Microsomal Suspensions Research Articles

Regional differences in microsomal lipid peroxidation and antioxidant levels in the guinea pig adrenal cortex

Lipid peroxidation (LP) and antioxidant levels were studied in the chromatically distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea pig adrenal cortex. Ferrous ion (Fe 2+) produced a concentration-dependent (10 −5 to 10 −3 M) stimulation of microsomal LP in both zones, but LP, as estimated by malonaldehyde production, was far greater in the inner zone. Although cytosolic ascorbic acid content was similar in the two zones, microsomal tocopherol levels were approx 4 times greater in the outer than inner zone. Subphysiological concentrations of ascorbic acid, like Fe 2+, initiated LP to a greater extent in inner than outer zone microsomes; optimal stimulation of LP by ascorbic acid occurred at concentrations of 100–200μ M in both zones. Physiological concentrations of ascorbic acid (1–5 mM), by contrast, did not initiate LP and, in fact, markedly inhibited Fe 2+-induced LP in both inner and outer zone microsomal preparations. Outer zone microsomes were more sensitive to the antioxidant effects of ascorbic acid than were inner zone preparations. Addition of α-tocopherol to inner zone microsomal suspensions inhibited Fe 2+-induced LP. The results indicate that there are regional differences in adrenocortical LP which may be caused by differences in tocopherol content. α-Tocopherol may serve important antioxidant functions within the adrenal cortex, thereby contributing to the functional zonation of the gland.

Effects of phenobarbital pretreatment on the in vivo metabolism of carbaryl in rats.

Phenobarbital (PB) pretreatment of animals is known to induce the activity of drug-metabolizing enzymes in liver microsomes. Previous studies showed that incubation of carbaryl with microsomes obtained from livers of untreated or PB-treated rats resulted in little or no oxidative metabolism of the substrate. In addition, no spectral interactions were observed when carbaryl was added to hepatic microsomal suspensions. The present study was carried out to determine the effect of PB pretreatment on the in vivo metabolism of carbaryl in rats.

Kinetic Analysis of Inhibition of Human Adrenal Steroidogenesis by Ketoconazole*

The kinetics of the inhibitory effects of the imidazole antimicrobial ketoconazole on the activities of the steroidogenic enzymes distal to cholesterol side-chain cleavage were studied in human adrenal microsomal and mitochondrial suspensions. Although ketoconazole was a competitive inhibitor of all five enzyme reactions, the effects on 17-hydroxylase, 17,20-desmolase, and 11-hydroxylase activities (Ki = 10(-8) M) were considerably greater than those on 21-hydroxylase and 3 beta-hydroxysteroid dehydrogenase/isomerase activities (Ki = 10(-4) M). These findings explain the clinical endocrine effects of ketoconazole in the usual therapeutic doses, which include inhibition of cortisol and androgen secretion, compensatory ACTH-mediated secretion of 17-desoxysteroids such as progesterone and aldosterone, and suppression of PRA.

Involvement of the esterase active site of egasyn in compartmentalization of β-glucuronidase within the endoplasmic reticulum

Organophosphorous compounds, which are potent inhibitors of egasyn-esterase activity, caused a rapid dissociation of the high molecular weight egasyn-microsomal β-glucuronidase complex when administered in vivo or when added in vitro to microsomal suspensions. The dissociation was relatively specific to phosphodiester inhibitors of the esterase active site. Also, the egasyn-esterase active site was inaccessible to substrates and to inhibitors when egasyn was complexed to β-glucuronidase. Dissociation of the egasyn-microsomal β-glucuronidase complex in vivo by organophosphorous compounds was followed by massive and rapid secretion of microsomal β-glucuronidase, but not egasyn, into plasma. These experiments implicate the egasyn-esterase active site in attachment of microsomal β-glucuronidase to egasyn by a novel mechanism that, in turn, compartmentalizes β-glucuronidase within the endoplasmic reticulum.

Stimulation of cyclophosphamide-induced pulmonary microsomal lipid peroxidation by oxygen

Cyclophosphamide (CP) causes lung toxicity in a wide variety of animals including humans. Recent reports suggest that CP increases lipid peroxide formation in the lung, and that oxygen (O 2) potentiates CP-induced lung toxicity. We hypothesized that CP, or one of its toxic metabolites, acrolein, stimulates lung lipid peroxide formation in the presence of high O 2 tensions. To test this, rat lung microsomes were treated in vitro with CP or acrolein in the presence of NADPH and 0–100% O 2 with and without superoxide dismutase (SOD), glutathione (GSH), dithiothreitol (DTT), and EDTA (agents which scavenge reactive O 2 species and/or detoxify reactive metabolites). Lipid peroxide formation in untreated microsomes was increased 40, 39, and 37% in 60, 80 and 100% O 2 respectively ( P < 0.02 vs. 21% O 2 air). Lipid peroxide formation in microsomes treated with CP increased 2–3-fold under 21% O 2 ( P < 0.05 vs. untreated under 21% O 2). However, increases in lipid peroxide formation were 3–4 fold in CP treated microsomes under 40–100% O 2 ( P < 0.001 vs. untreated at same % O 2). CP and acrolein-stimulated lipid peroxidation with and without O 2 exposure was significantly ( P < 0.05) reduced by prior addition of SOD, GSH, DTT, or EDTA to the lung microsomal suspension. These results indicate that lipid peroxide formation increases in CP and acrolein-treated lung microsomes, and high O 2 tensions stimulate CP-induced lipid peroxidation. Stimulation of CP-induced microsomal lipid peroxidation appears to be mediated by reactive O 2 species or metabolites.

Mechanisms mediating cephaloridine inhibition of renal gluconeogenesis

Incubation of renal cortical slices with cephaloridine (CPH) markedly inhibits pyruvate-supported gluconeogenesis, an effect which is independent of CPH-induced lipid peroxidation. CPH was found to inhibit pyruvate-supported gluconeogenesis in a time- and concentration-dependent manner. Pyruvate-supported gluconeogenesis was inhibited as early as 10 min following incubation of renal cortical slices with 5 m m CPH. Similarly, endogenous gluconeogenesis was impaired following CPH treatment. CPH depressed the renal cortical slice content of ATP by 50%, but only following 90 and 120 min of drug exposure, suggesting that mitochondrial dysfunction does not mediate the inhibition of gluconeogenesis by CPH. To identify the intracellular site(s) of CPH inhibition of gluconeogenesis, the effects of CPH on glucose production were evaluated using substrates catalyzed by rate-limiting reactions. CPH inhibited renal cortical slice gluconeogenesis when the following substrates were used: pyruvate (mitochondrial), oxaloacetate and fructose-1,6-diphosphate (FDP) (postmitochondrial), and glucose-6-phosphate (G6P, endoplasmic reticulum). Inhibition of G6P-supported gluconeogenesis occurred within 5 min of incubation with 5 m m CPH. Direct addition of CPH to microsomal suspensions inhibited G6Pase activity in a concentration-dependent fashion. By contrast, addition of CPH to cytosolic fractions did not affect FDPase activity. CPH increased the K m and decreased the V max of G6Pase, indicating mixed competitive and noncompetitive inhibition. These data indicate that the profound inhibition of renal cortical slice gluconeogenesis by CPH is mediated by inhibition of microsomal G6Pase activity.

Measurement of in vivo lipid peroxidation and toxicological significance

The quantitative determination of hydrocarbons exhaled by animals as an in vivo index of extensive lipid peroxidation is described. Advantages and limitations of this method are discussed. Acetaminophen-induced hepatic lipid peroxidation in mice is an example of oxidative stress, the extent of which is determined in vivo by the turnover of endoplasmic reticulum monooxygenase and the cofactor, e.g. glutathione status of the liver. In microsomal suspensions, none of the assay methods for lipid peroxidation identifies acetaminophen as a pro-oxidant. Rather, it acts like an antioxidant. The obvious limitations of in vitro experiments are emphasized. Cytosolic metabolism of allyl alcohol also leads, in a dose-dependent manner, to extensive lipid peroxidation. Evidence is presented that release of iron from intracellular stores following overproduction of NADH may be the primary cause of this lesion. The term reductive stress is suggested for this metabolic initiation of iron redox cycling. In experimental hepatitis induced, by galactosamine/ endotoxin, a leukotriene-mediated pathomechanism, no signs of lipid peroxidation are detectable. This means that ethane or pentane formation are definitively not late consequences of membrane deterioration but rather early causal events in special cases of hepatotoxicity.

The induction profile of three orally active imidazopyridine-containing cardiotonic agents in rat hepatic microsomes

The induction of hepatic cytochrome P-450-linked monooxygenases has been studied after the twice daily, oral administration of two imidazo[4,5- c]pyridine-containing compounds and one imidazo[4,5- b]pyridine-containing drug. The compounds were administered by the oral route, at different doses, for 6 days after which time hepatic microsomes were prepared. In vitro biochemical assays revealed that all three compounds increased the O-deethylation of 7-ethoxyresorufin in a dose-dependent manner while not significantly affecting either the O-dealkylation of 7-ethoxycoumarin or the levels of NADPH-cytochrome c reductase. Ethylmorphine N-demethylation was decreased after dosing with the imidazo[4,5- b]pyridine-containing drug. Levels of cytochrome(s) P-450 and liver-to-body weight ratios were not significantly altered. The imidazo[4,5- b]pyridine-containing compound was more potent in terms of the induction of 7-ethoxyresorufin than either of the imidazo[4,5- c]pyridine-containing compounds but was approximately fourfold less active in this regard than 3-methylcholanthrene. No induction of cytochrome- P-450-linked monooxygenase activities was evident at a twice daily dose of 5 mg/kg for 6 days for all three compounds tested, constituting a no-effect level. The imidazo[4,5- c]pyridine-containing compounds exhibited modified Type II difference spectra when added to a suspension of rat hepatic microsomes. The imidazo[4,5- b]pyridine-containing compound has previously been reported to be (i) a rapid and potent inducer of monooxygenase activity and (ii) have a Type II difference spectrum.

Studies on lipid peroxidation in normal and tumour tissues. The Novikoff rat liver tumour.

A study has been made of the factors that contribute to the decreased rates of lipid peroxidation under different pro-oxidant conditions in intact Novikoff tumour cells, and in microsomal suspensions prepared from Novikoff tumour cells, compared with isolated normal rat hepatocytes and microsomal suspensions prepared from normal rat liver. The pro-oxidant conditions were the addition of either NADPH, NADPH + ADP + iron, NADPH + CCl4 or ascorbate+iron to the experimental systems used, or exposure to gamma-radiation. Contributory factors to the lower rates of lipid peroxidation observed include: a significant decrease in the polyunsaturated fatty acid content of Novikoff cells or Novikoff microsomes; the decreases are especially marked for the C20:4 and C22:6 fatty acids; a very marked reduction in NADPH-cytochrome c reductase; and no detectable content of cytochrome P-450. Another, and in our opinion critical, contribution to the diminished rate of lipid peroxidation in the tumour material is the substantial increase in alpha-tocopherol relative both to total lipid and to methylene-interrupted double bonds in fatty acids. Moreover, the alpha-tocopherol is the major contributor to lipid-soluble chain-breaking antioxidant in lipid extracts of normal liver and of Novikoff tumour material.

Kinetics of the 5'-nucleotidase and the adenosine deaminase in subcellular fractions of rat brain.

Suspensions of rat brain microsomes, synaptosomes, and synaptic vesicles were able to convert adenosine to inosine by means of adenosine deaminase. Isosbestic points of this transformation, at 222, 250 and 281 nm, remained unchanged with time-course. This fact suggests that adenosine deaminase (ADA, E.C. 3.5.4.4) is located on the surface of the vesicles whereas purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4) is located inside the vesicles. Kinetic parameters of the particulate 5'-nucleotidase (5'N, E.C. 3.1.3.5) and adenosine deaminase were analogous to those of the cytosolic enzymes. These results suggest that soluble and particulate enzymes represent different pools of the same molecular species.

Enzymes of the purine metabolism in rat brain microsomes.

Rat brain microsomes, when they are suspended in moderate ionic strength medium, released enzyme activities of lactate dehydrogenase (LDH, E.C.1.1.1.27), malate dehydrogenase (MDH, E.C.1.1.1.37), adenosine deaminase (ADA, E.C.3.5.4.4), guanine deaminase (GAH, E.C.3.5.4.3), and purine nucleoside phosphorylase (PNP, E.C.2.1.2.4). The activities released decreased when the saline concentration of the medium was increased and the opposite occurred when 50 mM, pH 7.4 sodium phosphate medium was used. Rat brain microsomes that had been extracted previously by moderate ionic strength solutions still had activities of all the enzymes tested, and released these activities upon sonication or deoxycholate (DOC) treatment. The proportion of the activity released was similar for all the enzymes. DOC treatment released higher enzymic activities and a smaller amount of protein than sonication did. The proportion of activities released was similar to that found in the 105,000 g supernatant. The suspension of microsomes still retained activities of the above-mentioned enzymes after consecutive extractions with increasing concentrations of detergent solutions (DOC and Triton X-100). The amount of enzymic activities released from the microsomes by sonication or DOC treatment did not depend on the protein composition of the homogenization medium. Thus, on increasing the enzyme concentration in the homogenization medium, the activities released did not increase in parallel. The set of results obtained showed that the microsomal fraction is as useful as the cytosolic one for studying purine catabolism in rat brain. Furthermore, the conditions in which purine enzymes are attached to the microsomal fraction are probably closer to "in vivo" conditions than those in which these enzymes are found in the soluble fraction.

Intrahepatic sources of biliary-like micelles

To study the hepatocellular origins of biliary lipids, bile acid-lipid micelles were isolated from rat liver subfractions in vitro by Sephadex G-100 column chromatography, using 10 mM sodium taurocholate in the column eluant. Micelles of similar size to biliary micelles were obtained from microsome suspensions and from Golgi content that had been isolated in 5 mM sodium taurocholate-containing subcellular fractionation media. The phospholipid composition of these ‘biliary-like’ micelles was similar but not identical to that of biliary micelles; cholesterol was detected in the micelles from Golgi content but not microsomes. ‘Biliary-like’ micelles could not be isolated from plasma membranes (including highly purified canalicular membranes), Golgi membranes or liver cytosol. These data suggest that biliary lipid micelles (or micelle precursors) may originate from the endoplasmic reticulum and Golgi apparatus.

Effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin on lipid peroxidation in microsomal systems in vitro

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) when added to suspensions of rat hepatic microsomes in the presence of NADPH has little influence on the peroxidation of microsomal lipids unless the system also contains complexed ferric ion, in which case TCDD stimulates. This stimulation does not appear to require metabolism of the TCDD. Peroxidation was monitored by production of thiobarbiturate-reactive substances (malondialdehyde and dienals), production of conjugated dienes, and disappearance of polyunsaturated fatty acids. Stimulation of lipid peroxidation by TCDD in a mixed lysosome-microsome preparation resulted in significantly decreased ‘leakage’ of acid phosphatase into the medium, implying an effect on lysosomal membranes. Consideration both of the present results and data in the literature leads to the conclusion that it is premature to attempt to define the relationship between enzyme induction, lipid peroxidation and TCDD lethality.

The Induction Profile of Three Orally Active Imidazopyridine-Containing Cardiotonic Agents in Rat Hepatic Microsomes

The Induction Profile of Three Orally Active Imidazopyridine-Containing Cardiotonic Agents in Rat Hepatic Microsomes. BERNSTEIN, J. R, AND FRANKLIN, R. B., (1986). Fundam Appl. Toxicol. 7, 26-32. The induction of hepatic cytochrome P-450-linked monooxygenases has been studied after the twice daily, oral administration of two imidazo[4,5-c]pyridine-containing compounds and one imidazo[4,5-6]pyridine-containing drug. The compounds were administered by the oral route, at different doses, for 6 days after which time hepatic microsomes were prepared. In vitro biochemical assays revealed that all three compounds increased the O-deethylation of 7-ethoxyresorufin in a dose-dependent manner while not significantly affecting either the 0-de- alkylation of 7-ethoxycoumarin or the levels of NADPH-cytochrome c reductase. Ethylmorphine- N-demethylation was decreased after dosing with the imidazo[4,5-b]pyridine-containing drug. Levels of cytochrome(s) P-450 and liver-to-body weight ratios were not significantly altered. The imidazo[4,5-c]pyndine-containing compound was more potent in terms of the induction of 7-ethoxyresorufin than either of the imidazo[4,5-c]pyridine-containing compounds but was approximately fourfold less active in this regard than 3-methylcholanthrene. No induction of cy- tochrome-.P-450-linked monooxygenase activities was evident at a twice daily dose of 5 mg/kg for 6 days for all three compounds tested, constituting a no-effect level. The imidazo[4.5-c]pyridine-1 containing compounds exhibited modified Type II difference spectra when added to a suspension of rat hepatic microsomes. The imidazo[4,5-6]pyridine-containing compound has previously been reported to be (i) a rapid and potent inducer of monooxygenase activity and (ii) have a Type II difference Spectrum.(c) 1986 Society of Toxicology

Biological antioxidant activities of bromophenols and certain other antioxidants.

The biological antioxidant activities of bromophenols (5-bromo-3, 4-dihydroxy benzaldehyde and 5-bromo-3, 4-dihydroxy benzylalcohol), protocatechualdehyde and vanillyl octadecanamide were compared with α-tocopherol in vitro and in vivo. All of these antioxidants significantly inhibited the lipid peroxidation in microsomal suspensions induced by ascorbate/Fe(II) or NADPH. By removing the non-binding free antioxidants from the suspensions, the inhibitory activities markedly decreased in antioxidants other than α-tocopherol; however, considerable activity was still observed. The effects of orally administered antioxidants on the symptoms of vitamin E deficiency were examined using rats fed with a vitamin E-free diet and supplemented with oxidized oils. As a result and contrary to the effects in vitro, all of the antioxidants were much less effective in liver. However, some of the vitamin E-deficient symptoms such as organic solvent-soluble fluorescent materials in the heart and lung, and oxidative hemolysis were significantly suppressed by some antioxidants.

Effects of 2-mercaptopropionyl glycine on radiation-induced lipid peroxidation in liposomes and in rat liver microsomal suspensions.

gamma-Irradiation of rat liver microsomal suspensions resulted in the accumulation of both malondialdehyde (MDA) and lipid hydroperoxides. The presence of 2-mercaptopropionylglycine (MPG) during the irradiation period decreased the formation of MDA and lipid hydroperoxides in a dose (MPG)-dependent manner. This may be attributed to the ability of MPG to scavenge the free radicals produced by irradiation. Post-irradiation incubation of microsomes further enhanced the production of both MDA and lipid hydroperoxides; when high concentrations of MPG were present during the incubations the production of MDA and lipid hydroperoxides was substantially decreased. This antioxidant role of MPG was demonstrated for both pre-irradiated microsomes and liposomes and is thought to be due to the conversion of the hydroperoxy to hydroxy fatty acids within the lipid bilayer, as well as the scavenging action on initiating free radicals.

Carbon tetrachloride toxicity as a model for studying free-radical mediated liver injury.

A single dose of CCl4 when administered to a rat produces centrilobular necrosis and fatty degeneration of the liver. These hepatotoxic effects of CCl4 are dependent upon its metabolic activation in the liver endoplasmic reticulum to reactive intermediates, including the trichloromethyl free radical. Positive identification of the formation of this free radical in vivo, in isolated liver cells and in microsomal suspensions in vitro has been achieved by e.s.r. spin-trapping techniques. The trichloromethyl radical has been found to be relatively unreactive in comparison with the secondarily derived peroxy radical CCl3O2., although each free radical species contributes significantly to the biological disturbances that occur. Major early perturbations produced to liver endoplasmic reticulum by exposure in vivo or in vitro to CCl4 include covalent binding and lipid peroxidation; studies of these processes occurring during CCl4 intoxication have uncovered a number of concepts of general relevance to free-radical mediated tissue injury. Lipid peroxidation produces a variety of substances that have high biological activities, including effects on cell division; many liver tumours have a much reduced rate of lipid peroxidation compared with normal liver. A discussion of this rather general feature of liver tumours is given in relation to the liver cell division that follows partial hepatectomy.

Recessive X-linked ichthyosis: lack of immunologically detectable steroid sulfatase enzyme protein.

Patients with recessive X-linked ichthyosis (RXLI), one hereditary form of scaly skin, lack activity of the enzyme steroid sulfatase in all tissues studied. To investigate the molecular defect underlying the lack of enzyme activity, we prepared antisera against normal enzyme by injecting normal placental microsomal suspensions or partially purified steroid sulfatase into rabbits. Antibody activity was assessed by immunoprecipitation of detergent solubilized steroid sulfatase. In addition, we prepared rabbit antisera against RXLI placental microsomal suspensions. To detect immunologically cross-reactive material in patients' placentas, extracts were studied by immunoblot techniques and by competition with normal enzyme for antibody binding. Patients' extracts did not contain immunoreactive material co-migrating on electrophoresis with purified enzyme nor did they inhibit immunoprecipitation of normal enzyme. Sera from rabbits immunized with RXLI placental microsomes contain no antibodies to normal steroid sulfatase, as judged by their failure to immunoprecipitate normal enzyme or to react with normal steroid sulfatase on immunoblot. Thus the mutation in RXLI appears to reduce steroid sulfatase enzyme protein as well as enzyme activity.

Inhibition of the gastric (H + + K +)-ATPase by fenoctimine

The effects of fenoctimine, an inhibitor of gastric acid secretion, on the microsomal (H + + K +)-ATPase were studied. In the micromolar concentration range, fenoctimine inhibited hydrolysis of ATP and π-nitrophenyl phosphate by the (H + + K +)-ATPase. Inhibition was reversible and non-competitive with substrate. The apparent K i was dependent on the concentration of membranes, being increased by added liposomes or high microsomal membrane concentrations. Over the concentration range that (H + + K +)-ATPase was inhibited, fenoctimine increased by the turbidity of microsomal suspensions. The effects of fenoctimine were not specific for the gastric (H + + K +)-ATPase, since the hydrolytic activities of the (Na + + K +)-ATPase and mitochondrial ATPase were also inhibited by the drug. These results suggest that inhibition of hydrolysis may not be the direct result of an interaction between the (H + + K +-ATPase and fenoctimine but the secondary effect of a fenoctimine-induced perturbation of the microsomal membrane.

Cimetidine interaction with liver microsomes in vitro and in vivo: Involvement of an activated complex with cytochrome P-450

The O-deethylation of 7-ethoxycoumarin was inhibited in a mixed type manner by cimetidine in vitro and in microsomes isolated from rats treated with cimetidine in vivo. It was found that the inhibition was even greater if cimetidine was preincubated with the microsomal suspension in the presence of an NADPH-generating system prior to the addition of substrate. In vitro the decrease in activity was accompanied by a decrease in cytochrome P-450 content. This decrease was unaffected by the addition of EDTA to the microsomal suspensions, eliminating the possibility that free radical production was responsible for the decrease in cytochrome P-450. The decrease in activity and cytochrome P-450 content following preincubation of microsomal suspensions with cimetidine could be attenuated if potassium ferricyanide was added to the suspensions. The deethylation activity and cytochrome P-450 content of liver microsomes prepared from cimetidine-treated rats was decreased compared to control animals. The activity and cytochrome P-450 content of microsomes from cimetidine- treated rats could also be restored if microsomes were washed with potassium ferricyanide prior to incubation with substrate. It is proposed that an intermediate complex of cimetidine and cytochrome P-450 could be involved in the inhibition of microsomal metabolism by cimetidine.