Microsomal Electron Transport Components Research Articles

Chronic inhalation effects of tetrachloroethylene on hepatic and renal microsomal electron transport components and δ-aminolevulinic acid dehydratase in rats

Hepatic and renal microsomal electron transport components and erythrocyte δ-aminolevulinate dehydratase were investigated in adult male rats exposed to 0, 50, 200 and 600 ppm tetrachloroethylene for 4 weeks. Body weight and liver weight showed a significant decrease only in the 600 ppm group. A dosedependent decrease in erythrocyte δ-aminolevulinate dehydratase was observed at 200 and 600 ppm. Serum transaminase activity (SGPT) showed an increase in the 600 ppm group only. Hepatic and renal microsomal protein content showed an increase in all groups except in the kidneys of the 600 ppm group. Induction of hepatic cytochrome b 5 activity was observed in all groups. However, hepatic cytochrome P-450 showed an induction and slight inhibition at 200 and 600 ppm respectively, without any alteration at 50 ppm. Renal microsomal cytochrome P-450 activity was induced in all groups. Induction of hepatic and renal NADPH cytochrome c reductase activity was observed at 600 ppm, but no alteration was seen at 50 and 200 ppm. These results indicate that chronic inhalation of tetrachloroethylene at higher levels alters mixed-function oxidase and heme metabolism.

Microsomal enzyme activities, superoxide production, and antioxidant defenses in ribbed mussels ( Geukensia demissa) and wedge clams ( Rangia cuneata)

1. 1. Microsomes of hepatopancreae from the ribbed mussel, Geukensia demissa, and the wedge clam, Rangia cuneata, were examined for their ability to catalyze the reduction/oxidation cycling of xenobiotics, particularly paraquat, and subsequently stimulate superoxide anion (O − 2) production. 2. 2. Levels of the microsomal electron transport components cytochrome P-450, cytochrome b 5, NADH-cytochrome c reductase, and NADPH-cytochrome c reductase were similar in both bivalves and like those seen in other species of molluscs. 3. 3. in vitro studies indicated a dose-dependent increase in the rate of O − 2 generated in microsomal fractions incubated with paraquat; the highest concentration of paraquat employed (4 mM) elicited an 81% increase in cytochrome c reduction in mussels and a 135% increase in clams. In both species, cytochrome c reduction was inhibited by the addition of exogenous superoxide dismutase (SOD). 4. 4. Activities of SOD and catalase and the concentration of reduced glutathione were determined in the hepatopancreas of these bivalves. Similar values for these antioxidants were observed in both species.

Alterations in Hepatic Microsomal Mixed-Function Oxidase System during Different Levels of Food Restriction in Adult Male and Female Rats

Wistar strain adult male and female rats were given 25, 50 and 75% less food than an ad libitum-fed group of rats for 45 d and the effects of food restriction on hepatic drug metabolizing enzymes, microsomal electron transport components, NADPH-dependent lipid peroxidation and glutathione-S-transferase activities were studied. Compared to ad libitum-fed controls, the cytochrome P-450 levels were higher in food restricted male rats, while they were lower in food restricted females. The activities of NADPH cytochrome c reductase were lower in food restricted females than in ad libitum-fed controls. The activities of drug metabolizing enzymes, aminopyrine N-demethylase and acetanilide hydroxylase were higher in food restricted males, whereas in food restricted females these activities were lower than in respective groups fed ad libitum. Microsomal, NADPH-dependent lipid peroxidation was higher in 25 and 50% food restricted females while in 50 and 75% food restricted males it was lower than in ad libitum controls of the same sex. The cytosolic glutathione-S-transferase activities were lower in food restricted rats of both the sexes than in the same sexed controls. Another group of male and female rats were given 75% less food than the ad libitum-fed rats and refed for 3 d prior to killing. Here also, the effects of restriction were different between sexes. It is concluded that hepatic microsomal mixed-function oxidase system (MFOS) is altered due to feed restriction and food restriction followed by refeeding, in a sex-related manner.

Affinity chromatography of microsomal enzymes on immobilized detergent-solubilized cytochrome b5.

Highly selective chromatography of microsomal enzymes has been carried out on columns of immobilized cytochrome b5 that was obtained by detergent solubilization (d-b5) of the complete amphipathic molecule. Several partially purified isozymes of cytochrome P-450 are resolved on d-b5 columns, and one high-affinity isozyme has been readily purified to homogeneity. Chromatographic selectivity and correlation of elution order of isozymes of cytochrome P-450 with direct spectral measurements of affinity constants suggests affinity chromatography on d-b5 columns. Substantial one-step enrichments of NADH-cytochrome-b5 reductase and an unstable cytochrome b5-dependent oxidase of cholesterol synthesis, 4-methyl sterol oxidase, have been obtained on d-b5 columns which further supports this conclusion. Comparison of chromatographic behavior on columns of immobilized cytochrome b5 that was obtained by trypsin solubilization (t-b5) with d-b5 columns shows marked differences which must be attributed to the absence of the hydrophobic domain of the t-b5 molecule. NADH-cytochrome-b5 reductase and the high affinity isozyme of cytochrome P-450 purified by d-b5 affinity chromatography are poorly retained on t-b5 columns. A different cytochrome P-450 isozyme with lower affinity for cytochrome b5 is only retained on d-b5 columns. Cytochrome-P-450 reductase is not retained on either column. Because affinity chromatography is suggested on d-b5 columns, the procedure may be generally applicable for predicting protein-protein interactions of microsomal electron transport components that either donate electrons to, or receive electrons from, cytochrome b5. In addition, the procedure should have considerable utilitarian application for enzyme enrichment.

Effect of local anesthetics on stearoyl-CoA desaturase of Tetrahymena microsomes.

The effect of local anesthetics on the stearoyl-CoA desaturase activity was studied using Tetrahymena microsomal preparation. Dibucaine, tetracaine, and propranolol, a beta-blocking agent, nonspecifically inhibited the activities of NADPH-ferrihemoprotein reductase as well as of stearoyl-CoA desaturase and the terminal component, but lidocaine and procaine had no effect on these activities. The inhibitory potency was decreased in the order of dibucaine greater than propranolol greater than tetracaine much greater than lidocaine = procaine. According to the double-reciprocal plots of stearoyl-CoA desaturase, the inhibition by dibucaine appeared to be noncompetitive with respect to stearoyl-CoA as substrate. However, the activity of NADH-ferricyanide reductase was not significantly affected by concentrations of propranolol and tetracaine lower than 10mM, but by dibucaine. The terminal component, cyanide-sensitive factor, was most sensitive to local anesthetics among the microsomal electron transport components, suggesting a rate-limiting enzyme.

Benzo[a]pyrene oxidation and microsomal enzyme activity in the mussel (Mytilus edulis) and other bivalve mollusc species from the Western North Atlantic

Analysis of subcellular fractions revealed a complement of microsomal electron transport components including reductases and heme proteins in several organs of the three bivalve species Mytilus edulis, Macrocallista maculata and Area zebra. Dithionite difference spectroscopy of CO-treated microsomes yielded spectra typical of cytochrome P-450 in digestive gland and gill, with absorption maxima at 450 nm. A time-dependent reduction of cytochrome P-450 was also observed. The levels of these components and rates of microsomal benzo[a]pyrene (BP) metabolism were highest in the digestive gland, and were very similar between species. In M. edulis there was a suggested seasonal variation in BP metabolism but no population differences in this activity or in levels of other components. Digestive gland microsomal metabolites of BP identified by HPLC retention and UV spectroscopy included BP-1.6-quinone, BP-3,6-quinone and BP-6,12-quinone, which comprised 65% of the total metabolites, and dihydrodiols and phenols, the latter products consistent with cytochrome P-450 monooxygenation and expoxide hydrolase function. However, the inconsistent dependence of BP metabolism on NADPH, and inconsistent inhibition by CO suggest that catalyst(s) additional to cytochrome P-450 may be acting in BP metabolism. Based on these results and the prominent quinone formation, we speculate that peroxidative mechanism(s) may be involved. The role of peroxidative as well as well as monooxygenase reactions in the in-vivo disposition and effects of foreign chemicals in bivalves, and also the major function of cytochrome P-450 in these bivalves, remain to be established.

Effects of chronic ethanol consumption on male syrian hamster hepatic, microsomal mixed-function oxidases

Chronic alcohol consumption significantly increases the risk of drug interactions. We have described its effects on hamster microsomal monooxygenases. Male Syrian hamsters (85 g) were given 10% ethanol in water and food ad lib for up to 6 weeks. Microsomal electron transport components and metabolism of ethylmorphine, benzphetamine, aniline, and acetaminophen were measured. At 4 weeks, SDS-PAGE of ethanol microsomes showed an induced band with an Mr of 53,900 daltons and there was a 2–3 fold stimulation of aniline and acetaminophen metabolism. Cytochrome P-450 increase was not significant. For the six week period, Caloric intake (3 weeks, p<0.001), liquid consumption (3 weeks, p<0.05) and body weights (6 weeks, p<0.05) of ethanol animals were significantly greater than controls; kidney weights were significantly less ( p<0.05). Ethanol consumption increased from 20% of the daily caloric intake (week 1) to 31% (week 6). Induction of specific substrate metabolism without apparent deleterious physiological changes establishes hamsters fed 10% ethanol in drinking water as a biochemical model for the study of chronic alcohol consumption and specific drug interactions.

Mixed-function oxygenase activity and benzo[a]pyrene metabolism in the barnacle Balanus eburneus (Crustacea: Cirripedia)

1. Microsomal cytochrone P-450-dependent mixed-function oxygenases were observed in digestive gland, intestine, cirri and gonad of the cirriped crustacean (barnacle) Balanus eburneus. 2. Levels of the microsomal electron transport components cytochrome P-450, cytochrome b 5, NADPH-cytochrome c reductase and NADH-cytochrome c reductase in B. eburneus were lower than found in vertebrate liver, but like those seen in some decapod crustaceans. 3. The levels of benzo[a]pyrene hydroxylase activity were maximal at pH 7.5 and at temperatures between 15 and 30°C. Maximal activities ranged between 2 and 43 pmol 3-hydroxybenzo[a]pyrene min/mg microsomal protein as measured with a fluorescence assay. Activities were higher in summer than in winter. 4. Substrate saturation curves obtained for benzo[a]pyrene were hyperbolic, with none of the complexity seen in vertebrates. The K m S for benzo[a]pyrene metabolism in digestive gland and intestine were 6–7 × 10 −7 M. 5. Compounds eluting with 3-hydroxybenzo[a]pyrene in HPLC analyses constituted almost 60% of metabolites formed by barnacle digestive gland microsomes. 9-hydroxybenzo[a]pyrene, 7,8-dihydrodiol and 9,10-dihydrodiol were formed more slowly. 6. Benzo[a]pyrene hydroxylase activity in tissue microsomes from untreated animals was inhibited more than 50% by 10 −4 M 7,8-benzoflavone. 7. Cytochromes P-450 in barnacles might be functionally related to the status of hormones regulating development and reproduction.

Hepatic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish stenotomus versicolor

NADPH-cytochrome c reductase, benzo[ a]pyrene hydroxylase and aminopyrine demethylase activities in hepatic microsomes from the marine fish scup ( Stenotomus versicolor) were characterized according to dependence of Ph, temperature, ionic strength and Mg 2+. The kinetic properties of benzo[ a] pyrene hydroxylase were variable, depending on protein and substrate concentration, with measured K m values for benzo[ a]pyrene between 4 × 10 −7 M and 4 × 10 −5 M. The K m for aminopyrine was 7 × 10 −4 M, and NADPH-cytochrome c reductase had K m values of 2.1 × 10 −5 M and 1.3 × 10 −5 M for cytochrome c and NADPH. respectively. NADH supported benzo[ a]pyrene hydroxylation at 10 per cent of the rate seen with NADPH, and no synergism was observed. Aminopyrine demethylation proceeded at least as well with NADH as with NADPH, and there was synergism when combined. NADPH- and NADH-cytochrome c reductases were detected in “microsomes” from fourteen extrahepatic tissues, including kidney, testis, foregut, gill, heart, red muscle, hindgut, buccal epidermis, pyloric caecum, spleen, brain, lens, ovary and white muscle. Benzo[ a]pyrene hydroxylase was detected in all but white muscle, while cytochrome P-450 and aminopyrine demethylase were detectable in fewer tissues. Reduced, CO-ligated absorption maxima in the Soret region were 450 nm for all those but liver (occasionally 449 nm) and heart (about 447 nm). The estimated turnover numbers for benzo[ a]pyrene hydroxylase and aminopyrine demethylase, and the influence of 7,8-benzoflavone in vitro on benzo[ a]pyrene hydroxylase indicate that the cytochromes P-450 in different fish tissues are not catalytically equivalent.

Characterization of microsomal electron transport components from control, phenobarbital- and 3-methylcholanthrene-treated mice. III. Improved resolution and quantitation of major components in ammonium sulfate fractions from total liver microsomes

Quantitation of microsomal components in ammonium sulfate fractions using a high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and a comparison of these results with those from similar experiments on total liver microsomes has enabled us to identify and better characterize the interactions between microsomal electron transport components. It was found that: (1) phenobarbital decreased the amount of one protein component of approximately 50 000 molecular weight while increasing a component of very similar molecular weight; (2) only two proteins appeared to be associated with CO binding; (3) another protein of approximately 68 000 molecular weight, one of the glycoproteins found in liver microsomes, appears to be induced by phenobarbital pretreatment; (4) the induction of NADPH-cytochrome c reductase activity after phenobarbital pretreatment is not dependent on an increase in the known NADPH-dependent flavoprotein, but rather on the increase in some component found predominately in our most soluble sub-microsomal fraction. A very good separation of the above components was achieved by ammonium sulfate fractionation, e.g. simply on the basis of their solubility. This and the fact that the more-or-less soluble proteins were induced by phenobarbital or 3-methylcholanthrene respectively indicate that the solubility of membrane proteins plays a major role in the structure and function of microsomal membranes.

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

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

Involvement of cytochrome b5 in the oxidative desaturation of linoleic acid to gamma-linolenic acid in rat liver microsomes.

The effects of antibodies against microsomal electron-transport components on the in vitro activity of delta6-desaturation of linoleic acid to gamma-linolenic acid have been studied in intact microsomal membranes of rat liver. Reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (0.87 mM) served as electron donors, and effectively prompted the delta6-desaturase activities with yields of about 1.1 to 1.3 nmol per mg of protein in 10 min. Of the two antibodies studied under the same in vitro conditions, i.e., rabbit antisera preparations against rat liver microsomal hydrophilic parts of cytochrome b5 and NADPH-cytochrome c reductase, only the antibody against cytochrome b5 demonstrated a marked ability to inhibit the delta6-desaturase activity. This evidence supports a participation of cytochrome b5 in the delta6-desaturation of linoleic acid and suggests a pathway analogous to the delta9-desaturation of stearyl-CoA.

Biophysical and enzymological studies upon the interaction of trans-cinnamic acid with higher plant microsomal cytochromes P-450.

The interaction of trans-cinnamic acid with the cytochrome P-450 of microsomes derived from washed potato slices has been studied. The washing process increased the specific content of microsomal electron transport components and hence provided a useful material in which to study the interaction. Evidence is presented that the trans-cinnamic acid interacts with the cytochrome P-450, and that this interaction is analogous to "type 1" interactions of other cytochrome P-450 systems. This evidence includes the formation of a "type 1" substrate binding spectrum, an increased rate of reduction of cytochrome P-450 by NADPH in the presence of trans-cinnamic acid, an increased oxygen uptake and NADPH oxidation when trans-cinnamic acid is added to the microsomes in the presence of NADPH, and a close correlation between biophysical parameters of electron transport in the cytochrome P-450 system and enzymological parameters of the trans-cinnamic acid 4-hydroxulation reaction. The investigation has been extended to cytochrome P-450 systems of other tissues and it has been found that the trans-cinnamic acid 4-hydroxylation reaction cannot account for the presence of most of th cytochrome P-450 in several tissues. This suggests that other functions of higher plant cytochrome P-450 chains exist, and that the substrate specificityof the hemoprotein may vary in different plant tissues.

The relationship of cobaltous chloride-induced alterations of hepatic microsomal enzymes to altered carbon tetrachloride hepatotoxicity

The administration of cobaltous chloride to rats produced a selective dose-dependent decrease in the cytochrome P-450 component of the microsomal electron transport system. A concommitant of the alteration in microsomal electron transport components was a refractoriness to the carbon tetrachloride-induced elevation in serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), isocitric dehydrogenase (ICDH), and the decrease in hepatic microsomal glucose-6-phosphatase activity. Cobaltous chloride pretreatment also decreased the intensity of microsomal diene conjugation absorption observed after carbon tetrachloride administration. The results support the concept that cytochrome P-450 is the site of production of toxic metabolites following carbon tetrachloride intoxication.

Characterization of microsomal electron transport components from control, phenobarbital and 3-methylcholanthrene treated mice: II. Resolution and quantitation of cytochromes P-450 and P 1-450 and the so-called “Factor X” in SDS-polyacrylamide gels of total microsomes

By improving the resolution of microsomal proteins in SDS-polyacrylamide gels, we have succeeded in assigning one major protein not only to each of the four known components of microsomal electron transport but also to the proposed components, cytochrome P 1-450 and “Factor X”. The increases in P-450 protein after PB induction and P 1-450 protein after 3-MC induction were comparable to the respective increases in CO binding. They demonstrated molecular weights of 56,000 and 54,000 daltons respectively. The “Factor X” protein, molecular weight = 48,000, appears to be of non-heme iron character, and probably is an intermediated electron carrier between flavo- and hemoproteins.

Electron Paramagnetic Resonance Studies of Cytochrome P-450 in Plant Microsomes

The technique of electron paramagnetic resonnance spectrometry has been applied to the study of plant microsomal electron-transport components. Only tulip-bulb microsomes were found to give strong enough signals to allow detailed study. At 77 K in the oxidised state, signals were observed at g values of 2.40, 2.25 and 1.93, characteristic of cytochrome P-450 in the low-spin state, and also at g = 4.27, attributable to ferric iron in a rhombic environment. The signals at g = 2.40, 2.25 and 1.93 disappeared upon reduction with sodium dithionite. At 10 K in the oxidised state, signals at g = 8.3 and 3.3 appeared, and these were attributed to high-spin cytochrome P-450. At this temperature a further signal at g = 6, due to cytochrome P-420, was seen in aged tulip-bulb microsomes. Redox titration of both high-spin and low-spin cytochrome P-450 gave the same apparent midpoint potential of -315 +/- mV at pH 6.8 and 25 degrees C. The significance of this value is discussed. Addition of "type I" or "type II" ligands to oxidized cytochrome P-450 caused an increase and a decrease, respectively, in the ratio of the high-spin to the low-spin form. A second effect of aniline, a type II ligand of cytochrome P-450, was to remove the g = 6 signal, suggesting that it also interacts with cytochrome P-420. No iron-sulphur proteins similar to those found in some other cytochrome P-450 electron-transport chains could be detected in any of the microsomes analysed.

Characterization of microsomal electron transport components from control, phenobarbital, and 3-methylcholanthrene treated mice: I. Distribution of electron transport components in ammonium-sulfate fractions from mouse liver microsomes

Using a modified ammonium sulfate fractionation procedure, seven major components were found in various submicrosomal fractions. Four of these proteins could be assigned to known components of the microsomal electron transport system, cytochromes P-450 and b 5, and the NADH- and NADPH-cytochrome c reductases. The similarity of this system to that seen in the separation of mitochondrial enzymes suggests that the lipo-protein complexes of these fractions are particulate in nature and represent functional subunits of the microsomal membrane.

Ethanol metabolism in the vitamin C deficient guinea-pig

Vitamin C deficiency, although reducing microsomal aniline hydroxylase and NADPH oxidase activities and decreasing the amount of cytochrome P-450, had no effect on the rate in vivo of ethanol elimination. Ethanol given as a daily oral dose of 2.5g/kg for 14 days did not induce microsomal enzyme activities or increase the concentration of microsomal electron transport components. Drugs that are metabolized by the microsomal drug metabolizing system have increased plasma half lifes in scorbutic guinea-pigs, but no decrease in ethanol disappearance from the blood in the scorbutic animals used in the present study was noted. It is concluded that when ADH activity is normal, no further systems are required for ethanol metabolism.

Stimulation of drug metabolism by ascorbic acid in weanling guinea-pigs

Drug metabolism activities such as O-demethylase and N-demethylase and liver microsomal electron transport components including cytochrome P-450 and NADPH-cytochrome P-450 reductase were determined in weanling guinea-pigs under vitamin C deficiency as well as with normal and high intake of the vitamin. Animals depleted of the vitamin for only 8 days, with no signs of scurvy or weight loss, had decreased drug enzyme activities. Further depletion of the vitamin resulted in marked reduction of the microsomal drug-metabolizing system. Furthermore, administration of high amounts of ascorbic acid (2–200 mg/day) led to significant increases in liver drug oxidation activities and electron transport components. Studies of K m values in microsomes from vitamin C-deficient, normal and animals given high supplements of vitamin C showed no difference in the apparent affinity or V max of a typical drug oxidation reaction, N-demethylation. Specificity studies indicate that ascorbyl palmitate or d-isoascorbic acid can replace the vitamin, in part, in maintaining drug metabolism.

Species differences and d rug metabolism

In this report three types of drug oxidation reactions were studied in mice, rats and guinea pigs, e.g. aminopyrine N-demethylation, p-nitroanisole O-demethylation and aniline hydroxylation. The studies in vitro show that no species emerged as a rapid or slow metabolizer of these prototype drug substrates. The determination of the activity and quantity of liver microsomal electron transport components indicate that cytochrome P-450 and NADPH cytochrome P-450 reduetase are important factors in determining the activity of overall drug oxidation. However, the level of cytochrome b 5 and activity of NADPH cytochrome c reduetase as well as the apparent affinity of the microsomal system for NADPH could not account for the observed differences in drug metabolism. A study of the binding of drug substrates to cytochrome P-450 indicates that there are qualitative differences in this cytochrome which may contribute to species variation in drug metabolism. Species differences in cytochrome P-450 are not apparent in the reduced cytochrome P-450-CO complex or the typical type I (aminopyrine) and type II (aniline) substrate-cytochrome P-450 binding spectra. However, prolonged treatment of microsomes with aminopyrine or aniline results in an atypical type Ia or type IIa spectrum which has a maximum absorption at different wavelengths in various species. Studies employing phenobarbital or 3-methylcholanthrene to induce individual electron transport components indicate that N-demethylase and O-demethylase activities are dependent upon the level of cytochrome P-450 and that aniline hydroxylase activity is dependent upon the level of cytochrome P-450 reduetase activity.