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PubMed1983

Binding of a 4-methyl-4-aza-steroid to 5 alpha-reductase of rat liver and prostate microsomes.

A tritium labeled 5 alpha-reductase inhibitor, [1,2-3H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA), binds reversibly to a high affinity site (Kd, 6.5 nM) in liver microsomes from male rats. The binding requires a nicotinamide nucleotide coenzyme; NADH is at least 100 times less potent than NADPH, and NADP+, NAD+, flavin adenine dinucleotide, coenzyme A, and ADP are inactive. The relative potencies of 13 steroids as inhibitors of the binding of [3H]4-MA to liver microsomes correlate with their relative potencies as inhibitors of the conversion of [14C]testosterone to [14C]5 alpha-dihydrotestosterone by liver microsomes. Comparison of liver microsomes from mature female rats and microsomes from mature male rat liver, ventral prostate, spleen, kidney, and skeletal muscle shows that their NADPH-dependent [3H]4-MA binding capacities correlate with their levels of 5 alpha-reductase activity. These results suggest that [3H]4-MA binds specifically to 5 alpha-reductase in a NADPH-dependent manner. 5 alpha-Reductase was solubilized from liver microsomes with a detergent, Lubrol-WX, and the solubilized enzyme also binds [3H]4-MA. The relative potencies of 13 steroids as inhibitors of rat ventral prostate and liver 5 alpha-reductase are the same, strongly suggesting that the 5 alpha-reductases in the two tissues are the same.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1999

Biphasic kinetics of imipramine N-oxidation in rat brain microsomes.

Imipramine (IMI) N-oxidase activity in brain microsomes from rats of both sexes was determined by high performance liquid chromatography, and compared with the results in rat liver microsomes. Brain and liver microsomal IMI N-oxidation was sensitive to thermal inactivation and had an optimal pH at around 9.0. IMI N-oxidase activity (15.54 pmol/min/mg protein) in brain microsomes was about one-hundredth that of liver microsomes (2.08 nmol/min/mg protein) at a substrate concentration of 5 mM. IMI N-oxidase activities in both brain and liver microsomes displayed biphasic kinetics that associated a low Km-low Vmax element with a high Km-high Vmax component. Furthermore, a significant sex difference was observed in Vmax values (male>female) in both phases, but Km values were similar between male and female rats, resulting in a significant sex difference (male>female) in intrinsic clearance values (Vmax/Km) of the low-Km and the high-Km phases.

Animals↗
PubMed1983

Effects of starvation on microsomal cytochrome P-450 and laurate-omega-hydroxylation of rat kidney and liver.

Cytochrome P-450 (P-450) content and laurate-omega-oxidation activity in rat kidney and liver microsomes were investigated following starvation. Multiple forms of P-450 were analyzed by one dimensional separation using peroxidase stained SDS-continuous gradient polyacrylamide gel electrophoresis. Gels of the hepatic microsomes treated with phenobarbital showed three P-450 bands, and the renal microsomes showed one sharp band, which was induced remarkably by starvation and coincided with the middle molecular form of P-450 from the hepatic microsomes. Since laurate-omega-oxidation activity was induced specifically by starvation but not by drug treatment, in both the kidney and the liver microsomes, the middle molecular form of P-450 might catalyze laurate-omega-oxidation. It seemed, therefore, that a special P-450 subunit catalyzing laurate-omega-oxidation has a greater function in the renal rather than hepatic microsomes because the specific laurate-omega-oxidation activity per starvation induced P-450 content was relatively similar in both the kidney and the liver.

Animals↗
PubMed1981

Stimulation of protein-bound iodine formation by lipid extracts from hog thyroid microsomes.

The lipid materials extracted from hog thyroid microsomes were found to contain factors which stimulated both the NADPH oxidation by NADPH-cytochrome c reductase and PBI formation by a reconstituted system containing the reductase as a hydrogen peroxide generator and thyroid peroxidase. For both reactions, the stimulating activity in the extracts from the other subcellular fractions was less than that observed in the extracts from microsomal fraction. Lipids extracted from hog liver microsomes did not show any NADPH oxidation stimulating activity. Thin-layer chromatography of the lipid extracts from thyroid microsomes revealed the presence of at least two stimulating factors differing in chromatographic behaviour. At least one of the stimulating factors separated on TLC stimulated the oxygen consumption accompanied by generation of hydrogen peroxide due to the NADPH oxidation by NADPH-cytochrome c reductase. From these results it was suggested that in thyroid microsomes, but not in liver microsomes, there were stimulating factors extractable by chloroform-methanol. The factors could stimulate NADPH oxidation by NADPH-cytochrome c reductase followed by generation of hydrogen peroxide, resulting in the enhancement of PBI formation coupled with thyroid peroxidase.

Animals↗
PubMed2000

Inhibition of rat microsomal lipid peroxidation by the oral administration of D002.

The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS). When D002 (5-100 mg/kg body weight) was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46%) occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg) also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40%) and brain (28-44%) microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg) for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

Administration, Oral↗
PubMed1994

Changes in content of P450 isozymes in hepatic and renal microsomes of the male rat treated with cis-diamminedichloroplatinum.

1. The changes in the activity of drug-metabolizing enzymes and in the content of P450 isozymes in renal and hepatic microsomes after treatment of the male Sprague-Dawley rat with cis-diamminedichloroplatinum (Cisplatin, CDDP) were examined. 2. NADPH-P450 reductase activity in renal microsomes was significantly increased by treatment with CDDP, but lauric acid omega- and (omega-1)-hydroxylation activities of renal microsomes were not increased. 3. The level of P4502C23 was increased significantly and levels of P4504A2 and 4A8 tended to increase in renal microsomes. 4. In hepatic microsomes, lauric acid omega-hydroxylation activity was increased, but (omega-1)-hydroxylation activity was not. Levels of P4502C11 and 3A2, which are male-specific forms, were decreased, whereas levels of P4502A1, 2C7 and 2E1 were increased in hepatic microsomes. The levels of P4504A2 and 4A3 were increased by CDDP and the level of P4504A1 was not changed. Changes in the protein levels of P450 by CDDP were consistent with those in the mRNA levels reported previously (LeBlanc et al. 1992). 5. Male-specific forms in rat liver such as P4502C11 were decreased by CDDP, but those in the kidney such as P4504A2 was not. Therefore, CDDP has different influences on the regulation of hepatic and renal P450s.

Animals↗
PubMedDate not supplied

Comparison of the biosynthesis and composition of polyglycerophosphatides and phosphatidylinositols in mitochondria and microsomes isolated from neonatal and adult rat heart and liver.

The level of biosynthesis and the composition of polyglycerophosphatides (phosphatidylglycerol, phosphatidyglycerolphosphate, and diphosphatidylglycerol or cardiolipin) and phosphatidylinositols were examined in mitochondria and microsomes, respectively, isolated from neonatal and adult rat heart and liver. Biosynthesis of [3H]polyglycerophosphatides [( 3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate) was 4.5 times higher in neonatal than in adult heart mitochondria, whereas in the respective liver mitochondria this synthesis was only 15% higher in neonatal mitochondria. The biosynthesis of [3H]phosphatidylinositol was twice as high in neonatal as in adult heart microsomes, but very similar in the respective liver microsomes. The major biosynthesized polyglycerophosphatide was [3H]phosphatidylglycerol. The accumulation of [3H]phosphatidylglycerolphosphate depended on the origin of the mitochondria. Under our experimental conditions [3H]phosphatidylinositol was the only synthesized phosphoinositide in all microsomes. The biosynthesis of cardiolipin depended on the origin of the mitochondria and was highest in adult rat liver mitochondria and lowest in adult heart mitochondria. In all cases the biosynthesized [14C,3H] cardiolipin from [14C]phosphatidylglycerol and [3H]CDP-diglycerides had a ratio of 14C/3H around unity. The biosynthesis of [3H]CDP-diglycerides, the key precursor for the biosynthesis of phosphatidylglycerol, phosphatidylinositol, and cardiolipin, was 30% higher in neonatal than in adult heart microsomes and very similar in the respective liver microsomes. The subcellular localization of the enzymes required for the biosynthesis of the lipids and liponucleotides examined was found to be the same in membranes isolated from neonatal and adult rat heart and liver.

Aging↗
PubMed2000

The 1'-hydroxylation of Rac-bufuralol by rat brain microsomes.

The 1'-hydroxylation of rac-bufuralol, which is catalyzed by polymorphic CYP2D6 in humans, was studied in brain microsomes from male and female Wistar rats and from the female Dark Agouti rat, a model of the CYP2D6 poor metabolizer phenotype. The kinetics of the 1'-hydroxylation of bufuralol (1-1500 microM) by brain microsomes were biphasic. The activity of the high-affinity site of metabolism was consistent with Michaelis-Menten kinetics (apparent K(m1) = 0. 61-1.42 microM, V(max1) = 4.3-4.8 fmol/min/mg of protein), whereas the low-affinity activity was better described by a Hill function (K(50%(2)) = 253-258 microM, V(max2) = 817-843 fmol/min/mg of protein, n = 1.2-1.3). Values for kinetic constants were similar in all rat strains. Quinine was only a weak inhibitor of both the high- (apparent K(i) = 90 microM) and low-affinity (210 microM) sites of metabolism. In contrast, the kinetics of 1'-hydroxylation of bufuralol by rat liver microsomes were best described by a two-site Michaelis-Menten function. V(max) values were 3 to 5 orders of magnitude greater compared with those for brain microsomes (male and female Wistar), and liver microsomes from female Dark Agouti rats were significantly less active than those from Wistar rats. These data, together with the known potent inhibitory effect of quinine on bufuralol 1'-hydroxylation by rat liver microsomes, indicate tissue-specific differences in the enzymology of this reaction. The role of brain CYP2D enzymes remains to be clarified.

Animals↗
PubMed1976

Investigation of microsomal oxygenases of biosynthetic processes. Stearyl-CoA desaturase of adipose tissue and liver.

Porcine and rat microsomal stearyl-CoA desaturases require reduced pyridine nucleotide and oxygen, are cyanide sensitive, and are insensitive to carbon monoxide. The Km for stearyl-CoA is somewhat larger for liver than for the adipose desaturases, but, in general, assay conditions are quite similar. Adipose tissue microsomes contain cytochromes b5 and P-450, as well as the NADH- and NADPH-specific cytochrome reductases. Compared to liver, the specific contents and activities of electron carriers are much lower in adipose tissue, and activities of 4-methyl sterol oxidase of cholesterol biosynthesis, as well as the cytochrome P-450-dependent aminopyrene demethylase and benzypyrene hydroxylase, are negligible in adipose tissue microsomes. Furthermore, unlike hepatic desaturase, administration of insulin stimulates the adipose desaturase 3-fold without affecting either the amounts or activities of microsomal oxidation-reduction proteins; the changes in desaturase activities produced either by altering dietary fat or by fasting and/or fasting followed by refeeding are, in general, both more extensive and more permanent in adipose compared to liver microsomes. The effects produced by isotopic hydrogen substitution both in stearyl-CoA and in the medium (2H2O) are similar with microsomes from both tissues. The rate-determining step of desaturase appears to be similar in both tissues. The primary isotope effect, k H/Tr, observed with [9,10-3H2]stearyl-CoA is relatively small, 2.88. Since little, if any, primary isotope effect is associated with methyl sterol oxidase, these two mixed function oxidases of biosynthetic processes also appear to share this property in common.

Adipose Tissue↗
PubMedDate not supplied

Oxidation of butylated hydroxytoluene to toxic metabolites. Factors influencing hydroxylation and quinone methide formation by hepatic and pulmonary microsomes.

The effects of inducing agents and inhibitors on the cytochrome P-450-catalyzed oxidations of butylated hydroxytoluene (BHT) to form three metabolites were investigated with liver and lung microsomes from rats and mice. These compounds, the quinone methide (QM) formed by two-electron oxidation of BHT, the hydroxy-tert-butyl analog of BHT (BHT-OH) resulting from aliphatic hydroxylation, and the hydroxy-quinone methide (QM-OH) derived from BHT-OH, have been implicated previously as intermediates or products involved in BHT bioactivation and toxicity. Although there was little or no increase in BHT metabolism in pulmonary microsomes from either species following phenobarbital (PB) administration, 6- to 37-fold enhancements occurred in the transformation of BHT to QM, and the conversion of BHT-OH to QM-OH with hepatic microsomes from both species. The first step in QM-OH formation, hydroxylation of BHT to BHT-OH, is a minor pathway with hepatic microsomes from treated or untreated rats, thereby explaining the lack of QM-OH formation from BHT by that species. The two-step oxidation of BHT to QM-OH, however, is a relatively important metabolic pathway with hepatic microsomes from PB-treated mice, due to an unusually large (111-fold) PB-induced increase in the tert-butyl hydroxylation step. These results demonstrate that pulmonary microsomes from mice, but not rats, have relatively high constitutive P-450 activity for the formation of QM-OH from BHT, supporting the proposal that this metabolite is involved in BHT-induced pneumotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Mechanisms responsible for the thermal sensitivity of adrenal microsomal monooxygenases.

Studies were done to determine the mechanism(s) responsible for the thermal lability of adrenal microsomal monooxygenases. Preincubation of guinea pig adrenal microsomal suspensions at 37 degrees C caused large time-dependent declines in benzo(a)pyrene (BP) hydroxylase and benzphetamine (BZ) demethylase activities. Similar preincubations with hepatic microsomes had little effect on enzyme activities. The decreases in adrenal enzyme activities were completely prevented by co-incubation of microsomes with cytosol, but were not diminished by reduced glutathione, ascorbic acid, or bovine serum albumin. Partial protection was afforded by EDTA, suggesting that lipid peroxidation might be involved, but malonaldehyde production was not demonstrable and MnCl2, a potent inhibitor of lipid peroxidation, did not affect the decline in enzyme activities. The decreases in the rates of BP and BZ metabolism were prevented by including NADPH or NADP+ in the preincubation medium. The preincubation conditions causing losses of adrenal enzyme activities did not affect cytochrome P-450 concentrations or substrate binding to cytochromes P-450, as indicated by type I difference spectra. NADH-cytochrome c reductase activity also was not affected, but there were decreases in NADPH-cytochrome c reductase activity that were proportionately similar to the declines in drug-metabolizing activities. Direct assessment of NADPH-cytochrome P-450 reductase revealed similarly large decreases in enzyme activity resulting from preincubation of adrenal microsomes. The results demonstrate a need for extra caution when doing preincubation experiments with adrenal microsomal preparations, and suggest that the thermal lability of adrenal monooxygenases is attributable to effects at the active site of NADPH-cytochrome P-450 reductase.

Adrenal Glands↗
PubMed1991

Quantitation of microsomal alpha-hydroxylation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is activated to DNA alkylating species via two different alpha-hydroxylation pathways. Methylene hydroxylation leads to DNA methylation, whereas methyl hydroxylation yields DNA pyridyloxobutylation. We have developed a high-pressure liquid chromatography assay utilizing radiochemical detection that permits the determination of the extent of metabolism through each pathway in microsomal preparations. Levels of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) were used to measure the extent of methyl hydroxylation, whereas levels of the aldehyde, 4-oxo-1-(3-pyridyl)-1-butanone (OPB), were used to quantify the extent of methylene hydroxylation. Incubations of [5-3H]NNK with microsomes and cofactors were conducted in the presence of 5 mM sodium bisulfite to trap the reactive OPB. The inclusion of bisulfite did not affect the rate of NNK metabolism. Trapping the aldehyde also inhibited its further oxidation to the corresponding acid or reduction to HPB. Furthermore, the conversion of HPB to OPB made only a minor contribution to the OPB levels under our incubation conditions. Analysis of incubation mixtures containing [5-3H]NNK, cofactors, and either A/J mouse liver or lung microsomes demonstrated that OPB was a significant metabolite of NNK. The OPB:HPB ratio was greater in liver (1.5) than in lung (0.2-1) microsomal preparations. Apparent Km values for OPB and HPB formation in lung microsomes were 23.7 and 3.6 microM, respectively, whereas the corresponding values for liver microsomes were 19.1 and 73.8 microM, respectively. These data are consistent with the involvement of more than one cytochrome P-450 isozyme in the activation of NNK to DNA reactive species.

Animals↗
PubMed1975

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.

Adenylyl Cyclases↗
PubMed1986

Characterization of a protein fatty acylesterase present in microsomal membranes of diverse origin.

A microsomal activity of baby hamster kidney cells which cleaves ester-type bound fatty acids from acyl proteins in vitro has been characterized. This activity is also present in microsomal membranes from pig liver, calf kidney, and human mucous cells. Cell free deacylation is described for the Semliki Forest virus acyl proteins E1 and E2 and the precursor of E2 designated p62. Acyl chain cleavage operates with both exogenous and endogenous viral acyl protein substrates. The in vitro cleavage requires microsomes solubilized by detergents of which various kinds are equally effective (Nonidet P-40, Tween 20, sodium deoxycholate, Triton X-100, or octyl-beta-D-glucoside). If microsomes are boiled for 15 min prior to the incubation, deacylation is abolished completely and no radioactivity is released from the palmitoylated acyl proteins during incubation with either detergents or microsomes alone. No changes in the molecular structure of the deacylated Semliki Forest virus proteins were detected, and the cleavage product was identified as free fatty acid. Deacylation is time- and temperature-dependent and can be enhanced by increasing the concentration of microsomal protein in the incubation mixture. It is completely inhibited under acidic conditions (pH 5) and at low temperature (4 degrees C). Deacylation also occurs in the presence of EDTA and bivalent cations such as Mg2+, Mn2+, and Ca2+ which influence the reaction marginally. On the other hand, fatty acid release is drastically reduced with a mixture of Co2+, Zn2+, and Hg2+ ions. The activity is not identical with protein fatty acyltransferase operating in the reverse direction, since a partially purified preparation of this acyltransferase failed to cleave fatty acids from fatty acylated substrate proteins. Taken together, these data lead us to postulate an enzymatic activity which cleaves fatty acids from ester-type fatty acylated proteins, and we propose to designate this enzyme a protein fatty acylesterase.

Animals↗
PubMed1985

Photoaffinity labeling of steroid 5 alpha-reductase of rat liver and prostate microsomes.

21-Diazo-4-methyl-4-aza-5 alpha-pregnane-3,20-dione (Diazo-MAPD) inhibits steroid 5 alpha-reductase in liver microsomes of female rats with a Ki value of 8.7 +/- 1.7 nM, and the inhibition is competitive with testosterone. It also inhibits the binding of a 5 alpha-reductase inhibitor, [3H] 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one ([3H]4-MA), to the enzyme in liver microsomes. The inhibition of 5 alpha-reductase activity and of inhibitor binding activity by diazo-MAPD becomes irreversible upon UV irradiation. [1,2-3H]Diazo-MAPD binds to a single high affinity site (Kd 8 nM, 125 pmol binding sites/mg of protein) in liver microsomes of female rats, and this binding requires NADPH. Without UV irradiation, this binding is reversible, and it becomes irreversible upon UV irradiation. Both the initial reversible binding and the subsequent irreversible conjugation after UV irradiation are inhibited by inhibitors (diazo-MAPD and 4-MA) and substrates (progesterone and testosterone) of 5 alpha-reductase, but they are not inhibited by 5 alpha-reduced steroids (5 alpha-dihydrotestosterone and 5 alpha-androstan-3 alpha, 17 beta-diol). NADPH stimulates the binding of [3H] diazo-MAPD to microsomes of male rat liver and prostate. UV irradiation also induces conjugation of [3H] diazo-MAPD to these microsomes. Photoaffinity labeled liver microsomes of female rats were solubilized and fractionated by high performance gel filtration. The radioactive conjugate eluted in one major peak at Mr 50,000.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1985

Multiple forms of cytochrome P-450 from microsomes of rat liver, kidney, and lung resolved by high-performance liquid chromatography.

The amount of cytochrome P-450 in microsomes of rat kidney and lungs was 23 and 7%, respectively, of the amount in microsomes of the liver. HPLC profiles of solubilized microsomes showed that there were five or more forms of cytochrome P-450 in microsomes of kidneys and also of lungs of untreated rats. While 3-methylcholanthrene induced a new form of cytochrome P-450 in kidney and lung microsomes, phenobarbital caused no major change in the HPLC profiles of these microsomes.

Animals↗
PubMed1982

[Investigation of the lipid dependence of pyrophosphatase activity from rat liver microsomes and hepatoma by phospholipase C].

The lipid dependence of pyrophosphatase activity was studied by treatment of liver and hepatoma microsomes with phospholipase C from Cl. perfringens and B. cereus and a subsequent incorporation of various classes of phospholipids into the delipidated microsomes. Phospholipase C hydrolysis sharply lowers the pyrophosphatase activity of liver and hepatoma microsomes. The enzyme activity is restored after introduction of phospholipids into delipidated liver microsomes, the maximal effect being achieved on incorporation of phosphatidylcholine. All the phospholipids tested exerted the same reactivation effects on the delipidated microsomes of hepatoma. However, a more complete delipidation of hepatoma microsomes by phospholipase C hydrolysis and a subsequent organic solvent extraction revealed a specific dependence of the enzyme activity on phosphatidylserine.

Animals↗
PubMed1995

Disposition and metabolism of the angiogenic moderator O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM-1470) in human hepatocytes and tissue microsomes.

The biotransformation of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470; AGM 1470), a potent in vitro inhibitor of angiogenesis, was investigated in primary cultured human hepatocytes and microsomal fractions of various human tissues. Exposure of human hepatocytes to 5 microM [3H]TNP-470 led to a rapid metabolism of unchanged drug to six metabolic derivatives within 30 min. The predominant extracellular metabolites were M-II and M-IV, attaining a maximum level of 3.23 +/- 0.34 and 0.88 +/- 0.10 microM, respectively. M-II leveled off, while M-IV rapidly declined to 0.06 +/- 0.05 microM by 3 h. TNP-470 was undetectable after 60 min. M-V and M-VI slowly reached maximal concentrations of 0.26 +/- 0.12 and 0.32 +/- 0.16 microM, respectively. M-I only reached a concentration of 0.18 +/- 0.07 microM at 60 min and leveled at 0.13 +/- 0.06 microM for the remaining time of the experiment. The intracellular profile was different, with M-III and M-V representing the major metabolites detected. Studies using human liver microsomes demonstrated that M-IV formation was associated with an esterase-like enzymatic cleavage of TNP-470 and that this metabolite was then further metabolized by microsomal epoxide hydrolase to M-II, as evidenced by inhibition of this metabolic step by cyclohexene oxide, a microsomal epoxide hydrolase inhibitor. Extrahepatic metabolism of TNP-470 was also demonstrated using different sites of human intestinal, stomach, and kidney microsomes, with metabolite M-IV as the principal derivative detected in these tissues. Hepatic microsomal samples from seven different donors demonstrated large interindividual variations in the formation of both M-II and M-IV. In summary, this study demonstrates a rapid and extensive metabolism of TNP-470 in human tissues. The data emphasize the need to evaluate the in vivo formation and extent of TNP-470 metabolites to adequately assess the pharmacodynamic effects of this novel anticancer drug with a novel mechanism of action.

Antibiotics, Antineoplastic↗