NADPH-cytochrome P-450 Reductase Activity Research Articles

Exploring Cryptococcus neoformans CYP51 and Its Cognate Reductase as a Drug Target.

Cryptococcus remains a leading cause of invasive fungal infections in immunocompromised people. Resistance to azole drugs has imposed a further challenge to the effective treatment of such infections. In this study, the functional expression of full-length hexahistidine-tagged Cryptococcus neoformans CYP51 (CnCYP51-6×His), with or without its cognate hexahistidine-tagged NADPH-cytochrome P450 reductase (CnCPR-6×His), in a Saccharomyces cerevisiae host system has been used to characterise these enzymes. The heterologous expression of CnCYP51-6×His complemented deletion of the host CYP51 and conferred increased susceptibility to both short-tailed and long-tailed azole drugs. In addition, co-expression of CnCPR-6×His decreased susceptibility 2- to 4-fold for short-tailed but not long-tailed azoles. Type 2 binding of azoles to CnCYP51-6×His and assay of NADPH cytochrome P450 reductase activity confirmed that the heterologously expressed CnCYP51 and CnCPR are functional. The constructs have potential as screening tools and use in structure-directed antifungal discovery.

Effects of Echinococcus multilocularis metacestodes infection and drug treatment on the activities of biotransformation enzymes in mouse liver.

The changes of biotransformation enzymes will substantially affect the host's ability to metabolize drugs and other xenobiotic compounds. In order to further elucidate this process and promote the development in treatment of echinococcosis, we investigated the effects of Echinococcus multilocularis infection and drug treatment on biotransformation enzymes in mouse liver. In microsomal and cytosolic fractions, from the six activities assayed, significant decrease of glutathione S-transferases (GST) activity and significant increase of 7-pentoxyresorufin (PROD) and NADPH-cytochrome P450 reductase (CPR) activity were observed in the mice infected withE. multilocularismetacestodes. In addition, after six weeks treatment of albendazole inE. multilocularisinfected mice, significant decreased GST activity and significant increase of 7- ethoxyresorufin (EROD), PROD, and particularly 3-fold higher 7-methoxyresorufin (MROD) activity were observed. The 3-bromopyruvate treated mice only exhibited significantly lower GST activity. Our results demonstrate thatE. multilocularismetacestodes infection can affect the activities of main hepatic biotransformation enzymes and such alterations of activity may further affect the hepatic biotransformation of xenobiotics. Moreover, albendazole and 3-bromopyruvate, the promising potential drug againstEchinococcus, affected different hepatic biotransformation enzymes and may affect their metabolism. The findings will help to develop rational treatments with less side effects and promote the development of more efficient treatments againstE. multilocularis.

Plant species compositions alleviate toxicological effects of bisphenol A by enhancing growth, antioxidant defense system, and detoxification

Bisphenol A (BPA), a broadly disseminated endocrine disturbing chemicals in environment, is harmful to creatures and plants. Plants can uptake and metabolize BPA, but a single plant species ability is limited. Undeniably, plant species compositions have a more vital ability to remove pollutants than a single plant species. However, the mechanisms of plant species compositions alleviating toxicological effects of bisphenol A are poorly understood. Here, we administered plant species compositions, which based on a full-factorial design of Phragmites australis (A), Typha latifolia (B), and Arundo donax (C), to unveil their role in BPA exposure. The results illustrated that the root activity, biomass, and photosynthetic pigment contents of the mixed hydroponic group (e.g., sp(ABC)) were significantly increased under concentration of BPA(1.5, 5, and 10mg L-1), which showed that the root activity, fresh weight, dry weight, chlorophyll a, and total chlorophyll contents of shoots were increased. While mixed-hydroponic culture groups (e.g., sp(AB), sp(ABC)) significantly increased antioxidant enzyme activity and antioxidant substances under concentration of BPA(5 and 10mg L-1), it astoundingly diminished responsive oxygen species (ROS) and malondialdehyde (MDA) substance, proposing that mixed-hydroponic culture groups calmed oxidative stress. Further analysis revealed that mixed-hydroponic culture groups (e.g., sp(AB), sp(AC), sp(ABC)) of 1.5, 5, and 10mg L-1 BPA exposure significantly increased detoxification enzyme activity of NADPH-cytochrome P450 reductase (CPR), glutathione S-transferase (GST), and glycosyltransferase (GT). Moreover, mixed-hydroponic culture groups (e.g., sp(AB), sp(AC), sp(ABC)) decreased the BPA substance in leaves, proposing that mixed-hydroponic culture groups advanced BPA metabolism by improving CPR, GST, and GT enzyme activities. These results demonstrated that a mixed-hydroponic culture strategy can alleviate BPA phytotoxicity and possibly offer natural and potential phytoremediation methods for BPA.

Hepatic Scaling Factors for In Vitro-In Vivo Extrapolation of Metabolic Drug Clearance in Patients with Colorectal Cancer with Liver Metastasis.

In vitro-in vivo extrapolation (IVIVE) linked with physiologically based pharmacokinetics (PBPK) modeling is used to predict the fates of drugs in patients. Ideally, the IVIVE-PBPK models should incorporate systems information accounting for characteristics of the specific target population. There is a paucity of such scaling factors in cancer, particularly microsomal protein per gram of liver (MPPGL) and cytosolic protein per gram of liver (CPPGL). In this study, cancerous and histologically normal liver tissue from 16 patients with colorectal liver metastasis were fractionated to microsomes and cytosol. Protein content was measured in homogenates, microsomes, and cytosol. The loss of microsomal protein during fractionation was accounted for using corrections based on NADPH cytochrome P450 reductase activity in different matrices. MPPGL was significantly lower in cancerous tissue (24.8 ± 9.8 mg/g) than histologically normal tissue (39.0 ± 13.8 mg/g). CPPGL in cancerous tissue was 42.1 ± 12.9 mg/g compared with 56.2 ± 16.9 mg/g in normal tissue. No correlations between demographics (sex, age, and body mass index) and MPPGL or CPPGL were apparent in the data. The generated scaling factors together with assumptions regarding the relative volumes of cancerous versus noncancerous tissue were used to simulate plasma exposure of drugs with different extraction ratios. The PBPK simulations revealed a substantial difference in drug exposure (area under the curve), up to 3.3-fold, when using typical scaling factors (healthy population) instead of disease-related parameters in cancer population. These indicate the importance of using population-specific scalars in IVIVE-PBPK for different disease states. SIGNIFICANCE STATEMENT: Accuracy in predicting the fate of drugs from in vitro data using IVIVE-PBPK depends on using correct scaling factors. The values for two of such scalars, namely microsomal and cytosolic protein per gram of liver, is not known in patients with cancer. This study presents, for the first time, scaling factors from cancerous and matched histologically normal livers. PBPK simulations of various metabolically cleared drugs demonstrate the necessity of population-specific scaling for model-informed precision dosing in oncology.

Esterase, Glutathione S-Transferase and NADPH-Cytochrome P450 Reductase Activity Evaluation in Cacopsylla pyri L. (Hemiptera: Psyllidae) Individual Adults.

Simple SummaryThe resistance of pest insects to insecticides is a growing problem in all types of crops. Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain, and the large number of insecticide treatments necessary for its control may contribute to the emergence of resistance. Laboratory insecticide toxicity (mortality assays) and biochemical assays (to know the enzymatic mechanisms of the insects in acquiring resistance) are necessary to confirm the existence of this resistance. All the previous methodologies developed to evaluate enzyme activity in C. pyri to date have been performed using “pools” of adults. In this study, we determined the optimal working conditions for the evaluation of the enzymatic activities in individual insects. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed that one of the three resistance mechanisms studied had a high level of activity and was present in a high proportion of insects in the population selected with the insecticide as well as and in the field populations. These results validated the applied methodology.Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain. The large number of insecticide treatments necessary for control may be an important contributor to the emergence of resistance. Laboratory toxicity and biochemical assays are necessary to validate the existence of insecticide resistance and establish the underlying mechanisms. All the methodologies developed to evaluate enzyme activity in C. pyri to date have incorporated “pools” of adults to detect minimum activity ranges. In this study, we determined the optimal working conditions for evaluation of the activities of esterase, glutathione S-transferase and NADPH-cytochrome P450 reductase in individual insects via colorimetric methods using a microplate reader. The main factors affecting enzymatic analysis activity, such as enzyme source and substrate concentration, filter wavelength, buffer pH, reaction time and additives, were evaluated for optimization. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed high values and great variation in the activity ranges of esterase (EST) in the insecticide-selected population as well as in the field populations validating the applied methodology.

Interaction of Cp6-his and Cp6 with bovine serum albumin and liver microsomes: Spectroscopic and molecular docking studies

The conjugation of photosensitizer with a molecule specific to receptors over-expressed on cancer cells is a promising approach for improving tumor selectivity in photodynamic therapy (PDT). Since chemical nature of the photosensitizer is also affected by conjugation, this can have significant influence on its transport by serum proteins and clearance via liver microsomal enzyme system. In this report, the interaction of chlorin p6-histamine conjugate (Cp6-his) and free chlorin p6 (Cp6) with bovine serum albumin (BSA) and mouse liver microsomes was assessed by measuring the quenching of intrinsic tryptophan (Trp) fluorescence of proteins. The effect of photosensitizer binding on the activity of microsomal NADPH-cytochrome P-450 and NADH-cytochrome b5 reductase was also determined. The formation of protein carbonyl and lipid peroxides was measured to assess the photodynamic damage to proteins and lipids respectively. Further, the binding interaction between Cp6-his conjugate and parent Cp6 with BSA and cytochrome P-450 was validated using in-silico molecular docking approach. Results show that as compared to free Cp6, the affinity of conjugate to bind with serum albumin is 4 orders of magnitude less, which was also corroborated with finding of docking studies. In contrast, with microsomal proteins the binding affinity of Cp6-his was observed to be lower by 2 orders of magnitude as compared to that for Cp6. Further, the inhibition of the activities of microsomal enzymes induced by Cp6 conjugate and free Cp6 was nearly same (~15-20%). Measurements on quenching of photosensitizer fluorescence by iodide ions revealed that conjugate localized slightly deeper in microsomal membrane as compared to free Cp6 consistent with the slight difference in lipophilicity of the two photosensitizers. The amount of photodynamic damage to BSA induced by Cp6-his was significantly less as compared to that for Cp6, whereas no difference was observed in damage induced to microsomal protein and lipid. These results suggest that conjugation of Cp6 to histamine affects its binding to albumin but doses not significantly alters its interaction with microsomal enzyme system.

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies.

BACKGROUND:Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes.OBJECTIVE:For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes.METHODS:POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method.RESULTS:The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3(p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models.CONCLUSION:In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies.

Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats.

The major objective of the present study was to determine the ability of a triazole fungicide tebuconazole to induce cytochrome P450-dependent monooxygenases, oxidative stress, and endocrine-disrupting activity using male rats treated with tebuconazole at 10, 25, and 50 mg/kg p.o. once daily for 28 days. In liver, tebuconazole dose-dependently increased microsomal contents of cytochrome P450 and cytochrome b5 and the activities of NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, pentoxyresorufin O-dealkylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase. In kidney, tebuconazole increased 7-ethoxycoumarin O-deethylase activity without affecting other monooxygenase activities. In marked contrast to liver and kidney, tebuconazole decreased testicular 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities. The results of immunoblot analysis of liver microsomes of controls and tebuconazole-treated rats revealed that tebuconazole induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A proteins in liver. Additions of tebuconazole to liver microsomes inhibited microsomal 7-ethoxycoumarin O-deethylase activity in vitro (IC50 = 1.50-1.69 µM). Treatment of rats with tebuconazole decreased glutathione content and increased glutathione S-transferase, superoxide dismutase, catalase, and glutathione peroxidase activities in liver; increased superoxide dismutase activities in kidney and testis; but decreased glutathione S-transferase activity in testis. Treatments with tebuconazole decreased serum testosterone concentration and cauda epididymal sperm count. The present study demonstrates that tebuconazole induces a multiplicity of CYPs and oxidative stress in liver; inhibits testicular P450 and glutathione S-transferase activities; and produces anti-androgenic effects in male rats.

Thyrotoxic rubber antioxidants, 2-mercaptobenzimidazole and its methyl derivatives, cause both inhibition and induction of drug-metabolizing activity in rat liver microsomes after repeated oral administration

We examined the effects of thyrotoxic rubber antioxidants, 2-mercaptobenzimidazole (MBI, 0.3 mmol/kg/day) and its methyl derivatives, methyl-MBIs [4-methyl-MBI (4-MeMBI, 0.6 mmol/kg/day), 5-methyl-MBI (5-MeMBI, 0.6 mmol/kg/day), and 4(or 5)-methyl-MBI (4(5)-MeMBI, 0.6 or 1.2 mmol/kg/day)], on the drug-metabolizing activity in male rat liver microsomes by 8-day repeated oral administration. The weight of liver and thyroid were increased by all the test chemicals; MBI was most potent, and there was no additive or synergistic effect between 4-MeMBI and 5-MeMBI. MBI decreased the cytochrome P450 (CYP) content, NADPH-cytochrome P450 reductase (POR) activity, 7-ethoxycoumarin O-deethylation (ECOD) activity, and flavin-containing monooxygenase (FMO) activity, but increased the 7-pentoxyresorufin O-depentylation (PROD) activity, suggesting inhibition of the drug-metabolizing activity on the whole but induce some activities such as the CYP2B activity. On the contrary, all the methyl-MBIs increased the CYP content, CYB5 content, ECOD activity, 7-ethoxyresorufin O-deethylation (EROD) activity, and PROD activity, indicating that they are mostly inducible of the CYP activity. However, the methyl-MBIs decreased the FMO activity, and 5-MeMBI and 4(5)-MeMBI appeared inhibitory for CYPs 2C11 and 2C13. Between 4-MeMBI and 5-MeMBI, there was no additive or synergistic effect on the drug-metabolizing activity, but was counteraction. It was concluded that MBI and methyl-MBIs had both inhibitory and inducible effects on the drug-metabolizing activity in rat liver microsomes at thyrotoxic doses. The effects of 4(5)-MeMBI indicated that the increased liver weight alone can be a hepatotoxic sign but not an adaptive no-adverse response in toxicity studies. The present results were related to the toxicokinetic profiles of MBI and 4(5)-MeMBI in the repeated toxicity studies.

Effect of Age on The Hepatocellularity Number for Wistar rats.

Recently there has been a substantial increase in the number of juvenile animal toxicity studies that are conducted to support pediatric drug safety evaluation. Adequate design of juvenile toxicity studies in rats, for instance with respect to dose levels per age group, requires an understanding of age-dependent pharmacokinetics. In vitro-in vivo extrapolation (IVIVE) and physiologically based pharmacokinetic (PBPK) modeling can help to anticipate age-dependent drug exposure in juvenile toxicity studies provided age-dependent profiles for animal physiology and scaling factors are available. For instance, when hepatocytes are used to predict hepatic drug clearance, the hepatocellularity (number of hepatocytes per gram liver, HPGL) is required as one of the scaling factors. Although HPGL is known for adult rats, information on the influence of age on HPGL is missing. The present work profiles the hepatocellularity number in male Wistar rats as a function of age. Using the NADPH-cytochrome P450 reductase (NCR) activity method, the mean HPGL for the adult rat (8 weeks) was 104 × 10(6) cells/gram liver (relative standard deviation 17%). This value was calculated as a ratio between NRC activities in liver homogenates and suspended hepatocytes. The HPGL values were significantly higher (p <0.001) for rat pups until 3 weeks of age compared with adults. Our results revealed that the HPGL value showed a rapid decrease after 3 weeks (end of weaning), essentially reaching adult values by 4 weeks. This age-dependent HPGL profile will be instrumental for hepatic drug clearance prediction when designing juvenile toxicity studies in Wistar rats.

Enzyme Activities and Analysis of Susceptibility Levels in Turkish Tuta absoluta Populations to Chlorantraniliprole and Metaflumizone Insecticides

Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), tomato leaf miner, is one of the most devastating insect pests that attack tomato crops. T. absoluta larvae destroy tomato plants by producing large galleries in tomato leaves and burrowing stalks, apical buds, green and ripe fruits. This larva causes a major economic effect on tomato industry. Insecticides are commonly used to control this pest in tomato fields. The aim of this study is to determine the susceptibility levels of T. absoluta populations and biochemical analysis of mechanism(s) involved in chlorantraniliprole and metaflumizone metabolism, registered in Turkey for use against T. absoluta. IRAC bioassay method was used to determine the resistance ratios of these insecticides. Resistance ratios ranged between 7.2-and 1.7-folds for chlorantraniliprole, and 4.0- and 1.8-folds for metaflumizone in T. absoluta field populations. NADPH-Cytochrome P450 reductase, glutathione S-transferase, and esterase activities were determined by conducting biochemical assays in field populations of T. absoluta. While NADPH Cytochrome P450 reductase activity showed an increase only in Antalya population (1.9-fold), glutathione S-transferase activity showed an increase only in Adana population (1.2-fold). However, esterase activities were increased both in Adana (1.3-fold) and Ankara (2.2-fold) populations of T. absoluta.

A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants

Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification.

Effect of chondroitin sulfate on turpentine-induced down-regulation of CYP1A2 and CYP3A6

This study aimed to assess whether chronic administration of chondroitin sulfate (CS) affects baseline expression of cytochrome P450 isoforms and impedes the decrease in expression and activity of CYP1A2 and CYP3A6 in rabbits with a turpentine-induced inflammatory reaction (TIIR). Seven groups of 5 rabbits, 3 control groups and 4 receiving 20mg/kg/day of CS for 20 and 30days, were used. The rabbits of 1 control group and 2 groups receiving CS had a TIIR; finally, the rabbits of one of the control groups remained in the animal facilities for 30days to assess the effect of time and environment on cytochrome P450. In control rabbits, intake of CS for 20 and 30days did not affect CYP3A6, CYP1A2 and NADPH cytochrome P450 reductase (CPR) mRNA, protein expression and activity. Compared with control rabbits, the TIIR not only reduced mRNA, protein expression and activity of CYP3A6 and CYP1A2 but also that of CPR. In rabbits with TIIR, CS prevented the decrease of CYP3A6 expression but not the reduction in activity. CS did not impede TIIR-induced down-regulation of CYP1A2. Hepatic NO concentrations and NF-κB nuclear translocation were increased by the TIIR, effect reversed by CS. In vitro, in hepatocytes, CS did not alter the expression and activity of CYP3A6, CYP1A2, and CPR. In conclusion, oral CS elicits a systemic effect but does not affect CYP1A2, CYP3A6, and CPR in control rabbits, although in rabbits with TIIR, CS prevents CYP3A6 protein down-regulation but not that of CYP1A2.

Selenium and/or iodine deficiency alters hepatic xenobiotic metabolizing enzyme activities in rats

The objective of this study was to investigate the effects of iodine (I2) and/or selenium (Se) deficiency on thyroid hormones and hepatic xenobiotic metabolizing enzyme systems using a triple animal model. Three-week-old male Wistar rats were fed for seven weeks. Se deficiency was introduced by a diet containing <0.005mg/kg Se, and I2 deficiency was produced by sodium perchlorate containing drinking water. The levels of plasma thyroid hormones [total T4 (TT4), total T3 (TT3)], thyroid stimulating hormone (TSH); total microsomal cytochrome P450 (CYP450) and cytochrome b5 (CYP b5) levels; activities of microsomal NADPH-cytochrome P450 reductase (P450R), microsomal aniline hydroxylase (CYP2E1), microsomal 7-ethoxyresorufin O-deethylase (EROD), microsomal 7-pentoxyresorufin O-depentylase (PROD) and cytosolic glutathione S-transferase (GST) were determined. In I2 deficiency total CYP450 levels, activities of CYP2E1, EROD and GST decreased, and CYP b5 content increased significantly. In Se-deficient rats, total CYP450 level and CYP2E1 activity increased, and EROD and GST activities and CYP b5 level decreased significantly. In combined I2 and Se deficiency, except for CYP450 content and CYP2E1 activity, all enzyme activities and CYP b5 content decreased significantly compared to control group. Overall results of this study have suggested that metabolism of xenobiotics as well as endogenous compounds is affected by Se and I2 status.

A novel microplate‐based HPLC‐fluorescence assay for determination of NADPH‐cytochrome P450 reductase activity

A 96-well microplate-based HPLC endpoint assay is described for the determination of NADPH-cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB-C₁₈ analytical 4.6 × 150 mm, 5 µm column. To validate the method, recombinant human NADPH-P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow-rate of 0.8 mL/min. The CPR activity was quantified using NADPH fluorescence at λ(Ex) = 340 nm and λ(Em) = 450 nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 aparatus, is in the 250 nmol range. In addition, using this method it is possible to set up reactions in a high-throughput format.

Furazolidone induces the activity of microsomal enzymes that metabolize furazolidone in chickens

The nitrofuran antibacterial agent furazolidone (FZ) is still used in veterinary medicine in some countries in the Middle and Far East. The present study aimed to show the effect of FZ on the activity of microsomal enzymes that metabolize FZ, and to identify the enzyme that contributes to FZ metabolism in chickens. Wistar rats and White Leghorn chickens were administered FZ once a day for four consecutive days. FZ metabolism was accelerated by FZ administration in chickens, but not in rats. The elevation of FZ metabolism coincided with the induction of NADPH cytochrome P450 reductase (CPR) activity in chickens, but such induction was not observed in rats. FZ metabolizing activities were inhibited in the presence of a CPR inhibitor (diphenylene iodonium chloride) but not by the addition of archetypal cytochrome P450 inhibitors (CO or n-octylamine). The preset study concluded that FZ accelerated its own metabolism in the chicken by induction of the activity of CPR.

CYP3A5-mediated metabolism of midazolam in recombinant systems is highly sensitive to NADPH-cytochrome P450 reductase activity

Data from in vitro drug metabolism studies with recombinant enzyme systems are frequently used to predict human drug metabolism in vivo. However, for the CYP3A probe substrate midazolam (MDZ), considerable variability in enzyme kinetic parameters has been observed in different in vitro studies. The aim of this study was to explore the effect of varying activities of the electron donor NADPH-cytochrome P450 reductase (CPR) on CYP3A5-mediated metabolism of MDZ.Microsomes with similar levels of CYP3A5 but 12-fold difference in CPR activity showed a 30-fold difference in intrinsic clearance for the formation of 1′-OH-MDZ. Significantly higher Km and lower Vmax for the formation of 1′-OH-MDZ were found in microsomes with low CPR activity compared with microsomes with higher CPR activity (P = 0.024 and 0.001). In the microsomes with lowest CPR activity, the formation of 1′-OH-MDZ displayed Michaelis–Menten kinetics, whereas substrate inhibition was observed in the two preparations with higher CPR activity.The present study shows that the CPR activity in different recombinant enzyme preparations is crucial for in vitro CYP3A5-mediated clearance of MDZ. This suggests that the CPR activity of enzyme preparations could be an important factor for the ability of in vitro data to predict human drug metabolism in vivo.

Computational approach to characterization of human liver drug-metabolizing enzymes

Cytochromes P450 are the key enzymes for activating and inactivating many drugs; individual expression levels of CYPs may play a crucial role in drug safety and drug efficacy. Statistical comparison of biochemical profiles of 23 human liver microsomes have been used to characterize human liver samples. The profile included 12 parameters, namely activity of NADPH-cytochrome P450 reductase, cytochrome P450 content and cytochrome P450-dependent monooxygenase activities with marker substrates. Unsupervised statistical methods including cluster analysis and principal component analysis revealed with very high confidence the presence of two groups. Difference between the groups was explained by peculiarities of reductase activity and cytochrome P450 enzyme activities with 7-ethoxyresorufin, 7-methoxyresorufin, 7-methoxycoumarin, 7-benzyloxyresorufin and 7-benzyloxyquinoline. Results of biochemical assays coupled with multidimensional data analysis can be further used for targeted proteomic profiling of microsome oxidation mechanisms.

Mixture Toxicity Assessment of Nickel and Chlorpyrifos in the Sea Bass Dicentrarchus labrax

The present research work was designed to study Dicentrarchus labrax biotransformation and detoxification responses to acute exposure to nickel (Ni) and chlorpyrifos (CHP). Sexually immature sea bass were treated by intraperitoneal injection of nickel chloride (500 μg kg⁻¹), chlorpyrifos (10 mg kg⁻¹), and their binary mixture for 1, 3, and 7 days. Ni and CHP accumulation was quantified in liver after the exposure periods. The following biological responses were measured: (1) NADPH cytochrome P450 reductase (NCR) activity, as phase I biotransformation parameter; (2) gluthathione S-transferase (GST) activity as a phase II conjugation enzyme, acetylcholinesterase activity, and metallothionein (MT) content. Ni bioaccumulation in the liver resulted in an increasing uptake up to 15.48 μg g⁻¹ wet weight (Ni-treated animals) and 16.73 μg g⁻¹ wet weight (mixture-treated animals) after 7 days of exposure. CHP accumulation showed a distinct pattern in animals exposed to the mixture of chemicals in comparison with CHP-treated animals. NCR activity exhibited a marked activation in CHP and mixture-treated animals. GST activity was significantly increased starting from 1 day exposure in CHP-treated animals and after 3 days in Ni-treated animals. MT accumulation increased in all conditions, with a marked synergetic effect after 7 days of exposure. These data should be carefully considered in view of the biological effects of mixture pollutants, particularly in fish farming conditions.

Uptake and biochemical response to B[a]P in the sea anemone Anthopleura elegantissima

In order to evaluate the effects of benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH) in the sea anemone Anthopleura elegantissima at the biochemical level. NADPH cytochrome P450 reductase and cytochrome P450 were assayed in A. elegantissima under toxicant. One toxicity test was performed with 75 organisms distributed in 5 groups. Animals in groups G1, G2 and G3 were given increasing B[a]P. Two groups named GC and GS were used as controls. GC was treated with seawater and GS was treated with acetone. After 72 h of exposure, enzymatic activities were determined. Microsomes were isolated from the columnar tissue and exposed in vitro to the toxicant in order to explore their ability to incorporate B[a]P. Basal activity for this enzyme was 1.69 ± 0.18 (Mean ± standard deviation) nmol cyt C red min−1 mg−1 and there was no significant effect in GS organisms compared to GC organisms. Significant increases were observed in NADPH cytochrome P450 reductase in G3 organisms. In this group, the enzyme activity was 3.53 ± 0.40 nmol cyt C red min−1 mg−1. For cytochrome P450 content, a gradual increase was observed in organisms in groups G1 to G3. Basal content was 10.25 ± 0.49 pmol mg−1 microsomal protein. For G3 animals, P450 content was 27.51 ± 0.32 pmol mg−1 microsomal. For the test in vitro, it was found that microsomes isolated from G2 and G3 had the capacity to incorporate this substance when exposed to B[a]P at a level of 4 μ M in the surrounding medium. Spectrum recorded from 350 to 450 nm after a 40-min exposure for these groups showed significant difference from spectra obtained for microsomes in GC, GS and G1. It was concluded that the capacity to increase NADPH cytochrome P450 reductase activity as well as to increase NADPH cytochrome P450 reductase activity as well as to increase P450 content shows the ability of A. elegantissima to induce a mixed function oxidase activity in the presence of B[a]P.