Rat Liver Microsomal Activity Research Articles

Induction of the procarcinogen-activating CYP1A2 by a herbal dietary supplement in rats and humans

Herbal dietary supplements to promote health may be double-edge swords. A herbal dietary supplement, FastOne™, which contains extracts of kola nut, grape, green tea and Ginkgo biloba, and is used as an agent for weight management, was administered to rats to test whether it induced CYP1A2, a procarcinogen-activating enzyme. Western blot analysis indicated that treatments with 0.15, 0.3, 0.5, 1 and 2 g/kg of the supplement for 3 days increased CYP1A2 expression in rat liver microsomes in a dose-dependent manner. The 0.3, 0.5, 1 and 2 g/kg treatments increased rat liver microsomal CYP1A2 activity measures as the conversion of caffeine to paraxanthine to 166, 212, 331 and 473% of normal, respectively. In humans, the intake of 2 and 4 capsules of the supplement for 3 days increased CYP1A2 activity to 194 and 203%, respectively, as assessed by the change in the urinary ratio of 1,7-dimethylxanthine plus paraxanthine to unmetabolized caffeine. Intake of the herbal supplement increased CYP1A2 activity to levels higher than that observed from smoking (179%). This study suggests that the long-term intake of the dietary supplement inducing CYP1A2 may increase the incidence of colorectal cancers caused by procarcinogens activated by CYP1A2 in rapid N-acetyltransferase-2 acetylators and of lung adenocarcinoma in slow acetylators.

Inhibition of CYP 1A2-dependent MROD activity in rat liver microsomes: an explanation of the hepatic sequestration of a limited subset of halogenated aromatic hydrocarbons.

Many classes of halogenated aromatic compounds (HACs) are highly lipophilic environmental contaminants that exert toxic effects via the Ah receptor signal transduction pathway and whose metabolism generally involves monooxygenase enzymes of the CYP 1A family. Despite their lipophilicity, a high proportion of the body burden of certain polychlorinated dibenzo-p-dioxins and coplanar polychlorinated biphenyls is sequestered in liver, a process believed to involve CYP 1A2. In this work we examined HAC-induced inhibition of the demethylation of 7-methoxyresorufin, a process that is selectively catalyzed by CYP 1A2. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) were found to be strong competitive inhibitors of methoxyresorufin-O-demethylase activity, consistent with the high ability of hepatic tissue to sequester these compounds selectively.

Effect of Oxymetholone on SCE frequency in human lymphocyte chromosomes in vitro

Genotoxicity evaluation of a commonly used synthetic androgen, Oxymetholone, was carried out in human peripheral blood lymphocytes in vitro. Sister chromatid exchange (SCE) was used as genetic end point. The concentrations of the drug were determined after observing its effects on the mitotic index. A wide range of concentrations, i.e., 25, 50, and 100 micro g/ml of the drug, were used to determine the genotoxic effects in the absence as well as in the presence of rat liver microsomal activation system (S9 mix). The drug did not induce any significant increase in the SCE frequency at any of the concentrations either in the presence or in the absence of S9 mix. The maximum value of SCE was observed in the absence of S9 mix at 100 microg/ml concentration (i.e., 1.38+/-0.080/cell), which was not significant statistically. Moreover, the drug was not effective in increasing the SCE frequency even in the presence of S9 mix. The maximum value of SCE (i.e., 1.78+/-0.103/cell) was observed at 50 microg/ml of concentration in the presence of S9 mix. A dose relationship was also not observed. It was concluded that Oxymetholone does not affect the genetic material in human lymphocytes at a wide range of concentrations in the SCE assay.

Antioxidative components of Psoralea corylifolia (Leguminosae).

A meroterpene and four flavonoids were isolated from the seeds of Psoralea corylifolia as antioxidative components. Their structures were elucidated by spectral data and identified as bakuchiol (1), bavachinin (2), bavachin (3), isobavachin (4) and isobavachalcone (5). In particular, meroterpene 1 and flavonoids 4 and 5 showed broad antioxidative activities in rat liver microsomes and mitochondria. They inhibited NADPH-, ascorbate-, t-BuOOH- and CCl(4)-induced lipid peroxidation in microsomes. They also prevented NADH-dependent and ascorbate-induced mitochondrial lipid peroxidation. Bakuchiol (1) was the most potent antioxidant in microsomes and the inhibition of oxygen consumption induced by lipid peroxidation was time-dependent. Furthermore, bakuchiol (1) protected human red blood cells against oxidative haemolysis. These phenolic compounds in P. corylifolia were shown to be effective in protecting biological membranes against various oxidative stresses.

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.

Differential Effects of Fenofibrate or Simvastatin Treatment of Rats on Hepatic Microsomal Overt and Latent Diacylglycerol Acyltransferase Activities

Hepatic triacylglycerol secretion is elevated in insulin-resistant states. Microsomal diacylglycerol acyltransferase (DGAT) catalyzes the final reaction in the synthesis of triacylglycerol (TAG). We have previously described two DGAT activities in rat liver microsomes, one overt (cytosol-facing) and one latent (endoplasmic reticulum lumen-facing) (Owen MR, Corstorphine CG, Zammit VA: Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion. Biochem J 323:17-21, 1977). It was suggested that they are involved in the synthesis of TAG for the cytosolic droplet and VLDL lipidation, respectively. In the present study, we measured the overt and latent DGAT activities in rats fed diets containing one of two hypolipidemic drugs: fenofibrate (a peroxisome proliferator-activated receptor alpha [PPARalpha] agonist) and simvastatin (a 3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitor). We found that the activities of the two DGATs could be varied independently by these treatments. Fenofibrate raised overt DGAT activity but lowered that of latent DGAT. In contrast, simvastatin markedly lowered overt DGAT activity without affecting that of latent DGAT. The increase in overt DGAT activity induced by fenofibrate could not be mimicked by feeding a diet enriched in n-3 polyunsaturated fatty acids (PUFA), which lowered overt DGAT activity but did not affect latent DGAT, suggesting that n-3 PUFA act through a mechanism independent of PPARalpha activation. The fibrate-induced increase in overt DGAT activity and the inhibition of latent DGAT may provide a mechanism through which acyl moieties are retained within the liver for oxidation through the pathways concomitantly upregulated by PPARalpha activation.

Reductive metabolism of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one, by rat liver preparations.

The reduction of the triple bond and carbonyl group of an alpha,beta-ketoalkyne, 4-phenyl-3-butyn-2-one (PBYO), by rat liver microsomes and cytosol was investigated. The triple-bond-reduced product trans-4-phenyl-3-buten-2-one (PBO) and the carbonyl-reduced product 4-phenyl-3-butyn-2-ol (PBYOL) were formed when PBYO was incubated with rat liver microsomes in the presence of NADPH. The triple bond of 1-phenyl-1-butyne, deprenyl, ethynylestradiol, ethinamate, and PBYOL, in which the triple bond is not adjacent to a carbonyl group, were not reduced by liver microsomes even in the presence of NADPH. PBO was further reduced to 4-phenyl-2-butanone (PBA) by liver cytosol with NADPH. PBYOL was also formed from PBYO by liver cytosol in the presence of NADPH or NADH. The microsomal triple-bond reductase activity was inhibited by disulfiram, 7-dehydrocholesterol, and 18 beta-glycyrrhetinic acid but not beta-diethylaminoethyldiphenylpropylacetate or carbon monoxide. The triple-bond reductase activity in liver microsomes was not enhanced by several inducers of the rat cytochrome P450 system. These results suggested that the triple-bond reduction is caused by a new type of reductase, not cytochrome P450. The microsomal and cytosolic carbonyl reductase activities were not inhibited by quercitrin, indomethacin, or phenobarbital. Only S-PBYOL was formed from PBYO by liver cytosol. In contrast, liver microsomes produced R-PBYOL together with the S-enantiomer to some extent. Ethoxyresorufin-O-dealkylase activity in rat liver microsomes was markedly inhibited by PBYO and PBO, partly by PBYOL, but not by PBA.

Glucuronidation of acetaminophen catalyzed by multiple rat phenol UDP-glucuronosyltransferases.

Gunn rats glucuronidate acetaminophen (APAP) at reduced rates and show increased susceptibility to APAP-induced hepatotoxicity. This defect is presumed to involve UDP-glucuronosyltransferase (UGT) 1A6, which is nonfunctional in Gunn rats, but it is currently unclear whether other 1A family members are also involved. In humans, two 1A isoforms are known to be active (1A6 and 1A9) but 1A6 form has a 25-fold lower apparent K(m) (2 mM). Rat liver microsomal APAP UGT activity is induced by in vivo treatment with beta-naphthoflavone or oltipraz, an effect correlating with induction of 1A6 and 1A7. To address a possible role of 1A7 in APAP glucuronidation relative to other 1A forms, cDNAs encoding UGTs 1A1, 1A5, 1A6, 1A7, and 1A8 were expressed in human embryonic kidney cells and the contents of expressed enzyme in prepared membrane fractions determined by quantitative immunoblotting. At 2.5 mM APAP, 1A7 showed the highest specific activity (2.8 nmol/min/nmol 1A7 protein), followed by 1A6 (1.1 nmol/min/nmol), and 1A8 (0.27 nmol/min/nmol). 1A1 and 1A5 were essentially inactive. Kinetic comparisons indicated 1A7 had a similar apparent K(m) as 1A6 (4.7 versus 3.9 mM, respectively) but a 2.4-fold higher catalytic activity. These data suggest that in rats, 1A7 plays a major role in APAP glucuronidation and contributes to protection against APAP-induced hepatotoxicity. The involvement of other UGTs besides 1A6 is further underscored by the presence of significant residual APAP-glucuronidating activity by Gunn rat hepatocytes, indicating the activity of an unknown UGT2 family member.

Rat liver microsomal and nuclear activation of methanol to hydroxymethyl free radicals

Recent studies from other laboratories reported that during methanol intoxication lipid peroxidation and protein oxidation in liver occurred. Further, they detected free radicals-PBN adducts in bile and urine of methanol poisoned rats. In this work, we report the presence in liver microsomes and nuclei of NADPH dependent processes of hydroxymethyl (HMet) radical formation. The detection of HMet radicals was performed by GC/MS of the trimethylsilyl derivatives of the PBN (N- tert-butyl-a-phenylnitrone)-radical adducts. The formation of HMet radicals was observed only under nitrogen, in these in vitro conditions. Formation of formaldehyde from methanol was observed in aerobic incubation mixtures containing either microsomes or nuclei but also under nitrogen using microsomes. The latter process was not inhibited by diphenyleneiodonium while the anaerobic microsomal one producing HMet was strongly inhibited by it. This shows that they are independent processes. Results suggest that both, liver nuclei and microsomes are able to generate free radicals during NADPH-mediated methanol biotransformation.

Propranolol as an inhibitor of some cytochrome P450-dependent monooxygenase activities in native and induced rat liver microsomes.

The in vitro effect of propranolol (10(-3) M and 10(-4) M), a nonselective and extensively metabolized beta-adrenergic blocking agent, on rat liver drug metabolism in native and induced (with phenobarbital and beta-naphthoflavone [beta-NF]) microsomes was studied. The type of inhibition and the inhibitory constants of some cytochrome P450-dependent microsomal enzyme reactions (hexobarbital oxidation [HBO], ethylmorphine-N-demethylation [EMND], aniline hydroxylation [AH], ethoxycoumarin-O-deethylation [ECOD], ethoxyresorufin-O-dealkylation [EROD] and penthoxyresorufin-O-dealkylation [PROD]) were estimated. The results showed that propranolol competitively inhibited AH activity in native microsomes. The type of inhibition was changed from competitive to noncompetitive in all other enzyme activities studied. This inhibition was more pronounced after phenobarbital induction in PROD (Ki = 0.11 +/- 0.01 mM), ECOD (Ki = 0.40 +/- 0.09 mM) and EMND (Ki = 0.59 +/- 0.1 mM), and after beta-NF induction in AH (Ki = 0.28 +/- 0.05 mM) and in HBO (Ki = 0.35 +/- 0.1 mM) in native microsomes. It was assumed that the noncompetitive type of inhibition is due to the covalent binding of reactive metabolites derived from propranolol to hepatic microsomal proteins. The competitive type of inhibition of AH suggested a common P450 isoenzyme in the metabolism of propranolol and aniline. Thus, in this study, propranolol has been found to be not only a selective inhibitor of CYP2D6 isoenzyme-dependent reactions, but also a nonspecific inhibitor of other cytochrome P450 isoenzymes.

Substrate specificity for rat cytochrome P450 (CYP) isoforms: screening with cDNA-expressed systems of the rat

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1′-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4′-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.

Inhibition of UDP-glucuronosyltransferases in rat liver microsomes by natural mutagens and carcinogens.

Eight toxic compounds of natural origin present in the human diet or used as drugs were tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes with 1-naphthol (1-NA), phenolphthalein (PPh) and 4-nitrophenol (4-NP) as substrates. Strong inhibitory effects were observed with tannic acid (tannin) and the antifungal drug griseofulvin (GF): at a concentration of 1 mM, the two compounds completely suppressed the glucuronidation of 1-NA and PPh, respectively. A concentration of 0.1 mM still proved to be highly inhibitory, and even at a concentration as low as 50 microM, tannin produced nearly 50% inhibition of 1-NA conjugation. The UGT isoforms converting 4-NP were less sensitive to the tested compounds (with the exception of GF). Kinetic studies with tannin revealed an uncompetitive type of inhibition toward 1-NA, with an apparent Ki value of 20 microM. The inhibition by GF was non-competitive with respect to PPh and was of a mixed type toward UDP-glucuronic acid, with apparent Ki values of 40 microM and 30 microM, respectively. Tannin and GF did not act as substrates for rat microsomal UGT.

Rapid high-performance liquid chromatographic method for the separation of hydroxylated testosterone metabolites

A rapid high-performance liquid chromatography (HPLC) method is described for the quantitation of hydroxytestosterone metabolites. The method combines a Hypersil BDS C 18 analytical column (10 cm×0.46 cm) and a linear mobile phase (1.25 ml/min) gradient of tetrahydrofuran–acetonitrile–water (10:10:80, v/v) changing to tetrahydrofuran–acetonitrile–water (14:14:72, v/v) over 10 min then remaining isocratic for 3 min. The total run time for the chromatographic separation of eight metabolites of testosterone is 15 min. Detection by UV is linear between 300 ng/ml and 10 μg/ml with a limit of detection on column of 300 ng/ml. A method for the direct HPLC analysis of liver microsomal incubates of [ 14C]testosterone is also briefly described and when combined with the HPLC method, offers a distinct advantage over previously reported methods for the rapid screening of testosterone hydroxylase activity in rat and human liver microsomes.

Continuous Recording of Long-Chain Acyl-Coenzyme A Synthetase Activity Using Fluorescently Labeled Bovine Serum Albumin

The fluorescence-based long-chain fatty acid probe BSA-HCA (bovine serum albumin labeled with 7-hydroxycoumarin-4-acetic acid) is shown to respond to binding of long-chain acyl-CoA thioesters by quenching of the 450 nm fluorescence emission. As determined by spectrofluorometric titration, binding affinities for palmitoyl-, stearoyl-, and oleoyl-CoA (Kd = 0.2–0.4 μM) are 5–10 times lower than those for the corresponding nonesterified fatty acids. In the presence of detergent (Chaps, Triton X-100, n-octylglucoside) above the critical micelle concentration, acyl-CoA partitions from BSA-HCA and into the detergent micelles. This allows BSA-HCA to be used as a fluorescent probe for continuous recording of fatty acid concentrations in detergent solution with little interference from acyl-CoA. Using a calibration of the fluorescence signal with fatty acids in the C14 to C20 chain-length range, fatty acid consumption by Pseudomonas fragi and rat liver microsomal acyl-CoA synthetase activities are measured down to 0.05 μM/min with a data sampling rate of 10 points per second. This new method provides a very promising spectrofluorometric approach to the study of acyl-CoA synthetase reaction kinetics at physiologically relevant (nM) aqueous phase concentrations of fatty acid substrates and at a time resolution that cannot be obtained in isotopic sampling or enzyme-coupled assays.

Effects of Diabetes and Insulin on Hepatic Δ6 Desaturase Gene Expression

It has been recognized that rat liver microsomal Δ6 desaturation activity is defective in experimental diabetes, a fact that may be reverted by means of insulin treatment. In the present study, we used streptozotocin-induced diabetic rats in order to determine the regulatory role of insulin on the expression of hepatic Δ6 desaturase gene. The abundance of hepatic Δ6 desaturase mRNA in the diabetic rats is sevenfold lower than in the control. Insulin administration to diabetic rats induces Δ6 desaturase mRNA eightfold within 24 h. The effect of insulin on the Δ6 desaturase mRNA was inhibited 70% with dibutyryl-cAMP and theophylline administration and 90% by cycloheximide administration. Therefore, our data demonstrate that the activity of hepatic Δ6 desaturase in response to insulin is, at least in part, regulated by pretranslational events that require the synthesis of an unknown protein(s).

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 μ m) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe 2+/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe 3+-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe 2+/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 μ m, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe 3+-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 μ m), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe 3+-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5′-prenylxanthohumol, and NG, with no antioxidant property at 5 μ m concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.

Determination of UDP-glucuronosyltransferase UGT1A6 activity in human and rat liver microsomes by HPLC with UV detection.

A simple and sensitive method for the determination of UDP-glucuronosyltransferase UGT1A6 activity using 4-methylumbelliferone (4-MU) and 4-nitrophenol (4-NP) as substrates in human and rat liver microsomes by high-performance liquid chromatography (HPLC) with uv detection is reported. The method was validated for the determination of 4-methylumbelliferyl beta-D-glucuronide (4-MUG) and 4-nitrophenyl beta-D-glucuronide (4-NPG) with respect to specificity, linearity, detection limit, recovery, stability, precision and accuracy. There was no interference from matrix and non-enzymatic reactions. Calibration curves for 4-MUG and 4-NPG are linear from 0.5 to 500 microM. Average recoveries ranged from 98 to 100% in spiked liver microsomes samples. 4-MUG and 4-NPG were stable at 4 degrees C for at least 72 h in spiked liver microsomes samples. The method was found to be more sensitive than previous methods using a spectrophotometer, a spectrofluorometer and HPLC. The detection limit for 4-MUG and 4-NPG (signal-to-noise ratio of 3) was 14 and 23 nM, respectively. The intra- and inter-day precision (relative S.D. (RSD)) and accuracy (relative mean error (RME)) was <5 and 9%, respectively. The intra- and inter-day reproducibility (RSD) of UGT1A6 enzyme assay in liver microsomes was <6%. With this improved sensitivity, the kinetics of UGT activities toward 4-MU and 4-NP in human and rat liver microsomes could be determined more precisely. In addition, the method could determine the non-inducible, and 3-methylcholanthrene- and phenobarbital-inducible activities of UGT1A6 in rat liver microsomes under the same assay conditions. Therefore, this method is applicable to in vivo and in vitro studies on the interaction of xenobiotic chemicals with UGT1A6 isoform in mammals using small amounts of biological samples.

Immunolocalization of a novel cholesteryl ester hydrolase in the endoplasmic reticulum of murine and human hepatocytes.

We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissue-restricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism.

Influence of subchronic administration of catechol estrogens on the formation of reactive oxygen species in rat liver microsomes

Metabolic pathways of estrogens are the formation of catechol estrogens (CE; 2- and 4-hydroxy-estrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. In previous investigations subchronic administration of estrogens showed prooxidative and antioxidative activities in rat liver microsomes (BARTH et al. 1999). To find out whether or not catechol metabolites are responsible for prooxidative activity, we checked 2- and 4-hydroxy-estradiol (2OH-E2 and 4OH-E2) and the non-catechol metabolite 6alpha-hydroxy-estradiol (6alpha-OH-E2) for formation of reactive oxygen species in liver microsomes of 30-day-old male Wistar rats after 5 days treatment (1, 10 mg/kg b. wt. orally, once a day). The results were compared with those after treatment of the rats with estradiol (E2), estradiol valerate (E2V) and ethinylestradiol (EE2). In liver homogenates glutathione and lipid peroxides were determined, in microsomes NADPH-Fe++-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) were investigated. In liver 9000 x g supernatants monooxygenase activities were measured. The two catechol estrogens did not show any antioxidative activity, whereas 6alpha-OH-E2 significantly diminished lipid peroxides in the liver as well as LPO and LUM-CL in liver microsomes. Among estrogens, only EE2 showed antioxidative activity. Both CE inhibited ethoxycoumarin O-deethylation. Peroxidative activity as enhanced LUC-CL was found after 2OH-E2 (1 mg/kg b.wt.) and E2, but 10 times higher doses of both CE did not change LUC-CL. Microsomal H2O2 generation was enhanced by E2, E2V and both CE, not by 6alpha-OH-E2. The lower level of H2O2 enhancement caused by CE in comparison to E2 and E2V together with unchanged LUC-CL after high CE doses did not unequivocally prove the CE to be mainly responsible for the prooxidative activities of E2 and E2V in liver microsomes, at least in 30-day-old male rats. Unchanged GSH in the liver after CE administration supports this hypothesis.

Synthesis of new potent and selective aromatase inhibitors based on long-chained diarylalkylimidazole and diarylalkyltriazole molecule skeletons

A series of long-chained diarylalkylimidazoles and diarylalkyltriazoles were synthesized and evaluated for the inhibitory potency for aromatase (estrogen synthetase) activity in human placental microsomes. The relative specificity of inhibition was evaluated by measuring the inhibition of cholesterol side-chain cleavage enzyme (desmolase) in human placental mitochondria and the inhibition of 7-ethoxycoumarin O-deethylase (a typical drug-metabolizing enzyme activity) in rat liver microsomes. The structural requirements including substituent effects for the strongest potency and for the highest specificity were delineated. α,ω-Diarylalkyltriazoles and imidazoles were the most interesting molecules, in which the geometric and optical isomerism displayed remarkable selectivity for aromatase inhibition.