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PubMed1996

Effects of maternal vitamin E and selenium status during the perinatal period on age-related changes in heart, lung and liver microsomal lipid peroxidation in rat pups.

Female Long-Evans rats of breeding age were acclimated to one of four experimental diets differing in their vitamin E (E) and selenium (Se) contents. These animals were bred and the offspring produced were nursed by their respective dams from birth through 21 days postpartum. Rat pups were killed at postpartum intervals of 4, 9, 14 and 21 days and heart, lung and liver tissue used for isolation of microsomes. Microsomes were used for quantitation of E content as well as for assays of lipid peroxidation (thiobarbituric acid reactive products formation) using an ascorbate/ADP nonenzymatic assay system. Microsomal E content was lower in heart and lung tissues of rat pups at each postpartum interval compared with maternal levels. Liver microsomal E was higher in rat pups at day 4 postpartum compared with maternal levels. Significant differences (P < 0.05) in microsomal E content of rat pup tissues were associated with maternal vitamin E status during the perinatal period and not to differences in dietary Se. In vitro lipid peroxidation was highest in rat pups nursing dams of low E status, the magnitude of which was greatest in heart and liver microsomes. Lung microsomes, in contrast, peroxidized at very low rates compared with heart and liver microsomes. These effects were independent of maternal Se status. Microsomal lipid peroxidation in tissues of rat pups was lowest at the earliest postpartum interval studied (4 days) and, in general, increased with postpartum age. Reduced glutathione did not inhibit lipid peroxidation in heart or lung microsomes but was effective in this respect in liver microsomes of rat pups as early as 4 days postpartum.

Aging↗
PubMed1993

Oxidative stress-induced activation of microsomal glutathione S-transferase in isolated rat liver.

The activation of microsomal glutathione (GSH) S-transferase in isolated rat liver by oxidative stress was investigated using both ischemia/reperfusion and perfusion with hydrogen peroxide. When the isolated liver was reperfused for 30 min and 60 min after 90 min ischemia, microsomal GSH S-transferase activity, but not cytosolic transferase activity, was increased 1.2-fold and 1.3-fold, respectively. In addition, microsomal GSH peroxidase activity was also significantly increased after 60 min reperfusion following ischemia. The increase in microsomal GSH S-transferase activity by ischemia/reperfusion was reversed by dithiothreitol. When N-ethylmaleimide, which activates microsomal GSH S-transferase by covalent binding to the cysteine residue of the enzyme, was incubated with microsomes, transferase activity was increased to 526% in control microsomes and to 399% in liver that underwent ischemia/reperfusion liver. These data indicate that microsomal GSH S-transferase is activated by ischemia/reperfusion of the liver by means of disulfide bond formation. When rats were pretreated with a catalase inhibitor 3-amino-1,2,4-triazole for 6 weeks, microsomal GSH S-transferase activity was increased 1.4-fold by ischemia/reperfusion, corresponding to a 1.8-fold increase as compared to the non-perfused liver of untreated rats. Perfusion of the isolated liver with hydrogen peroxide (1 mM, 15 min) also caused a significant increase in microsomal GSH S-transferase activity with a concomitant decrease in GSH content, confirming that liver microsomal GSH S-transferase in rats was activated in vivo by oxidative stress.

Animals↗
PubMed2000

Stereoselective propranolol metabolism in two drug induced rat hepatic microsomes.

AIM:To study the influence of inducers BNF and PB on the stereoselective metabolism of propranolol in rat hepatic microsomes.METHODS:Phase I metabolism of propranolol was studied by using the microsomes induced by BNF and PB and the non induced microsome as the control.The enzymatic kinetic parameters of propranolol enantiomers were calculated by regression analysis of Lineweaver-Burk plots. Propranolol concentrations were assayed by HPLC.RESULTS:A RP-HPLC method was developed to determine propranolol concentration in rat hepatic microsomes. The linearity equations for R(+)propranolol and S(-) propranolol were A = 705.7C+311.2C (R = 0.9987) and a = 697.2C+311.4C (R = 0.9970) respectively. Recoveries of each enantiomer were 98.9%, 99.5%, 101.0% at 60&mgr;mol/L, 120&mgr;mol/L, 240&mgr;mol/L respectively. At the concentration level of 120&mgr;mol/L, propranolol enantiomers were metabolized at different rates in different microsomes. The concentration ratio R(+)/S(-) of control and PB induced microsomes increased with time, whereas that of microsome induced by BNF decreased. The assayed enzyme parameters were: 1. Km. Control group: R(+)30 plus minus 8, S(-)18 plus minus 5; BNF group: R(+)34 plus minus 3, S(-)39 plus minus 7; PB group: R(+)38 plus minus 17, S(-)36 plus minus 10. 2. Vmax. Control group: R(+)1.5 plus minus 0.2, S(-)2.9 plus minus 0.3; BNF group: R(+)3.8 plus minus 0.3, S(-)3.3 plus minus 0.5; PB group: R(+)0.07 plus minus 0.03, S(-)1.94 plus minus 0.07. 3. Clint. Control group: R(+)60 plus minus 3, S(-)170 plus minus 30; BNF group: R(+)111.0 plus minus 1, S(-) 84 plus minus 5; PB group: R(+)2.0 plus minus 2, S(-)56.0 plus minus 1. The enzyme parameters compared with unpaired t tests showed that no stereoselectivity was observed in enzymatic affinity of three microsomes to enantiomers and their catalytic abilities were quite different and had stereoselectivities.Compared with the control, microsome induced by BNF enhanced enzyme activity to propranolol R(+)enantiomer, and microsome induced by PB showed less enzyme activity to propranolol S(-)enantiomer which remains the same stereoselectivities as that of the control.CONCLUSION:Enzyme activity centers of the microsome were changed in composition and regioselectivity after the induction of BNF and PB, and the stereoselectivities of propranolol cytochrome P450 metabolism in rat hepatic microsomes were likely due to the stereoselectivities of the catalyzing function in enzyme.CYP1A subfamily induced by BNF exhibited pronounced contribution to propranolol metabolism with stereoselectivity to R(+)enantiomer.CYP2B subfamily induced by PB exhibited moderate contribution to propranolol metabolism, but still had the stereoselectivity of S(-)enantiomer.

Journal Article↗
PubMed1994

CYP2C11 and CYP2B1 are major cytochrome P450 forms involved in styrene oxidation in liver and lung microsomes from untreated rats, respectively.

The contribution of cytochrome P450s (P450s) to the formation of styrene glycol from styrene in rat liver microsomes was investigated using monoclonal antibodies to P450s. Anti-CYP2E1 inhibited the formation to a similar extent in ethanol-treated microsomes and in control microsomes in terms of percentage inhibition, whereas to a greater extent in the former than the latter in terms of net inhibition, and only at low substrate concentration. Anti-CYP2C11/6 also inhibited the formation in control and in ethanol-treated microsomes at both low and high concentrations of styrene, and the net degree of inhibition was greater than that obtained with anti-CYP2E1, even in ethanol-treated microsomes where CYP2E1 was induced. Anti-CYP2B1/2 and anti-CYP1A1/2 inhibited the formation only in phenobarbital (PB)- and 3-methylcholanthrene (MC)-induced microsomes, respectively. These results suggest that (1) at least four P450s, CYP2C11/6, CYP2E1, CYP2B1/2 and CYP1A1/2, contribute to the metabolism of styrene, (2) CYP2C11/6, which probably corresponds to CYP2C11, is the major form of P450 responsible for the metabolism in untreated rat liver microsomes, and also in those treated with ethanol. Anti-CYP2E1 inhibited styrene oxidation more prominently in microsomes from styrene-treated rats than in those from control rats at a low substrate concentration. Although styrene treatment did not influence the total metabolism of styrene in liver microsomes at a high substrate concentration, inhibition of the metabolism by anti-CYP2C11/6 decreased with increasing styrene dose, whereas that by anti-CYP2B1/2 increased, suggesting that styrene treatment increases CYP2B1/2 but decreases CYP2C11/6 in rat liver, and the major form of P450 which mediates styrene oxidation is CYP2B1/2 after the treatment. Only anti-CYP2B1/2, which probably corresponds to CYP2B1, inhibited styrene oxidation in lung microsomes from untreated and even styrene-treated rats. Thus, the major form of P450 responsible for the metabolism of styrene is different in each tissue.

Animals↗
PubMed1988

Studies on the ability of rat lung and liver microsomes to facilitate transfer and metabolism of benzo[a]pyrene from diesel particles.

Little is known about the bioavailability of inhaled organic compounds that are associated with particles. It is known that certain particle-associated organic compounds, such as polycyclic aromatic hydrocarbons (PAH) adsorbed on diesel soot particles, are retained in the lung longer than PAH inhaled in pure form. If such particle-associated compounds are available for tissue interaction, their prolonged retention may result in an increased potential for a toxic effect. To determine the factors affecting the bioavailability of particle-associated PAH, we have studied the ability of microsomes to facilitate transfer of benzo[a]pyrene (B[a]P) adsorbed on the surface of diesel exhaust soot particles to the microsomes and the ability of the microsomes to metabolize the transferred B[a]P. Our results indicate that rat lung and liver microsomes were able to facilitate the transfer of small amounts of B[a]P from diesel particles (less than 3%), but only a fraction of the amount transferred (1-2%) was metabolized. Under the same incubation conditions without soot, free B[a]P was extensively metabolized by microsomes, principally to B[a]P-9,10-diol. Lung microsomes were about twice as effective as liver microsomes for the transfer of the B[a]P. The ability to transfer B[a]P to the microsomes was independent of metabolism or the presence of protein, but was related to the lipid content of the microsomal fraction. There was no metabolism of the B[a]P coated on diesel particles as analyzed by high-performance liquid chromatography. These findings suggest that microsomes are able to enhance the slow transfer of only a small amount of B[a]P from diesel particles in a form that can be metabolized. However, over a long period of time, this slow release might be significant.

Adsorption↗
PubMed1992

Metabolism of [4-14C]estrone in hamster and rat hepatic and renal microsomes: species-, sex- and age-specific differences.

The metabolism of [4-14C]estrone (E1) was examined in liver and kidney microsomes of adult castrated male and ovariectomized female hamsters and rats and in neonatal and immature hamster renal microsomes. In castrated male hamster liver microsomes, E1 was metabolized extensively to six major metabolites; 15 beta-hydroxyestrone, 7 alpha-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone, 2-hydroxyestrone, and delta(9,11)-dehydroestrone, and a nonpolar fraction. Six minor metabolites of E1 were also detected. In contrast, kidney microsomes derived from castrated male hamsters metabolized E1 to mainly 17 beta-estradiol, 2- and 4-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone and one monohydroxyestradiol metabolite. However, 16 alpha-hydroxyestrone was not detected. A variable, but low amount of estriol was also found. Interestingly, the quantity of 2-hydroxyestrone found in kidney microsomes of the hamster represented 26% of the total amount of metabolites formed, whereas in liver microsomes, only 9% of the overall metabolism resulted in the formation of 2-hydroxyestrone. The ability of kidney microsomes of female ovariectomized hamsters and two different rat strains to metabolize E1 was 5.9- and 9.4-fold lower, respectively, compared to renal microsomes of male castrated hamsters. The onset of oxidative metabolism in newborn hamster kidneys during development was also assessed. The results indicate that the oxidative metabolism of [14C]E1 in renal microsomes of newborn hamsters was 20-fold less than in kidney microsomes of adult hamsters. While catechol E1 metabolites were essentially negligible in hamster kidneys of these ages, it was evident that the conversion of E1 to estradiol via 17 beta-hydroxysteroid dehydrogenase resembles levels seen in the adult animals. Between the age of one and two months, the male hamster kidney exhibited the capacity to metabolize E1 at levels seen in fully mature adult hamsters.

Age Factors↗
PubMed2001

Aging promotes a different phosphatidic acid utilization in cytosolic and microsomal fractions from brain and liver.

Among the morphological and biochemical changes taking place in the membranes of aged tissues, we reported in previous studies on alterations in phospholipid synthesis and phospholipid-specific fatty acid composition. Phosphatidic acid (PA) and diacylglycerol (DAG) are central intermediates in phosphoglyceride and neutral lipid biosynthetic pathways and have also recently been implicated in signal transduction. The present paper shows the effect of aging on phosphatidate phosphohydrolase (PAPase) activiy, which operates on phosphatidic acid to synthesize diacylglycerol. Two forms of mammalian PAPase can be indentified on the basis of subcellular localization and enzyme properties, one involved predominantly in lipid synthesis (PAP 1) and the other in signal transduction (PAP 2). Microsomal and cytosolic fractions of brain and liver from 3.5-month-old (adult) and 28.5-month-old (aged) rats were used. PAPase isoform activities were differentiated on the basis of N-ethylmaleimide (NEM) sensitivity and Mg(2+)-dependency. Our results demonstrate that aging caused PAP 2 to increase in brain microsomal fractions but did not affect PAP 1, whereas in brain cytosolic fractions, it caused a strong decrease in PAP 1 (57%). The distribution of enzymes between microsomes and cytosol changed in aged rats with respect to adult rats, showing a translocation of PAP 1 from cytosol to microsomes. In addition, an increase in the production of monoacylglycerol (MAG) was observed in microsomes from aged brain. PAP 2 activity in liver microsomal fractions from aged rats showed no changes with respect to adult rats whereas PAP 1 activity increased 228% in microsomal fractions and 76% in cytosolic fractions in this tissue. The distribution of PAP 1 activity between microsomal and cytosolic fractions in liver tissue was also affected in aged rats, indicating a translocation of this form of the enzyme from cytosolic to microsomal fractions. The production of monoacylglycerol in liver microsomes also increased, whereas there was a decrease in MAG formation from cytosolic fraction. The changes observed in the two PAPase forms in brain and liver of aged rats with respect to adult rats suggest that PA is differently utilized by the PAPase isoforms, probably generating aging-related DAGs different to those present in adults and required for specific cellular functions. The changes observed in liver PAP 1 from aged with respect to adult rats suggest that such changes could be related with modifications in lipid homeostasis induced by age-altered hormonal balance. However, PA-modified utilization during aging through PAP 2 activity could be related to alterations in neural signal transduction mechanisms.

Aging↗
PubMed1991

Species comparison of acrylonitrile epoxidation by microsomes from mice, rats and humans: relationship to epoxide concentrations in mouse and rat blood.

Acrylonitrile (ACN) has been shown to cause tumors of the brain, stomach and Zymbal's gland in rats in several bioassays, but it has not been tested in other species. The carcinogenic risk of humans exposed to ACN is unclear. ACN is metabolized in the liver to 2-cyanoethylene oxide (CEO), which is believed to be the proximate or ultimate carcinogenic species. Therefore, the kinetics of CEO formation were studied with liver and lung microsomes from mice and humans using a GC-MS assay for CEO, and the data were compared with previously obtained kinetic parameters for rat microsomal enzymes. The rate of CEO formation by human liver microsomes was comparable to that of rat liver microsomes, but less than that of mouse liver microsomes. Liver microsomes produced more CEO than lung microsomes with all three species. CEO formation by microsomes from mice was approximately 4 times greater than that by microsomes from rats or humans, suggesting that mice would have higher CEO concentrations in blood than rats after ACN exposure. However, after oral administration of ACN, the concentration of CEO in mouse blood was one-third that in rat blood at all doses and time points examined. These results show that CEO circulates via the blood, providing exposure to distant sites. The blood concentrations of CEO do not appear to correlate with rates of microsomal CEO formation. This suggests that species differences in the detoxication of CEO may play an important role in determining circulating CEO concentrations and distant organ exposure.

Acrylonitrile↗
PubMed1991

Fatty acid elongation and desaturation enzyme activities of bovine liver and subcutaneous adipose tissue microsomes.

Assay conditions were established for the fatty acid elongation and the delta 9 desaturase enzyme systems of bovine liver and adipose tissue microsomes; rat liver microsomes were used as a reference. Overall fatty acid elongation was determined by measuring the incorporation of [2-14C]malonyl-coenzyme A (CoA) to 14C-labeled stearate. Rat liver elongation activity was .50 +/- .02 nmol.min-1.mg protein-1; bovine liver microsomal elongation activity was substantially lower (P less than .05), with a mean value of .15 +/- .02 nmol.min-1.mg protein-1. The elongation activity of bovine s.c. adipose tissue microsomes (.42 +/- .10 nmol.min-1.mg protein-1) was not different (P greater than .05) from the activity observed in rat liver microsomes. To determine the fatty acid delta 9 desaturase activity, microsomes were incubated in the presence of [1-14C]stearoyl-CoA and nicotinamide adenine dinucleotide (reduced form) (NADH), and the production of radioactively labeled oleate was quantified. Microsomal delta 9 desaturase activity was similar in rat liver and bovine s.c. adipose tissue microsomes with rates of .15 +/- .04 and .21 +/- .05 nmol.min-1.mg protein-1, respectively. However, no desaturase activity was detected in bovine liver microsomes, indicating that the liver is not a major site of oleate synthesis in this species. To investigate differences in fatty acid metabolism relative to breed type, eight Angus and seven Braford heifers were slaughtered at approximately 12 mo of age. Subcutaneous fat thickness over the 12th-13th thoracic vertebrae was greater in the Angus heifers than in the Braford heifers. However, no differences (P greater than .05) were observed in mean adipocyte size or number of cells per gram of adipose tissue between the Angus and Braford heifers. Similarly, there were no significant differences between the Angus and Braford s.c. adipose tissues for microsomal fatty acid elongation or delta 9 desaturation, or for nicotinamide adenine dinucleotide phosphate (NADP)-malate dehydrogenase, fatty acid synthetase, or the pentose cycle reductases. The inability of bovine liver to convert stearate to oleate was in agreement with the fatty acid composition of the liver lipid, which had a smaller percentage of oleate and a higher percentage of stearate than s.c. adipose tissue.

Acetyltransferases↗
PubMed1992

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in human lung and liver microsomes and cytochromes P-450 expressed in hepatoma cells.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent tobacco-specific carcinogen in animals, has been linked to tobacco-related cancers in humans. The cytochrome(s) P-450 (P-450) responsible for the metabolic activation of NNK in humans has not been identified. The present work investigated the ability of human lung and liver microsomes and 12 forms of human P-450, expressed in Hep G2 (hepatoma) cells, to metabolize NNK. Of the 12 P-450 forms, P-450 1A2 had the highest activity in catalyzing the conversion of NNK to the keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone. P-450s 2A6, 2B7, 2E1, 2F1, and 3A5 also had measurable activities in the formation of keto alcohol. The apparent Km and Vmax for the formation of keto alcohol in the P-450 1A2-expressed Hep G2 cell lysate were 309 microM and 55 pmol/min/mg protein, respectively. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol, a reductive product, was the major metabolite formed, whereas the formation of keto alcohol and its aldehyde and acid derivatives (all alpha-hydroxylation products) constituted approximately 1% of the initial amount of NNK in P450-expressed Hep G2 cell lysate. A similar metabolite pattern was observed with human lung or liver microsomes. In human lung microsomes, the apparent Kms for the formation of 4-hydroxy-4-(3-pyridyl)butyric acid, 4-oxo-1-(3-pyridyl)-1-butanone, NNK-N-oxide, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were 526, 653, 531, and 573 microM, respectively; the formation of keto alcohol was not observed. For human lung microsomes, there was no sex-related difference in NNK metabolism. Carbon monoxide (90% atmosphere) significantly inhibited the metabolism of NNK in human lung and liver microsomes. 7,8-Benzoflavone, an inhibitor of P-450s 1A1 and 1A2, had no effect on NNK metabolism in human lung microsomes but decreased the formation of keto alcohol by 47% in human liver microsomes. Similarly, antibodies against human P-450s 1A2 and 2E1 decreased keto alcohol formation by 42% and 53%, respectively, in human liver microsomes but did not affect NNK metabolism in lung microsomes. Inhibitory antibodies against P-450s 2A1, 2C8, 2D1, or 3A4 had little or no effect on the metabolism of NNK in human liver or lung microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies↗
PubMedDate not supplied

Characteristics of a microsomal cytochrome P-448-mediated reaction. Ethoxyresorufin O-de-ethylation.

Certain characteristics of ethoxyresorufin O-de-ethylation, as catalyzed by microsomes of liver, lung, and intestine of control and pretreated rats and hamsters, were studied. The results support previous suggestions that the reaction is catalyzed primarily by a 3-methyl-cholanthrene (MC)-inducible mono-oxygenase which has a 448-nm absorption maximum in the reduced-CO difference spectrum. Ethoxyresorufin exhibited a type I binding spectrum with liver microsomes from MC-induced rats, but there was no clear interaction with microsomes from control or phenobarbital (PB)-induced rats. Maximum MC-induction of type I binding and de-ethylase activity coincided with the appearance of a cytochrome P-450 spectrum whose absorption maximum was shifted to 448 nm. Low concentrations of alpha-naphthoflavone (ANF) or benzo[a]pyrene (BP) inhibited the de-ethylation with liver microsomes of MC-treated rats (150 approximately 10(-9)M) but not those of control rats. A kinetic analysis of BP inhibition of this reaction showed it to be competitive. Inhibition of the MC-induced liver microsomal reaction by low concentrations of BP or ANF diminished rapidly with time. MC-induced rat liver microsomal de-ethylation of ethoxyresorufin was less sensitive than the PB-induced reaction to inhibition by metyrapone or SKF 525-A (I50 approximately 10(-6) - 10(-4)M). However, microsomes from rat liver, lung, and intestine had very low constitutive activities (less than 0.1 nmol/min/mg of protein). MC greatly induced the de-ethylation reaction in liver (200 X), intestine (40 X) and lung (10 X). De-ethylation of ethoxyresorufin in microsomes from control and MC-induced rat lung was inhibited by low concentrations of either ANF or BP (I50 approximately 10(-8) M). Control hamster liver microsomes were several times more active in de-ethylation than control rat liver microsomes, but MC-induction of hamster liver was only 1/10 of that in rat liver. Control hamster lung activity was similar to that of control rat lung, but was not appreciably induced by MC.

Animals↗
PubMed1990

Cyclosporine A induced lipid peroxidation and influence on glucose-6-phosphatase in rat hepatic and renal microsomes.

The in vitro effect of cyclosporine A (CsA) on lipid peroxidation (LPO) in hepatic and renal microsomes (male Wistar rats) were investigated either with different CsA concentrations (0.3-1000 micrograms/ml), incubation time 3 h or for different periods of time (0.5-3.0 h) at a CsA concentration of 1000 micrograms/ml. LPO was monitored by measuring the formation of malondialdehyde (MDA) using the thiobarbituric acid assay. Furthermore the influence of CsA on the microsomal enzyme glucose-6-phosphatase was investigated. CsA caused a time- and concentration-dependent increase of LPO in hepatic and renal microsomes. The lowest CsA concentration which produced a significant increase in MDA production amounted to 1 microgram/ml for hepatic microsomes and 3 micrograms/ml for renal microsomes. Under identical experimental conditions, the MDA production by hepatic microsomes was 3 to 5 fold higher than by renal microsomes. Addition of the radical scavenger alpha-tocopherol (1 mM) to the incubation medium decreased the CsA-caused microsomal MDA production. Regarding the microsomal enzyme, CsA decreased the specific activity of glucose-6-phosphatase in a time- and concentration-dependent fashion. Compared to microsomal MDA production, higher CsA concentrations were necessary to effect on specific enzyme activity. The results suggest, that production of free radicals and subsequently lipid peroxidation could participate in cyclosporine A induced hepato- and nephrotoxicity.

Animals↗
PubMed1990

Immunochemical analysis of a cytochrome P-450IA1 homologue in human lung microsomes.

The monoclonal antibody MAb 1-7-1, which specifically binds to cytochromes P-450IA1 and P-450IA2 in 3-methylcholanthrene-induced rat liver microsomes, was used to identify a cytochrome P-450IA1 homologue in human lung microsomes. Although MAb 1-7-1 had similar affinity constants for human and rat microsomes, the amount bound to human lung microsomes was severalfold lower than that bound to microsomes from untreated rat or rabbit lung and much lower than the amount bound to 3-methylcholanthrene-induced rat lung or liver microsomes. The amount bound to untreated baboon lung microsomes was similar to that bound to human lung microsomes. Three cytochrome P-450IA1-catalyzed activities, 7-ethoxyresorufin O-deethylase, 7-ethoxycoumarin, O-deethylase, and aryl hydrocarbon hydroxylase, were measurable in human lung microsomes, but the cytochrome P-450IA2-dependent activity acetanilide 4-hydroxylase was not. MAb 1-7-1 inhibited, and its binding correlated strongly with, 7-ethoxyresorufin O-deethylase activity (r = 0.92, p less than 0.01) in human lung microsomes. 7-Ethoxyresorufin O-deethylase activities in human lung were similar to those measured in untreated baboon lung but considerably lower than those present in untreated rabbit lung, untreated or 3-methylcholanthrene-induced rat lung and liver, or human liver. We conclude that MAb 1-7-1 recognizes a cytochrome P-450IA1 homologue in human lung and that no cytochrome P-450IA2 homologue is detected. Cytochrome P-450IA1 is expressed in human lung at relatively low levels, similar to those observed in untreated primate (baboon) lung. The majority of the 19 human lung samples examined do not exhibit a permanent polycyclic aromatic hydrocarbon-induced state with respect to this isozyme.

Animals↗
PubMed1980

The environment of cytochrome P-450 in testicular microsomes.

The effects of trypsin, phospholipase A, and chymotrypsin on NADPH-cytochrome c reductase and cytochrome P-450 of microsomes from cryptorchid mouse testes and liver were compared. Trypsin released both enzymes almost completely from testis microsomes, while it readily released only NADPH-cytochrome c reductase from liver microsomes. Chymotrypsin alone, even under conditions where 30-40% of the microsomal protein was hydrolyzed, had little effect on localization or activity of either enzyme in either tissue. Phospholipase A destroyed cytochrome P-450 in testicular microsomes but had little effect on this enzyme in hepatic microsomes or on NADPH-cytochrome c reductase in either preparation. When, however, the microsomes were incubated with chymotrypsin in the presence of a detergent, the effects were similar to those of trypsin alone; testicular cytochrome P-450 was destroyed, while hepatic cytochrome P-450 was only slightly solubilized, and NADPH-cytochrome c reductase from both types of microsomes was both solubilized and activated. From these results we conclude that arginyl and/or lysyl bonds may play a significant role in the junction between the hydrophobic region of the membrane and the anchor region of the reductase molecule and that cytochrome P-450 of testicular microsomes is more superficially located in the lipid bilayer than is hepatic microsomal cytochrome P-450.

Animals↗
PubMed1975

Characterization of a rat lung microsomal fraction obtained by sepharose 2B ultrafiltration.

A new procedure for obtaining rat lung microsomes essentially free of interfering hemoproteins has been developed. The method includes Sepharose 2B column chromatography of the 12,000 X g supernatant of lung homogenates, followed by ultracentrifugation of the material eluted in the void volume. Microsomes isolated in this manner contain specific levels of cytochromes b5 and P-450 and of NADPH-cytochrome c reductase that are among the highest ever reported for a rat lung microsomal fraction. After treatment of rats with 3-methylcholanthrene, the specific content of cytochrome P-450 in lung microsomes is doubled and that of cytochrome b5 increases 1.5 times. Several spectral differences between hepatic and lung microsomal cytochrome P-450 are apparent. In lung microsomes, the maximum of the reduced CO-bound cytochrome complex in a difference spectrum is at 453 nm for the noninduced hemoprotein and shifts to 451 nm after 3-methylcholanthrene induction. In contrast, no significant change in the ethylisocyanide difference spectra of reduced microsomes is obtained after induction; moreover, the spectra obtained with induced and noninduced cytochrome P-450 are similar to the one shown by hepatic microsomes from polycyclic hydrocarbon-treated rats. Furthermore, spectrophotometric studies on n-octylamine binding to control and induced lung cytochrome P-450 yielded results different from those previously obtained with rabbit liver microsomes. It is concluded that the cytochrome P-450 present in rat lung microsomes before and after 3-methylcholanthrene treatment of the animals is distinctly different from the liver hemoprotein.

Animals↗
PubMed1993

Further characterization and purification of the flavin-dependent S-benzyl-L-cysteine S-oxidase activities of rat liver and kidney microsomes.

Previously, we provided evidence that cysteine conjugate S-oxidase (S-oxidase) activities of rat liver and kidney microsomes may be associated with flavin-containing monooxygenases (FMOs). In this study, the biochemical properties of these activities were further investigated. When NADPH was replaced by NADH, the S-oxidase activities were reduced significantly. Removal of the flavin moiety from microsomes significantly reduced the S-oxidase activities; however, addition of exogenous FAD or FMN restored the activities of the flavin-depleted microsomes. Solubilization of hepatic or renal microsomes with Emulgen 911, Nonidet P-40, Triton X-100, or 3-[(3-cholamidopropyl)dimethyl-ammonio]-1- propane sulfate or inclusion of the sulfhydryl-reactive agents Hg2+, N-ethylmaleimide, or iodoacetamide did not affect the S-oxidase activities, whereas solubilization of either hepatic or renal microsomes by cholate or heating of renal microsomes in the absence of NADPH significantly reduced the S-oxidase activities. In addition to male rat hepatic and renal microsomes, the S-oxidase activities were detected in lung microsomes of male rats and hepatic and renal microsomes of male mice and female rats and rabbits. The male rat kidney maintained the highest S-oxidase activity of all species and tissues examined. Whereas the aforementioned results provided further evidence for the S-oxidase activities being associated with FMOs, unambiguous evidence for this hypothesis was provided by the purification of the activities from rat liver (580-fold) and kidney (700-fold) microsomes and by the use of the isolated proteins in polyacrylamide gel electrophoresis, flavin content determinations, amino-terminal amino acid sequence analysis, amino acid composition analysis, and substrate kinetic studies. The findings that the S-oxidases were immunoreactive with antibodies raised against the pig liver 1A1 isozyme but not with antibodies raised against the rabbit lung 1B1 isozyme and that the liver S-oxidase amino-terminal amino acid sequence was more comparable to the amino-terminal amino acid sequences of pig and rabbit liver 1A1 isozymes than to those of rabbit lung 1B1 and liver 1D1 isozymes provide evidence that the S-oxidases are related to the known FMO 1A1 isozymes.

Adenine Nucleotides↗
PubMedDate not supplied

Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity.

The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.

Animals↗
PubMed1992

Role of cytochrome P-450 2E1 in ethanol-, carbon tetrachloride- and iron-dependent microsomal lipid peroxidation.

This study investigated the role of cytochrome P-450 2E1 in enhanced microsomal lipid peroxidation in experimental alcoholic liver disease. We also examined the contribution of this isoform to the increased microsomal injury in alcoholic liver disease caused by carbon tetrachloride-induced or iron-induced oxidant stress. Adult male Wistar rats were intragastrically infused with a high-fat diet and ethanol or glucose for 16 wk; this resulted in hepatic lipid peroxidation and fibrogenesis in the ethanol-fed animals. Microsomes were isolated by differential centrifugation in the presence of 100 mumol/L deferoxamine, washed twice in buffer without deferoxamine and incubated in the absence or presence of ethanol (50 mmol/L), carbon tetrachloride (150 mumol/L), ferric citrate (50 mumol/L) or ferric citrate plus ethanol at 37 degrees C for 30 min in an NADPH-generating system. The basal rate of lipid peroxidation in microsomes isolated from ethanol-fed rats was increased by 52% compared with that in microsomes from controls. Carbon tetrachloride-induced and ferric citrate-induced lipid peroxidation were also accentuated in microsomes from ethanol-fed rats, by 76% and 108%, respectively. Ethanol added in vitro significantly reduced basal (-58%) and ferric citrate-induced (-48%) lipid peroxidation in microsomes from ethanol-fed rats, whereas it had an insignificant effect on that in control microsomes. In fact, this protective effect of ethanol on microsomes from ethanol-fed rats resulted in attenuation of the difference in the level of microsomal lipid peroxidation between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗