pretreatment also potentiated in vitro¹⁴CHCl₃ metabolism to ¹⁴CO₂ and covalently bound radioactivity in rabbit renal cortical slices and microsomes. Addition of l-cysteine significantly reduced covalent binding in renal microsomes from both phenobarbital-treated and control rabbits and was associated with the formation of the radioactive phosgene-cysteine conjugate 2-oxothiazolidine-4-carboxylic acid (OTZ). Formation of OTZ was enhanced in renal microsomes from phenobarbital-pretreated rabbits. Thus, this in vitro model supports the hypothesis that the kidney metabolizes CHCl₃ to the nephrotoxic metabolite, phosgene.     

Effects of steroid hormones in vitro on adrenal xenobiotic metabolism in the guinea pig

Studies were carried out to determine the effects of steroid hormones in vitro on adrenal and hepatic microsomal benzphetamine demethylation and benzo[a] pyrene hydroxylation. Testosterone inhibited adrenal drug metabolism but had no effect on hepatic enzymes, whereas 6β-hydroxytestosterone had no effect in either tissue. All of the corticosteroids tested (cortisol, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, progesterone, and 17-hydroxyprogesterone) produced a concentration-dependent inhibition of adrenal drug metabolism, but had little or no effect on hepatic metabolism. The 17-deoxy-steroids were more potent inhibitors of adrenal metabolism than were their 17-hydroxylated counterparts. Cortisol was a potent inhibitor of adrenal benzphetamine and benzo[a]pyrene metabolism, produced a type I difference spectrum in adrenal microsomes, and diminished the magnitude of the benzphetamine-induced spectrum; 6 β-hydroxycortisol had none of these effects. Prior addition of benzphetamine to adrenal microsomes reduced the size of cortisol-induced spectral change. The results demonstrate that the effects of corticosteroids in vitro are relatively specific for adrenal enzymes and established a close association between the 6 β-hydroxylase and some drug-metabolizing enzymes. Adrenal steroids may have an important role in the regulation of adrenal xenobiotic metabolism.     

Studies on the activation of a model furan compound—toxicity and covalent binding of 2-(N-ethylcarbamoylhydroxymethyl)furan

2-(N-ethylcarbamoylhydroxymethyl)furan was studied as a simple model for the entire class of toxic furans. This compound is toxic to both the lung and liver of the rat—covalent binding occurs predominantly in these target organs. The model was covalently bound to protein and nucleic acids throughout the cell; the enzymes responsible for activating the compound to a form(s) capable of covalently binding to these tissue macromolecules are localized in the microsomal and, to a much lesser extent, nuclear fractions of the cells of the target organs. Binding and toxicity appear to involve the furan moiety of the compound; this binding can be inhibited by added nucleophiles, but not by epoxide hydrase inhibitors. Studies utilizing both microsomes and highly purified reconstituted cytochrome P-450 systems for activation indicate that the reactive metabolite(s) possesses a certain amount of stability, in agreement with the observed distribution of the compound in vivo.     

Formation of inactive cytochrome P-450 Fe(II)-metabolite complexes with several erythromycin derivatives but not with josamycin and midecamycin in rats

The effects of some macrolides (4mmoles·kg^?1 p.o. daily for 4 days in vivo; 0.3mM in vitro) on hepatic drug-metabolizing enzymes in rats were compared. One group of macrolides including previously studied compounds (oleandomycin, erythromycin and troleandomycin), as well as several other erythromycin derivatives, showed induction of microsomal enzymes and formation of inactive cytochrome P-450-metabolite complexes in vivo; this formation increased in the order: oleandomycin, erythromycin ethylsuccinate, erythromycin stearate, erythromycin itself, erythromycin propionate, erythromycin estolate and troleandomycin. Troleandomycin and, to a lesser extent, erythromycin and oleandomycin formed cytochrome P-450-metabolite complexes when incubated in vitro with 1 mM NADPH and microsomes from rats pretreated with troleandomycin or phenobarbital, but not with microsomes from control rats or rats treated with 3-methylcholanthrene. In contrast, two other macrolides, josamycin and midecamycin, showed no induction of microsomal enzymes and no detectable formation of cytochrome P-450-metabolite complexes in vivo. In vitro, these macrolides failed to form detectable complexes even with microsomes from rats pretreated with troleandomycin or phenobarbital. Hexobarbital sleeping time was unaffected by preadministration of josamycin or midecamycin (4 mmoles·kg^?1 p.o.) 2 hr earlier; the in vitro activity of hexobarbital hydroxylase was not inhibited by 0.3 mM josamycin or midecamycin. We conclude that, unlike several erythromycin derivatives, josamycin and midecamycin do not form inactive cytochrome P-450-metabolite complexes in rats.     

Species and strain differences in target organ alkylation and toxicity by 4-ipomeanol. Predictive value of covalent binding in studies of target organ toxicities by reactive metabolites.

The organ specificities of the in vivo covalent binding of 4-ipomeanol were closely correlated with the patterns of organ-specific damage by 4-ipomeanol in several different animal species and strains. In all species tested, the lung was a major target for 4-ipomeanol covalent binding and toxicity. In the hamster and the mouse, 4-ipomeanol caused liver necrosis and kidney necrosis, respectively, in addition to pulmonary damage. Correspondingly, high levels of covalent binding of 4-ipomeanol occurred in these target organs in these species. These in vivo results, in addition to studies of the in vitro covalent binding of 4-ipomeanol in microsome preparations from the various target tissues, were consistent with the view that the organ-specific toxicities of 4-ipomeanol were caused by a highly reactive 4-ipomeanol metabolite(s) primarily produced in situ in the respective target tissues. The present results suggest that studies of both the in vivo and the in vitro covalent binding of 4-ipomeanol may have some utility in predicting the target organ specificity of 4-ipomeanol toxicity in other species. The present investigations also have identified some relevant new in vivo toxicity models for future studies of the relationships between the metabolism and the toxicity of 4-ipomeanol.     

Inhibition of hepatic microsomal drug-metabolizing enzymes by habu snake (trimeresurus flavoviridis) venom fractions

An inhibitor of hepatic microsomal drug-metabolizing enzyme activity was isolated from the venom of the Habu snake (Trimeresurus flavoviridis) by gel filtration through Sephadex G-100 and Amberlite CG-50 column chromatography. The inhibitor, designated R-CG-50-2, gave one band on sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis and caused high hemorrhagic activity when administered i.c. to rabbits. R-CG-50-2 inhibited the drug-metabolizing enzyme system even after being heated at 70° for 5 min, in spite of a complete loss of hemorrhagic activity. Cytochrome P-450 content and NADPH-cytochrome c reductase activity of rat hepatic microsomes were decreased by administration in vivo either R-CG-50-2 or heated R-CG-50-2. The extent of the decrease was greater with unheated R-CG-50-2 than with heated R-CG-50-2. In both cases, cytochrome P-420, the inactive form of cytochrome P-450, was not detected. Lipid peroxidation in hepatic microsomes was also decreased by administration of unheated R-CG-50-2 but the decrease was not significant. In an in vitro experiment, both heated and unheated R-CG-50-2 decreased the cytochrome P-450 content and the NADPH-cytochrome c reductase activity of microsomes, but, unlike the results in vivo, cytochrome P-450 was converted to cytochrome P-420. Microsomal lipid peroxidation was greatly inhibited by either heated or unheated R-CG-50-2 in vitro. It was concluded that the inhibition of the hepatic microsomal drug-metabolizing enzyme system by either heated or unheated R-CG-50-2 may have been due to the decrease in the cytochrome P-450 content and the NADPH-cytochrome c reductase activity, and that lipid peroxidation may not have had an effect on the inhibition.     

Varying effects of sulfhydryl nucleophiles on acetaminophen oxidation and sulfhydryl adduct formation

The effects of glutathione, cysteine, N-acetylcysteine, cysteamine, α-mercaptopropionylglycine and methionine on the NADPH-dependent metabolism and covalent binding of acetaminophen have been examined in mouse liver microsomal incubations. With the exception of methionine, all of the nucleophiles decreased covalent binding by forming adducts with the electrophilic metabolite of acetaminophen. The adducts were measured quantitatively by high pressure liquid chromatography. In contrast to glutathione, N-acetylcysteine and α-mercaptopropionylglycine, both cysteamine and cysteine in high concentrations also decreased covalent binding of acetaminophen through another mechanism, inhibition of the formation of the reactive acetaminophen metabolite. These results indicate that both inhibition of metabolite formation and detoxification of metabolite by sulfhydryl adduct formation are mechanisms that can be important in reducing acetaminophen toxicity in overdosed patients treated with these nucleophiles.     

Covalent binding of procainamide in vitro and in vivo to hepatic protein in mice

Procainamide (PA) formed reactive metabolites capable of covalently binding to protein both in vivo and in vitro. The in vitro covalent binding of PA to washed hepatic microsomal protein prepared from control male mice was dependent upon mixed-function oxidase activity. The binding was proportional with time and protein concentration. Glutathione and SKF 525-A inhibited the in vitro covalent binding by 88 and 51%, respectively. The addition of NaF to the incubation medium produced a concentration-dependent decrease in covalent binding. Covalent binding of N-acetylprocainamide in vitro was 90% less than that of procainamide and was not increased by NaF. The in vivo covalent binding of PA to hepatic protein in male mice was increased with phenobarbital and 3-methylcholanthrene pretreatment, resulting in increase in binding of 29 and 56%, respectively, compared to control mice. Pretreatment of mice with SKF 525-A inhibited binding by 39%. Depletion of hepatic glutathione with diethyl maleate pretreatment increased the amount of covalent binding in vivo. Bioactivation of PA by hepatic microsomal enzymes in the mouse produces a metabolite capable of covalent interactions with cellular macromolecules.     

Effects of ethanol on hepatic microsomal drug-metabolizing enzymes in the rat

The activities of several hepatic microsomal drug-metabolizing enzymes were determined in male and female rats after administration of 20% ethanol or an isocaloric amount of glucose in drinking water for 10–49 days. The aniline hydroxylase activity increased, whereas the activities of both pentobarbital hydroxylase and benzphetamine N-demethylase were decreased in male rats given ethanol and killed without ethanol withdrawal. Twenty-four hr after removal of ethanol, the aniline hydroxylase remained elevated but a striking increase of both pentobarbital hydroxylase and benzphetamine N-demethylase above control values occurred. Six days later, all three of these microsomal enzymes returned to normal control values. The reduction in pentobarbital hydroxylase and benzphetamine N-demethylase could not be attributed simply to high endogenous ethanol levels since: (1) addition of high concentrations of ethanol in vitro inhibited microsomal aniline hydroxylase and pentobarbital hydroxylase but did not reduce benzphetamine N-demethylase; and (2) acute administration of ethanol by gastric tube, which markedly elevated the blood ethanol level, did not result in a decline in the enzyme activities. These two findings suggest that the observed decrease in microsomal enzymes in male rats required persistent ethanol exposure. In contrast to male rats, female rats given ethanol for 28 days showed a significant increase in aniline hydroxylase activity, but the activities of pentobarbital hydroxylase and benzphetamine N-demethylase were not decreased. Moreover, administration of ethanol for 28 days to female rats did not reduce the response of these three enzyme activities to pentobarbital administration. It is concluded that the effects of chronic ethanol ingestion on the hepatic microsomal drug-metabolizing enzymes are complex and depend on sex, exposure to other agents and, most importantly, on the duration and proximity of ethanol intake.     

Vinylidene chloride: Its metabolism by hepatic microsomal cytochrome P-450 in vitro

The effects of inducing agents on the binding and metabolism of vinylidene chloride by hepatic microsomal cytochrome P-450 are reported. Hanes plots for the Type I binding of vinylidene chloride to cytochrome P-450 were biphasic with hepatic microsomes from untreated and β-naphthoflavone- or phenobarbital-treated male rats. Neither pretreatment affected the value of the K_s (ca. 0.22 mM) for the high-affinity binding site for vinylidene chloride, while phenobarbital induction, but not β-naphthoflavone treatment, decreased the value of the K_s for the low-affinity site by 3-fold to ca. 1.6 mM. The maximum extents of binding (ΔA_max or ΔA_max/nmole cytochrome P-450) of vinylidene chloride were decreased or not affected by β-naphthoflavone induction, while ΔA_max but not ΔA_max/ nmole cytochrome P-450 was elevated following phenobarbital induction. The rate of vinylidene chloride stimulated CO-inhibitable hepatic microsomal NADPH oxidation was not affected by β-naphthoflavone induction, but was increased significantly following phenobarbital induction. Vinylidene chloride was converted to monochloroacetate and to the previously unreported metabolite, dichloroacetaldehyde, by hepatic microsomes plus NADPH-generating system. Measurable levels of 2-mono- and 2,2-dichloroethanol, and of chloroacetaldehyde and dichloroacetic acid, were not produced from vinylidene chloride under these conditions. SKF-525A and CO:O₂ (80:20, v/v) inhibited the conversion of vinylidene chloride to monochloroacetate and dichloroacetaldehyde by approximately 60%. The rates of production of monochloroacetate and dichloroacetaldehyde in the presence of NADH were ca. 15% of the rates seen with NADPH-generating system. The rate of monochloroacetate production per mg microsomal protein was not affected by β-naphthoflavone induction but was increased slightly following phenobarbital induction. In contrast, the V_max values per mg microsomal protein for the metabolism of vinylidene chloride to dichloroacetaldehyde were not elevated by either pretreatment. Incubation of vinylidene chloride, NADPH-generating system, EDTA and hepatic microsomes from untreated and β-naphthoflavone- or phenobarbital-treated rats did not result in any significant alterations in the levels of microsomal cytochrome P-450 and heme or in the covalent binding of the mono- or dichloroacetyl moieties to microsomal or buffer constituents, but it did result in significant production of H₂O₂. It is concluded that multiple forms of cytochrome P-450 bind and metabolize vinylidene chloride. However, the form of the enzyme elevated by phenobarbital plays, at most, a minor role in these processes, while the form induced by β-naphthoflavone is not involved in either process. The effect of metabolism of vinylidene chloride by cytochrome P-450 on the relationship between the metabolism and toxicity of vinylidene chloride in vivo and its mutagenicity in vitro is considered.     

Hexobarbital sleeping time and drug metabolism in rats with ligated bile ducts—A lack of correlation

Hexobarbital sleeping time is commonly used as an index of the activity of hepatic microsomal drug-metabolizing enzymes in animals. This report describes anomalies between hexobarbital sleeping time and the rate of metabolism in vitro by microsomal enzymes in rats after bile duct ligation (BDL). The duration of hexobarbital sleeping time, 2–24 hr after BDL, was approximately twice that of sham-operated controls. No significant decrease in the activity of microsomal aminopyrine demethylase, aniline hydroxylase, hexobarbital oxidase or the amount of cytochrome P-450 was detected during this period. A further prolongation of hexobarbital sleeping time was observed 48–72 hr after BDL, and this was accompanied by a significant impairment of drug metabolism in vitro. The effect of BDL on hexobarbital sleeping time was independent of the route of administration. Thiopental sleeping time was prolonged at 12 and 72 hr after BDL. Zoxazolamine paralysis time was prolonged at 72 hr after BDL, but not at 12 hr. Plasma protein binding of hexobarbital and thiopental was not altered by hyperbilirubinemia. These data suggest that changes in drug metabolism are not responsible for the prolongation of hexobarbital sleeping time during the early phase of cholestasis caused by BDL.     

Studies on the effects of l-ascorbic acid on acetaminophen-induced hepatotoxicity: 1. Inhibition of the covalent binding of acetaminophen metabolites to hepatic microsomes in vitro

The covalent binding of [¹⁴C]acetaminophen metabolites to male mouse hepatic microsomes was inhibited by the sulfhydryl compounds, reduced glutathione, cysteamine, and l-cysteine, and also by l-ascorbic acid (vitamin C, LAA). Although the sulfhydryl compounds were more effective inhibitors of macromolecular binding than LAA, the combination of LAA with any of the thiol agents resulted in additive inhibition of covalent binding of [¹⁴C]acetaminophen metabolites. Similar results were obtained in studies with hepatic microsomes from female mice and male hamsters. Investigations into the mechanism of inhibition of covalent binding of [¹⁴C]acetaminophen metabolites indicated that LAA probably acts by scavenging the reactive intermediates generated by the microsomal mixed-function oxidase enzymes rather than by the inhibition of their formation. The results suggest that LAA, at concentrations found in rodent and human liver, may supplement the endogenous protective mechanisms (such as reduced glutathione) which operate in vivo to prevent the covalent binding of reactive acetaminophen metabolites and hence hepatic necrosis. The possible application of this study to the use of LAA in the prevention and treatment of acetaminophen-induced hepatotoxicity in man is discussed.     

Effect of parathion and its metabolites on calcium uptake activity of rat skeletal muscle sarcoplasmic reticulum in vitro.

The insecticide parathion and its potent anticholinesterase metabolite paraoxon induce skeletal muscle necrosis when administered in vivo to rats. In the present study the effects in vitro of parathion and its metabolites on microsomal (sarcoplasmic reticular) calcium uptake activity in rat diaphragm skeletal muscle are examined. Parathion (0.05 mM) is a potent inhibitor of this calcium uptake. The inhibition is apparently competitive with calcium in the system. Parathion (0.05 mM) is also shown to inhibit calcium-dependent ATPase activity associated with the microsomal calcium uptake. Paraoxon, the active anticholineslerase metabolite of parathion, and p-nitrophenol, a hydrolytic metabolite of paraoxon, have no inhibitory effects at this level. At 1.5 mM levels they do inhibit the skeletal muscle microsomal calcium uptake. Eserine, a chemically unrelated anticholinesterase agent, also has inhibitory effects at 1.5 mM. When these same compounds are incubated with isolated rat hemidiaphragms they antagonize the muscle contraction elicited by direct stimulation of the muscle. The skeletal muscle necrosis caused by parathion and paraoxon appear to relate to the anticholinesterase activity in vivo. The relatively potent inhibition of calcium uptake activity of sarcoplasmic reticulum in vitro seen with parathion appears to be an independent action and not related to cholinesterase inhibition.