Microsomal Monooxygenase System Research Articles

Comparative Analysis of Bioelectrocatalytic Cytochrome P450 3A4 Systems

This article describes the approaches developed by the authors with the aim to increase the efficiency of electro enzymatic reactions catalyzed by cytochrome P450 3A4. A comparative analysis of cytochrome P450 3A4 systems was carried out during the formation of the functional complexes hemoprotein-flavin nucleotides as low-molecular models of NAD(P)H-dependent cytochrome P450 reductase. The formation of a productive enzyme-substrate complex before the stage ofaccepting electrons from the modified electrode was studied from the electocatalytic viewpoint. Incorporation of the enzyme into nanopores of different nature on the electrode (2D-3D transition) was also studied. The results on the electrochemical reduction of bactosomes as the functionally active models of the microsomal monooxygenase system are also considered. The electrochemical and electrocatalytic parameters of cytochrome P450 3A4 were compared for different models of the electrocatalytic generation of metabolites.

Electron transfer between cytochrome c and microsomal monooxygenase generates reactive oxygen species that accelerates apoptosis

Generation of reactive oxygen species (ROS) are possibly induced by the crosstalk between mitochondria and endoplasmic reticula, which is physiologically important in apoptosis. Cytochrome c (Cyt c) is believed to play a crucial role in such signaling pathway by interrupting the coupling within microsomal monooxygenase (MMO). In this study, the correlation of ROS production with the electron transfer between Cyt c and the MMO system is investigated by resonance Raman (RR) spectroscopy. Binding of Cyt c to MMO is found to induce the production of ROS, which is quantitatively determined by the in-situ RR spectroscopy reflecting the interactions of Cyt c with generated ROS. The amount of ROS that is produced from isolated endoplasmic reticulum depends on the redox state of the Cyt c, indicating the important role of oxidized Cyt c in accelerating apoptosis. The role of electron transfer from MMO to Cyt c in the apoptotic mitochondria-endoplasmic reticulum pathway is accordingly proposed. This study is of significance for a deeper understanding of how Cyt c regulates apoptotic pathways through the endoplasmic reticulum, and thus may provide a rational basis for the design of antitumor drugs for cancer therapy.

Influence of cranioskeltal trauma, complicated with blood loss, on the bile-forming function of liver during the early period of traumatic disease among rats with different resistance to hypoxia and its correction

Introduction. The development of multiorgan dysfunction and insufficiency is one of the serious complications of severe multiple and combined lesions. In their pathogenesis, a key role is played by secondary lesions of organs remote from the site of direct injury, which is caused by the systemic response of the body to inflammation and is a characteristic feature of traumatic disease. As a model of the development of organ damage in experimental polytrauma, the biliary function of liver is often investigated. However, the features of systemic disorders, in particular, the biliary function of liver, in conditions of severe skeletal injury, depending on the resistance to hypoxia, have not been studied enough.Purpose: to determine the dynamics of biliary function of liver in case of cranioskeletal trauma complicated with blood loss among rats with different resistance to hypoxia during the early period of traumatic disease and to evaluate the effectiveness of thiocetam in the correction of identified disorders.Materials and methods. 108 nonlinear white male rats weighing 180-200 g were used in the experiments. Previously, individual resistance to hypoxia was determined, rats were divided into two groups: with high- and low-resistance (HR and LR) animals to hypoxia. Subsequently, HR and LR rats were divided into 4 groups: one control and three experimental. Under conditions of thiopentale sodium anesthesia (40 mg kg-1) the first experimental group HR and LR of rats was had cranioskeletal trauma, the second - acute blood loss in the amount of 20-22% of circulating blood volume, the third experimental group after application of cranioskeletal injuries and acute blood loss in order to correct HR and LR-rats were thrown Thiocetam intraperitoneally, dose 250 mg kg-1 of body weight 1 time per day. Animals of the control and experimental groups in 1, 3 and 7 days after injury were catheterized the common bile duct, collected bile, in which the content of total bile acids was determined.It was found out that laboratory white rats, which are genetically different in resistance to hypoxia, also differ in the intensity of biliary function of liver. Among HR rats, the bile content of total bile acids was higher than that among LR rats. Under the influence of cranioskeletal injury, the bile content of total bile acids decreased among both HR and LR rats. Despite the lower initial content of total bile acids in the bile of LR rats, under the influence of cranioskeletal trauma in this group, the degree of the studied indicator reduction was significantly greater than among HR rats, indicating greater sensitivity of LR rats to pathogens of traumatic disease, which lead to damage to the microsomal monooxygenase system of hepatocytes. Under the conditions of acute blood loss additional modeling, the disorders were more visible, but the result was statistically significant only among LR rats in 3 days of the experiment. In addition, LR rats had a greater degree of reduction of the studied indicator in 3 and 7 days of the experiment. Under the influence of seven-day-usage of Thiocetam in the groups of LR and HR rats with cranioskeletal trauma, the violation of total bile acids content in the bile became significantly lower compared to animals without correction. A similar result was found under conditions of additional acute blood loss. Under these conditions, the degree of increase of total bile acids content in the bile was greater among LR rats than among HR rats, which indicates a higher efficiency of Thiocetam among LR rats.Conclusions: 1. The content of total bile acids in the bile among intact HR-rats is significantly higher than among LR-rats. Under the influence of cranioskeletal trauma, the content of total bile acids in the bile is reduced compared to the control, but the degree of reduction is significantly greater among LR rats after 7 days of the experiment. Complication of cranioskeletal injury with acute blood loss causes a significantly greater reduction in the bile content of total bile acids among LR rats compared with HR rats after 3 and 7 days of the experiment. The use of Thiocetam is accompanied by a decrease of liver biliary dysfunction, which is more visible among LR rats with acute blood loss.

Biosensor selection of small compounds, modulating a complex formation between cytochrome P450s and NADPH-dependent P450 oxidoreductase

The study of the effect of low-molecular-weight compounds (substrates, endogenous metabolites, drugs and xenobiotics) on the kinetic and equilibrium parameters of functionally significant binary protein-protein interactions (PPIs) is of both fundamental and clinical importance. The surface plasmon resonance (SPR) is the method of the first choice for studying PPIs. Earlier, SPR analysis revealed the modulating effect of steroidal substrates on the affinity of interactions between steroidogenic microsomal cytochromes P450 (CYP) and their redox partner (cytochrome b5). In this work, we have shown the suitability of the experimental approach for assessing the selective effect of the cofactor NADPH on the interaction between cytochromes CYP3A4 or CYP2E1 with NADPH-dependent P450 oxidoreductase (CPR). Experiments have shown that the CYP3A4/CPR complex is not modulated by NADPH, while the dissociation rate of the CYP2E1/CPR complex in the presence of NADPH significantly decreased: the koff values in the absence and presence of NADPH were (3.6 ± 0.2) • 10-3 s-1 and (3.8 ± 0.2) • 10-4 s-1, respectively. Thus, in the presence of NADPH, an increase in the affinity of CYP2E1/CPR complex formation by approximately one order of magnitude was observed, while NADPH did not affect the kon value of this complex. Co-injection of NADPH at the CYP2E1/CPR complex preformed in the absence of NADPH had minor influence on the koff values (<10%). This suggests a stabilizing role of NADPH for the CYP2E1/CPR complex formation. Thus, the use of our approach made it possible to assess the effect of the main electron supplier for the microsomal cytochrome P450 monooxygenase system on the kinetic rate constants of CYP/CPR complexes.

Viral-mediated gene delivery of TMBIM6 protects the neonatal brain via disruption of NPR-CYP complex coupled with upregulation of Nrf-2 post-HI

BackgroundOxidative stress, inflammation, and endoplasmic reticulum (ER) stress play a major role in the pathogenesis of neonatal hypoxic-ischemic (HI) injury. ER stress results in the accumulation of unfolded proteins that trigger the NADPH-P450 reductase (NPR) and the microsomal monooxygenase system which is composed of cytochrome P450 members (CYP) generating reactive oxygen species (ROS) as well as the release of inflammatory cytokines.We explored the role of Bax Inhibitor-1 (BI-1) protein, encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, in protection from ER stress after HI brain injury. BI-1 may attenuate ER stress-induced ROS production and release of inflammatory mediators via (1) disruption of the NPR-CYP complex and (2) upregulation of Nrf-2, a redox-sensitive transcription factor, thus promoting an increase in anti-oxidant enzymes to inhibit ROS production. The main objective of our study is to evaluate BI-1’s inhibitory effects on ROS production and inflammation by overexpressing BI-1 in 10-day-old rat pups.MethodsTen-day-old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5 h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48 h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). BI-1 and Nrf-2 silencing were achieved by icv injection at 48 h pre-HI using siRNA to elucidate the potential mechanism. Percent infarcted area, immunofluorescent staining, DHE staining, western blot, and long-term neurobehavior assessments were performed.ResultsOverexpression of BI-1 significantly reduced the percent infarcted area and improved long-term neurobehavioral outcomes. BI-1’s mediated protection was observed to be via inhibition of P4502E1, a major contributor to ROS generation and upregulation of pNrf-2 and HO-1, which correlated with a decrease in ROS and inflammatory markers. This effect was reversed when BI-1 or Nrf-2 were inhibited.ConclusionsOverexpression of BI-1 increased the production of antioxidant enzymes and attenuated inflammation by destabilizing the complex responsible for ROS production. BI-1’s multimodal role in inhibiting P4502E1, together with upregulating Nrf-2, makes it a promising therapeutic target.

A large-scale comparative analysis of affinity, thermodynamics and functional characteristics of interactions of twelve cytochrome P450 isoforms and their redox partners

The aim of the present work was to establish the thermodynamic and functional differences in the protein-protein interactions between the components of the P450-dependent mitochondrial (mit) and microsomal (mic) monooxygenase systems using 12 different isoforms of cytochromes P450 and two redox partners, NADPH-dependent cytochrome P450 reductase (CPR) and adrenodoxin (Adx). Comparative analysis of the affinity, thermodynamics, enzymatic activity and the ability for one-electron reduction has been carried out. The study of protein-protein interactions to determine the equilibrium dissociation constants (Kd) was performed using surface plasmon resonance (SPR) biosensor Biacore 3000. We demonstrated that CPR and Adx interacted with both, micCYPs and mitCYPs, with different affinities (Kd values ranged from 0.01 to 2 μM). All complexes of microsomal (micCYP) and mitochondrial (mitCYP) cytochrome P450 with redox partners can be divided into three groups depending on the prevalent role of either enthalpy or entropy contribution. About 90% of CYP/redox partner complexes were entropy-driven, while the contribution of enthalpy and entropy differed significantly in case of mitCYP/Adx complexes. The CYP11A1/Adx complex was enthalpy-driven, while CYP11B1/Adx and CYP11B2/Adx complexes were entropy-driven. Thermodynamic discrimination of mitCYPs/Adx complexes is likely associated with the different functional impact of CYP11A1 and CYP11B. The exception was the enthalpy-entropy-driven (mixed type) CYP21A2/Adx complex. CPR and Adx were able to transfer the first electron to micCYPs while mitCYPs demonstrated high specificity to Adx. Productive catalysis for mitCYPs observed only in the presence of Adx/AdR pair, while in case of steroidogenic micCYPs (CYP17A1, CYP19A1, and CYP21A2) it was found either in the presence of a CPR or an Adx/AdR pair. From the evolutionary point of view, the type 1 electron transport system (mitCYPs, Adx and NADPH-dependent adrenodoxin reductase (AdR)) increased the specialization of protein-protein interactions (PPI) significantly, which was accompanied by an increase in the specificity of electron transfer. In contrast, the evolution of the type 2 electron transport system (micCYPs and CPR) led to an increase in versatility of PPI as demonstrated for steroidogenic microsomal cytochrome P450s. Our data enhance the current understanding of molecular recognition and summarize qualitative and thermodynamic characteristics of protein-protein interactions in the P450-dependent mitochondrial and microsomal monooxygenase systems.

Structural and Functional Studies of the Membrane-Binding Domain of NADPH-Cytochrome P450 Oxidoreductase.

NADPH-cytochrome P450 oxidoreductase (CYPOR), the essential flavoprotein of the microsomal cytochrome P450 monooxygenase system, is anchored in the phospholipid bilayer by its amino-terminal membrane-binding domain (MBD), which is necessary for efficient electron transfer to cytochrome P450. Although crystallographic and kinetic studies have established the structure of the soluble catalytic domain and the role of conformational motions in the control of electron transfer, the role of the MBD is largely unknown. We examined the role of the MBD in P450 catalysis through studies of amino-terminal deletion mutants and site-directed spin labeling. We show that the MBD spans the membrane and present a model for the orientation of CYPOR on the membrane capable of forming a complex with cytochrome P450. EPR power saturation measurements of CYPOR mutants in liposomes containing a lipid/Ni(II) chelate identified a region of the soluble domain interacting with the membrane. The deletion of more than 29 residues from the N-terminus of CYPOR decreases cytochrome P450 activity concomitant with alterations in electrophoretic mobility and an increased resistance to protease digestion. The altered kinetic properties of these mutants are consistent with electron transfer through random collisions rather than via formation of a stable CYPOR-P450 complex. Purified MBD binds weakly to cytochrome P450, suggesting that other interactions are also required for CYPOR-P450 complex formation. We propose that the MBD and flexible tether region of CYPOR, residues 51-63, play an important role in facilitating the movement of the soluble domain relative to the membrane and in promoting multiple orientations that permit specific interactions of CYPOR with its varied partners.

THE DEVELOPMENT OF MEMBRANOUS PATHOLOGY OF HEPATOCYTES THE INFLUENCE OF INTOXICATION

The study was performed at the Department of Clinical Pathophysiology, Topographical Anatomy and Operative Surgery at Kharkiv Medical Academy of Postgraduate Education as a part of research project "Radiotoxins' pathochemical mechanisms and methods of early diagnostics and correction", state registration No. 0117U000589.
 We studied the subtoxic effect of small doses of sodium fluoride on the activity of microsomal hepatocytes on 30 Wistar rats' populations in subacute experiment. The intensity of lipid peroxidation (LPO) in liver of rats which were administered sodium fluoride orally for a long time at doses of 1/10 and 1/100 LD50, was evaluated by the content of its molecular products - diene conjugates (DC), and MDA-reagents Schiff bases. We found that oral administration of sodium fluoride to rats at doses of 1/10 and 1/100 LD50 promotes a statistically significant increase (r≤0,002) relative to the control group of animals in DC content during the entire period of observation. At a dose 1/10 LD50 we observed the most significant increase in this indicator on the 10th day of the experiment - at 265%, and at a dose 1/100 LD50 – on the 20th day an average of 234%. In rats’ liver by the action of subtoxical dose of sodium fluoride at a dose of 1/10 LD50, starting from the 20th day, we detected a gradual increase (r≤0.001) of TBA-reagents relative to control - for 27, 41, 78, 133%. Secondary end products and lipid peroxidation, which are defined under the long-term of sodium fluoride, somehow contribute to the disruption of the microstructure of hepatocytes membranes, their permeability, reduce their division, regeneration and inhibition of mitochondrial respiratory chain enzymes and microsomal monooxygenase system.

Thermodynamics of interactions between mammalian cytochromes P450 and b5

Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent.

Liver and Its Lymph Region at Benzo[a]pyrene Effects in an Experiment

Benzo[a]pyrene (BaP) is the widespread environmental toxicant with carcinogenic activity. BaP undergoes metabolic activation in the liver microsomal monooxygenase system, and its regional lymph nodes act as peripheral filters, purifying lymph formed in the liver. It remains an open question about the significance of the integral liver and lymphatic system work in supporting processes of adaptation and resistance to the BaP effects. The purpose of our investigation was to study structural and metabolic changes in the liver and its lymph region in males Wistar rats weighing 180-220g. Animals of the experimental group (n =20) daily for 3 days was performed BaP injections: intraperitoneal with 2 mg per 100g of body weight in 0.2-0.3 ml of olive oil. Rats in the control group (n =20) received in the same mode of injection of olive oil. The light and electron microscopy morphometric study of the liver and its regional lymph nodes morphometric analysis were performed. The intensity of lipid peroxidation in the liver was measured by the number of diene conjugates (DC), ketotriene conjugates (KC) and malondialdehyde (MDA). Simultaneous increase of the hepatic sinusoids relative area and the specific area of the regional lymph nodes sinus system under the BaP effect was found. At ultrastructural level dilatation of the Disse spaces, filling of cell detritus and collagen fibers bundles of these spaces were noted. Damage of nuclear apparatus, balloon transformation of granular endoplasmic reticulum profiles were found in hepatocytes, condensation of the mitochondrial matrix was observed. The relative squares of B-dependent zones increased and T-dependentparacortical zone reduced in the regional lymph nodes. The increase in the content of KC and MDA in liver was identified. benzo[a]pyrene causes interrelated cascade of reactions in the liver and in its lymphatic region: a disturbance of the blood-lymph barrier morphological organization, and so obstruction of the lymphatic drainagefrom the organ. These promotes development of tissue hypoxia and apoptosis, protein synthesizing and energy cell apparatus disruptions, formation of lymphoid tissue temporary infiltrates in the liver.

CYP-13A12 of the nematode Caenorhabditis elegans is a PUFA-epoxygenase involved in behavioural response to reoxygenation.

A specific behavioural response of Caenorhabditis elegans, the rapid increase of locomotion in response to anoxia/reoxygenation called the O2-ON response, has been used to model key aspects of ischaemia/reperfusion injury. A genetic suppressor screen demonstrated a direct causal role of CYP (cytochrome P450)-13A12 in this response and suggested that CYP-eicosanoids, which in mammals influence the contractility of cardiomyocytes and vascular smooth muscle cells, might function in C. elegans as specific regulators of the body muscle cell activity. In the present study we show that co-expression of CYP-13A12 with the NADPH-CYP-reductase EMB-8 in insect cells resulted in the reconstitution of an active microsomal mono-oxygenase system that metabolized EPA (eicosapentaenoic acid) and also AA (arachidonic acid) to specific sets of regioisomeric epoxy and hydroxy derivatives. The main products included 17,18-EEQ (17,18-epoxyeicosatetraenoic acid) from EPA and 14,15-EET (14,15-epoxyeicosatrienoic acid) from AA. Locomotion assays showed that the defective O2-ON response of C20-PUFA (polyunsaturated fatty acid)-deficient, Δ-12 and Δ-6 fatty acid desaturase mutants (fat-2 and fat-3 respectively) can be restored by feeding the nematodes AA or EPA, but not ETYA (eicosatetraynoic acid), a non-metabolizable AA analogue. Short-term incubation with 17,18-EEQ was sufficient to rescue the impaired locomotion of the fat-3 strain. The endogenous level of free 17,18-EEQ declined during anoxia and was rapidly restored in response to reoxygenation. On the basis of these results, we suggest that CYP-dependent eicosanoids such as 17,18-EEQ function as signalling molecules in the regulation of the O2-ON response in C. elegans. Remarkably, the exogenously administered 17,18-EEQ increased the locomotion activity under normoxic conditions and was effective not only with C20-PUFA-deficient mutants, but to a lesser extent also with wild-type worms.

Oxidative stress in entomopathogenic fungi grown on insect-like hydrocarbons.

Entomopathogenic fungi mostly attack their insect hosts by penetration through the cuticle. The outermost insect surface is covered by a lipid-rich layer, usually composed of very long chain hydrocarbons. These fungi are apt to grow on straight chain hydrocarbons (alkanes) as the sole carbon source. Insect-like hydrocarbons are first hydroxylated by a microsomal P450 monooxygenase system, and then fully catabolized by peroxisomal β-oxidation reactions in Beauveria bassiana. In this review, we will discuss lipid metabolism adaptations in alkane-grown fungi, and how an oxidative stress scenario is established under these conditions. Fungi have to pay a high cost for hydrocarbon utilization; high levels of reactive oxygen species are produced and a concomitant antioxidant response is triggered in fungal cells to cope with this drawback.

Reponses of the Hepatic Microsomal Cytochrome P450 Monooxygenase System in Rock Bream Oplegnathus fasciatus Exposed to Tributyltin (TBT)

Abstract The study was conducted to investigate the responses of the hepatic microsomal cytochrome P450 monooxygenase system in the rock bream Oplegnathus fasciatus after chronic exposure to 0, 1, 2, 4, and 8 μg/L tributyltin (TBT) concentrations for 4 weeks. Hepatic cytochrome 450 content and ethoxyresorufin O -deethylation (EROD) activity were found to significantly increase in fish treated with the higher concentration of TBT (≥4 μg/L); however, no significant changes were observed in penthoxyresorufin O -deethylation (PROD) activity in all treated groups compared to the control group. These findings suggest that exposure to a low TBT concentration (≥4 μg/L) has the potential to induce cytochrome 450 content and EROD enzyme activity in hepatic tissue in the rock bream. Key words: Oplegnathus fasciatus , Tributyltin, Cytochrome 450, EROD, PROD Introduction Many aquatic ecosystems are faced with spatially or tem-porally alarmingly high levels of xenobiotic chemicals (Brack et al., 2002; Diez et al., 2002). Tributyltin (TBT) is a typi-cal synthetic pollutant, which was selected for investigation in this study. TBT is an organotin compound used primarily as a biocide in antifouling paints for ships, boats, and fishing nets. The use of TBT as an antifouling agent in paint has been banned for small boats and fishing nets in most countries as it has been shown to be toxic to aquatic life and is an endo-crine disrupting chemical that generates severe reproductive effects in aquatic animals (Horiguchi et al., 1994). Reproduc-tive impairment in gastropods has been reported at low TBT concentrations (Gibbs et al., 1988). TBT has been shown to induce imposex and intersex in brosobranchs (Oehlmann et al., 1998), and impaired reproductive function in fish has also been described (Mc Allister and Kime, 2003).Some studies have reported toxic effects of TBT such as morphological and functional alterations of teleost gills in aquatic media (Byrne et al., 1989; Schwaiger et al., 1992; Tsu -da et al., 1992; Wang and Huang, 1998). TBT has also been shown to markedly inhibit fish hepatic CYP and in particu-lar the isozyme cytochrome P4501A1 (CYP1A), both in vivo and vitro (Fent and Bucheli, 1994; Morcillo and Porte, 1997). The CYP1A isozyme plays a key role in the biotransformation of xenobiotic compounds, leading to either their detoxifica-tion or bioactivation, while other CYP forms are important

Cross-Linking Mass Spectrometry and Mutagenesis Confirm the Functional Importance of Surface Interactions between CYP3A4 and Holo/Apo Cytochrome b5

Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.

Eicosanoid formation by a cytochrome P450 isoform expressed in the pharynx of Caenorhabditis elegans

Caenorhabditis elegans harbours several CYP (cytochrome P450) genes that are homologous with mammalian CYP isoforms important to the production of physiologically active AA (arachidonic acid) metabolites. We tested the hypothesis that mammals and C. elegans may share similar basic mechanisms of CYP-dependent eicosanoid formation and action. We focused on CYP33E2, an isoform related to the human AA-epoxygenases CYP2C8 and CYP2J2. Co-expression of CYP33E2 with the human NADPH-CYP reductase in insect cells resulted in the reconstitution of an active microsomal mono-oxygenase system that metabolized EPA (eicosapentaenoic acid) and, with lower activity, also AA to specific sets of regioisomeric epoxy- and hydroxy-derivatives. The main products included 17,18-epoxyeicosatetraenoic acid from EPA and 19-hydroxyeicosatetraenoic acid from AA. Using nematode worms carrying a pCYP33E2::GFP reporter construct, we found that CYP33E2 is exclusively expressed in the pharynx, where it is predominantly localized in the marginal cells. RNAi (RNA interference)-mediated CYP33E2 expression silencing as well as treatments with inhibitors of mammalian AA-metabolizing CYP enzymes, significantly reduced the pharyngeal pumping frequency of adult C. elegans. These results demonstrate that EPA and AA are efficient CYP33E2 substrates and suggest that CYP-eicosanoids, influencing in mammals the contractility of cardiomyocytes and vascular smooth muscle cells, may function in C. elegans as regulators of the pharyngeal pumping activity.

Sex-related differences in rat liver microsomal enzymes and their induction by doxapram.

The effects of doxapram on the hepatic microsomal mono-oxygenase system of male and female rats were investigated. Male and female rats were administered doxapram (10-120 mg kg-1 day-1, i.p.) for 4 days. In female rats, administration of doxapram (20, 40, 60, 80, 100 and 120 mg kg-1) elevated the parameters in a dose-dependent manner while doxapram (100 and 120 mg kg-1) elevated the levels of cytochrome P450 and hexobarbitone hydroxylase in male rats. Doxapram (40 mg kg-1) caused induction of hepatic drug metabolism typified by an increase of hepatic microsomal cytochrome P450 content and activities of hexobarbitone hydroxylase, benzphetamine N-demethylase and ethylmorphine N-demethylase in female rats, but no change in male rats. These findings were supported by the results of SDS/polyacrylamide-gel electrophoresis. However, 7-ethoxycoumarin O-de-ethylase and arylhydrocarbon hydroxylase activities were significantly increased in male rats. NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities, and cytochrome b5 content were unaffected in rats of both sexes. The sex-dependent cytochrome P450 species may be selectively sensitive to the action of doxapram.

Interactions of monoamine oxidase inhibitors, N-acetylenic analogues of tryptamine, with rat liver microsomal cytochrome P450.

Interactions between some novel and potent monoamine oxidase inhibitors (MAOIs), acetylenic analogues of tryptamine, and rat liver microsomal cytochrome P450 (P450) as evidenced by visible spectra analysis were analysed. Compounds with a secondary aliphatic amine moiety throughout induced type II difference spectra and exhibited the highest affinity for P450, whereas tertiary amines induced type I spectral changes and showed diminished affinity. P450 dependent aniline hydroxylase activity was inhibited by all compounds in an irreversible time-dependent manner. Only tertiary aliphatic amines constituted the substrate for P450-dependent N-demethylase activity, with comparable kinetic parameters. The N-demethylated metabolites were identified by thin-layer chromatography and mass-spectrometric analyses. These findings describe the role of P450-dependent microsomal mono-oxygenase systems in the metabolism of some MAOI acetylenic tryptamine derivatives and the possible hepatic contribution to adverse interactions between MAOIs, endobiotics and sympathomimetic compounds.

Combined use of mass spectrometry and heterologous expression for identification of membrane-interacting peptides in cytochrome P450 46A1 and NADPH-cytochrome P450 oxidoreductase

Cytochrome P450 46A1 (CYP46A1) and NADPH-cytochrome P450 oxidoreductase (CPR) are the components of the brain microsomal mixed-function monooxygenase system that catalyzes the conversion of cholesterol to 24-hydroxycholesterol. Both CYP46A1 and CPR are monotopic membrane proteins that are anchored to the endoplasmic reticulum via the N-terminal transmembrane domain. The exact mode of peripheral association of CYP46A1 and CPR with the membrane is unknown. Therefore, we studied their membrane topology by using an approach in which solution-exposed portion of heterologously expressed membrane-bound CYP46A1 or CPR was removed by digestion with either trypsin or chymotrypsin followed by extraction of the residual peptides and their identification by mass spectrometry. The identified putative membrane-interacting peptides were mapped onto available crystal structures of CYP46A1 and CPR and the proteins were positioned in the membrane considering spatial location of the missed cleavage sites located within these peptide as well as the flanking residues whose cleavage produced these peptides. Experiments were then carried out to validate the inference from our studies that the substrate, cholesterol, enters CYP46A1 from the membrane. As for CPR, its putative membrane topology indicates that the Q153R and R316W missense mutations found in patients with disordered steroidogenesis are located within the membrane-associated regions. This information may provide insight in the deleterious nature of these mutations.

The P450 oxidoreductase, RedA, controls development beyond the mound stage in Dictyostelium discoideum

BackgroundNADPH-cytochrome-P450 oxidoreductase (CPR) is a ubiquitous enzyme that belongs to a family of diflavin oxidoreductases and is required for activity of the microsomal cytochrome-P450 monooxygenase system. CPR gene-disruption experiments have demonstrated that absence of this enzyme causes developmental defects both in mouse and insect.ResultsAnnotation of the sequenced genome of D. discoideum revealed the presence of three genes (redA, redB and redC) that encode putative members of the diflavin oxidoreductase protein family. redA transcripts are present during growth and early development but then decline, reaching undetectable levels after the mound stage. redB transcripts are present in the same levels during growth and development while redC expression was detected only in vegetative growing cells. We isolated a mutant strain of Dictyostelium discoideum following restriction enzyme-mediated integration (REMI) mutagenesis in which redA was disrupted. This mutant develops only to the mound stage and accumulates a bright yellow pigment. The mound-arrest phenotype is cell-autonomous suggesting that the defect occurs within the cells rather than in intercellular signaling.ConclusionThe developmental arrest due to disruption of redA implicates CPR in the metabolism of compounds that control cell differentiation.

Acute depletion of reduced glutathione causes extensive carbonylation of rat brain proteins

This study was aimed at establishing whether oxidative stress induced by acute depletion of brain glutathione (GSH) is sufficient to generate protein carbonyls (PCOs). To this end, rat brain slices were incubated separately with the GSH depletors 1,3-bis[2-chloroethyl]-1-nitrosourea (BCNU) and diethyl maleate (DEM), and protein carbonylation was assessed on Western blots after derivatization with dinitrophenyl hydrazine. Incubation with 1 mM BCNU or 10 mM DEM for 2 hr decreased GSH levels by > 70%. Under these conditions the carbonylation of several proteins (40-120 kDa) increased by 2-3 fold. Isolation of carbonylated proteins showed that augmented PCOs represents a rise in the amount of oxidized protein. The iron chelator deferoxamine, the superoxide scavenger rutin and the H2O2 quencher dimethylthiourea all prevented DEM-induced protein carbonylation and lipid peroxidation (TBARS), indicating that the underlying mechanism involves the iron-catalyzed generation of hydroxyl radicals from H(2)O(2) (Fenton reaction). Inhibition of catalase activity with sodium azide and aminotriazole, and glutathione peroxidase activity with mercaptosuccinic acid did not increase PCOs or TBARS, suggesting that increased production of reactive oxygen species (ROS) rather than compromised cellular antioxidant defenses is the cause for the accumulation of H2O2 after GSH depletion. PCO formation was not affected by the xanthine oxidase inhibitor oxypurinol but it was reduced by SKF-525A and carbonyl cyanide 3-chlorophenylhydrazone, indicating that the microsomal monooxygenase system and the mitochondrial electron transport system are the major sources of ROS. Consistent with these findings, subcellular fractionation studies showed that mitochondria and synaptosomes are the major PCO-containing organelles. These results were also supported by the anatomic distribution of PCOs in brain. Our observations may be important in the context of multiple sclerosis where decreased GSH, mitochondrial dysfunction, excessive production of ROS, and increased protein carbonylation have all been reported.