Microsomal Suspensions Research Articles

Acute inhibition of oxidative drug metabolism by propoxyphene (Darvon)

Propoxyphene is a potent inhibitor of the hepatic, microsomal mixed-function oxidases, acting in a manner similar to the prototype inhibitor of drug metabolism, SKF 525-A (β-diethylaminoethyl-2, 2-diphenylvalerate), which it resembles chemically. Propoxyphene ( K i = 4.6 ± 0.9 × 10 −5 M) and SKF 525-A ( K i = 4.0 ± 1.1 × 10 −6 M) inhibited competitively the activity of aminopyrine N-demethylase in washed microsomes from mouse liver. Both drugs were considerably weaker, noncompetitive inhibitors of the hydroxylation of aniline by microsomes. They manifested typical type I binding spectra to cytochrome P-450, propoxyphene binding approximately 15 per cent as avidly as SKF 525-A ( K s = 6.9 ± 0.5 × 10 −5 M for propoxyphene; 9.1 ± 1.8 × 10 −6 M for SKF 525-A). When propoxyphene and SKF 525-A were injected i.p. in equimolar (0.26 m-mole/kg) doses, both depressed the N-demethylation of aminopyrine and the hydroxylation of aniline by the 10, 000 g supernatant fraction of mouse liver removed 0.5 hr post-administration. Propoxyphene was 30–40 per cent as potent as SKF 525-A as an inhibitor of these activities. When potassium ferricyanide (50 μM) was added to suspensions of hepatic microsomes from animals pretreated with propoxyphene or SKF 525-A, the complexes formed between the drugs and cytochrome P-450 were destroyed. This same equimolar dose of propoxyphene and SKF 525-A prolonged hexobarbital sleeping time and zoxazolamine paralysis time, propoxyphene again being 30–40 per cent as potent as SKF 525-A in these tests. Chronically, propoxyphene increased the rate of its own metabolism as well as that of aminopyrine and aniline, and levels of cytochrome P-450 and microsomal protein. Propoxyphene thus seems to act in a manner very similar to that of SKF 525-A, acting as a potent inhibitor of microsomal drug metabolism when given acutely and as an inducer when given chronically.

The destruction of pulmonary and hepatic cytochrome P-450 by phthalaldehyde

Addition of phthalaldehyde to pulmonary and hepatic microsomes resulted in destruction of cytochrome P-450 at all concentrations of phthalaldehyde tested. Phthalaldehyde destruction of pulmonary and hepatic cytochrome P-450 did not require NADPH. Addition of phthalaldehyde to pulmonary and hepatic microsomal suspension also resulted in inhibition of p-xylene hydroxylase and benzphetamine N-demethylase activities. The in vitro inhibition of p-xylene hydroxylase and benzphetamine N-demethylase as well as the destruction of P-450 could be prevented by supplementing microsomes with hepatic cytosol or purified aldehyde dehydrogenase. Lipid peroxidation did not occur when phthalaldehyde was added to either pulmonary or hepatic microsomes. It is suggested that phthalaldehyde can be used to study the in vitro drug metabolism reactions in these tissue preparations.

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 m m 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 CS 2 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.

Optical probe responses on sarcoplasmic reticulum. Merocyanine and oxonol dyes.

The fluorescence and absorbance of merocyanine 540 in suspensions of skeletal muscle microsomes is altered by the binding of Ca2+ and other cations to the membrane. The order of effectiveness of various cations in causing this effect is La greater than Ca congruent to Mg greater than K. Competition between Ca2+, Mg2+, and K+ suggests the involvement of low affinity, relatively nonspecific cation binding sites in the process. Changes in the fluorescence and absorbance of merocyanine were also observed during ATP-dependent accumulation of calcium into sarcoplasmic reticulum. These changes are satisfactorily explained by the binding of accumulated calcium to binding sites on the interior of sarcoplasmic reticulum membrane. The small absorbance response of the oxonol dye bis[1,3-dibutylbarbituric acid-(5)]trimethinoxonol to Ca2+ and ATP is qualitatively similar to that of merocyanine 540 and can be readily explained by the same mechanism. We have found no clear evidence that any of the observed dye responses are due to changes in the diffusion potential across the sarcoplasmic reticulum membrane generated by an electrogenic transport mechanism. The possibility is considered that merocyanine and oxonol dyes respond to changes in electrostatic surface potential caused by the binding of cations.

Superoxide and hydrogen peroxide production and NADPH oxidation stimulated by nitrofurantoin in lung microsomes: Possible implications for toxicity

The addition of nitrofurantoin to aerobic incubation mixtures containing rat lung microsomes strongly enhanced the generation of adrenochrome from epinephrine. Adrenochrome formation in this system was blocked by superoxide dismutase, but not by catalase. Hydrogen peroxide production was also strongly enhanced by nitrofurantoin in these preparations; superoxide dismutase did not significantly alter the amount of H 2O 2 measured, but no H 2O 2 was detected in incubation mixtures in the presence of catalase. Nitrofurantoin enhanced the oxidation of NADPH in lung microsomal suspensions under aerobic conditions; the enhancement was unaffected by catalase but was partially prevented by superoxide dismutase. Neither adrenochrome formation nor H 2O 2 production were enhanced by nitrofurantoin under anaerobic (N 2) conditions, but NADPH oxidation in the presence of nitrofurantoin was greater under anaerobic conditions than under aerobic conditions. These results are consistent with the view that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen species, emphasizing some in vitro metabolic similarities with the lung-toxic herbicide, paraquat.

Effect of lysolecithin in solubilizing membrane-bound glycosyltransferases

Microsomal membranes were solubilized by incubation with lysolecithin which caused considerable release of galactosyl- and N-acetylglucosaminyl-transferase into a high-speed supernatant fraction. With a critical concentration of lysolecithin (2.5 mg/10 mg protein in 1 mL microsome suspension), there was a maximal binding of radioactive lysolecithin to the sediment fraction obtained after high-speed centrifugation. Increase of lysolecithin concentration (above 2.5 mg/mL) in the incubation mixture caused a progressive release of the enzymes into the supernatant fraction. Lysolecithin binding to the membrane was greatly inhibited by 1 M NaCl, and high salt concentration also inactivated galactosyltransferase in the sediment, suggesting an electrostatic interaction between lysolecithin and membrane enzyme. In contrast, high NaCl concentration had no inhibitory effect on the enzyme activity in the sediment when the fraction was prepared by treatment with Triton X-100. Lysolecithin-treated microsomal sediment and supernatant galactosyltransferase was inactivated by oleoyllysophosphatidic acid but not by palmitoyllysophosphatidic acid or egg yold lysophosphatidic acid. Triton X-100 treated microsomal fractions were also similarly affected by different species of lysophosphatidic acid. The results suggested a similarity of interactions of lysophosphatidic fatty acyl species with lysolecithin and Triton-treated galactosyltransferase.

EFFECT OF LINOLEIC ACID HYDROPEROXIDEON LIVER MICROSOMAL ENZYMES IN VITRO

Rat liver microsomes incubated with linoleic acid hydroperoxide (LAHPO) lost cytochrome P-450 specifically among the enzymes of microsomal electron transport systems. The loss of cytochrome P-450 content and glucose-6-phosphatase activity by LAHPO was accompanied by an increase in malondialdehyde (MDA) production. Turbidity of microsomal suspensions was decreased with increasing MDA production, but not proportionately. Diethyldithiocarbamate (DTC), N,N'-diphenyl-p-phenyl- enediamine and α-tocopherol inhibited almost completely the LAHPO-induced MDA production of microsomes, however no perfect protection against the loss of cytochrome P-450 content and glucose-6-phosphatase activity was observed. The decrease of microsomal turbidity by LAHPO was little affected in the presence of DTC. Purified cytochrome P-450 was destroyed by LAHPO, with minimal protection by the compounds described above. These results suggest the possibility that the loss of microsomal enzyme activities during lipid peroxidation may be attributed largely to a direct attack on enzyme proteins by lipid peroxides rather than indirectly to a structural damage of microsomal membranes resulting from peroxidative breakdown of membrane lipids.

ANDROGEN AROMATIZATION IN GYNECOMASTIA: IN VITRO STUDIES

The etiology of durable pubertal gynecomastia remains unexplained. Hormonal studies are controversial and receptor measurements inconclusive. A local conversion of androgens has been suggested. We have studied glandular and adipose fractions obtained from mastectomy of 3 adolescent boys. Microsomal suspension of the tissues was incubated with 14C testosterone or 3H-Δ4-androstenedione. Aromatization activity was measured by tritiated water formed during the incubation. Placental microsomes served as control. The identification of estrogen products was performed as follows: sodium partition, filtration on isomerase column, elution of the radioactivity corresponding to estrogens (E3) and finally gel chromatography of that extract. In placental microsomes, aromatase activity was: 0.66 pmoles/mg protein/min; in adipose tissues: 0.14 fmoles/mg protein/min; in glandular tissues: 0.13 fmoles/mg protein/min. We conclude that in vitro gynecomastic glandular tissue transforms androgen to the same extent as adipose tissue. An exaggerated local estrogen formation can most likely be excluded.

An in vivo-in vitro comparison of the effects of bile acids on the structural organization and functional activity of liver microsomal monooxygenases

The study deals with changes in the binding and metabolism of type I (aminopyrine and 3,4-benzpyrene) and type II (aniline) substrates by liver microsomal monooxygenases, the activity of NADPH-dependent reductases and lipid peroxidation systems in microsomes, and with the kinetics of ANS binding to microsomal membranes during the development of cholestasis produced by bile duct ligation. The changes observed were compared with the effects of di- and trihydroxy bile acids added to the control microsomal suspension in vitro. It was found that, in contrast with cholic acid, deoxycholic acid (both were sodium salts) added at final concentrations of 0.06–0.07% and 0.09–0.12%, simulates changes in the liver microsomal fraction on 4th and 7th day of cholestasis development, respectively. The corresponding conjugated derivatives of the bile acids used have been characterized by much less effect upon microsomal monooxygenases.

Removal of exogenous haem or haemoglobin from rat liver microsomes

Procedures to remove noncytochrome haem or haemoglobin from rat liver microsomal preparations are described. Over 80% of the radioactive label due to exogenous [ 14C]haemoglobin could be removed by washing the microsomal suspension. In contrast, only about 50% of noncytochrome [ 14C]haem was removed by washing microsomes with either buffer or albumin solutions at 0 to 4°C. The most effective procedure to remove noncytochrome haem was to incubate the microsomal suspension for 5 to 6 min at 37°C in the presence of human serum albumin and dithioerythritol. Under these conditions, over 90% of the radioactive label was removed. This procedure brought about no reduction in the concentration of microsomal cytochrome b 5 or P-450, or the integrity of the mixed function oxidases, as judged by the hydroxylation of aniline or the N-demethylation of aminopyrene. Under similar conditions human apohemopexin was no more effective than albumin in bringing about the removal of haem from the microsomal suspension.

Determination of oxide synthetase and hydratase activities by a new highly sensitive gas chromatographic method using styrene and styrene oxide as substrates

A new highly sensitive gas chromatographic method for the separate measurement of microsomal oxide synthetase (aniline, reduced-flavoprotein: oxygen oxidoreductase (4-hydroxylating), EC 1.14.14.1) and oxide hydratase (glycol hydro-lyase (epoxide-forming), EC 4.21.63) activities by using styrene or styrene oxide as substrate is presented. The assay has been developed in order to permit the evaluation of the true kinetic parameters of the two enzymes, respectively, catalysing the activation and desactivation processes of styrene. In the presence of a microsomal suspension and a NADPH generating system, styrene is converted, under the influence of oxide synthetase, into styrene oxide, a certain amount of which is hydrated into styrene glycol, either spontaneously or enzymatically by the microsomal oxide hydratase; the remaining fraction of the oxide is chemically hydrated in acidic conditions. The total amount of styrene glycol formed is quantitated by electron-capture gas chromatography after its conversion into pentafluobenzoyl derivative. For the measurement of oxide hydratase activity, styrene glycol formed after incubation of styrene oxide with the microsomal fraction is estimated as before. The kinetic data obtained in this study demonstrate that, at low protein concentrations, the affinity of oxide synthetase for styrene is about 20 times as low as that of the oxide hydratase for styrene oxide.

Formation of microsomal cytochrome P-450 complexes studied by the NMR relaxation of water

Cytochrome P-450 in microsomes from liver of phenobarbital treated and control rats has been studied by light absorption and by magnetic resonance methods (EPR and NMR). The nuclear relaxation rate of water protons was measured for microsomal suspensions in the presence of various reactants of Type I and II. The change of relaxation rates correlates well with the spin state conversion of the heme iron. No competition between eventual inner-sphere water molecules and the reactants seems to occur. The temperature dependence of the low spin to high spin equilibrium was studied by light absorption and was accounted for in the temperature variation of the molar relaxation rates of the two spin states.

Solubilization and partial purification of prostaglandin endoperoxide synthetase of rabbit kidney medulla

The microsomes of rabbit kidney medulla converted arachidonic acid into prostaglandin E 2 in the presence of hemoglobin, tryptophan and glutathione as activators. When the microsomal suspension was treated with 1% Tween 20, a solubilized enzyme was obtained which catalyzed the conversion of arachidonic acid to prostaglandins G 2 and H 2. The solubilized enzyme was adsorbed to and then eluted from an ω-aminooctyl Sepharose 4B column, resulting in about 10-fold purification over the microsomes. The partially purified enzyme produced predominantly prostaglandin G 2 in the presence of hemoglobin, while prostaglandin H 2 was produced in the presence of both hemoglobin and tryptophan. The stimulation of prostaglandin endoperoxide formation was also observed with other heme and aromatic compounds. Prostaglandin H 2 synthesis was inhibited by a variety of compounds including non-steroidal anti-inflammatory drugs, thiol compounds and prostaglandin analogues with a thiol group(s).

Effects of carbon tetrachloride on isolated rat liver cells: stimulation of lipid peroxidation and inhibitory action of free-radical scavengers [proceedings

The biochemical mechanisms underlying the toxic effects of carbon tetrachloride on rat liver have been studied extensively (for references, see Recknagel, 1967; Slater, 1972). The earliest morphological disturbances described have concerned the endoplasmic reticulum; biochemical studies in vitro with microsomal fractions have emphasized the rapidity with which carbon tetrachloride damages many of the enzymic activities of this important hepatocellular structure, Many of the damaging effects of carbon tetrachloride on the liver require a preliminary metabolism or 'activation' of carbon tetrachloride t o a highly reactive intermediate that is probably the trichloromethyl radical (CC13*). One consequence of the production of CC&' in the liver endoplasmic reticulum is a stimulation of lipid peroxidation, which is usually measured by the formation of thiobarbituric acidreactive substances (see Slater, 1972). In liver microsomal fractions, the stirnulation of lipid peroxidation by carbon tetrachloride is dependent on a supply of NADPH, is inhibited by a variety of free-radical scavengers (Slater & Sawyer, 1971c), and is not decreased by concentrations of p-chloromercuribenzoate or CO that strongly inhibit drug metabolism (Slater & Sawyer, 19716). Such results have led to the suggestion (Slater & Sawyer, 19716) that carbon tetrachloride is metabolized through dissociative electron capture involving the NADPH-flavoprotein and, possibly, a non-haem iron/ thiol protein rather than cytochrome P-450. However, these previous studies were all done with liver microsomal suspensions, and it might be argued that the disruption of endoplasmic reticulum to produce these suspensions may result in pathways of carbon tetrachloride reduction that are artifacts. In this study we have repeated results obtained previously with liver microsomal suspensions, but this time using isolated whole liver cells, where the structural integrity of the endoplasmic reticulum is maintained. Male albino Wistar rats were used from Charles River, Margate, Kent, U.K. (body wt. 250-3008); they were fed on diet C.R.M.X. (C. Hill Group, Poole, Dorset, U.K.) and water ad libitum. Liver cells were isolated by a small modification of the method described by Gravela ctal. (1 977). Since substances that react with thiobarbituric acid were detectable only when cells were suspended in saline media, we used the following incubation medium: 60m~-NaC1/40m~-KC1/5Om~-Hepes buffer ( p H 7 . 4 ) / 2 m ~ MgSOJl m ~ C a C l , / l m ~ N a , H P 0 , / 5 m~-glucose/0.58m~-amino acid mixture. Cell numbers were measured with a haemocytometer; cell viability was routinely assessed by the Trypan Blue-exclusion procedure. Samples ofcell suspension (3mI, 2 x lo7 cells each) were incubated for 60niin a t 37°C in thedark, withorwithout carbon tetrachloride(2.5~1/3ml)inaclosedsystem(seelegend t o Fig. 1 ; E. Gravela, G. Poli, E. Albano and M. U. Dianzani, unpublished work). Thiobarbituric acid-reacting material was measured (Slater & Sawyer, 1 9 7 1 ~ ) on cell suspensions after protein precipitation with trichloroacetic acid. Aminopyrene metabolism was measured in the cell homogenate by the method described by Slater & Sawyer (19716). Cytochrome P-450 was determined in whole cells as described by Kupfer & Orrenius (1970) by using a millimolar extinction coefficient of 91 litre. mmolz1.cm-'. Promethazine and compound S K F 525A were gifts from May and Baker, Dagenham, Essex, U.K. and Smith, Kline and French, Welwyn Garden City, Herts., U.K., respectively. Fig. 1 gives some of the results obtained for the effects of a number of metabolic inhibitors and free-radical scavengers on the production of thiobarbituric acid-positive

Ethionine-induced alterations in hepatic microsomal cytochromes in vivo and in vitro: Sexual dimorphism

Investigation of the effects of ethionine intoxication on hepatic microsomal cytochromes in rats revealed that b 5 and P-450 concentrations dropped to less than control values in female animals within 8 hr. Loss of the two hemoproteins, however, did not occur simultaneously. We observed reduction of b 5 earlier than the decrease in P-450. Moreover, restitution of cytochrome b 5 to control amounts became apparent by the seventh day after administration of the intoxicant. Male rats had only a loss of b 5; at 16 hr, P-450 values remained unchanged. Adding ethionine in vitro to microsomal suspensions (with or without NADH supplementation) failed to reproduce the in vivo alterations. Also, we were unable to correlate changes in peroxidation with cytochromal modifications, although male and female animals differed markedly in ethionine-stimulated peroxidation.

Effects of PBBs on cattle. III. Target organ modification as shown by renal function and liver biochemistry

Efforts were made to more clearly delineate target organs and mechanisms of toxicity for PBBs in cattle. Methods were developed to obtain sequential liver biopsies on bovine heifers which yield 0.5 to 1.0 g of tissue. PBB was fed at a dose of 250 mg/head/day to Holstein heifers for 202 days. This dose produced no clinical signs of toxicity in any of the heifers, yet this produced tissue PBB concentration of greater than 100 times the FDA tolerance in body fat of 0.3 ppm. Liver biopsies (0.5-1.0 g each) were taken at days 0, 90, and 180. The liver tissue was homogenized and microsomes were prepared. Dithionite difference spectra were determined on the carbon monoxide treated microsome suspension and the cytochrome P-450 content determined. Also, the 100,000g supernatant was saved for ornithine decarboxylase analysis as a measure of hepatocyte proliferative activity. Results of the cytochrome P-450 analysis showed a significant (p < 0.05) two-fold elevation (per gram of wet liver) by day 90 and remained significantly (p < 0.05) elevated on day 180. The cytochrome P-450 values of control animals not receiving PBBs showed no such increase with time. The biopsy procedure appeared not to adversely affect the liver cytochrome P-450 concentration in the control heifers. These results show that PBBs at a dose of 250 mg/day induced the drug metabolism system of the liver, of which the cytochrome P-450 is a part, indicating that the liver is a potential target organ for PBBs. However, this has not been shown to cause clear signs of hepatotoxicity in the cow as determined from histopathology or serum enzyme analyses. The observed elevation of gross liver weights of the PBB-treated animals might be an expected consequence of the cytochrome P-450 induction. In contrast to rodents, the kidney has been identified by histopathology as a target organ for PBB toxicity in cattle. However, renal function studies with (131)I-sodium-iodohippurate and (125)I-sodium iodothalamate in PBB treated cows indicated that PBB toxicity to the kidney did not affect glomerular filtration rate or effective renal plasma flow even though nephrotoxic effects were produced. From these studies, both liver (as expected) and kidney (unexpected) were affected by PBBs. For liver this did not result in hepatotoxicity while for kidney nephrotoxicity was produced but could not be mechanistically explained.

Fusion of microsomal vesicles.

Studies which indicate the fusion of rat liver microsomal vesicles show that the rate of fusion of microsomal vesicles, as revealed by electron microscopic examinations, is dependent on the fusion temperature and the amount of detergent present in the microsomal suspension.

Sensitive gas chromatographic methods for the determination of vinyl epoxide synthetase activity using trichloroethylene as a model substrate

Two specific and very sensitive methods for the determination of vinyl epoxide synthetase activity in liver microsomes are described. Trichloroethylene, which is used as a substrate, is converted into trichloroethylene oxide by a hepatic epoxide synthetase. Chloral hydrate, the final rearrangement product of trichloroethylene oxide, is determined by electron-capture gas chromatography, either after derivatization with pentafluorophenylhydrazine or after its conversion into chloroform under alkaline conditions. The kinetic parameters of the epoxidation reaction were determined on rat hepatic microsomal suspensions.

Microsomal suspension test with testes homogenate: non-enzymatic influence on the mutation frequency of the indicator organism

Microsomal suspension test with testes homogenate: non-enzymatic influence on the mutation frequency of the indicator organism

A study of microsomal oxidative functions with the aid of a series of N-colchiceyl derivatives.

N-Colchiceyl derivatives are presented as a class of substances which, provided certain structural requirements are met, are oxidatively degraded by microsomal suspensions both at the nitrogen-linked side chain at ring C and at the oxygen-linked side chain(s) at Ring A. The chemical structures of unknown metabolites have been elucidated by comparison with authentic samples whose syntheses are described. Comparative studies indicate structure-dependent preferred sites for the introduction fo oxygen, whose extent correlates rather closely with the partition coefficient in the system n-octanol/ethanol/water. The results demonstrate, with the inclusion of some inhibition data, the suitability of N-colchiceyl derivatives for detailed investigations of microsomal degradative processes.