Microsomal System Research Articles

In Vitro and In Vivo Metabolic Activation of Obacunone, A Bioactive and Potentially Hepatotoxic Constituent of Dictamni Cortex.

Obacunone is one of the major bioactive constituents from Dictamni cortex, a traditional Chinese medicine widely used in China. Oral administration of obacunone or Dictamni cortex extract has been shown to cause liver injury in rats. Given that obacunone contains a furan ring, which is a structural alert, metabolic activation might be responsible for obacunone-induced liver injury. In this study, bioactivation pathways of obacunone in rat and human liver microsomes were investigated. Obacunone was first metabolized into cis-butene-1,4-dial, and then cis-butene-1,4-dial was captured by glutathione, N-acetyl-cysteine, and N-acetyl-lysine in the microsomal incubation system. A total of 13 adducts derived from the reaction of cis-butene-1,4-dial with glutathione and/or N-acetyl-lysine were detected and structurally identified by liquid chromatography coupled to high-resolution tandem mass spectrometry. The major metabolite (M7) was identified to be the cyclic mono-glutathione conjugate of cis-butene-1,4-dial, which was detected in bile and urine of obacunone-treated rats. M9 and M10, obacunone-derived glutathione-cis-butene-1,4-dial-NAL conjugates, were detected in the microsomal incubations of obacunone fortified with glutathione and N-acetyl-lysine as trapping agents. M3 and M4, pyrroline-2-one derivatives, were also detected in microsomal incubations. Further phenotyping studies indicated that ketoconazole showed a strong inhibitory effect on the production of cis-butene-1,4-dial in a concentration-dependent manner. CYP3A4 was demonstrated to be the primary enzyme responsible for the bioactivation of obacunone by using individual recombinant human CYP450 enzymes. The current study provides an overview of CYP450-dominated bioactivation of obacunone and contributes to the understanding of the role of bioactivation in obacunone-induced liver injury.

Role of SUMOylation in Ethanol‐Modulated Mitochondria Homeostasis

ProposeAlcohol‐induced liver disease (ALD) is a major health concern of alcohol abuse and a leading cause of liver‐related morbidity and mortality. Alterations in mitochondrial morphology and function are a hallmark of ALD. Mitochondria synthesize most of the adenosine triphosphate (ATP) needed by mammalian cells and are intimately involved in the generation of and defense against reactive oxygen species (ROS). Evidence support the importance of ROS production and oxidative stress in the development of ALD. SUMOylation is a dynamic and reversible posttranscriptional modification involved in many cellular pathways including transcription, intracellular transport, DNA repair, replication, and cell signaling. We recently demonstrated that UBC9, the sole E2 protein required by SUMOylation machinery, is upregulated in Intragastric ethanol‐fed mouse model (IE) and cirrhotic tissues. We also found that UBC9 is phosphorylated and this is correlated with high level of SUMOylation in lipopolysaccharides‐activated Kupffer cells that lead inflammation development. In addition, we described a novel key role of SUMOylation inducing the enzymatic activity and protein stability of Cytochrome P450 2E1 (CYP2E1), that is the major component of the microsomal ethanol‐oxidizing system (MEOS), in NIAAA mouse model. This study aims to investigate whether ethanol induces mitochondrial dysfunction in ALD regulating the SUMOylation status of key respiratory chain proteins and explore the molecular mechanisms.MethodsPrimary mouse hepatocytes and livers from NIAAA mouse model were used. mRNA and protein levels were analyzed by Real‐Time PCR and Western Blotting, respectively. OCR (oxygen consumption rate) and Δψm (mitochondrial membrane potential) were measured by Seahorse mito stress test and JC‐1 staining by confocal microscopy, respectively.ResultsIn order to explore the role of SUMOylation in ALD, Mass Spectrometry (MS) was performed to identify SUMOylated proteins in 10‐day ethanol‐feeding+1 Binge ethanol (NIAAA) model, where SUMOs binding columns were used to purify SUMOylated proteins from total livers. Interestingly, we found that ethanol induces changes in SUMOylation state of several mitochondrial proteins involved in ATP synthesis (ATP5B, SOD2, CLC25A5, HSPD1, FBP1, CYCS), ATP metabolism (ATP5A1, ATP5B, ATP5C1, ATP5F1, ATP5J2, ASPA8, AK3), and mitochondria disorder (UQCRC2, PC, HMGCS2, ECHS1, COX6B1, NDUFV3, COX6C). These finding could suggest a potential role of SUMOylation in oxidative phosphorylation, electron transport chain (ETC) and respiratory control ratio may be modulating the ability of these proteins to form the complex above and/or regulating their activity. Specifically, ethanol treatment decreases ATP production and maximal respiration in primary mouse hepatocytes, while UBC9 knockdown prevents the loss of respiration. Furthermore, UBC9 silencing prevents the drop of Δψm when ethanol is added. This finding clearly indicates that SUMOylation is involved in mitochondrial physiology.Conclusionsthese novel finding provides highly novel information on the role of SUMOylation in the development of ALD and may provide new therapeutic strategies, which is of high public health relevance.Support or Funding InformationNIH/NIAAA R21AA027325‐01A1

Isolation of Caffeine from Carbonated Beverages

The work presented on the isolation of naturally occurring alkaloid from carbonated beverages. The extensive presence of caffeine in different plants plays an important role in the long-standing acceptance of caffeine-containing products. Caffeine (3,7-dihydro-1, 3,7-trimethyl-1H-purine-2,6-dione or 1,3,7-trimethylxanthine) is an alkaloid belongs to Methylxanthine family. Liquid-liquid extraction methods were used in the assay of research work. Chloroform was taken as extracting solvent. Solid residue of caffeine was recrystallized from 95% ethanol using 5ml/gram (5ml per gram). It is declared to raise caffeine, effects a number of different drugs include Paracetamol, Benzodiazepines and Aspirin and amount of plasma free Fatty acids increases. While inform that in regular sleeping interaction caffeine take place and raise the absorption of certain drugs. Changes in drug metabolizing enzymes, acts as an agent in a microsomal system of the body. The highest amount of caffeine dry crystal is extracted in sting sample while the 7up sample is free from caffeine.

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.

A new approach for prevention the oxidations and mutations: zinc borate

Zinc borate is a white crystalline powder substance with variable composition (34% B 2 O 3 , 45% ZnO and 20% H 2 O). It is used as a fungus and mildew inhibitor, to fire proof textiles, and for other uses. Although there are limited studies about the biologic properties of this compound. The present study was aimed to investigate the antioxidant and antimutagenic activity of zinc borate and also to figure out its cytotoxic effect. Antioxidant activity of zinc borate was determined with β- carotene linoleic acid and DPPH radical scavenging assays. The mutagenicity and antimutagenic activity was determined with AMES/ Salmonella microsomal test systems using Salmonella typhimurium TA98 and TA100 strains. The cytotoxic effect of zinc borate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay using mouse fibroblast L929 cell line. When compared to the control, % inhibition values of 1 mg/mL zinc borate for DPPH radical scavenging and β- carotene linoleic acid tests were calculated as 13.50% and 30%, respectively. At the tested concentrations, zinc borate did not exhibit any mutagenic effect. Zinc borate exhibited low antimutagenic activity at 0.2 and 0.04 mg/plate concentrations on S. typhimurium TA98 strain while it exhibited no antimutagenic activity on S. typhimurium TA100 strain. In vitro toxicity against L929 cell line with IC 50 values of zinc borate were found to be 40.70 µg/mL and 32.93 µg/mL for 24h and 72h, respectively. In spite of the low DPPH free radical scavenging activity, zinc borate has found to have moderate total antioxidant activity besides low antimutagenic potential. Zinc borate has a potential to use for medical purpose with its antioxidative and antimutagenic properties.

Metabolic activation of TM5441 in vitro and in vivo: Formation of reactive metabolites and human enzymes involved

TM5441, a furan-containing drug, is an inhibitor of plasminogen activator inhibitor-1 (PAI-1), which can induce intrinsic apoptosis of human cancer cell lines. The aim of this study was to identify the reactive metabolites of TM5441 and to reveal the bioactivation pathways that are associated with its hepatotoxicity. The reactive metabolites were trapped by using glutathione (GSH) or N-acetyl-lysine (NAL) in rat, dog, and human liver microsomal incubation system after exposure to TM5441. Two metabolic activation pathways were disclosed. The first bioactivation pathway was dominated by Cytochrome P450 enzymes (CYP450s); TM5441 was metabolized into cis-2-butene-1,4-dial derivative dependent on NADPH, which can be trapped in the liver microsomal incubations fortified with GSH or NAL as trapping agents. Five metabolites (M1, M2, M9, M12 and M13) associated with GSH and three metabolites (M4, M7 and M14) associated with NAL were identified by liquid chromatography-high resolution mass spectrometry. The second bioactivation pathway was catalyzed by UDP-glucuronosyltransferases (UGTs); TM5441 was conjugated with glucuronide to form acyl-glucuronide (M10), which further reacted with GSH, resulting in the identification of a TM5441-S-acyl-GSH adduct (M11) in liver microsomal incubations fortified with uridine-5′-diphosphoglucuronidc acid (UDPGA) and GSH. M9, M10, M11, M12 and M13 were also detected in bile samples of rats given TM5441. Compared with rat, dog would display closer bioactivation profiles to human. The CYP450 enzyme responsible for the bioactivation of TM5441 was mainly identified as CYP3A4, using human recombinant CYP450 enzymes and specific inhibitory studies. The UGT enzymes responsible for the bioactivation of TM5441 mainly involved UGT2B7, 1A1 and 1A4. These results facilitate the understanding of the bioactivation of TM5441 and potential toxicological implications.

Cytochrome P450 and flavin-containing monooxygenase enzymes are responsible for differential oxidation of the anti-thyroid-cancer drug vandetanib by human and rat hepatic microsomal systems

We studied the in vitro metabolism of the anti-thyroid-cancer drug vandetanib in a rat animal model and demonstrated that N-desmethylvandetanib and vandetanib N-oxide are formed by NADPH- or NADH-mediated reactions catalyzed by rat hepatic microsomes and pure biotransformation enzymes. In addition to the structural characterization of vandetanib metabolites, individual rat enzymes [cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO)] capable of oxidizing vandetanib were identified. Generation of N-desmethylvandetanib, but not that of vandetanib N-oxide, was attenuated by CYP3A and 2C inhibitors while inhibition of FMO decreased formation of vandetanib N-oxide. These results indicate that liver microsomal CYP2C/3A and FMO1 are major enzymes participating in the formation of N-desmethylvandetanib and vandetanib N-oxide, respectively. Rat recombinant CYP2C11 > >3A1 > 3A2 > 1A1 > 1A2 > 2D1 > 2D2 were effective in catalyzing the formation of N-desmethylvandetanib. Results of the present study explain differences between the CYP- and FMO-catalyzed vandetanib oxidation in rat and human liver reported previously and the enzymatic mechanisms underlying this phenomenon.

Pharmacokinetics and pharmacodynamics analysis of XQ-1H and its combination therapy with clopidogrel on cerebral ischemic reperfusion injury in rats

Ischemic stroke is the main cause of disability and mortality worldwide. 10-O-(N N-dimethylaminoethyl)-ginkgolide B methane-sulfonate (XQ-1 H) is a novel drug based on the remedial approach for ischemic stroke. Clopidogrel, a widely used anti-platelet drug, is often co-prescribed in the clinic. In this study, we established an UPLC-MS/MS spectrometry method for the determination of XQ-1H and investigated the pharmacokinetic effect of clopidogrel on XQ-1H in rats subjected to middle cerebral artery occlusion (MCAO). Meanwhile, the anti-apoptotic and neuroprotective effects of XQ-1H and its combination with clopidogrel were also studied. The results revealed that XQ-1H and its combination with clopidogrel abridged brain infarct volume, cerebral edema and alleviated neurological dysfunction caused by cerebral ischemic reperfusion injury. Further study demonstrated that XQ-1H combined with clopidogrel lessened TUNEL positive cells, up-regulated bcl-2 expression notably and down-regulated bax expression as compared to both XQ-1H and clopidogrel individually. In addition, a rapid, sensitive UPLC-MS/MS method was developed to quantify the concentration of XQ-1H in MCAO/R rats. Our pharmacokinetic results showed that clopidogrel significantly increased the exposure of XQ-1H, increased the peak plasma concentration (Cmax), area under the curve (AUC) and slowed elimination of XQ-1H in the co-administered group. Besides, for further exploring which CYP450 isoforms are involved in the XQ-1H metabolism, XQ-1H was incubated in human liver microsomes (HLMs) system with or without P450 isoform-selective inhibitors. Our results revealed that clopidogrel altered pharmacokinetics of XQ-1H potentially and subsequently enhanced the pharmacological effect of XQ-1H. Moreover, XQ-1H could be applied as an efficacious neuroprotective agent for ischemic stroke because of its considerable effect on averting neuronal apoptosis.

Effects of atorvastatin on pharmacokinetics of amlodipine in rats and its potential mechanism

Atorvastatin combined with amlodipine (ALDP) can efficiently treat the hypertension with coronary heart disease. However, the drug-drug interaction between atorvastatin and ALDP are still unknown.This study investigates the effects of atorvastatin on the pharmacokinetics of ALDP in rats and clarifies its main mechanism.The pharmacokinetic profiles of oral administration of ALDP (1 mg/kg) in Sprague-Dawley rats, with or without pretreatment of atorvastatin (1.5 mg/kg/d for 7 d) were investigated. The effects of atorvastatin on the metabolism of ALDP were also investigated using rat liver microsomes.The results showed that atorvastatin could significantly increase the peak plasma concentration (from 18.28 ± 2.65 to 24.13 ± 1.96 ng/mL) and decrease the oral clearance (from 4.57 ± 1.15 to 1.79 ± 0.28 L/h/kg) of ALDP. In the rat liver microsome systems, the intrinsic clearance rate of ALDP was decreased by the pretreatment with atorvastatin (39.26 ± 2.1 vs. 33.24 ± 3.3 μL/min/mg protein).Those results indicated that atorvastatin could significantly affect the pharmacokinetic of ALDP, via inhibiting the metabolism of ALDP in rats.

Evaluation of days-dependent chloramphenicol dosage on rat liver microsomal lipid peroxidation and catalase activity

Drugs and chemical agents can alter the cellular functions associated with the oxidative metabolism, thereby stimulating ROS production. The objective of the this study was the in vivo study of the inhibitory effect of chloramphenicol on hepatic microsomal enzyme system and the effect on lipid peroxidation using the thiobarbituric acid (TBA) reactive as index of peroxidation damage was investigated. The rats were randomly divided into 3 groups; group 1 serves as the control receiving saline water, group 2 receive chloramphenicol at a dose of 28.6 mg/kg body weight/day for 5 days and group 3 for 7 days. Liver protein content, lipid hydroperoxides and catalase activity were all determined. TBARS concentration increased statistically significantly with time of exposure. After 5 days of treatment, the level of TBARS increased by 103.56% with exogenous oxidant and by 141.54% for without oxidant, as compared to the respective control. On day 7, TBARS level in the liver was approximately 90.75% higher than in the control group with oxidant and 117.03% higher than the control without oxidant. The antibiotic elicited significant increase in rat liver lipid peroxidation compared to control. There were decreases in microsomal protein content of the liver in the drug-treated rats when compared with the control rats. It also showed that chloramphenicol treatment affects cytosolic catalase activities. It decreases catalase levels by 15.75% in 5 days dosage treatment and 17.81% in 7 days dosage treatment respectively. There were significance difference (P<0.05) in the treated and control irrespective of the days of dosage. The catalase level in hepatocyte in 5 days and 7 days group was significance at p<0.01. In conclusion, it was evident that treatment of rats for 5 days and 7 days with the therapeutic doses of Chloramphenicol altered antioxidant system and the intensity of effects was concentration/dosage days dependent, it resulted in membrane lipid peroxidation, protein damage, and inhibition of microsomal catalase due to increased generation of free radicals, reactive oxygen species and reactive nitrogen speciesKeywords: Chloramphenicol, rat liver, lipid peroxidation, catalase activity, antioxidant

Alcoholic Liver Disease: Current Mechanistic Aspects with Focus on Their Clinical Relevance.

The spectrum of alcoholic liver disease (ALD) is broad and includes alcoholic fatty liver, alcoholic steatohepatitis, alcoholic hepatitis, alcoholic fibrosis, alcoholic cirrhosis, and alcoholic hepatocellular carcinoma, best explained as a five-hit sequelae of injurious steps. ALD is not primarily the result of malnutrition as assumed for many decades but due to the ingested alcohol and its metabolic consequences although malnutrition may marginally contribute to disease aggravation. Ethanol is metabolized in the liver to the heavily reactive acetaldehyde via the alcohol dehydrogenase (ADH) and the cytochrome P450 isoform 2E1 of the microsomal ethanol-oxidizing system (MEOS). The resulting disturbances modify not only the liver parenchymal cells but also non-parenchymal cells such as Kupffer cells (KCs), hepatic stellate cells (HSCs), and liver sinusoidal endothelial cells (LSECs). These are activated by acetaldehyde, reactive oxygen species (ROS), and endotoxins, which are produced from bacteria in the gut and reach the liver due to gut leakage. A variety of intrahepatic signaling pathways and innate or acquired immune reactions are under discussion contributing to the pathogenesis of ALD via the five injurious hits responsible for disease aggravation. As some of the mechanistic steps are based on studies with in vitro cell systems or animal models, respective proposals for humans may be considered as tentative. However, sufficient evidence is provided for clinical risk factors that include the amount of alcohol used daily for more than a decade, gender differences with higher susceptibility of women, genetic predisposition, and preexisting liver disease. In essence, efforts within the last years were devoted to shed more light in the pathogenesis of ALD, much has been achieved but issues remain to what extent results obtained from experimental studies can be transferred to humans.

Prediction of potential drug interactions of apigenin based on molecular docking and in vitro inhibition experiments

The purpose of this study was to investigate the effect of apigenin on UGT1 A1 enzyme activity and to predict the potential drug-drug interaction of apigenin in clinical use. First,on the basis of previous experiments,the binding targets and binding strength of apigenin to UGT1 A1 enzyme were predicted by computer molecular docking method. Then the inhibitory effect of apigenin on UGT1 A1 enzyme was evaluated by in vitro human liver microsomal incubation system. Molecular docking results showed that apigenin was docked into the active region of UGT1 A1 enzyme protein F,consistent with the active region of bilirubin docking,with moderate affinity. Apigenin flavone mother nucleus mainly interacted with amino acid residues ILE343 and VAL345 to form hydrophobic binding Pi-Alkyl. At the same time,the hydroxyl group on the mother nucleus and the amino acid residue LYS346 formed an additional hydrogen bond,which increased the binding of the molecule to the protein. These results suggested that the flavonoid mother nucleus structure had a special structure binding to the enzyme protein UGT1 A1,and the introduction of hydroxyl groups into the mother nucleus can increase the binding ability. In vitro inhibition experiments showed that apigenin had a moderate inhibitory effect on UGT1 A1 enzyme in a way of competitive inhibition,which was consistent with the results of molecular docking. The results of two experiments showed that apigenin was the substrate of UGT1 A1 enzyme,which could inhibit the activity of UGT1 A1 enzyme competitively,and there was a risk of drug interaction between apigenin and UGT1 A1 enzyme substrate in clinical use.

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.

Azo dyes degradation and mutagenicity evaluation with a combination of microbiological and oxidative discoloration treatments

This work evaluated the degradation of the Acid Blue 161 and Procion Red MX-5B dyes in a binary solution by the filamentous fungus Aspergillus terreus and the yeast Saccharomyces cerevisiae in systems with and without electrochemical oxidation as the pretreatment process. UV–Vis spectrophotometry, high-performance liquid chromatography with (HPLC), Fourier transform infrared (FT-IR) spectroscopy and Salmonella/microsome assay (Ames test) were applied towards the degradation analysis of the dyes. Adsorption tests with white clay immobilized on alginate were also conducted after the discoloration treatments to remove intermediate metabolites formed during the degradation of the dye molecules. The discoloration treatments led to the complete color removal of the solutions in all the systems tested. The clay demonstrated affinity for the metabolites formed after discoloration treatments, the removal rates were variable, but the all systems has proved efficient. The Salmonella/microsome assay (Ames test) with strains TA98 and TA100 in the absence and presence of exogenous metabolism (S9 microsomal system, Moltox) revealed that the initial molecules and by-products of the metabolism of the dyes were direct mutagens. The electrochemical/A. terreus/clay system was able to discolor the solutions and transform the direct mutagens into non-mutagenic compounds in addition to reducing the mutagenic potency of the pro-mutagens to the Salmonella strain TA100/S9, which demonstrates the high efficiency of this system with regard to discoloring and degrading azo dye molecules and their by-products. Therefore, this study showed that although not having standard treatment system for this type of pollutant, the combination of treatments can be considered promising. The use of electrochemical oxidation along with microbiological treatment may lead to the degradation and mineralization of these compounds, reducing or eliminating the environmental impact caused by the improper disposal of these dyes in aquatic environments.

LC-Q-TOF/MS-oriented systemic metabolism study of pedunculoside with in vitro and in vivo biotransformation

As a triterpene saponin, pedunculoside is one of the most abundant, representative and active components in plants of genus Ilex (Aquifoliaceae). Pedunculoside has been used to treat myocardial ischemia, ameliorate hyperlipidemia and prevent liver injury. In this paper, a systemic in vitro liver microsomes / S9 and intestinal bacteria incubation, and in vivo animal experiment were performed, using LC-Q-TOF/MS analysis and a three-step data processing protocol. As a result, Bifidobacterium adolescentis and Bifidobacterium breve were identified to potentially metabolize pedunculoside among the intestinal bacteria tested. A total of 11 metabolites were found and tentatively identified, with 6 in both microsomal and bacterial incubation systems, and 9 after rats orally administered with pedunculoside. The metabolites detected involving both phase I and phase II metabolism, mainly through deglycosylation (hydrolyzation), dehydrogenation, hydroxylation and conjugation, and some of them underwent more than one-step metabolic reactions. Most of the metabolites have not been reported before. In vitro, liver microsome and intestinal bacteria prefer to metabolize pedunculoside in totally different ways; while in vivo, intestinal tract is the most important site for the metabolism and excretion of pedunculoside, where both intestinal bacteria and the host metabolic enzymes participate in its metabolism and disposition. The importance of intestinal bacteria should be highlighted. This study would contribute to a better understanding of pedunculoside metabolism, which can provide scientific evidence for its pharmacodynamic mechanism research and prove its clinical application.

Demethylbellidifolin isolated from Swertia bimaculate against human carboxylesterase 2: Kinetics and interaction mechanism merged with docking simulations

In this study, forty-nine kinds of traditional Chinese medicines (TCMs) were evaluated for their inhibitory activities against human carboxylesterase 2 (HCE 2) using a human liver microsome (HLM) system. Swertia bimaculata showed significant inhibition on HCE 2 at 10 μg/mL among forty-nine kinds of TCMs. The extract of Swertia bimaculata was separated by preparative HPLC to afford demethylbellidifolin (1) identified by MS, 1H NMR, and 13C NMR spectra. Demethylbellidifolin (1) was assayed for its inhibitory HCE 2 effect by HCE 2-mediated DDAB hydrolysis, and its potential IC50 value was 3.12 ± 0.64 μM. Demethylbellidifolin (1) was assigned as a mixed-type competitive inhibitor with the inhibiton constant Ki value of 6.87 µM by Lineweaver-Burk and slope plots. Living cell imaging was conducted to corroborate its inhibitory HCE 2 activity. Molecular docking indicated potential interactions of demethylbellidifolin (1) with HCE 2 through two hydrogen bonds of the C-3 and C-5 hydroxy groups with amino acid residues Glu227 and Ser228 in the catalytic cavity, respectively.

On the trail of steroid aromatase: The work of Kenneth J. Ryan

On the trail of steroid aromatase: The work of Kenneth J. Ryan

ADME pharmacogenetics: future outlook for Russia.

This systematic review reflects the results of pharmacogenetic studies in the Russian Federation aimed at studying the genes involved in the drug biotransformation system. The works of Russian researchers found by us are mostly devoted to microsomal liver oxidation enzymes (metabolism) and membrane transporter systems (absorption and excretion). This review presents population-ethnic and associative clinical studies on the genes of the CYP450 system, noncytochrome oxidation enzymes (SULT1A1, CES1), membrane transporter system genes (ABCB1, SLCO1B1) and warfarin biotransformation enzymes (VKORC1, GGCX). The information is structured in the form of 11 tables, divided by regions of the Russian Federation.

Importance of the identification of the CYP3A4*2 polymorphism for theprescription of pharmacological treatment

Variability in response to drugs is a problem in clinical practice. The rate of patients who respond adequately to pharmacological therapy is generally around 60. The CYP450 multienzyme complex is a microsomal system located in the endoplasmic reticulum. The enzymes participate in phase I metabolism of xenobiotics. CYP3A4 is the isoenzyme mostly expressed in liver. Its gene is polymorphic, of which CYP3A4*1A is the wild allele and CYP3A4*2 is a polymorphism which consists of the S222P substitution in the amino acid sequence, affecting the activity of the enzyme. Methods: In this study, we determine the frequency of CYP3A4*2 polymorphism in Mexican individuals. 62 apparently healthy individuals, from Toluca de Lerdo, Mexico. The sample was 3 mL of peripheral blood. The DNA was extracted and PCR-RFLPs were performed. Results: 58 individuals possess the wild allele in homozygosity CYP3A4*1A/CYP3A4*1A (94%) and 4 individuals were heterozygous CYP3A4*1A/CYP3A4*2 (6%). The homozygous polymorphic CYP3A4*2/CYP3A4*2 was not found in any individual. Conclusion: The CYP3A4*2 polymorphism is rare in the population studied. Given the low frequency of CYP3A4*2 polymorphisms found in homozygous or heterozygous condition, it is advisable to consider the genotype of the patient before prescribing drugs metabolized by this gene.

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.