2,3,7,8 Tetrachlorodibenzo-p-dioxin-induced RNA abundance changes identify Ackr3, Col18a1, Cyb5a and Glud1 as candidate mediators of toxicity.

2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD) is an aromatic, long-lived environmental contaminant. While the pathogenesis of TCDD-induced toxicity is poorly understood, it has been shown that the aryl hydrocarbon receptor (AHR) is required. However, the specific transcriptomic changes that lead to toxic outcomes have not yet been identified. We previously identified a panel of 33 genes that respond to TCDD treatment in two TCDD-sensitive rodent species. To identify genes involved in the onset of hepatic toxicity, we explored 25 of these in-depth using liver from two rat strains: the TCDD-resistant Han/Wistar (H/W) and the TCDD-sensitive Long-Evans (L-E). Time course and dose-response analyses of mRNA abundance following TCDD insult indicate that eight genes are similarly regulated in livers of both strains of rat, suggesting that they are not central to the severe L-E-specific TCDD-induced toxicities. The remaining 17 genes exhibited various divergent mRNA abundances between L-E and H/W strains after TCDD treatment. Several genes displayed a biphasic response where the initial response to TCDD treatment was followed by a secondary response, usually of larger magnitude in L-E liver. This secondary response was most often an exaggeration of the original TCDD-induced response. Only cytochrome b5 type A (microsomal) (Cyb5a) had equivalent TCDD sensitivity to the prototypic AHR-responsive cytochrome P450, family 1, subfamily a, polypeptide 1 (Cyp1a1), while six genes were less sensitive. Four genes showed an early inter-strain difference that was sustained throughout most of the time course (atypical chemokine receptor 3 (Ackr3), collagen, type XVIII, alpha 1 (Col18a1), Cyb5a and glutamate dehydrogenase 1 (Glud1)), and of those genes examined in this study, are most likely to represent genes involved in the pathogenesis of TCDD-induced hepatotoxicity in L-E rats.

In vitro effects of myricetin, morin, apigenin, (+)-taxifolin, (+)-catechin, (-)-epicatechin, naringenin and naringin on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase.

The microsomal NADH-dependent electron transport system consisting of cytochrome b5 reductase and cytochrome b5 participates in a number of physiologically important processes including lipid metabolism as well as is involved in the metabolism of various drug and xenobiotics. In the present study, we assessed the inhibitory effects of eight dietary flavonoids representing five distinct chemical classes on cytochrome b5 reduction by purified cytochrome b5 reductase. From the flavonoids tested, myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC50 value of 0.35µM. Myricetin inhibited b5 reductase noncompetitively with a Ki of 0.21µM with respect to cofactor NADH, and exhibited a non-linear relationship indicating non-Michaelis-Menten kinetic binding with respect to cytochrome b5. In contrast to the potent inhibitory activity of myricetin, (+)-taxifolin was found to be a weak inhibitor (IC50=9.8µM). The remaining flavonoids were inactive within the concentration range tested (1-50µM). Analysis of structure-activity data suggested that simultaneous presence of three OH groups in ring B is a primary structural determinant for a potent enzyme inhibition. Our results suggest that inhibition of the activity of this system by myricetin or myricetin containing diets may influence the metabolism of therapeutic drugs as well as detoxification of xenobiotics.

Assessment of competitive and mechanism-based inhibition by clarithromycin: use of domperidone as a CYP3A probe-drug substrate and various enzymatic sources including a new cell-based assay with freshly isolated human hepatocytes.

Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 µM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9-19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug.

Characterization of CYP3A isozymes involved in the metabolism of domperidone: role of cytochrome b5 and inhibition by ketoconazole.

Previous studies have indicated that CYP3As are involved in the metabolism of the prokinetic agent domperidone.The objectives of our study were to characterize further the role of specific CYP3A isoforms in the metabolism of domperidone and to compare the kinetic parameters of domperidone to those of the CYP3A probe drug midazolam. Intrinsic clearance for the formation of domperidone major metabolite (5-hydroxydomperidone) was the highest with rCYP3A4 (0.4 mL/min/nmol CYP450) compared to rCYP3A5 (0.04 mL/min/nmol CYP450). The addition of cytochrome b5 to recombinant enzymes (rCYP3A4 or rCYP3A5) increased up to 6-fold the Vmax for the formation of 5-hydroxydomperidone. In contrast, much similar intrinsic clearance values for rCYP3A4 and rCYP3A5 were determined in the respective formation of either 1-hydroxmidazolam (1.28 and 1.57 mL/min/nmol CYP450) or 4-hydroxymidazolam(0.04 and 0.06 mL/min/nmol CYP450). Vmax for the formation of midazolam metabolites was increased to a lesser extent(1.5-3-fold) by the addition of cytochrome b5. Ketoconazole more potently inhibited CYP3A4 than CYP3A5 for both domperidone and midazolam. However, the addition of cytochrome b5 to the incubation mixture appeared to decrease the inhibitory potency of ketoconazole towards CYP3A4 for domperidone but not for midazolam. Our results indicate that CYP3A4 plays major role in the metabolism of domperidone. We demonstrated a modulatory role of cytochrome b5mostly for the metabolism of domperidone and confirmed selective inhibition of CYP3A4 over CYP3A5 by ketoconazole. Comparison of domperidone kinetic parameters to those of the CYP3A probe drug midazolam suggests that domperidone exhibits a much higher CYP3A4/CYP3A5 selectivity ratio than midazolam.

NADH cytochrome b5 reductase and cytochrome b5 catalyze the microsomal reduction of xenobiotic hydroxylamines and amidoximes in humans.

Hydroxylamine metabolites, implicated in dose-dependent and idiosyncratic toxicity from arylamine drugs, and amidoximes, used as pro-drugs, are metabolized by an as yet incompletely characterized NADH-dependent microsomal reductase system. We hypothesized that NADH cytochrome b5 reductase and cytochrome b5 were responsible for this enzymatic activity in humans. Purified human soluble NADH cytochrome b5 reductase and cytochrome b5, expressed in Escherichia coli, efficiently catalyzed the reduction of sulfamethoxazole hydroxylamine, dapsone hydroxylamine, and benzamidoxime, with apparent Km values similar to those found in human liver microsomes and specific activities (Vmax) 74 to 235 times higher than in microsomes. Minimal activity was seen with either protein alone, and microsomal protein did not enhance activity other than additively. All three reduction activities were significantly correlated with immunoreactivity for cytochrome b5 in individual human liver microsomes. In addition, polyclonal antibodies to both NADH cytochrome b5 reductase and cytochrome b5 significantly inhibited reduction activity for sulfamethoxazole hydroxylamine. Finally, fibroblasts from a patient with type II hereditary methemoglobinemia (deficient in NADH cytochrome b5 reductase) showed virtually no activity for hydroxylamine reduction, compared with normal fibroblasts. These results indicate a novel direct role for NADH cytochrome b5 reductase and cytochrome b5 in xenobiotic metabolism and suggest that pharmacogenetic variability in either of these proteins may effect drug reduction capacity.

Effects of chronic exposure to cadmium on renal cytochrome P450-dependent monooxygenase system in rats.

The aim of this study was to evaluate the effects of chronic exposure to cadmium (Cd) on the renal cytochrome P450-dependent monooxygenase system. For this purpose, male Wistar rats were intoxicated with Cd administered in drinking water at a concentration of 5 or 50 mg Cd/l for 6, 12 and 24 weeks. Concentrations of cytochrome P450 and cytochrome b(5) as well as activities of NADPH-cytochrome P450 reductase and NADH-cytochrome b(5) reductase were determined in the kidney microsomal fraction. Protein content of CYP1A1, CYP2E1 and CYP3A1 cytochrome P450 isoforms was evaluated as well. In the rats exposed to 5 mg Cd/l, the concentration of cytochrome P450 decreased (by 41%) after 24 weeks of the experiment. The activity of NADPH-cytochrome P450 reductase decreased (by 24%) after 6 and 12 weeks, whereas after 24 weeks it remained unchanged, compared with the control group. Moreover, a decrease in the concentration of cytochrome b(5) (by 25, 15 and 26% at 6, 12 and 24 weeks, respectively) and the activity of its NADH reductase (by 26 and 31% at 6 and 24 weeks, respectively) was noted in these animals. At the exposure to 50 mg Cd/l, the concentrations of cytochrome P450 and cytochrome b(5) and the activities of their corresponding reductases were decreased at each time-point. Western blot analysis revealed that all isoforms of cytochrome P450 studied were affected by Cd and the effect was dependent on the level and the duration of exposure. The results of this study indicate that chronic exposure to Cd in a dose- and time-dependent manner affects the kidney cytochrome P450-dependent monooxygenase system by decreasing the concentrations of cytochrome P450 and cytochrome b(5) and inhibiting the activities of their corresponding reductases. The effect of Cd on the cytochrome P450 content is associated with its ability to stimulate or inhibit of various P450 isoforms. A very important finding of this study is that Cd affects the kidney cytochrome P450-dependent monooxygenase system at relatively low exposure and low kidney Cd accumulation (2.40+/-0.15 microg/g). As the experimental model used reflects human exposure to Cd, we conclude that Cd can affect the kidney cytochrome P450-dependent monooxygenase system in environmentally exposed humans. Previously we have reported disorders in the system in the liver of rats at the same levels of exposure as in this study. Thus, we hypothesize that the metabolism and detoxification of many substances, including xenobiotics, may be seriously affected in Cd-exposed subjects.

Propofol inhibits renal cytochrome P450 activity and enflurane defluorination in vitro in hamsters.

PURPOSE: To determine the effect of propofol on renal cytochrome P450 activity and defluorination of enflurane. METHODS: Renal microsomes were prepared by homogenization and differential centrifugation from pooled hamster kidneys. Defluorination of enflurane was assessed by measuring free fluoride metabolites after reacting enflurane with renal microsomes incubated with various concentrations, 0.05 - 1.0 mmol x L(-1) propofol in the NADPH-generating system. Drug metabolizing activities of renal cytochrome P450 mono-oxygenase enzymes were evaluated within microsomes preincubated with propofol and reacted with the specific marker substrates, aniline, benzo(a)pyrene, erythromycin and pentoxyresorufin, for cytochrome P450 2E1, 1A1, 3A4 and 2B1, respectively. RESULTS: Renal defluorination of enflurane was inhibited by clinical concentrations, 0.05 mmol x L(-1) of propofol (P < 0.05). Dose-dependent inhibition of defluorination, aniline and benzo(a)pyrene hydroxylase within kidney microsomes was related to propofol concentration. Propofol demonstrated a profound inhibition of renal pentoxyresorufin dealkylase activity even at low concentrations, 0.05 mmol x L(-1) (P < 0.01). Propofol did not exhibit inhibition of erythromycin N-demethylation of kidney microsomes except at high concentration, 1.0 mmol x L(-1). Spectral analyses of key coenzymes of renal cytochrome P450 monooxygenase, cytochrome b5 and cytochrome c reductase, demonstrated an inhibition when incubated with high concentrations of propofol (P < 0.05). CONCLUSION: In an in vitro study in an NADPH-generating system of hamster kidney microsomes, propofol, in clinical concentrations, exhibited a broad-spectrum of inhibition to renal monooxygenase activities and enflurane defluorination.

In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver.

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.

Zinc ethylene-bis-dithiocarbamate (Zineb)-mediated inhibition of monooxygenases and lipid peroxidation in bovine liver microsomes.

Hepatic bovine microsomes were incubated with Zineb concentrations ranging from 2.5 mM to 2.5 microM. Only the higher concentrations of the fungicide (2.5 and 0.25 mM) elicited a sharp decline in cytochrome P450, cytochrome b5 and total sulphydryl groups content as well as in the activities of NADPH cytochrome c reductase, aminopyrine N-demethylase and aniline 4-hydroxylase. The loss of cytochrome P450 was matched by a concomitant increase in the amount of cytochrome P420, which represents a catalytically inactive form of cytochrome P450. The same concentrations of the fungicide, either alone or in the presence of NADPH 1 mM, failed to increase the amount of thiobarbituric reactive substances with respect to control incubations, thereby excluding the possibility of lipid peroxidation as a contributing factor in the loss of cytochrome P450 and in the inhibition of cytochrome P450-mediated metabolism. It is concluded that Zineb can depress monooxygenase activity in bovine hepatic microsomes mainly through the denaturation of cytochrome P450 and the impaired transfer of reducing equivalents to the complex cytochrome P450-substrate. These mechanisms might also account for the inhibition in lipid peroxidation brought about by the fungicide.

A protein inhibitor of hepatic drug metabolizing enzymes from Ehrlich ascites cells.

Hepatic drug metabolizing enzymes were significantly decreased in Ehrlich ascites tumour-bearing mice. A protein inhibitor of hepatic drug metabolizing enzymes was isolated from Ehrlich ascites cells and purified. This involved ammonium sulphate fractionation (60-80%), DEAE, phosphocellulose, Sephadex G-100 and hydroxyapatite column chromatography. Purification attained was 800-fold. The inhibitory protein was effective in decreasing all the components of hepatic mixed-function-oxidase system and drug metabolizing enzymes both in vivo and in vitro. This novel inhibitor may have potential applications in chemical carcinogenesis.

Requirements for cytochrome b5 in the oxidation of 7-ethoxycoumarin, chlorzoxazone, aniline, and N-nitrosodimethylamine by recombinant cytochrome P450 2E1 and by human liver microsomes.

NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.

Involvement of cytochrome b5 in the metabolism of tetrachlorobiphenyls catalyzed by CYP2B1 and CYP1A1.

The role of cytochrome b5 in the cytochrome P450 (CYP)-dependent hydroxylation of tetrachlorobiphenyl (TCB) isomers was examined using a reconstituted mixed function oxygenase (MFO) system containing purified CYP2B1 or 1A1, and rat liver microsomes. Hydroxylations of 2,2',5,5'- and 3,3',4,4'-TCBs were catalyzed mainly by CYP2B1 and 1A1, respectively, in the reconstituted MFO system and those of 2,3',4',5- and 2,3',4,4'-TCBs were mediated by both cytochrome P450 systems. The activity toward 2,2',5,5'- and 2,3',4',5-TCB was significantly increased 6.5- and 5.5-fold, respectively, by addition of cytochrome b5 in the reconstituted MFO system containing of CYP2B1. Either hydroxylation activity toward 2,3',4,4'-TCB with the CYP2B1 system was very low or decreased by addition of cytochrome b5. These results suggest that the involvement of cytochrome b5 to the hydroxylation of TCBs is dependent on the TCB congener being metabolized, and the cytochrome P450 isoform involved in its metabolism.

[Correction of disorders in the monooxygenase system function with diethylnicotinamide (cordiamine) in tetrachloromethane- induced and viral hepatitis]. / Korrektsiia diétilnikotinamidom (kordiaminom) narusheniia funktsii monooksigenaznoi sistemy pri tetrakhlormetanovom i virusnom gepatitakh.

Content of cytochromes P-450 and b5 and the rate of oxidative dealkylation in liver microsomes as well as the antipyrine pharmacokinetics were normalized in rats with acute CCl4-induced hepatitis after treatment with cordiamine (diethyl nicotinamide) at a dose of 40 mg, subcutaneously, 2 times daily within 4 days. Cordiamine (30 drops 3 times daily within 8 days) contributed to normalization of the hydroxylating reaction in liver tissue of patients with viral hepatitis A, estimated by the "antipyrine" test. The drug exhibited stabilizing effect on hydrophobic interactions in microsomal membranes; diethyl nicotinamide possessed antiradical and vitamin properties.

Interaction of aflatoxin B1 with the rat monooxygenase system and relationships to its acute toxic effect.

The effect of aflatoxin B1 (AFB1) on the liver microsomal and nuclear mixed function oxidase system (MFO) of adult male rats was studied at oral doses of 1 and 3 mg/kg. At first both doses increased the activities and concentrations of all P450 components followed. The maximum values were observed between 24 and 48 h and were dose- and enzyme-dependent. After 72 h the values dropped to 15-55% of the initial values. Inhibition of the MFO system lasted even after 120 h, when a trend to return to the normal values was already noticeable. We assume that AFB1 acts as an inductor of the monooxygenase system and the reactive electrophilic intermediate(s) bind irreversibly to or autocatalytically inactivate P450.

[The state of the microsomal oxidative system in the rat liver during fecal peritonitis]. / Sostoianie mikrosomal'noi okislitel'noi sistemy pecheni krys pri ostrom kalovom peritonite.

Content and activity of cytochrome P-450, activity of main microsomal enzymes as well as content of glycogen, lactic and pyruvic acids were studied in liver tissue of rats within 24, 48 and 72 hrs after simulation of acute fecal peritonitis. Inhibition of the enzymatic activity of microsomal oxidative system correlated with hypoxia which developed in liver tissue within all the periods of acute fecal peritonitis studied. At the same time, catalytic activity of cytochrome P-450 was increased towards substrates of the I and II types. Endotoxemia and hypoxia of liver tissue, resulting in activation of phospholipases, appear to be mainly responsible for inhibition of microsomal enzymes activity in liver tissue under conditions of acute peritonitis.

Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: a proposed molecular model for a cytochrome P-450cam electron-transfer complex.

Cytochrome b5 has been genetically engineered to afford a fluorescent derivative capable of monitoring its association with cytochrome P-450cam from Pseudomonas putida [Stayton, P. S., Fisher, M. T., & Sligar, S. G. (1988) J. Biol. Chem. 263, 13544-13548]. In the mutant cytochrome b5, threonine is replaced by a cysteine at position 65 (T65C) and has been labeled with the environmentally sensitive fluorophore acrylodan. In this paper, the physiological P-450cam reductant putidaredoxin, an Fe2S2.Cys4 iron-sulfur protein, is shown to competitively inhibit the cytochrome b5 association, suggesting that cytochrome b5 and putidaredoxin bind to a similar site on the cytochrome P-450cam surface. Since the crystal structures for both cytochrome b5 and cytochrome P-450cam have been solved to high resolution, the complex has been computer modeled, and a good fit was found on the proximal surface of nearest approach to the P-450cam heme prosthetic group. The proposed model includes electrostatic contacts between conserved cytochrome b5 carboxylates Glu-44, Glu-48, Asp-60, and the exposed heme propionate with cytochrome P-450cam basic residues Lys-344, Arg-72, Arg-112, and Arg-364, respectively. Putidaredoxin has similarly been shown to contain a carboxylate-based binding surface, and the current results suggest that if the model is correct, then it also interacts at the proposed site, probably utilizing similar P-450cam electrostatic contacts.