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PubMed1995

Interaction of tetrachloroethylene with rat hepatic microsomal P450-dependent monooxygenases.

1. We have studied the effects of tetrachloroethylene (PCE) on the kinetics of the P450-dependent monooxygenases in rat liver microsomes. 2. 7-Pentoxyresorufin O-depentylase (PROD) and 7-benzyloxyresorufin O-debenzylase (BROD) activities in phenobarbital (PB)-treated rat liver microsomes were substantially inhibited by PCE. The inhibition profiles were non-competitive for both enzyme activities; Ki's from Eadie-Hofsee plots were 0.16 and 0.29 mM for PROD and BROD respectively. In contrast, the enzyme activities in untreated, beta-naphthoflavone (BNF)-, isoniazid (ISN)- and pregnenolone-16 alpha-carbonitrile (PCN)-induced microsomes were not affected by PCE. 3. 7-Ethoxycoumarin O-deethylase (ECOD) activity in PB-induced microsomes was competitively inhibited by PCE, with a Ki that was lower than those of other microsomes. 4. PCE inhibited 7-ethoxyresorufin O-deethylase (EROD) activities in some microsomes slightly. The Ki for PCE was the lowest in untreated, followed by ISN-treated microsomes. 5. No effect of PCE upon aniline 4-hydroxylase (AN4H) and testosterone 6 beta-hydroxylase (TS6BH) activities was evident in any microsomal preparation. 6. These results indicate that PCE inhibits PB-inducible, P450-dependent monooxygenases in vitro non-competitively or competitively, and that the P450 enzymes of the P4502B subfamily may contribute to PCE toxicity.

Animals↗
PubMed1994

Oxygen radical formation in well-washed rat liver microsomes: spin trapping studies.

The generation of hydroxyl radicals by rat liver microsomes was monitored by spin trapping with 5,5-dimethylpyrroline N-oxide (DMPO). The results confirm and extend previous data which demonstrated that hydroxyl radicals are produced by microsomes in the presence of NADPH and O2, and without the exogenous addition of iron. No EPR signals could be detected unless catalase activity which was associated with the microsomes could be substantially diminished. Addition of azide was the most effective means of eliminating catalase activity, but azide also reacted rapidly with hydroxyl radicals, forming azidyl radicals which were in turn trapped by DMPO. Extensive washing and preincubation of microsomes with 3-amino-1,2,4-triazole in the presence of H2O2 were evaluated as alternative methods of decreasing the catalase contamination of microsomes. Although neither method completely eliminated microsomal catalase activity, addition of azide was no longer necessary for hydroxyl radical detection with DMPO. When highly washed microsomal preparations were tested, weak signals of the superoxide radical adduct of DMPO could also be detected. These data indicate that the sensitivity of spin trapping in microsomal systems can be improved substantially when care is taken to eliminate cytosolic contaminants such as catalase.

Animals↗
PubMed1983

Vitamin K-dependent carboxylase: effect of endogenous microsomal protein precursors on the rate of exogenous substrate carboxylation.

Rat liver microsomes contain a Triton X-100 solubilizable vitamin K-dependent carboxylase activity that converts specific glutamyl residues of a microsomal prothrombin precursor to gamma-carboxyglutamyl residues. This activity has been studied in partially (0.25% Triton X-100) and completely (1.0% Triton X-100) solubilized rat liver microsomal preparations. The rate of vitamin K-dependent carboxylation of endogenous microsomal protein precursors was very rapid in the completely solubilized liver microsomal preparation, and carboxylation of an exogenous peptide substrate (Phe-Leu-Glu-Glu-Leu) proceeded at the same time. In the partially solubilized liver microsomal preparation, the rate of protein carboxylation was greatly reduced, and a lag in carboxylation of the exogenous substrate was observed. When microsomal preparations which were depleted of endogenous precursors were used, this lag was eliminated. These data suggest that both substrates utilize the same microsomal pool of carboxylase and that the fraction of the carboxylase bound to the endogenous precursors is not immediately available to exogenous substrates.

Animals↗
PubMed1984

Cholesterol esterification by mammary gland microsomes from the lactating rat.

Cholesterol esterification by fatty acyl-CoA:cholesterol acyltransferase (ACAT, EC 2.3.1.26) has been demonstrated in microsomes prepared from breast tissue of the lactating rat, employing the incorporation of [1-14C]oleoyl coenzyme-A into cholesteryl[14C]oleate. The regulation of this activity in vitro was studied by supplementing microsomes with unesterified cholesterol supplied either as a dispersion in acetone or by incubating microsomes with cholesterol-enriched serum lipoproteins prior to enzyme assay. ACAT activity was increased by more than 80% when the ratio of unesterified cholesterol to microsomal protein was increased from 29 to 48 micrograms/mg protein, indicating that the normal cholesterol content of mammary gland microsomes does not saturate this enzyme. Cholesterol esterification could be inhibited nearly completely in vitro by addition of the polar steroid progesterone (93% decrease in ACAT activity with 75 microM progesterone) and, to lesser extents, by estradiol and retinol. Enzyme assays were also performed after incubating microsomes with N-acylamides that inhibit cholesteryl ester synthesis in other systems. Under conditions where the mammary gland ACAT reaction was inhibited by more than 82%, the esterification of retinol was reduced by less than 30% and incorporation of [14C]oleoyl-CoA into triglycerides was not inhibited at all. These studies indicate that the lactating mammary gland contains ACAT activity having properties similar to ACAT in other organs. The presence of ACAT activity in the lactating mammary gland provides a possible mechanism for the synthesis of cholesteryl esters found in milk. It can also be inferred that ACAT in mammary gland microsomes is likely to be distinct from the microsomal acyltransferases that catalyze the esterification of retinol and glycerides.

Acyltransferases↗
PubMed1999

Brain microsomal calcium accumulation in rats with increasing age: involvement of thapsigargin-sensitive Ca2+-ATPase.

The alteration in calcium content and Ca2+-ATPase activity in the brain microsomes of rats with increasing age was investigated. Brain microsomal calcium content was significantly increased in aged rats (50 weeks old) as compared with that of young rats (5 weeks old). Such an increase was not seen in the brain mitochondria of aged rats. Increasing age caused a significant elevation of Ca2+-ATPase activity in the brain microsomes. The microsomal Ca2+-ATPase activity of young rats was significantly decreased by the addition of thapsigargin (10-7-10-5 M), a specific inhibitor of microsomal Ca2+ pump enzyme (Ca2+-ATPase), in the enzyme reaction mixture. The inhibitory effect of thapsigargin (10-5 M) was also seen in the brain microsomes of aged rats. Moreover, the effect of calcium (5 and 10 microM) addition in elevating Ca2+-ATPase activity was not revealed in the microsomes of aged rats, whereas the metal could increase the enzyme activity in young rats, suggesting an involvement of activatory factor on the enzyme with increasing age. The present study demonstrates that ageing induces an increase in thapsigargin-sensitive Ca2+-ATPase activity and a corresponding elevation of calcium content in the brain microsomes. This finding suggests a cellular mechanism by which ageing causes calcium accumulation in brain.

Aging↗
PubMed2000

Midazolam and triazolam biotransformation in mouse and human liver microsomes: relative contribution of CYP3A and CYP2C isoforms.

Midazolam (MDZ) and triazolam (TRZ) hydroxylation, reactions considered to be cytochrome P-4503A (CYP3A)-mediated in humans, were examined in mouse and human liver microsomes. In both species, alpha- and 4-hydroxy metabolites were the principal products. Western blotting with anti-CYP3A1 antibody detected a single band of immunoreactive protein in both human and mouse samples: 0.45 +/- 0. 12 and 2.02 +/- 0.24 pmol/mg protein (mean +/- S.E., n = 3), respectively. Ketoconazole potently inhibited MDZ and TRZ metabolite formation in human liver microsomes (IC(50) range, 0.038-0.049 microM). Ketoconazole also inhibited the formation of both TRZ metabolites and of 4-OH-MDZ formation in mouse liver microsomes (IC(50) range, 0.0076-0.025 microM). However, ketoconazole (10 microM) did not produce 50% inhibition of alpha-OH-MDZ formation in mouse liver microsomes. Anti-CYP3A1 antibodies produced concentration-dependent inhibition of MDZ and TRZ metabolite formation in human liver microsomes and of TRZ metabolite and 4-OH-MDZ formation in mouse liver microsomes to less than 20% of control values but reduced alpha-OH-MDZ formation to only 66% of control values in mouse liver microsomes. Anti-CYP2C11 antibodies inhibited alpha-OH-MDZ metabolite formation in a concentration-dependent manner to 58% of control values in mouse liver microsomes but did not inhibit 4-OH-MDZ formation. Thus, TRZ hydroxylation appears to be CYP3A specific in mice and humans. alpha-Hydroxylation of MDZ has a major CYP2C component in addition to CYP3A in mice, demonstrating that metabolic profiles of drugs in animals cannot be assumed to reflect human metabolic patterns, even with closely related substrates.

Animals↗
PubMed1979

Mutant and immunochemical studies on the involvement of cytochrome b5 in fatty acid desaturation by yeast microsomes.

The involvement of cytochrome b5 in palmitoyl-CoA desaturation by yeast microsomes was studied by using yeast mutants requiring unsaturated fatty acids and an antibody to yeast cytochrome b5. The mutants used were an unsaturated fatty acid auxotroph (strain E5) and a pleiotropic mutant (strain Ole 3) which requires either Tween 80 and ergosterol or delta-aminolevulinic acid for growth. Microsomes from the wild-type strain possessed both the desaturase activity and cytochrome b5, whereas those from mutant E5 contained the cytochrome but lacked the desaturase activity. Microsomes from mutant Ole 3 grown with Tween 80 plus ergosterol were devoid of both the desaturase activity and cytochrome b5, but those from delta-aminolevulinic acid-grown mutant Ole 3 contained cytochrome b5 and catalyzed the desaturation. The cytochrome b5 content in microsomes from mutant Ole 3 could be varied by changing the delta-aminolevulinic acid concentration in the growth medium, and the desaturase activity of the microsomes increased as their cytochrome b5 content was increased. The antibody to yeast cytochrome b5, but not the control gamma-globulin fraction, inhibited the NADH-cytochrome c reductase and NADH-dependent desaturase activities of the wild-type microsomes. It is concluded that cytochrome b5 is actually involved in the desaturase system of yeast microsomes. The lack of desaturase activity in mutant Ole 3 grown with Tween 80 plus ergosterol seems to be due to the absence of cytochrome b5 in microsomes, whereas the genetic lesion in mutant E5 appears to be located at ther terminal desaturase.

Cytochromes↗
PubMed2000

Phenacetin deacetylase activity in human liver microsomes: distribution, kinetics, and chemical inhibition and stimulation.

Microsomal and cytosolic phenacetin deacetylase activities were examined in human liver and kidneys. Kinetic properties of the activities were also studied in human liver microsomes. Phenacetin deacetylase activity was predominantly localized in the liver microsomal fraction. The specific activities of phenacetin deacetylation in liver cytosol and in kidney microsomes and cytosol were all less than 5% of that in liver microsomes. In human liver microsomes, Eadie-Hofstee plots for phenacetin deacetylation were monophasic, indicating a single-enzyme catalytic reaction. The Michaelis-Menten parameters, K(m) and V(max), for the deacetylation were 4.7 mM and 5.54 nmol/min/mg of protein, respectively. The intrinsic clearance, calculated as V(max)/K(m), was 1.18 microl/min/mg of protein. Although the organophosphate bis(4-nitrophenyl)phosphoric acid markedly inhibited the reaction in human liver microsomes, the activity has a tolerance to the treatment of phenylmethylsulfonyl fluoride, a serine hydrolase inhibitor. Prazosin, a peripheral alpha(1)-adrenergic antagonist, noncompetitively inhibited the phenacetin deacetylation with a K(i) value of 19.0 microM. Flutamide, a nonsteroidal androgen receptor antagonist, stimulated the activity by up to 349%. This increase was accompanied by a decrease in the K(m) value and no change in the V(max) value, resulting in an increase in the intrinsic clearance by up to 700% of the control. These results suggest that the phenacetin deacetylase localized in human liver microsomes has not only a catalytic site but also a negative and/or positive modulation site or sites.

Carboxylic Ester Hydrolases↗
PubMed2001

Metabolism of a disulfiram metabolite, S-methyl N,N-diethyldithiocarbamate, by flavin monooxygenase in human renal microsomes.

S-Methyl N,N-diethyldithiocarbamate (MeDDC), a metabolite of the alcohol deterrent disulfiram, is converted to MeDDC sulfine and then S-methyl N,N-diethylthiocarbamate sulfoxide, the proposed active metabolite in vivo. Several isoforms of CYP450 and to a lesser extent flavin monooxygenase (FMO) metabolize MeDDC in the liver. The human kidney contains FMO1 and several isoforms of CYP450, including members of the CYP3A, CYP4A, CYP2B, and CYP4F subfamilies. In this study the metabolism of MeDDC by the human kidney was examined, and the enzymes responsible for this metabolism were determined. MeDDC was incubated with human renal microsomes from five donors or with insect microsomes containing human FMO1, CYP4A11, CYP3A4, CYP3A5, or CYP2B6. MeDDC sulfine was formed at 5 microM MeDDC by renal microsomes at a rate of 210 +/- 50 pmol/min/mg of microsomal protein (mean +/- S.D., n = 5) and by FMO1 at 7.6 +/- 0.2 nmol/min/nmol (n = 3). Oxidation of 5 microM MeDDC was negligible by all CYP450 tested (< or =0.03 nmol/min/nmol). Inhibition of FMO by methimazole or heat diminished MeDDC sulfine formation 75 to 89% in renal microsomes. Inhibition of CYP450 in renal microsomes by N-benzylimidazole or antibody to the CYP450 NADPH reductase had no effect on MeDDC sulfine production. Benzydamine N-oxidation, a probe for FMO activity, correlated with MeDDC sulfine formation in renal microsomes (r = 0.951, p = 0.013). The K(M) values for MeDDC sulfine formation by renal microsomes and recombinant human FMO1 were 11 and 15 microM, respectively. These results demonstrate a role for the kidney and FMO1 in the metabolism of MeDDC in humans.

Chromatography, High Pressure Liquid↗
PubMed2001

Kinetics of testosterone 6beta-hydroxylation in the reconstituted system with similar ratios of purified CYP3A4, NADPH-cytochrome p450 oxidoreductase and cytochrome B5 to human liver microsomes.

Kinetics of testosterone 6beta-hydroxylation were determined using a reconstituted system that consisted of CYP3A4, cytochrome b5 and NADPH-cytochrome P450 oxidoreductase (OR) with similar ratios as those seen in human liver microsomes and compared with those determined using human liver microsomes. Two reconstituted systems were constructed in accordance with two human liver microsomal samples that showed extremely high and low ratios of OR/CYP3A4. The Km values of testosterone 6beta-hydroxylation obtained from the reconstituted systems with high and low OR/CYP3A4 ratios were 29.3 and 35.2 microM, respectively, which were similar to that of the corresponding human liver microsomal samples (23.2 and 40.0 microM, respectively). However, Vmax values obtained from the reconstituted systems (3.7 and 0.8 pmol/min/pmol CYP3A4) were much lower than those from the human liver microsomes (44.2 and 31.1 pmol/min/pmol CYP3A4). The results suggest that the interaction between substrate and CYP3A4 in the reconstituted systems appear to be similar to human liver microsomes but that the velocity of the substrate metabolism in the reconstituted systems is different from that in human liver microsomes. In conclusion, our reconstituted systems could be used for the determination of affinity but not for the determination of the maximum velocity of substrate metabolism. Further studies on the protein-protein interactions between CYP3A4, OR, cytochrome b5 and/or a specific lipid environment are required to establish a reconstituted system showing similar kinetic properties to those of human liver microsomes.

Cytochrome P-450 CYP3A↗
PubMed1976

Ethanol metabolism by a transplantable hepatocellular carcinoma. Role of microsomes and mitochondria.

1. Ethanol metabolism in slices or homogenates of transplantable hepatocellular carcinoma HC-252 (HC-252) was 50 to 60% of the rate found in host liver slices or homogenates when they were expressed per gram of tissue wet weight and 70 to 80% of the liver when the rates were expressed per milligram of tissue protein. At 10 mM ethanol, the activities of alcohol dehydrogenase in tumor and liver supernatants were comparable. 2. Tumor microsomes did not oxidize ethanol in the presence of a NADPH-generating system, indicating the absence of the microsomal ethanol-oxidizing system and catalase-mediated peroxidation of ethanol. The HC-252 microsomes were contaminated with catalase, and acetaldehyde production occurred in the presence of a H2O2-generating system (xanthine oxidase). The virtual absence of ethanol oxidation and drug metabolism (aminopyrine demethylase and aniline hydroxylase) in HC-252 microsomes may be due to the low activities of NADPH-cytochrome c reductase, NADPH oxidase, and NADPH-dependent oxygen uptake. 3. Microsomal oxidation of ethanol was present in Morris hepatoma 5123C, a well-differentiated tumor of intermediate growth rate, while activity was negligible in microsomes from Morris hepatoma 7288CTC, a less differentiated tumor. Microsomal NADPH oxidase was present in the well differentiated tumor 5123C but was lacking in the less differentiated tumor 7288CTC. Several microsomal, mitochondrial, and cytosolic properties of HC-252 are similar to those of Morris hepatoma 7288CTC but differ from those of the more differentiated 5123C tumor and normal liver. 4. The content of mitochondrial protein in HC-252 was only 25% that of liver, and oxygen consumption per gram of tumor was only 28% that of the liver. When corrected for the mitochondrial protein content, oxygen uptake in tumor HC-252 and liver homogenates was comparable. Isolated tumor and liver mitochondria displayed comparable State 4 and 3 rates of oxygen consumption with succinate and glutamate as substrates. The activities of the reconstituted malate-aspartate and alpha-glycerophosphate shuttles were only slightly lower in isolated HC-252 mitochondria compared to liver mitochondria, when shuttles were reconstituted with purified enzymes. 5. Antimycin inhibited alcohol metabolism,and pyruvate stimulated alcohol metabolism, much less in tumor slices than in liver slices, suggesting the presence of an augmented mitochondria-independent, cytosolic mechanism for oxidizing reducing equivalents in the tumor. These factors suggest that oxidation of NADH is the limiting factor in ethanol metabolism. Whereas, in the liver mitochondrial reoxidation is predominant, in HC-252, cytosolic reoxidation of NADH also plays a major role.

Adenosine Triphosphatases↗
PubMed2004

Rabbit liver microsomal system: study of interaction with two model N-nitrosamines and their metabolism.

Rabbit liver microsomes of control (non-treated) or animals induced either by ethanol (EtOH) or phenobarbital (PB) were incubated with N-nitrosodimethylamine (NDMA) or N-nitrosomethylaniline (NMA). Difference spectroscopy showed that NMA is bound to the substrate-binding site of cytochrome P-450 (CYP) isoforms as heme ligand in control and EtOH pre-treated microsomes. On the other hand, PB-induced microsomes exhibit with NMA substrate type of spectra. NDMA does not provide any type of binding spectra with used microsomal systems. Oxidative bio-activation of N-nitrosamines by the microsomal CYP isoforms was measured as formaldehyde formation. Analysis of reaction kinetics in control microsomes revealed, for both substrates, two values of Michaelis-Menten constant (K(m)) for, K(m) values of 0.03 and 0.13 mmol/l for NDMA, and 0.30 and 0.82 mmol/l for NMA. Induction of animals with EtOH resulted in a decrease in the K(m) value for both substrates. In contrast, PB treatment caused an elevation of K(m) value for NDMA. Based on these data, we conclude that EtOH-inducible microsomal CYP isoforms (mainly CYP2E1) are responsible for binding and N-demethylation metabolism of both studied N-nitrosamines in rabbit liver microsomal system. The role of the other CYP isoforms involved in the metabolism of mentioned N-nitrosamines is discussed.

Animals↗
PubMedDate not supplied

Human microsomal N-oxidative metabolism of cocaine.

The N-oxidative metabolism of cocaine has been described previously in some detail in the mouse, and is associated with hepatotoxic effects of cocaine in this species. As part of an effort to determine whether this metabolism-dependent toxicity is relevant to clinical reports of liver injury from cocaine, the in vitro N-oxidative metabolism of cocaine was examined in microsomal suspensions obtained from human liver specimens. Human hepatic microsomal suspensions were capable of metabolizing cocaine to norcocaine, and norcocaine to N-hydroxynorcocaine, which is the sequential oxidative metabolism observed in mice to lead to toxic metabolite formation. Additionally, incubation of 4-[3H]cocaine with human hepatic microsomal suspensions resulted in the formation of a metabolite that bound irreversibly to microsomal protein. Each of these reactions was inhibited or abolished by gassing the incubation mixture with carbon monoxide, omitting NADPH, or adding the cytochrome P-450 inhibitors SKF 525-A (50 microM) or n-octylamine (3 mM). Competing microsomal esterase activity reduced the apparent rate of N-demethylation of cocaine by 50%, but had much less effect on the N-hydroxylation of norcocaine. In general, apparent KM values for both the N-demethylation and N-hydroxylation reactions in human liver microsomal suspensions were greater than those observed in comparable incubations using mouse hepatic microsomal suspensions, whereas Vmax rates were lower. The extent of irreversible (i.e. nonextractable) binding of cocaine following its metabolism in vitro was comparable between human and mouse microsomal suspensions, however. Similarities in the N-oxidative metabolism of cocaine suggest that humans and mice share the same bioactivation mechanism of cocaine-induced liver injury.

Adolescent↗
PubMedDate not supplied

Oxidation of ethylene glycol to formaldehyde by rat liver microsomes. Role of cytochrome P-450 and reactive oxygen species.

Rat liver microsomes oxidized ethylene glycol to formaldehyde in a NADPH-dependent, carbon monoxide-sensitive manner. Formaldehyde production was inhibited by substrates and ligands for cytochrome P-450 such as aniline, p-nitrophenol, pyrazole, and 4-methylpyrazole, and inhibitors such as tryptamine, cimetidine, and miconazole. The apparent Km for ethylene glycol was about 25 mM and the apparent Vmax was about 6 nmol/min/mg protein. Microsomes isolated from rats treated with pyrazole or 4-methylpyrazole to induce cytochrome P-450IIE1 oxidized ethylene glycol at rates which were about twice those found with control microsomes or microsomes isolated from rats treated with phenobarbital or 3-methylcholanthrene, although significant rates were found with all microsomal preparations. Antibody raised against the pyrazole-induced P-450IIE1 inhibited formaldehyde production from ethylene glycol in microsomes from pyrazole-treated rats. H2O2 itself did not oxidize ethylene glycol to formaldehyde; however, the microsomal reaction was inhibited by catalase or glutathione plus glutathione peroxidase and was stimulated by added H2O2 in the presence of NADPH. Nonheme iron also appeared to be required for ethylene glycol oxidation in view of the inhibition of formaldehyde production by desferrioxamine, EDTA, and DTPA. Microsomal oxidation of ethylene glycol was not sensitive to superoxide dismutase, hydroxyl radical scavengers, or Trolox, suggesting that the oxidant derived from H2O2 and iron and responsible for the production of formaldehyde from ethylene glycol was not superoxide, hydroxyl radical, or lipid hydroperoxide. These results suggest that ethylene glycol is oxidized to formaldehyde by an oxidant derived from H2O2 and nonheme iron, and that cytochrome P-450 may function to generate the H2O2 and to catalyze reduction of the nonheme iron.

Animals↗
PubMed1976

Characterization of microsomal methyl sterol demethylase in two Morris hepatomas.

Previously, we reported that the rate of metabolism of methyl sterol intermediates of cholesterol biosynthesis by broken-cell preparations of Morriss hepatoma 7777 is very slow, whereas the intact tumors are known to synthesize cholesterol quite efficiently. Active preparations have now been obtained by substitution of pyrophosphate for phosphate buffer. Although substitution of pyrophosphate buffer markedly enhances microsomal methyl sterol demethylation rates 3- to 4-fold in hepatoma 7777, other microsomal enzymes and electron carriers in either liver or a more slowly growing hepatoma appear to be unaffected by pyrophosphate. Several properties of the active microsomal methyl sterol demethylase have now been compared for control rat liver, host liver, tumor 7777, and tumor 5123C. Conditions necessary for the assay of initial velocities of enzymic reactions in the tumor microsomes have been established with respect to the amount of protein, time-course, concentrations of cofactors and substrate, pH, and other variables. The K'm and the responses to the variables studied above are very similar for methyl sterol demethylase of microsomes isolated from control liver, host liver, tumor 5123C, and tumor 7777. The multienzymic demethylase in the various preparations has been found to be inhibited similarly by in vitro additions of cyanide, cytochrome c, and bile salts. Thus, the enzymes of the microsomal-bound 4-methyl sterol demethylase of cholesterol biosynthesis appear to be very similar in liver and these 2 Morris hepatomas. When xenobiotic inducers of microsomal oxidases, such as phenobarbital and methylcholanthrene, are administered to normal and tumor-bearing rats, elevated rates of methyl sterol demethylation are observed with isolated liver microsomes obtained from both normal and tumor-bearing rats. Similar increases are not observed in the tumors. Furthermore, daily administration of an intestinal bile acid sequestrant elevates hepatic methyl sterol demethylase, but statistically significant changes were not observed in tumors 7777 and 5123C. Since the enzymes of methyl sterol demethylase appear to be grossly similar in liver and these hepatomas, regulation of the activity of the multienzymic system contained in the tumors may be altered. On the other hand, these agents in vivo simply may not affect liver and the hepatomas similarly, due to a lack of uptake of the foreign substances by the tumor that has been transplanted to the thighs.

Animals↗
PubMed1977

Investigation of the rate-determining microsomal reaction of cholesterol biosynthesis from lanosterol in Morris hepatomas and liver.

Previously, we reported that the properties or microsomal 4-methyl sterol demethylase isolated from liver and Morris hepatomas 5123C and 7777 are grossly similar. The individual enzymic steps of this multicomponent system have now been studied, and the rate-determining step has been determined and shown to be identical for liver and these hepatomas. The rates of microsomal oxidative attacks of the 4alpha-methyl, 4alpha-hydroxymethyl, and 4-aldehydic groups are similar for microsomes prepared from rat liver and hepatoma 7777. The rates of mixed-function oxidative attack appear to increase in the order;--CH3 less than --CH2OH less than --CHO. Furthermore, the hepatic and hepatoma NAD-dependent decarboxylase, which catalyzes the reaction following the three oxidative attacks is similar in properties and velocity. The fifth step, an NADPH-dependent reduction of the 3 ketosteroid that is produced by decarboxylation, is also similar. For both tissues, the latter two reactions, under in vitro conditions, proceed at rates that exceed the initial oxidative process. Thus, for elimination of both of the 4-methyl groups of lanosterol, the 10 individual reactions catalyzed in this multicomponent system are identical in liver and hepatoma 7777 microsomes, and the rate-determining stop for both liver and hepatoma is the inital oxidative attack on the 4alpha-methyl group of cholesterol procursors. When the rate-determining reaction of both liver and hepatoma 7777 microsomes is assayed at different temperatures, the same activation energies and the same characteristic breaks in the arrhenius plots are observed. Thus, for both liver and hepatoma, both the nature and the site of rate determination in this multienzymic system must be similar. Since the microsomal enzymes of liver nad hepatoma appear to be catalytically similar and rate determination appears to be similar, too, the characteristic lact of response of tumor microsomes to treatments in vivo that alter host liver microsomal demethylation activity suggests that the insensitivity of these tumors to dietary cholesterol should not be ascribed to alterations in the catalytic proteins. Evidence in this report suggests that the postmicrosomal supernatant fraction of both liver and hepatoma contains a cytosolic protein that may participate in the regulation of the rate-determining attack of 4alpha-methyl sterol substrates. Thus, either qualitative or quantitative differences between the postmicrosomal supernatant fractions obtained from liver and heptomas may account for the observed differences in rates of cholesterol biosynthesis.

Animals↗
PubMedDate not supplied

Role of P-450c in the formation of a reactive intermediate of chlorotrianisene (TACE) by hepatic microsomes from methylcholanthrene-treated rats.

A previous study has shown that chlorotrianisene is metabolized by hepatic microsomal cytochrome P-450 monooxygenase(s) to a reactive intermediate that binds covalently to microsomal proteins [Juedes, Bulger, and Kupfer: Drug Metab. Dispos. 15, 786 (1987)]. Covalent binding of chlorotrianisene in hepatic microsomes is dramatically stimulated by treatment of rats with methylcholanthrene (MC), which is known to induce two major P-450 isozymes, P-450c (IA1) and P-450d (IA2). To determine whether P-450c and/or P-450d are involved in catalysis of covalent binding of chlorotrianisene, antibodies to P-450c and P-450d were used. Incubations of chlorotrianisene were conducted with liver microsomes from MC-treated rats (MC microsomes) and a monoclonal antibody (mAb) raised to the major MC-induced isozyme P450c, mAb 1-7-1, or a polyclonal monospecific antibody (pAb) to P-450d, pAb anti-d (-c). At a 5:1 ratio of antibody to microsomal protein, mAb 1-7-1 inhibited covalent binding by 67%, whereas pAb anti d (-c) showed a 10% inhibition. Maximal inhibition by mAb 1-7-1 was 89% at a 100:1 ratio of antibody to microsomal protein. From these findings it was concluded that P-450c is the major isozyme responsible for the metabolism of chlorotrianisene to the covalently binding reactive intermediate in MC microsomes. Additionally, it was observed that potentiation of covalent binding occurred with the noninhibitory mAbs used in these incubations. Substituting bovine serum albumin (BSA) for antibodies showed that this increase in binding is probably due to an increase in acceptor sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

On the role of lipid peroxidation and protein-bound aldehydes in the haloalkane-induced inactivation of microsomal glucose 6 phosphatase.

The inactivation of liver microsomal glucose 6 phosphatase induced either by Fe2+ or by haloalkanes (CCl4, CBrCl3) was investigated in NADPH-microsomes systems. In the case of haloalkanes, EDTA was included in the incubation mixtures, so to exclude participation of free Fe2+ in the ensuing lipid peroxidation. Microsomal glucose 6 phosphatase activity was measured along with the release of malonic dialdehyde and the appearance of carbonyl products bound to microsomal protein, taken as indices of the peroxidative process. Fe2+ was added to NADPH-microsomes at different concentrations, one (6 microM) resulting in an extent of lipid peroxidation comparable with that induced by haloalkanes, the other (60 microM) representing a situation of excess Fe2+, leading to massive lipid peroxidation. Inhibition of glucose 6 phosphatase caused by 6 microM Fe2+ was comparable to that induced by haloalkanes in EDTA-microsomes systems, which supports the view that lipid peroxidation--rather than covalent binding of free radical metabolites--represents the main event leading to the inactivation of glucose 6 phosphatase caused by haloalkanes. The production of 4-hydroxynonenal--the known toxic product of lipid peroxidation--was also studied. A remarkable accumulation of 4-hydroxynonenal was observed in the microsomal membranes after peroxidation induced by 6 microM Fe2+ or haloalkanes, as compared to the incubation medium. In addition, experiments carried out with CCl4 and CBrCl3 in vivo suggested the possible existence of a cytosolic detoxification system able to remove lipid-derived carbonyls bound to microsomal protein.

Aldehydes↗