Isolated Rat Liver Microsomes Research Articles

Synthesis, in silico prediction of sites of metabolism and in-vitro hepatotoxicity evaluationofnew seriesN’-substituted 3-(1,3,7-trimethyl-xanthin-8-ylthio)propanehydrazides

New series of 3-(1,3,7-trimethyl-xanthin-8-ylthio)propanehydrazide derivatives were designed and synthesized. The targed compounds were obtained in yields of 54 to 100% and their structures were elucidated by FTIR,1H NMR,13C NMR, MS and microanalyses. The tested compounds were subjected to in silico prediction of sites of metabolism (SOMs). The predictions show thatthe main metabolic changes will be primarily related to oxidation of the sulfur atom in the side chain, carried out under the action of CYP2C19, as well as O-demethylation of compounds containing methoxy groups.The N-demethylation of the xanthine fragment was determined to be regulated by CYP1A2, CYP2C9, CYP2D6 and CYP3A4. Theperformed in vitro studies confirmed for two of the tested compounds to be low hepatotoxic, due to the presented prooxidant effects at subcellular level (isolated rat liver microsomes). These results highlight these molecules as promising hydrazide-hydrazone structures for the design of compounds with low hepatotoxicity.

Synthesis and preliminary hepatotoxicity evaluation of new caffeine-8-(2-thio)-propanoic hydrazid-hydrazone derivatives

New series of caffeine-8-(2-thio)-propanoic hydrazid-hydrazone derivatives were designed and synthesized. The targed compounds were obtained in yields of 51 to 96% and their structures were elucidated by FTIR,1H NMR,13C NMR, MS and microanalyses. All of the compounds were found to be “drug-like” as they fulfill the criteria of drug-likeness, which includes the MDDR-like rule. The tested compounds were subjected toin silicoprediction of substrate/metabolite specificity and Drug Induced Liver Injury (DILI). The prediction for indicated that the evaluated compounds would most probably act as CYP1A2 substrates. The performedin vitrostudies didn’t reveal statistically significant hepatotoxicity of the tested compounds, probably due to the pro-oxidant effects expressed on sub-cellular (isolated rat liver microsomes) level. The obtained experimental results confirmed the predicted low hepatotoxicity for the tested structures. Based on these results the compounds may be considered as promising structures for design of future molecules with low hepatotoxicity.

Activation versus inhibition of microsomal glutathione S-transferase activity by acrolein. Dependence on the concentration and time of acrolein exposure

The toxicity of acrolein, an α,β-unsaturated aldehyde, is due to its soft electrophilic nature and primarily involves the adduction of protein thiols. The thiol glutathione (GSH) forms the first line of defense against acrolein. The present study confirms that acrolein added to isolated rat liver microsomes can increase microsomal GSH transferase (MGST) activity 2–3 fold, which can be seen as a direct adaptive increase in the protection against acrolein. At a relatively high exposure level, acrolein appeared to inhibit MGST. The activation is due to adduction of thiol groups, and the inactivation probably involves adduction of amino groups in the enzyme by acrolein. The preference of acrolein to react with thiol groups over amino groups can explain why the enzyme is activated at a low exposure level and inhibited at a high exposure level of acrolein. These opposite forms of direct adaptation on the level of enzyme activity further narrow the thin line between survival and promotion of cell death, governed by the level of exposure.

Evaluation of the impact of 16-dehydropregnenolone on the activity and expression of rat hepatic cytochrome P450 enzymes

16-dehydropregnenolone (DHP) is a promising novel antihyperlipidemic agent developed and patented by Central Drug Research Institute (CDRI), India. The purpose of the present study was to investigate whether DHP influences the activities and mRNA expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D2, CYP2E1 and CYP3A1) in Sprague-Dawley (SD) rats. A cocktail suspension of CYP probe substrates which contained caffeine (CYP1A2), tolbutamide (CYP2C11), dextromethorphan (CYP2D2), chlorzoxazone (CYP2E1) and dapsone (CYP3A1) was administered orally on eighth- or fifteenth-day to rats pre-treated with DHP intragastrically at a dose of 36 and 72mg/kg for one week and two weeks. The concentrations of probe drugs in plasma were estimated by liquid chromatography-tandem mass spectrometry (LC–MS/MS). Alongside, the effect of DHP on CYPs activity and mRNA expression levels were assayed in isolated rat liver microsomes and by real-time reverse transcription-polymerase chain reaction (RT-PCR), respectively. DHP had significant inducing effects on CYP1A2, 2C11, 2D2 and 2E1 with no effect on CYP3A1 in dose- and time-dependent manner, as revealed from the pharmacokinetic profiles of the probe drugs in rats. In-vitro microsomal activities and mRNA expression results were in good agreement with the in-vivo pharmacokinetic results. Collectively, the results unveiled that DHP is an inducer of rat hepatic CYP enzymes. Hence, intense attention should be paid when DHP is co-administered with drugs metabolized by CYP1A2, 2C11, 2D2 and 2E1, which might result in drug-drug interactions and therapeutic failure.

Cortisol promotes endoplasmic glucose production via pyridine nucleotide redox.

Both increased adrenal and peripheral cortisol production, the latter governed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), contribute to the maintenance of fasting blood glucose. In the endoplasmic reticulum (ER), the pyridine nucleotide redox state (NADP/NADPH) is dictated by the concentration of glucose-6-phosphate (G6P) and the coordinated activities of two enzymes, hexose-6-phosphate dehydrogenase (H6PDH) and 11β-HSD1. However, luminal G6P may similarly serve as a substrate for hepatic glucose-6-phophatase (G6Pase). A tacit belief is that the G6P pool in the ER is equally accessible to both H6PDH and G6Pase. Based on our inhibition studies and kinetic analysis in isolated rat liver microsomes, these two aforesaid luminal enzymes do share the G6P pool in the ER, but not equally. Based on the kinetic modeling of G6P flux, the ER transporter for G6P (T1) preferentially delivers this substrate to G6Pase; hence, the luminal enzymes do not share G6P equally. Moreover, cortisol, acting through 11β-HSD1, begets a more reduced pyridine redox ratio. By altering this luminal redox ratio, G6P flux through H6PDH is restrained, allowing more G6P for the competing enzyme G6Pase. And, at low G6P concentrations in the ER lumen, which occur during fasting, this acute cortisol-induced redox adjustment promotes glucose production. This reproducible cortisol-driven mechanism has been heretofore unrecognized.

Formation of a Predominant Metabolite of Hydroxydihydrocarvone Evaluated by a Biomimetic Oxidative Model and in Rat Liver Microsomes

This paper reports the biomimetic oxidation of hydroxydihydrocarvone by iodosylbenzene using tetraphenyl-porphine iron(III) chloride as the catalyst in ethyl acetate. Mass spectrometry fragmentation maps of hydroxydihydrocarvone (obtained by gas chromatography-mass spectrometry analyses) allowed for the identification of the major product as 4-hydroxy-5-(2-hydroxypropan-2-yl)-2-methylcyclohex-2-en-1-one (4-hydroxy-hydroxydihydrocarvone). This compound was also observed in an in vitro metabolism assay that employed isolated rat liver microsomes, thereby validating the biomimetic procedure.

Antihyperglycemic activity of Caralluma tuberculata in streptozotocin-induced diabetic rats

The current study aimed at evaluating the potential and mechanisms of the antidiabetic activity of the methanolic extract (ME) of Caralluma tuberculata as well as its chloroform (CF), n-butanol (BF) and the remaining water fractions (RFs) in streptozotocin-induced diabetic rats. The antidiabetic activity was evaluated through assessing fasting blood glucose (FBG), insulin levels, oral glucose tolerance test (OGTT), glucose utilization by isolated rat psoas muscle, gut glucose absorption and G-6-Pase activity in isolated rat liver microsomes. Both ME and RF showed the highest potency, where ME had superior activity. The mechanism underlying the observed antihyperglycemic activity of ME could be attributed, at least in part, to enhanced skeletal muscle utilization of glucose, inhibition of hepatic gluconeogenesis and stimulation of insulin secretion. ME was standardized through LC-MS analysis for its major pregnanes.

Manifold effects of palmitoylcarnitine on endoplasmic reticulum metabolism: 11β-hydroxysteroid dehydrogenase 1, flux through hexose-6-phosphate dehydrogenase and NADPH concentration

With the exception of the oxidation of G6P (glucose 6-phosphate) by H6PDH (hexose-6-phosphate dehydrogenase), scant information is available about other endogenous substrates affecting the redox state or the regulation of key enzymes which govern the ratio of the pyridine nucleotide NADPH/NADP. In isolated rat liver microsomes, NADPH production was increased, as anticipated, by G6P; however, this was strikingly amplified by palmitoylcarnitine. Subsequent experiments revealed that the latter compound, well within its physiological concentration range, inhibited 11β-HSD1 (11β-hydroxysteroid dehydrogenase 1), the bidirectional enzyme which interconnects inactive 11-oxo steroids and their active 11-hydroxy derivatives. Notably, palmitoylcarnitine also stimulated the antithetical direction of 11β-HSD1 reductase, namely dehydrogenase. This stimulation of H6PDH may have likewise contributed to the NADPH accretion. All told, the result of these enzyme modifications is, in a conjoint fashion, a sharp amplification of microsomal NADPH production. Neither the purified 11β-HSD1 nor that obtained following microsomal sonification were sensitive to palmitoylcarnitine inhibition. This suggests that the long-chain amphipathic acylcarnitines, given their favourable partitioning into the membrane lipid bilayer, disrupt the proficient kinetic and physical interplay between 11β-HSD1 and H6PDH. Finally, although IDH (isocitrate dehydrogenase) and malic enzyme are present in microsomes and increase NADPH concentration akin to that of G6P, neither had an effect on 11β-HSD1 reductase, evidence that the NADPH pool in the endoplasmic reticulum shared by the H6PDH/11β-HSD1 alliance is uncoupled from that governed by IDH and malic enzyme.

The stereoselective metabolism of halofantrine to desbutylhalofantrine in the rat: Evidence of tissue‐specific enantioselectivity in microsomal metabolism

The pharmacokinetics of the antimalarial drug (+/-)-halofantrine are stereoselective in humans and rats. To better understand the stereoselective metabolism of the drug to its primary metabolite, desbutylhalofantrine (DHF), a series of in vitro and in vivo experiments were undertaken in the rat. Formation of (-)-DHF exceeded that of (+)-DHF in liver microsomes [(-):(+) ratio of intrinsic formation clearances = 1.4]. In contrast, in intestinal microsomes no significant stereoselectivity was noted in the formation of the DHF enantiomers. Intestinal microsomes were also less efficient at producing the DHF enantiomers than were liver microsomes. Based on kinetic analysis of the DHF formation, there appeared to be more than one enzyme involved in the biotransformation. (+/-)-Ketoconazole (KTZ) effectively inhibited the formation of both DHF enantiomers by both liver and intestinal microsomes, although the reduction was more marked in liver microsomes. Through a combination of the use of CYP antibodies and recombinant CYP isoenzymes, the involvement of CYP 2B1/2, 3A1, 3A2, 1A1, 2C11, 2C6, 2D1, and 2D2 were implicated in the metabolism of halofantrine to DHF. Of these, CYP3A1/2 and CYP2C11 appeared to be the primary isoenzymes involved, although CYP2C11 showed greater (+)-DHF than (-)-DHF formation, whereas for CYP3A1 it was similar to the isolated rat liver microsomes. In vivo, oral (+/-)-KTZ caused significant increases in plasma halofantrine and decreases in DHF enantiomer plasma concentrations.

Reductive metabolism of the nitroimidazole-based hypoxia-selective cytotoxin NLCQ-1 (NSC 709257).

The enzymatic cell-free metabolism of the novel hypoxia-selective cytotoxin 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1) was investigated under hypoxic or aerobic conditions in the presence of purified reductive enzymes or isolated rat liver microsomes by monitoring the parent compound with HPLC-UV analysis. Enzymatic reduction of NLCQ-1 with isolated rat liver microsomes and NADPH or NADH showed that, only under hypoxic conditions, ca. 45% and 60% of the parent compound was reduced, respectively, within 1 h of incubation (37 degrees C). Under identical conditions but in the presence of 2'-AMP (a P450 reductase inhibitor), 6-propyl-2-thiouracil or p-hydroxymercuribenzoate (cytochrome b5 reductase inhibitors), NLCQ-1 reduction was inhibited. Enzymatic cell-free metabolism of NLCQ-1 with recombinant human DT-diaphorase (DTD) and NADPH or NADH under hypoxic or aerobic conditions showed that < or = 5% of the compound was reduced within 2 h. Reduction kinetics with human P450 reductase-expressing microsomes showed ca. 75% or 50% reduction of NLCQ-1 under hypoxic or aerobic conditions, respectively, after 2 h incubation. These results suggest that DTD is not involved in the initial steps of the bioreductive metabolism of NLCQ-1, although it could be involved with metabolites of NLCQ-1, and that cytochrome P450 and cytochrome b5 reductases play a significant role in the bioreductive metabolism of NLCQ-1.

In vitro desaturation and elongation of rumenic acid by rat liver microsomes.

Various nutritional studies on CLA, a mixture of isomers of linoleic acid, have reported the occurrence of conjugated long-chain PUFA after feeding experimental animals with rumenic acid, 9c,11t-18:2, the major CLA isomer, probably as a result of successive desaturation and chain elongation. In the present work, in vitro studies were carried out to obtain information on the conversion of rumenic acid. Experiments were first focused on the in vitro delta6-desaturation of rumenic acid, the regulatory step in the biosynthesis of long-chain n-6 PUFA. The conversion of rumenic acid was compared to that of linoleic acid (9c,12c-18:2). Isolated rat liver microsomes were incubated with radiolabeled 9c,12c-18:2 and 9c,11t-18:2 under desaturation conditions. The data indicated that [1-(14)C]9c,11t-18:2 was a poorer substrate for delta6-desaturase than [1-(14)C]9c,12c-18:2. Next, in vitro elongation of 6c,9c,11t-18:3 and 6c,9c,12c-18:3 (gamma-linolenic acid) was investigated in rat liver microsomes. Under elongation conditions, [1-(14)C]6c,9c,11t-18:3 was 1.5-fold better converted into [3-(14)C]8c,11c,13t-20:3 than [1-(14)C]6c,9c,12c-18:3 into [3-(14)C]8c,11c,14c-20:3. Finally, in vitro delta5-desaturation of 8c,11c,13t-20:3 compared to 8c,11c,14c-20:3 was investigated. The conversion level of [1-(14)C]8c,11c,13t-20:3 into [1-(14)C]5c,8c,11c,13t-20:4 was 10 times lower than that of [1-(14)C]8c,11c,14c-20:3 into [1-(14)C]5c,8c,11c,14c-20:4 at low substrate concentrations and 4 times lower at the saturating substrate level, suggesting that conjugated 20:3 is a poor substrate for the delta5-desaturase.

The protection of long chain polyunsaturated fatty acids by melatonin during nonenzymatic lipid peroxidation of rat liver microsomes.

The in vivo and in vitro effects of melatonin (N-acetyl-5-methoxytryptamine) on lipid peroxidation of long chain polyunsaturated fatty acids (PUFA) located in rat liver microsomes were determined. The effect of intraperitoneal administration of melatonin (10 mg/kg weight/24 hr) on ascorbate-Fe++ induced lipid peroxidation of isolated rat liver microsomes was first examined. The ascorbate induced light emission in hepatic microsomes was inhibited by melatonin treatment [control group: 10.714 +/- 0.894; melatonin group: 3.162 +/- 0.515, counts per minute (cpm) x 10(-5)]. Significant differences in the content of arachidonic C20:4 n-6 and docosahexaenoic acid C22:6 n-3 were observed when control microsomes were lipid peroxidized in the presence of ascorbic acid. These changes were less pronounced in liver microsomes isolated from melatonin treated rats. In vitro assays showed that after incubation of rat liver microsomes in an ascorbate-Fe++ system, at 37 degrees C for 210 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of melatonin. The fatty acid composition of total lipids isolated from rat liver microsomes was substantially modified when subjected to nonenzymatic lipid peroxidation with a considerable decrease of docosahexaenoic acid 22:6 n-3 and arachidonic acid 20:4 n-6. The inhibition of the lipid peroxidation process evaluated as chemiluminescence (total cpm at selected times) was melatonin concentration dependent. Melatonin, at a concentration 1.2 mm, inhibited almost completely the lipid peroxidation process. Arachidonic and docosahexaenoic acids were more affected than docosapentaenoic acid during the lipid peroxidation process. Not all fatty acids were equally protected after the addition of melatonin to the incubation medium. Our results indicate that melatonin may act in vivo and in vitro as an antioxidant protecting long chain PUFA present in rat liver microsomes from the deleterious effect by a selective mechanism that reduces the loss of docosahexaenoic and arachidonic acids.

β-Carotene: interactions with α-tocopherol and ascorbic acid in microsomal lipid peroxidation

β-Carotene, α-tocopherol, and ascorbic acid were tested for their ability to inhibit, enhance, or react synergistically with O2 (15, 150, 760 torr) and, 2,2′-azobis (2-amidino-propane) dihydrochloride (AAPH) or 1,1′-azobis (cyclohexane-carbonitrile) (ACCN) in isolated rat liver microsomes. β-Carotene did not protect against lipid peroxidation, i.e., malondialdehyde (MDA) formation, in microsomal samples incubated at 37°C with aqueous soluble AAPH at all added β-carotene concentrations and oxygen tensions. More MDA (16%, p < 0.001) was produced at 15 torr of O2, and 160 nmol/mg protein of β-carotene compared to respective vehicle control. Individually, α-tocopherol and ascorbic acid exhibited antioxidant protection (ascorbic acid ≫ α-tocopherol); however, a mixture of both compounds was no more protective than ascorbic acid alone. β-Carotene demonstrated a concentration-dependent antioxidant affect at 15 torr O2 (p < 0.01); but a prooxidant effect at higher O2 at 150 and 760 torr (>57%, p < 0.001) by lipid-soluble ACCN. α-Tocopherol exhibited concentration-dependent inhibitory effects on microsomal MDA formation at all oxygen tensions, but was most effective under 150 torr. Ascorbic acid demonstrated a concentration-dependent antioxidant effect only at 150 torr. ACCN-induced lipid peroxidation was no greater for the combination of the three compounds than ascorbic acid added alone. Thus, antioxidant or prooxidant activities for β-carotene, α-tocopherol, and ascorbic acid in microsomal suspensions are related to O2 tension, solubility, antioxidant concentrations and are governed by complex interactions. Differences between AAPH- and ACCN-induced lipid peroxidation are related to differences in lipid solubility.

Role of regucalcin as an activator of Ca2‐ATPase activity in rat liver microsomes

The effect of Ca2+-binding protein regucalcin on Ca2+-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1–1.0 μM) in the enzyme reaction mixture led to a significant increase in Ca2+-ATPase activity. Regucalcin significantly stimulated ATP-dependent 45Ca2+ uptake by the microsomes. Thapsigargin (10−6 M), a specific inhibitor of microsomal Ca2+ pump enzyme (Ca2+-ATPase), clearly inhibited regucalcin (0.5 μM)-increased microsomal Ca2+-ATPase activity. Liver microsomal Ca2+-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 μM) in increasing Ca2+-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10−2 %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca2+ ionophore (A23187; 10−7 M). The present study demonstrates that regucalcin can stimulate Ca2+ pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca2+-ATPase. J. Cell. Biochem. 74:663–669, 1999. © 1999 Wiley-Liss, Inc.

Role of regucalcin as an activator of Ca2+-ATPase activity in rat liver microsomes

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver microsomes was investigated. The presence of regucalcin (0.1-1.0 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. Thapsigargin (10(-6) M), a specific inhibitor of microsomal Ca(2+) pump enzyme (Ca(2+)-ATPase), clearly inhibited regucalcin (0.5 microM)-increased microsomal Ca(2+)-ATPase activity. Liver microsomal Ca(2+)-ATPase activity was markedly decreased by N-ethylmaleimide (NEM; 2.5 mM), while the activity was clearly elevated by dithiothreitol (DTT; 2.5 mM), indicating that the sulfhydryl (SH) group of the enzyme is an active site. The effect of regucalcin (0.5 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of NEM (2.5 mM) or digitonin (10(-2) %), a solubilizing reagent of membranous lipids. Moreover, the effect of regucalcin on enzyme activity was seen in the presence of Ca(2+) ionophore (A23187; 10(-7) M). The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver microsomes, and that the protein may act the SH groups of microsomal Ca(2+)-ATPase.

Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference.

Decreased triacylglycerol synthesis within hepatocytes due to decreased diacylglycerol acyltransferase (DGAT) activity has been suggested to be an important mechanism by which diets rich in fish oil lower plasma triacylglycerol levels. New findings suggest that eicosapentaenoic acid (EPA), and not docosahexaenoic acid (DHA), lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis. To contribute to the understanding of the triacylglycerol-lowering mechanism of fish oil, the different metabolic properties of EPA and DHA were studied in rat liver parenchymal cells and isolated rat liver organelles. EPA-CoA was a poorer substrate than DHA-CoA for DGAT in isolated rat liver microsomes, and in the presence of EPA, a markedly lower value for the triacyl[3H]glycerol/diacyl[3H]glycerol ratio was observed. The distribution of [1-14C]palmitic acid was shifted from incorporation into secreted glycerolipids toward oxidation in the presence of EPA (but not DHA) in rat liver parenchymal cells. [1-14C]EPA was oxidized to a much greater extent than [1-14C]DHA in rat liver parenchymal cells, isolated peroxisomes, and especially in purified mitochondria. As the oxidation of EPA was more effective and sensitive to the CPT-I inhibitor, etomoxir, when measured in a combination of both mitochondria and peroxisomes, we hypothesized that both are involved in EPA oxidation, whereas DHA mainly is oxidized in peroxisomes. In rats, EPA treatment lowered plasma triacylglycerol and increased hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity in both the presence and absence of malonyl-CoA. Whereas only EPA treatment increased the mRNA levels of CPT-I, DHA treatment increased the mRNA levels of peroxisomal fatty acyl-CoA oxidase and fatty acid binding protein more effectively than EPA treatment. In conclusion, EPA and DHA affect cellular organelles in relation to their substrate preference. The present study strongly supports the hypothesis that EPA, and not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation.

Metabolites of acetaminophen trigger Ca 2+ release from liver microsomes

Release of mitochondrial calcium is believed to play a key role in the toxicity of acetaminophen in biological systems. Elevated cytosolic Ca 2+ may also result from activation of calcium releasing channels. The major metabolites of acetaminophen, benzoquinone imine and 1,4-benzoquinone, induced Ca 2+ release in isolated rat liver microsomes. The 1,4-benzoquinone-induced release of calcium was suppressed by ryanodine and fully inhibited by reduced glutathione. Concentrations of 1,4-benzoquinone that induced Ca 2+ release did not affect the activity of the microsomal Ca 2+, Mg 2+–APTase. The binding of [ 3H]ryanodine to liver microsomes, however, was significantly decreased by 1,4-benzoquinone, suggesting a direct interaction of this metabolite with the ryanodine-binding protein (ryanodine receptor). These results suggest that cellular Ca 2+ levels may be elevated by acetaminophen by pathways involving, in part, activation of Ca 2+ releasing channels such as the ryanodine receptor.

Formation of fatty acid ethyl esters in rat liver microsomes. Evidence for a key role for acyl-CoA: ethanol O-acyltransferase.

Fatty acid ethyl esters have been detected in high concentrations in organs commonly damaged by alcohol abuse and are regarded as being important non-oxidative metabolites of ethanol. The formation of fatty acid ethyl esters (FAEEs) has been ascribed to two enzymic activities, acyl-CoA : ethanol O-acyltransferase (AEAT) and FAEE synthase. In the present study we determined AEAT and FAEE synthase activities in isolated rat liver microsomes and further characterized the microsomal AEAT activity in more detail. The determined AEAT and FAEE synthase activities were found to be similar (about 1.7 nmol.min-1.mg-1). However, the AEAT activity was increased about sixfold by the addition of 250 microm bis-(4-nitrophenyl) phosphate (a serine esterase inhibitor) to the incubation whereas FAEE synthase activity was completely inhibited. p-Hydroxymercuribenzoic acid (a cysteine-reacting compound) also stimulated AEAT activity (about fourfold) but had no effect on FAEE synthase activity. The effects of the inhibitors suggest that the formation of FAEEs by AEAT was severely counteracted by enzymic hydrolysis of the substrate (acyl-CoA) and to a lesser extent the product by serine esterases. dl-Melinamide, a hypocholesterolaemic drug, was found to be a very potent inhibitor of AEAT activity with an IC50 value of about 2.5 microm. Furthermore, we compared the activities of two purified microsomal carboxylesterases, ES-4 and ES-10, and identified ES-4 as the enzyme responsible for hydrolysis of FAEEs. The two carboxyesterases were also tested for FAEE synthase activity, but neither had any detectable activity. Esterase ES-4 was found to have some AEAT activity, but it was low. When measured under optimal conditions without competing hydrolysis the capacity of AEAT is thus considerably higher than FAEE synthase and the results are consistent with an important role for AEAT in the formation of ethyl esters. As the ratio acyl-CoA/non-esterified fatty acids is high under normal conditions, AEAT is probably the most important enzyme in fatty acid ethyl ester formation.

Expression of autoantibodies to specific cytochromes P450 in a case of disulfiram hepatitis

Background/Aims: Immunological mechanisms are involved in many adverse drug reactions. In certain forms of drug-induced hepatitis, patients have been reported to express specific autoantibodies to hepatic drug-metabolising enzymes. The alcohol deterrent disulfiram is associated with a low frequency of severe liver toxicity, including hepatitis, but the mechanism of the toxicity is unknown. We investigated whether autoantibodies to cytochrome P450 enzymes were expressed in the serum of a 28-year-old male patient, who developed hepatitis after 7 weeks of disulfiram treatment and in whom possible causes of hepatitis other than disulfiram had been ruled out. Methods: Patient serum IgG reactivity was analysed by immunoblotting or ELISA against test antigens consisting of recombinant/purified human or rat liver P450 enzymes, or isolated rat liver microsomes. Results: A significant serum reactivity was found in immunoblotting against human cytochromes P450 1A2 and rat P450 3A1, using serum dilutions of up to 1:900 and 1:2400, respectively. In contrast, the reactivity against cytochromes P450 2E1, 2C9, 2D6, 3A4, and rat liver P450 reductase was either very low or undetectable. ELISA reactivity was low in general, indicating that the P450 epitopes were not surface exposed. Immunoblotting of rat liver microsomes revealed that autoantibodies recognised one major polypeptide corresponding to P450 3A. Autoantibody titres remained stable for at least 6 months after acute hepatitis. A similar reactivity was not found in any of ten control sera. Conclusions: The expression of autoantibodies directed against specific cytochromes P450 in a case of disulfiram hepatitis suggests that immunological mechanisms are involved in this adverse drug reaction, and that these P450 proteins should be evaluated as possible diagnostic test antigens in disulfiram hepatotoxicity.

Modulation of P450-dependent ifosfamide pharmacokinetics: a better understanding of drug activation in vivo.

The anti-cancer prodrug ifosfamide (IF) is metabolized by liver P450 enzymes by two alternative pathways. IF is activated to 4-hydroxy IF (4-OH-IF), which ultimately yields the alkylating mustard isophosphoramide, whereas IF N-dechlororethylation inactivates the drug and produces the neurotoxic metabolite chloroacetaldehyde (CA). Both reactions are catalysed by multiple liver P450 enzymes in vitro in isolated rat liver microsomes. The present pharmacokinetic study investigates the potential for modulation of these alternative pathways of IF metabolism in vivo using the adult male Fischer 344 rat model. Rats were treated with IF alone or in conjunction with various P450 inducers and inhibitors in an effort to improve the balance between drug activation and drug inactivation. Plasma concentrations, areas under the curve (AUC) and half-lives were calculated for 4-OH-IF and CA, allowing estimations of the extent of IF activation and deactivation/toxification. Induction of liver P450 2B enzymes by 4-day high-dose phenobarbital (PB) pretreatment significantly decreased the fraction of IF undergoing 4-hydroxylation (AUC(4-OH-IF)/AUC(4-OH-IF)+AUC(CA)), from 37% to 22% of total metabolism (P < 0.05), consistent with in vitro findings that the PB-inducible P450 enzyme 2B1 plays a major role in IF N-dechloroethylation. Pretreatment with the P450 3A inducer dexamethasone proportionally decreased the AUC for both IF metabolites, without any net impact on the fraction of IF undergoing metabolic activation. By contrast, the P450 2B1 inhibitor metyrapone preferentially increased the AUC for the 4-hydroxylation pathway in 3-day low-dose PB-induced rats, thereby increasing the total fraction of IF metabolized via the activation pathway from 36% to 54% (P < 0.05), whereas the P450 inhibitors orphenadrine and troleandomycin had no significant affect on AUC values. These findings demonstrate specific roles for P450 2B and 3A enzymes in catalysing these pathways of IF metabolism in vivo, and demonstrate the potential for modulation of IF's alternative metabolic pathways in a therapeutically useful manner. These studies also highlight several clinically relevant drug interactions that may occur during concomitant administration of IF with drugs and other compounds that modulate hepatic P450 enzyme levels.