Rat Liver Microsomal Proteins Research Articles

Pharmacokinetic, metabolic stability, plasma protein binding and CYP450s inhibition/induction assessment studies of N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide as potential type 5 phosphodiesterase inhibitors

ABSTRACTN-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide (NHPPC) is a new potential of type 5 phosphodiesterase (PDE5) inhibitors, synthesized from the avanafil analogue for the treatment of erectile dysfunction. The targets of this article were to assess plasma protein binding, liver microsomal metabolic stability, inhibition and induction on cytochrome P450 isozymes and the pharmacokinetics of NHPPC. Equilibrium dialysis technique was applied to determine Plasma protein binding (PPB) and NHPPC was evaluated in male Sprague–Dawley rats and Beagle dogs in vivo pharmacokinetic. The NHPPC was highly bound to plasma proteins in rats, dogs and human tested and the mean values for PPB rate were 96.2%, 99.6% and 99.4%, respectively. After in vitro liver microsomes incubated for 60 min, the percent remaining of NHPPC was 42.8%, 0.8% and 42.0% in rats, dogs and human, respectively. In vitro intrinsic clearance was found to be 0.0233, 0.1204 and 0.0214 mL/min/mg protein in rat, dog and human liver microsomes of NHPPC, respectively. NHPPC showed no significant inhibitory effects on major CYP450 enzymes, and had no significant induction potential on CYP1A2 and CYP3A4. Following oral administration in rats and dogs, tmax was 6 and 0.5 h, respectively. The clearance for NHPPC was 1.19 and 1.46 L/h/kg in rats and dogs, respectively. And absolute bioavailability in rat and dog were approximately 34.5% and 53.1%, respectively. These results showed that NHPPC has a good development prospect.

Time-dependent inhibition of carbamazepine metabolism by piperine in anti-epileptic treatment

AimsThe first-line anti-epileptic agent carbamazepine has narrow therapeutic index and can potentially interact with piperine, the major component from black pepper. The present study aimed to delineate the mechanism of such interaction for safe usage of carbamazepine during epilepsy control. Materials and methodsThe effect of piperine on carbamazepine hepatic metabolism was examined using rat or human liver microsomes. Mechanistic static model was applied to predict the extent of interaction. In addition, liver microsomal activities, mRNAs and protein expressions of genes regulating carbamazepine metabolism were evaluated after two weeks oral administrations of 3.5 and 35 mg/kg piperine in rats. Moreover, the effect of piperine on the xenobiotic receptor constitutive androstane receptor (CAR) was further accessed. Key findingsTime-dependent inhibitory effect of piperine on carbamazepine metabolism was observed, with kinact and KI of 0.0153 min−1 and 18.34 μM for rat, and 0.0093 min−1 and 9.45 μM for human. Based on such in-vitro metabolic parameters, further estimation using mechanistic static model indicated that piperine could increase the AUC of CBZ by 7% and 11% in rat and human, respectively. Significant inhibition on rat liver microsomal activity, Cyp3a2 mRNA and protein expression, CAR mRNA were demonstrated with piperine at 35 mg/kg. Yet, no direct effect on the activity of CAR for piperine was found. SignificanceWe have demonstrated the time-dependent inhibition by piperine on carbamazepine metabolism as the interaction mechanism. Prolonged use of piperine at high dose could increase carbamazepine concentrations through inhibiting metabolic enzyme activities and their related genes expressions.

Comparison of In Vitro Hepatic Scoparone 7-O-Demethylation between Humans and Experimental Animals.

Scoparone is a natural bioactive compound in Chinese herbal medicines. It has numerous pharmacological actions, including liver protective, hypolipidemic, antitumor, and anti-inflammatory effects. The primary metabolism route of scoparone is O-demethylation to scopoletin or isoscopoletin catalyzed by CYP enzymes. The aims of our study were to identify the human CYP enzymes catalyzing scoparone 7-O-demethylation to scopoletin and to compare this oxidation reaction in liver microsomes among different species. A high throughput fluorescent-based assay method was developed to determine the scoparone 7-O-demethylation to scopoletin rate. The rate was 100 - 400 nmol/(min×g protein) in mouse and rabbit liver microsomes, 10 - 20 nmol/(min×g protein) in pig microsomes, 1 - 3 nmol/(min×g protein) in human and less than 1 nmol/(min×g protein) in rat liver microsomes. Human CYP1A1 (Km13 µM and Vmax0.8 min-1), CYP1A2 (Km48 µM and Vmax0.3 min-1), and CYP2A13 (Km10 µM and Vmax22 min-1) were the most efficient catalysts of the reaction. The CYP2A6 selective inhibitor pilocarpine and an antibody against mouse CYP2A5 inhibited scoparone 7-O-demethylation to scopoletin in rabbit, mouse, and pig liver microsomes, indicating involvement of CYP2A enzymes in the reaction. Hepatic scoparone 7-O-demethylation to scopoletin differed between species both with respect to the rate of reaction and catalyzing enzymes. These species differences need to be taken into account when testing scoparone pharmacokinetics in animals and humans.

Assessment of in vitro metabolic stability, plasma protein binding, and pharmacokinetics of E- and Z-guggulsterone in rat.

Guggulsterone is a racemic mixture of two stereoisomers (E- and Z-), obtained from the gum resin of Commiphora mukul and it is marketed as an antihyperlipidemic drug. The aim of our study was to assess the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties namely solubility, in vitro metabolism, plasma protein binding and oral pharmacokinetic studies of E- and Z-guggulsterone. In vitro metabolism experiments were performed by using rat liver and intestinal microsomes. In vitro intrinsic clearance (CLint ) was found to be 33.34 ± 0.51 and 39.23 ± 8.12 μL/min/mg protein in rat liver microsomes for E- and Z-isomers, respectively. Plasma protein binding was determined by equilibrium dialysis method and in vivo pharmacokinetic studies were performed in male Sprague Dawley (SD) rats. Both isomers were highly bound to rat plasma proteins (>95% bound). Plasma concentration of E- and Z-isomers decreased rapidly following oral administration and were eliminated from systemic circulation with a terminal half-life of 0.63 ± 0.25 and 0.74 ± 0.35 h, respectively. The clearance (CL) for E-isomer was 2.79 ± 0.73 compared to 3.01 ± 0.61 L/h/kg for Z-isomer, indicating no significant difference (student t test; p <0.05) in their elimination.The pharmacokinetics of both isomers was characterized by extensive hepatic metabolism as seen with rat liver microsomes with high clearance and low systemic availability in rats. In brief, first-pass metabolism seems to be responsible factor for low bioavailability of guggulsterone. Copyright © 2015 John Wiley & Sons, Ltd.

Identification of Acetaminophen Adducts of Rat Liver Microsomal Proteins using 2D-LC-MS/MS.

Xenobiotic metabolism in the liver can give rise to reactive metabolites that covalently bind to proteins, and determining which proteins are targeted is important in drug discovery and molecular toxicology. However, there are difficulties in the analysis of these modified proteins in complex biological matrices due to their low abundance. In this study, an analytical approach was developed to systematically identify target proteins of acetaminophen (APAP) in rat liver microsomes (RLM) using two-dimensional chromatography and high-resolution tandem mass spectrometry. In vitro microsomal incubations, with and without APAP, were digested and subjected to strong cation exchange (SCX) fractionation prior to reverse-phase UHPLC-MS/MS. Four data processing strategies were combined into an efficient label-free workflow meant to eliminate potential false positives, using peptide spectral matching, statistical differential analysis, product ion screening, and a custom-built delta-mass filtering tool to pinpoint potential modified peptides. This study revealed four proteins, involved in important cellular processes, to be covalently modified by APAP. Data are available via ProteomeXchange with identifier PXD002590.

Effect of borneol on cytochrome P450 3A enzyme and midazolam pharmacokinetics in rats

Borneol is a commonly used herbal medication in China and Japan. Previous studies have indicated that borneol could reduce the plasma concentrations of oneself and concomitant drugs, and its first-pass metabolism could be catalyzed by the cytochrome P450 3A (CYP3A) enzyme as well. The impact of borneol on CYP3A activity and efficacy in influencing the pharmacokinetics of co-administrated drugs is currently unknown. Therefore, the purpose of the current study is to investigate the effect of borneol on CYP3A enzyme in vivo. After treatment with borneol twice daily for 3days, rat liver microsomes were exposed to probe substrates to determine CYP3A enzyme activity, protein, and RNA harvested using microsomal testosterone 6β-hydroxylation as a marker of enzyme activity. To verify the result, the effect of borneol on the pharmacokinetics of the CYP3A model substrate midazolam was further examined. The results showed that borneol treatment had increased CYP3A expression at the mRNA, protein, and activity (testosterone 6β hydroxylase activity) level in rat liver microsomes. In addition, borneol accelerated the metabolism of midazolam, which was consistent with the enhancement in CYP3A metabolic capacity. The hepatic clearance (Cl) of midazolam injected via the caudal vein in rats following borneol co-administration was higher; however, the area under the curve (AUC0-∞) was lower than the solvent. Hence, it was proposed that borneol could increase the metabolic activity of the CYP3A enzyme, which might cause drug-drug interactions in humans when using Chinese herbal or Western medicine with borneol.

HPLC-fluorescence Determination of EROD Activity in Wistar Rat Liver Microsomes Obtained by Two Different Extraction Procedures

This work determines the total protein concentration present in microsome fractions obtained by two distinct extraction procedures: ultracentrifugation and calcium aggregation. Additionally, the batch to batch variability within the extraction procedures used and the microsome stability and activity at -80°C during 90 days were determined using 7- ethoxyresorufin as a marker substract. An HPLC method to determine the activity of the cytochrome CYP450 1A1/2 was developed and fully validated by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity by in vitro microsomal mixed-function biotransformation. The biotransformation product, resorufin, was obtained by incubation of 500 μg of rat liver microsomal proteins. The incubation time was 5 min and the assay was monitored by using an HPLC coupled to a fluorescence detector (λexc 530 nm and λem 582 nm). The total analysis time was 20 min and the sample preparation was a fast and simple protein precipitation. The results show that both extraction procedures are useful tools for the extraction of subcellular fractions allowing the recovery of appropriate protein concentration for further in vitro metabolism studies by cytochrome P450, specifically CYP1A1 and CYP1A2. Keywords: EROD activity, Calcium aggregation, Ultracentrifugation, Rat liver microsomes, HPLC-fluorescence, CYP1A1, CYP1A2, protein, ethoxyresorufin, microsomal

Anthraquinones from the Roots of Knoxia valerianoides

To investigate the chemical constituents of the roots of Knoxia valerianoides and their biological activities. The anthraquinones were isolated by using a combination of various chromatographic techniques including column chromatography over silica gel, Sephadex LH-20, and reversed-phase HPLC. Structures of the isolates were identified by their physical-chemical properties and spectroscopic analysis including 2D NMR and MS. Antioxidant, anti-HIV, neuroprotective, and cytotoxic activities were screened by using cell-based models. Twenty-two constituents were isolated from an ethanolic extract of the roots of K. valerianoides. Their structures were identified as nordamnacanthal (1), ibericin (2), rubiadin (3), damnacanthol (4), 2-ethoxymethylknoxiavaledin (5), 3-hydroxymorindone (6), knoxiadin (7), 2-formyl knoxiavaledin (8), lucidin (9), xanthopurpurin (10), 1, 3-dihydroxy-2-methoxy-9, 10- anthraquinone (11), lucidin(-methyl ether (12), digiferruginol (13), 3-hydroxy-2-methyl-9,10-anthraquinone (14), rubiadin-1-methyl ether (15), 6-methoxylucidin (-ethyl ether (16), 1,3,6-trihydroxy-2-methyl-9,10-anthraquinone (17), 1,3-dihydroxy-2-hydroxy methyl-6-methoxy-9,10-anthraquinone (18), 1,3,6-trihydroxy-2-methoxymethyl-9,10- anthraquinone (19), 3,6-dihydroxy-2- hydroxymethyl-9,10-anthraquinone (20), and 1,6-dihydroxy-2-methyl-9,10-anthra quinone (21). In the in vitro assays, at a concentration of 1 x 10(-5) mol x L(-1), no compounds were active against human cancer cell lines (HCT-8, Bel7402, BGC-823, A549, and A2780), deserum and glutamate induced PC12-syn cell damage, LPS induced NO production in macrophage, Fe2+-cystine induced rat liver microsomal lipid peroxidation, HIV-1 replication, and protein tyrosine phosphatase 1B (PTP1B). Compounds 9-21 were obtained from the roots of K. valerianoides for the first time.

Quantitative shot-gun proteomics and MS-based activity assay for revealing gender differences in enzyme contents for rat liver microsome

Liver microsomes are subcellular fractions that contain many metabolizing enzymes for drugs and endogeneous compounds. Some of these enzymes are regulated by sex hormonal control and exhibit sex-dependent expression pattern and metabolizing speed. Studying these enzymes, however, are complicated by the presence of isoforms such as cytochrome P450 (CYP450), which families share more than 50% amino acid identities. In this study, we applied quantitative shot-gun proteomics approach coupled with stable-isotope dimethyl labeling, two-dimensional reversed-phase peptide separation and tandem mass spectrometry (MS) to explore the gender-dependent expression of rat liver microsomal proteins. A total of 391 proteins were identified and quantified by this approach, and 56% of quantified proteins were enzymes. Although shot-gun approach is rarely used for identifying protein isoforms, we identified 53 isoforms by at least one unique peptide including 21 isoforms of CYP450s. Moreover, by quantitative and statistics assessment, we were able to classify them into 28 male dominant enzymes including CYP2C12 CYP2C11, CYP2C13, CYP2B3, CYP2C11, CYP2C70 and CYP3A2 which are known to be male specific, 21 female dominant enzymes including CYP2A1, CYP2C7, CYP2C12, CYP2D26, alcohol dehydrogenase 1, carboxylesterase 3, glutathione S-transferase, liver carboxylesterase 4, UDP-glucuronosyltransferase 2B1, and glyceraldehyde-3-phosphate dehydrogenase which are known to be female specific; and 125 sex-independent enzymes. However, most of the sex specificities revealed from this study, such as the male specificity of CYP2D1, were novel and not yet reported. We then conducted a mass spectrometry-multiple reaction mode (MS-MRM) based enzyme activity method to determine the catalyzing rate of CYP2D1 in male and female liver microsomes using carteolol as its specific substrate. The reaction rate catalyzed by CYP2D1 in female rats was determined to differ significantly with the rate in male rats. Moreover, the ratio (female/male) of reaction rate (0.68) was found to correlate with their relative protein abundance (0.72). This study revealed novel sex dependences of many rat liver enzymes and also demonstrated a unique MS-based analytical platform that could identify novel iso-enzymes and further quantify their abundance and enzyme activity.

Highly sensitive quantification of key regulatory oxysterols in biological samples by LC-ESI-MS/MS

We describe a highly sensitive and specific method for the quantification of key regulatory oxysterols in biological samples. This method is based upon a stable isotope dilution technique by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After alkaline hydrolysis of human serum (5 microl) or rat liver microsomes (1 mg protein), oxysterols were extracted, derivatized into picolinyl esters, and analyzed by LC-MS/MS using the electrospray ionization mode. The detection limits of the picolinyl esters of 4beta-hydroxycholesterol, 7alpha-hydroxycholesterol, 22R-hydroxycholesterol, 24S-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, and 24S,25-epoxycholesterol were 2-10 fg (5-25 amol) on-column (signal-to-noise ratio = 3). Reproducibilities and recoveries of these oxysterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.8% to 12.7% and 2.9% to 11.9%, respectively. The recovery experiments were performed using rat liver microsomes spiked with 0.05 ng to 12 ng of oxysterols, and recoveries of the oxysterols ranged from 86.7% to 107.3%, with a mean recovery of 100.6%. This method provides reproducible and reliable results for the quantification of oxysterols in small amounts of biological samples.

Establishment of a PF2D‐MS/MS platform for rapid profiling and semiquantitative analysis of membrane protein biomarkers

Current proteome profiling techniques have identified relatively few mammalian membrane proteins despite their numerous important functions. To establish a standard throughput-potential profiling platform for membrane proteins, Triton X-100-solubilized rat liver microsomal proteins were separated on a 2-D separation system (2-D liquid phase fractionation (PF2D)) in two different pH ranges (4.0-8.5 and 7.0-10.5). This system produced 182 proteins with more than two transmembrane domain (TMD), including 16 TMDs with high confidence. Comparative 2-D liquid maps with high resolution and reproducibility have been constructed for liver microsome from the phenobarbital (PB) treated rats. PF2D was also found to be useful for the semiquantification of some representative cytochrome P450 family proteins (e.g., cytochrome P450 2B2) that were induced by PB treatment compared with untreated controls. Thus, the combination of both high-detection capacity and rapid preliminary semiquantification in a PF2D platform could become a standard system for the routine analysis of membrane proteins.

Precolumn derivatization of cysteine residues for quantitative analysis of five major cytochrome P450 isoenzymes by liquid chromatography/tandem mass spectrometry

The development of a novel method for absolute quantification of the five most clinically relevant CYP450 isoenzymes is described based on chemical derivatization of cysteine residues. The sulfhydryl-reactive reagents, 2-bromo-4'-chloroacetophenone (p-CPB) and 2-bromo-4'-bromoacetophenone (p-BPB), are proposed for use in quantitative proteomics. After reducing and denaturing, the P450s are derivatized with p-CPB for sulfhydryl alkylation then subjected to trypsin digestion. The resulting p-CPB-attached peptides are enriched using a phenyl resin solid-phase cartridge, then separated on a Zorbax 300SB reversed-phase column, and detected under positive electrospray ionization in the multiple reaction monitoring mode. Quantification is achieved using p-BPB-modified peptides as internal standards. Validation results demonstrated that this method showed good linearity between the concentration range of 10 fmol/microg to 5 pmol/microg for the six selected peptides in a complex matrix (rat liver microsomal protein). Intra-day and inter-day precision, expressed by relative standard deviation, were all less than 18%. Assay accuracy was within +/- 20% in terms of relative error. The quantitative derivatization approach proved to be reproducible, cost-effective and readily suitable for high-throughput assays. The reliability of this method for quantification of intact P450s was demonstrated through comparing with the well-applied isotope-coded affinity tag (ICAT) method.

Thin film voltammetry of metabolic enzymes in rat liver microsomes

We report herein thin film voltammetry and kinetics of electron transfer for redox proteins in rat liver microsomes for the first time. Films were made layer-by-layer from liver microsomes and polycations on pyrolytic graphite electrodes. Cyclic voltammograms were chemically reversible with a midpoint potential of −0.48 V vs SCE at 0.1 V s −1 in pH 7.0 phosphate buffer. Reduction peak potentials shifted negative at higher scan rates, and oxidation–reduction peak current ratios were ∼1 consistent with non-ideal quasireversible thin film voltammetry. Analysis of oxidation–reduction peak separations gave an average apparent surface electron transfer rate constant of 30 s −1. Absence of significant electrocatalytic reduction of O 2 or H 2O 2 and lack of shift in midpoint potential when CO is added that indicates lack of an iron heme cofactor suggest that peaks can be attributed to oxidoreductases present in the microsomes rather than cytochrome P450 enzymes.

Metabolic activation of indole-containing prostaglandin D2 receptor 1 antagonists: Impacts of glutathione trapping and glucuronide conjugation on covalent binding

Some DP1 receptor antagonists from an indole-containing series were shown to cause in vitro covalent binding to protein in rat and human liver microsomes. Glutathione trapping experiments along with in vitro labeling assays confirmed that the presence of a strong electron withdrawing group was necessary to abrogate in vitro covalent binding, leading to the discovery of MK-0524. Hepatocyte incubations and in vivo studies showed that acyl-glucuronide formation did not translate into covalent binding.

Use of a fluorescent substrate for the selective quantification of rat CYP3A in the liver and the intestine

Introduction Quantification of cytochrome P450 is a major issue in the development of new drugs. Different assays have been reported, but few are very selective for the 3A isoform or cytochrome P450. The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor 3-[(3, 4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-one has recently been used successfully to probe isoform 3A of cytochrome P450 in the liver. However, its selectivity for the rat isoform remains to be established as well as its applicability in other tissue, such as the intestine. The purpose of this study was to ascertain the specificity of this substrate for the rat 3A isoform of cytochrome P450 using Supersomes and its application in non-hepatic tissue (e.g., intestine). Methods: Specificity of the 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one for the isoform 3A of rat cytochrome P450 was established by using either isoform-specific inhibitory antibody or microsomes expressing only one cytochrome P450 isoform. Activity was assayed in rat liver and intestinal microsomal protein preparations. Results: Experiments with inhibitory antibodies revealed that in liver and intestinal microsomes, more than 90% of the substrate metabolism was inhibited by antibodies against isoform 3A. Selectivity of the substrate for rat 3A isoform was further determined by testing the metabolic activity of various Supersomes™ preparations. Discussion: In conclusion, our results validate the usefulness of 3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-methylsulfonyl)phenyl] furan-2(5H)-one as a simple and specific substrate to study the activity of the isoform 3A of cytochrome P450 in the rat liver and intestine.

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in Km (2 microM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation

Background/Aims : Although sublingual buprenorphine is safely used as a substitution drug in heroin addicts, large overdoses or intravenous misuse may cause hepatitis. Buprenorphine is N-dealkylated to norbuprenorphine by CYP3A. Methods : We investigated the mitochondrial effects and metabolic activation of buprenorphine in isolated rat liver mitochondria and microsomes, and its toxicity in isolated rat hepatocytes and treated mice. Results: Whereas norbuprenorphine had few mitochondrial effects, buprenorphine (25–200 μM) concentrated in mitochondria, collapsed the membrane potential, inhibited β-oxidation, and both uncoupled and inhibited respiration in rat liver mitochondria. Both buprenorphine and norbuprenorphine (200 μM) underwent CYP3A-mediated covalent binding to rat liver microsomal proteins and both caused moderate glutathione depletion and increased cell calcium in isolated rat hepatocytes, but only buprenorphine also depleted cell adenosine triphosphate (ATP) and caused necrotic cell death. Four hours after buprenorphine administration to mice (100 nmol/g body weight), hepatic glutathione was unchanged, while ATP was decreased and serum transaminase increased. This transaminase increase was attenuated by a CYP3A inducer and aggravated by a CYP3A inhibitor. Conclusions: Both buprenorphine and norbuprenorphine undergo metabolic activation, but only buprenorphine impairs mitochondrial respiration and ATP formation. The hepatotoxicity of high concentrations or doses of buprenorphine is mainly related to its mitochondrial effects.

Identification of a reactive metabolite of terbinafine: insights into terbinafine-induced hepatotoxicity.

Oral terbinafine treatment for superficial fungal infections of toe and fingernails is associated with a low incidence (1:45000) of hepatobiliary dysfunction. Due to the rare and unpredictable nature of this adverse drug reaction, the mechanism of toxicity has been hypothesized to be either an uncommon immunological or metabolically mediated effect. However, there is little evidence to support either mechanism, and toxic metabolites of terbinafine have not been identified. We incubated terbinafine with both rat and human liver microsomal protein in the presence of GSH and were able to trap an allylic aldehyde, 7,7-dimethylhept-2-ene-4-ynal (TBF-A), which corresponds to the N-dealkylation product of terbinafine. TBF-A was also prepared synthetically and reacted with excess GSH to yield conjugates with HPLC retention times and mass spectra identical to those generated in the microsomal incubations. The major GSH conjugate, characterized by (1)H NMR, corresponds to addition of GSH in a 1,6-Michael fashion. There remains a second electrophilic site on this metabolite, which can bind either to a second molecule of GSH or to cellular proteins via a 1,4-Michael addition mechanism. Moreover, we demonstrated that the formation of the GSH conjugates was reversible. We speculate that this allylic aldehyde metabolite, formed by liver enzymes and conjugated with GSH, would be transported across the canalicular membrane of hepatocytes and concentrated in the bile. The mono-GSH conjugate, which is still reactive, could bind to hepatobiliary proteins and lead to direct toxicity. Alternatively, it could modify canalicular proteins and lead to an immune-mediated reaction causing cholestatic dysfunction.

Electron paramagnetic resonance spectrometry evidence for bioreduction of tirapazamine to oxidising free radicals under anaerobic conditions

Tirapazamine (SR 4233) is a bioreductive antitumour drug in Phase III clinical trial which is activated in hypoxic tumour regions to generate a cytotoxic species. Electron paramagnetic resonance (EPR) spectrometry was used to investigate directly the formation of free radicals as the result of tirapazamine reduction by NADPH-supplemented liver microsomes. Under anaerobic conditions, the tirapazamine nitroxide free radical EPR signal was not evident over a range of rat or human liver microsomal protein (1–5 mg) concentrations. However, in combination with 1,1′,5,5′-dimethylpyrolline-1-N-oxide (DMPO), a spin trap for short-lived free radicals, tirapazamine resulted in formation of a 1:1:1:1:1:1 spectrum with hyperfine splitting A N = 15.8 G A H = 22.3 G consistent with generation of DMPO-R, a carbon-centered radical adduct. Addition of DMSO increased the signal intensity of the carbon-centred radical by at least twofold. The hyperfine splitting constants associated with DMPO-R could be indicative of a tirapazamine carbon-centred radical per se or, more likely, carbon radicals from endogenous materials (or DMSO) in the biological matrix as a result of oxidative attack by the tirapazamine primary radical. Formation of DMPO-OH, the hydroxyl radical spin adduct, by tirapazamine in the absence of air indicates that liberation of a hydroxyl radical may be a consequence of tirapazamine bioreduction under anaerobic conditions. The reactivity of tirapazamine free radicals with endogenous microsomal substances to generate reactive carbon-centred radicals indicates that tirapazamine may disrupt a wide range of cellular activities.

Further immunochemical and biocatalytic characterization of CYP1A1 from feral leaping mullet liver (Liza saliens) microsomes.

CYP1A is known to play important roles in the metabolism, detoxification and bioactivation of carcinogens and other xenobiotics in animals including fish. In our laboratory, CYP1A1 was obtained in a highly purified form with a specific content of 15-17 nmol P450 per mg protein from liver microsomes of feral fish, leaping mullet (Liza saliens). Purified mullet CYP1A1 showed a very high substrate specificities for 7-ethoxyresorufin and 7-methoxyresorufin in a reconstituted system containing purified fish P450 reductase and lipid. In addition, effects of each individual components of the reconstituted system, i.e., CYP1A1 and P450 reductase on 7-methoxyresorufin O-demethylase (MROD) activity were studied. 7-ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by alpha-naphthoflavone (ANF). At 0.5 and 2.5 microM. ANF inhibited EROD activity by 90 and 98%, respectively. Mullet CYP1A1 did not catalyze monooxygenations of other substrates such as aniline, ethylmorphine, N-nitrosodimethylamine and p-nitrophenol. Antibodies produced against CYP1A1 orthologues in fish such as trout and scup showed strong cross-reactivity with the purified mullet CYP1A1. In addition, anti-L. saliens liver CYP1A1 produced in our laboratory inhibited both the EROD and MROD activities catalyzed by L. saliens liver microsomes but stronger inhibition was observed with EROD activity. On the other hand, anti-mullet CYP1A1 antibodies showed very weak cross-reactivity with two proteins (presumably CYP1A1 and CYP1A2) in 3MC-treated rat liver microsomes. Moreover, 3MC-treated rat liver microsomal EROD activity was weakly inhibited by the anti-L. saliens liver CYP1A1. These results strongly suggested that the purified mullet CYP1A1 is structurally, functionally and immunochemically similar to the CYP1A1 homologues purified from other teleost species but functionally and immunochemically distinct from mammalian CYP1A1.