Brassica napus cytochrome P450 superfamily: Origin from parental species and involvement in diseases resistance, abiotic stresses tolerance, and seed quality traits.

Cytochromes P450 monooxygenases (CYP450s) constitute the largest enzymic protein family that is widely present in plants, animals, and microorganisms, participate in numerous metabolic pathways, and play diverse roles in development, metabolism, and defense. Rapeseed (Brassica napus) is an important oil crop worldwide and have many versions of reference genome. However, there is no systemically comparative genome-wide analysis of CYP450 family genes in rapeseed and its parental species B. rapa and B. oleracea. In this study, we identified 765, 293 and 437 CYP450 genes in B. napus, B. rapa and B. oleracea, respectively, which were unevenly located in A01-A10 and/or C01-C09 chromosomes in corresponding species. Phylogenetic relationship analysis indicated that 1745 CYP450 proteins from three Brassica species and Arabidopsis were divided into 4 groups. Whole genome duplication (WGD) or segmental duplication resulted in gene expansion of CYP450 family in three Brassica species. There were 33-83 SSR loci in CYP450 genes of three Brassica species, and numerous transcription factor binding sites were identified in their promoters. A total of 459-777 miRNAs were predicted to target 174-426 CYP450 genes in three Brassica species. Based on transcriptome data, BnCYP450s, BrCYP450s and BoCYP450s were differentially expressed in various tissues. There existed numerous BnCYP450 DEGs in response to pathogens and abiotic stresses. Besides, many BnCYP450 DEGs were involved in the regulation of important traits, such as seed germination, seed ALA content, and yellow-seed. The qRT-PCR experiment confirmed the transcriptome analysis results by validating two representative Sclerotinia-responsive BnCYP450 DEGs as an example. Three BnCYP450s genes (CYP707A1, CYP81F1, CYP81H1) might be regulated by seed-specific transcription factors BnTT1 and BnbZIP67 to participate in the development and metabolism of seed coat and embryo by undertaking related metabolic reactions.

Live to die another day: novel insights may explain the pathophysiology behind smoker's paradox in SARS-CoV-2 infection.

The severe acute respiratory coronavirus 2 (SARS-CoV-2) infection demonstrates a highly variable and unpredictable course. Several reports have claimed a smoker's paradox in coronavirus disease 2019 (COVID-19), in line with previous suggestions that smoking is associated with better survival after acute myocardial infarction and appears protective in preeclampsia. Several plausible physiological explanations exist accounting for the paradoxical observation of smoking engendering protection against SARS-CoV-2 infection. In this review, we delineate novel mechanisms whereby smoking habits and smokers' genetic polymorphism status affecting various nitric oxide (NO) pathways (endothelial NO synthase, cytochrome P450 (CYP450), erythropoietin receptor (EPOR); ß-common receptor (ßcR)), along with tobacco smoke modulation of microRNA-155 and aryl-hydrocarbon receptor (AHR) effects, may be important determinators of SARS-CoV-2 infection and COVID-19 course. While transient NO bioavailability increase and beneficial immunoregulatory modulations through the above-mentioned pathways using exogenous, endogenous, genetic and/or therapeutic modalities may have direct and specific, viricidal SARS-CoV-2 effects, employing tobacco smoke inhalation to achieve protection equals self-harm. Tobacco smoking remains the leading cause of death, illness, and impoverishment.

Impact of Pomegranate Juice on the Pharmacokinetics of CYP3A4- and CYP2C9-Mediated Drugs Metabolism: A Preclinical and Clinical Review.

The Punica granatum L. (pomegranate) fruit juice contains large amounts of polyphenols, mainly tannins such as ellagitannin, punicalagin, and punicalin, and flavonoids such as anthocyanins, flavan-3-ols, and flavonols. These constituents have high antioxidant, anti-inflammatory, anti-diabetic, anti-obesity, and anticancer activities. Because of these activities, many patients may consume pomegranate juice (PJ) with or without their doctor's knowledge. This may raise any significant medication errors or benefits because of food-drug interactions that modulate the drug's pharmacokinetics or pharmacodynamics. It has been shown that some drugs exhibited no interaction with pomegranate, such as theophylline. On the other hand, observational studies reported that PJ prolonged the pharmacodynamics of warfarin and sildenafil. Furthermore, since it has been shown that pomegranate constituents inhibit cytochrome P450 (CYP450) activities such as CYP3A4 and CYP2C9, PJ may affect intestinal and liver metabolism of CYP3A4 and CYP2C9-mediated drugs. This review summarizes the preclinical and clinical studies that investigated the impact of oral PJ administration on the pharmacokinetics of drugs that are metabolized by CYP3A4 and CYP2C9. Thus, it will serve as a future road map for researchers and policymakers in the fields of drug-herb, drug-food and drug-beverage interactions. Preclinical studies revealed that prolonged administration of PJ increased the absorption, and therefore the bioavailability, of buspirone, nitrendipine, metronidazole, saquinavir, and sildenafil via reducing the intestinal CYP3A4 and CYP2C9. On the other hand, clinical studies are limited to a single dose of PJ administration that needs to be protocoled with prolonged administration to observe a significant interaction.

Roles of the ubiquitin ligase CUL4B and ADP-ribosyltransferase TiPARP in TCDD-induced nuclear export and proteasomal degradation of the transcription factor AHR.

The aryl hydrocarbon receptor (AHR) is a transcription factor activated by exogenous halogenated polycyclic aromatic hydrocarbon compounds, including the environmental toxin TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and naturally occurring dietary and endogenous compounds. The activated AHR enhances transcription of specific genes including phase I and phase II metabolism enzymes and other targets genes such as the TCDD-inducible poly(ADP-ribose) polymerase (TiPARP). The regulation of AHR activation is a dynamic process: immediately after transcriptional activation of the AHR by TCDD, the AHR is exported from the nucleus to the cytoplasm where it is subjected to proteasomal degradation. However, the mechanisms regulating AHR degradation are not well understood. Here, we studied the role of two enzymes reported to enhance AHR breakdown: the cullin 4B (CUL4B)AHR complex, an E3 ubiquitin ligase that targets the AHR and other proteins for ubiquitination, and TiPARP, which targets proteins for ADP-ribosylation, a posttranslational modification that can increase susceptibility to degradation. Using a WT mouse embryonic fibroblast (MEF) cell line and an MEF cell line in which CUL4B has been deleted (MEFCul4b-null), we discovered that loss of CUL4B partially prevented AHR degradation after TCDD exposure, while knocking down TiPARP in MEFCul4b-null cells completely abolished AHR degradation upon TCDD treatment. Increased TCDD-activated AHR protein levels in MEFCul4b-null and MEFCul4b-null cells in which TiPARP was knocked down led to enhanced AHR transcriptional activity, indicating that CUL4B and TiPARP restrain AHR action. This study reveals a novel function of TiPARP in controlling TCDD-activated AHR nuclear export and subsequent proteasomal degradation.

Transport and metabolism of tyrosine kinase inhibitors associated with chronic myeloid leukemia therapy: a review.

Imatinib, nilotinib, dasatinib, bosutinib, ponatinib, and asciminib are FDA-approved tyrosine kinase inhibitors (TKIs) for chronic myeloid leukemia (CML), each of which has a specific pharmacological profile. Asciminib has been recently (2021) approved for patients resistant to former TKIs, and because the binding site of this drug (the myristoyl pocket in the ABL1 kinase) is different from that of other TKIs (ATP-binding sites), it is, therefore, effective against T315I mutation of BCR-ABL oncoprotein. All TKIs have a different pharmacological profile due to different chemical structures. Imatinib is the only TKI whose absorption depends on both influx (OCT1 and OATP1A2) and efflux (ABCB1 and ABCG2) transporters, whereas the others rely only on efflux transporters. The efflux of dasatinib is also regulated by ABCC4 and ABCC6 transporters. Nilotinib and ponatinib are transported passively, as no role of transporters has been found in their case. A phenomenon common to all in the metabolic aspect is that the CYP3A4 isoform of CYP450 primarily metabolizes TKIs. Not only does CYP3A4, flavin-containing monooxygenase 3 (FMO3), and uridine 5'-diphospho-glucuronosyltransferase (UGT) also metabolize dasatinib, and similarly, by glucuronidation process, asciminib gets metabolized by UGT enzymes (UGT1A3, UGT1A4, UGT2B7, and UGT2B17). Additionally, the side effects of TKIs are categorized as hematological (thrombocytopenia, neutropenia, anemia, and cardiac dysfunction) and non-hematological (diarrhea, nausea, vomiting, pleural effusion, and skin rash). However, few toxicities are drug-specific, like degradation of biomolecules by ponatinib-glutathione (P-GSH) conjugates and clinical pancreatitis (dose-limited toxicity and manageable by dosage alterations) are related to ponatinib and asciminib, respectively. This review focuses on the pharmacokinetics of approved TKIs related to CML therapy to comprehend their specificity, tolerability, and off-target effects, which could help clinicians to make a patient-specific selection of CML drugs by considering concomitant diseases and risk factors to the patients.

Characterisation of seven medications approved for attention-deficit/hyperactivity disorder using in vitro models of hepatic metabolism.

The metabolism of most medications approved for the treatment of attention deficit/hyperactivity disorder (ADHD) is not fully understood.In vitro studies using cryopreserved, plated human hepatocytes (cPHHs) and pooled human liver microsomes (HLMs) were performed to more thoroughly characterise the metabolism of several ADHD medications.The use of enzyme-specific chemical inhibitors indicated a role for CYP2D6 in atomoxetine (ATX) metabolism, and roles for CYP3A4/5 in guanfacine (GUA) metabolism.The 4-hydroxy-atomoxetine and N-desmethyl-atomoxetine pathways represented 98.4% and 1.5% of ATX metabolism in cPHHs, respectively. The 3-OH-guanfacine pathway represented at least 2.6% of GUA metabolism in cPHHs, and 71% in HLMs.The major metabolising enzyme for methylphenidate (MPH) and dexmethylphenidate (dMPH) could not be identified using these methods because these compounds were too unstable. Hydrolysis of these medications was spontaneous and did not require the presence of protein to occur.Clonidine (CLD), amphetamine (AMPH), and dextroamphetamine (dAMPH) did not deplete substantially in cPHHs nor HLMs, suggesting that these compounds may not undergo considerable hepatic metabolism. The major circulating metabolites of AMPH and dAMPH (benzoic acid and hippuric acid) were not observed in either system, and therefore could not be characterised. Additionally, inhibition experiments suggested a very minimal role for CYP2D6 in CLD and AMPH metabolism.

Temperature and solvent exposure response of three fatty acid peroxygenase enzymes for application in industrial enzyme processes.

Free fatty acids (FFAs) are a useful feedstock for a range of industrial chemical synthesis applications. However, efficiently converting FFAs to molecules for biofuel and other high-value chemicals requires more efficient and cost-effective catalysts. Cytochrome P450 fatty acid peroxygenases (CYP152) have a unique chemistry that allows use of the peroxide shunt pathway for biochemical conversion of FFAs. Known CYP152s are heat labile, however, requiring characterization of more thermotolerant versions for use in industrial applications. A fatty acid peroxygenase from Bacillus methanolicus (CYP152K6) was shown here to have a higher optimal reaction temperature than OleT (CYP152L1). CYP152K6 was stable up to 50 °C and showed great stability in 3% acetone and dimethylformamide. Stability in solvents helps the enzyme's substrates remain soluble in solution for more efficient catalysis, and heat stability allows enzymes to remain active longer during industrial processes.

Drug-drug interaction of acetaminophen and roxithromycin with the cocktail of cytochrome P450 and hepatotoxicity in rats.

Acetaminophen (APAP) and roxithromycin (ROX) are often used in combination in clinical practice. To evaluate their drug-drug interactions (DDIs) and the hepatotoxicity of co-administration, rats were randomly separated into four groups: Control, APAP (50 mg/kg), ROX (5.5 mg/kg) and APAP-ROX (50 mg/kg and 5.5 mg/kg, respectively). The pharmacokinetic parameters between APAP and ROX were assayed by HPLC, and a cocktail method was used to evaluate the activities of cytochrome (CYP) 450. The liver microsome CYP2E1 protein was detected using Western blot. The levels of plasma parameters, mRNA levels of inflammatory factors (TNF-α, INF-γ, VCAM-1, CXCL-1 and STAT-3) and antioxidant factors (Nrf-2, GSTA, GCLC-1, HO-1 and NQO1) were determined using real-time PCR, along with the observation on histopathological changes in the liver tissue. APAP and ROX co-treatment significantly increased CYP2E1 activity, decreased CYP2D6 activity and prolonged APAP and ROX clearance. Co-treatment increased mRNA expressions of TNF-α, NQO1 and MDA while decreasing GPX and SOD levels. Histopathological evidence showed the changes of liver tissues in terms of structure, size and tight arrangement. This study confirmed that a combination of APAP and ROX inhibited APAP metabolism and that the peak concentration of ROX was delayed. The resulting high level of CYP2E1 may induce oxidative stress and cause liver damage.

Boosting 11-oxo-ß-amyrin and glycyrrhetinic acid synthesis in Saccharomyces cerevisiae via pairing novel oxidation and reduction system from legume plants.

Glycyrrhetinic acid (GA) and its precursor, 11-oxo-ß-amyrin, are typical triterpenoids found in the roots of licorice, a traditional Chinese medicinal herb that exhibits diverse functions and physiological effects. In this study, we developed a novel and highly efficient pathway for the synthesis of GA and 11-oxo-ß-amyrin in Saccharomyces cerevisiae by introducing efficient cytochrome P450s (CYP450s: Uni25647 and CYP72A63) and pairing their reduction systems from legume plants through transcriptome and genome-wide screening and identification. By increasing the copy number of Uni25647 and pairing cytochrome P450 reductases (CPRs) from various plant sources, the titers of 11-oxo-ß-amyrin and GA were increased to 108.1 ± 4.6mg/L and 18.9 ± 2.0mg/L, which were nearly 1422-fold and 946.5-fold higher, respectively, compared with previously reported data. To the best of our knowledge, these are the highest titers reported for GA and 11-oxo-ß-amyrin from S. cerevisiae, indicating an encouraging and promising approach for obtaining increased GA and its related triterpenoids without destroying the licorice plant or the soil ecosystem.

The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes.

1. The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes. 2. 100 µM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC50) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00 µM, and the inhibition kinetic parameters (Ki) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11 µM, respectively. 3. Metabolism of morusin exhibited significant species differences. The quantities of M1 from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The Km values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83 µM, and Vmax for these species were 0.09, 1.23, 1.43, 0.15, and 0.75 nmol/min/mg, respectively. CLint (intrinsic clearance) values (Vmax/Km) for morusin obeyed the following order: monkey > rat > minipig > dog > human. CLH (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12 mL/min/kg body weight, respectively. 4. This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.

The Impact of Anti-Epileptic Drugs on Growth and Bone Metabolism.

Epilepsy is a common neurological disorder worldwide and anti-epileptic drugs (AEDs) are always the first choice for treatment. However, more than 50% of patients with epilepsy who take AEDs have reported bone abnormalities. Cytochrome P450 (CYP450) isoenzymes are induced by AEDs, especially the classical AEDs, such as benzodiazepines (BZDs), carbamazepine (CBZ), phenytoin (PT), phenobarbital (PB), and valproic acid (VPA). The induction of CYP450 isoenzymes may cause vitamin D deficiency, hypocalcemia, increased fracture risks, and altered bone turnover, leading to impaired bone mineral density (BMD). Newer AEDs, such as levetiracetam (LEV), oxcarbazepine (OXC), lamotrigine (LTG), topiramate (TPM), gabapentin (GP), and vigabatrin (VB) have broader spectra, and are safer and better tolerated than the classical AEDs. The effects of AEDs on bone health are controversial. This review focuses on the impact of AEDs on growth and bone metabolism and emphasizes the need for caution and timely withdrawal of these medications to avoid serious disabilities.

Association of CYP2C9*2 Allele with Sulphonylurea-Induced Hypoglycaemia in Type 2 Diabetes Mellitus Patients: A Pharmacogenetic Study in Pakistani Pashtun Population.

Polymorphism in cytochrome P450 (CYP) 2C9 enzyme is known to cause significant inter-individual differences in drug response and occurrence of adverse drug reactions. Different alleles of the CYP2C9 gene have been identified, but the notable alleles responsible for reduced enzyme activity are CYP2C9*2 and CYP2C9*3. No pharmacogenetic data are available on CYP2C9*2 and CYP2C9*3 alleles in the Pakistani population. In Pakistan, pharmacogenetics, which examines the relationship between genetic factors and drug response, are in the early stages of development. We, for the first time, investigated the association between the CYP2C9 variant alleles CYP2C9*2 and CYP2C9*3 and the incidence of hypoglycaemia in patients with Type 2 diabetes mellitus (T2DM) receiving sulphonylurea medications. A total of n = 400 individuals of Pashtun ethnicity were recruited from 10 different districts of Khyber Pakhtunkhwa, Pakistan to participate in the study. The study participants were divided into two distinct groups: the case group (n = 200) and the control group (n = 200). The case group consisted of individuals with T2DM who were receiving sulphonylurea medications and experienced hypoglycaemia with it, whereas the control group included individuals with T2DM who were receiving sulphonylurea medication but did not experience sulphonylurea-induced hypoglycaemia (SIH). Blood samples were obtained from study participants following informed consent. DNA was isolated from whole blood samples using a Wiz-Prep DNA extraction kit. Following DNA isolation, CYP2C9 alleles were genotyped using MassARRAY sequencing platform at the Centre of Genomics at the Rehman Medical Institute (RMI). The frequency of CYP2C9*2 (low-activity allele) was more frequent in the diabetic patients with SIH compared to the control group (17.5% vs. 6.0%, p = 0.021). The frequency of its corresponding genotype CYP2C9*1/*2 was higher in cases compared to the control group (10% vs. 6% with p = 0.036); the same was true for genotype CYP2C9*2/*2 (7% vs. 3.5% with p = 0.028). Logistic regression analysis evidenced potential association of CYP2C9*2 allele and its genotypes with SIH. When adjusted for confounding factors such as age, weight, sex, mean daily dose of sulphonylurea, and triglyceride level, the association between the CYP2C9*2 allele and hypoglycaemia remained consistent. Confounding factors played no role in SIH (insignificant p-value) because both groups (cases and controls) were closely matched in term of age, weight, sex, mean daily dose of sulphonylurea, and triglyceride levels. Our study suggests that genetic information about a patient's CYP2C9 gene/enzyme can potentially assist physicians in prescribing the most suitable and safest drug, based on their genetic make-up.

Gene polymorphism of cytochrome P450 significantly affects lung cancer susceptibility.

BACKGROUND: Cytochrome P450 (CYPs) are heme proteins involved in the metabolism of a variety of endogenous and exogenous substances and play an important role in the carcinogenesis mechanisms of environmental and hereditary factors. The objective of this study was to investigate how polymorphisms of CYPs correlate with lung cancer (LC) susceptibility. METHODS: Six single nucleotide polymorphisms (SNPs) were genotyped in this study. The chi-square test and unconditional logistic regression model were used to evaluate the correlation between SNPs and LC susceptibility. The expressions and survival data of genes in patients with LC were mined using Oncomine and Kaplan-Meier Plotter database. RESULTS: Four SNPs were found to be significantly associated with the risk of LC development (P < 0.05). The most significant correlation was that the A allele and AA genotype of CYP2D6 rs1065852 were associated with increased risk of LC development (adjusted odds ratio [OR] = 1.35, 95% confidence interval [95%CI] = 1.13-1.60, P = 9.04e-4; OR = 1.83, 95%CI = 1.29-2.59, P = 0.001 respectively). Similar association of this variant was also found in the subgroups of male patients, cases in III-IV stages, positive lymph node, squamous cell carcinomas and adenocarcinomas. Whereas rs1065852 was considered as protective factor in females (adjusted OR = 0.33, 95% CI = 0.16-0.70, P = 0.004). In stratified analyses, the association of CYP24A1 rs2762934, CYP24A1 rs6068816, CYP20A1 rs2043449 polymorphism with LC risk appeared stronger in some subgroups. CYP2D6, CYP24A1 and CYP20A1 are overexpressed in some pathological types of LC (P < 0.05), and high levels of CYP2D6 and CYP20A1 indicate poor and good prognosis of LC, respectively. CONCLUSION: This study revealed that rs1065852, rs2043449, rs2762s934, and rs6068816 of CYPs were associated with LC susceptibility in the Northwestern Chinese Han population; CYP2D6 and CYP20A1 were overexpressed and correlated with prognosis of LC.

Cabozantinib and apixaban: an hitherto unreported interaction.

The use of direct oral anticoagulant in cancer patients is an emerging issue, which seems to be an alternative to low molecular weight heparin. Every year several new drugs are approved as anticancer treatment with possible drug-drug interaction with other drugs such as oral anticoagulant. We describe, for the first time, a case of neutropenia and thrombocytopenia in a patient in treatment with cabozantinib, a novel anticancer treatment used in metastatic renal cell carcinoma, and apixaban with promptly resumption of the toxicity after the interruption of cabozantinib. This case suggest a possible interaction between these two pharmaceutical agents, which merit caution considering the spreading of the two drugs.

The Cytochrome P450-Mediated Metabolism Alternation of Four Effective Lignans From Schisandra chinensis in Carbon Tetrachloride-Intoxicated Rats and Patients With Advanced Hepatocellular Carcinoma.

It is highly valuable to study the pharmacokinetics of herbal components under the pathological condition of liver dysfunction for safe and rational use of herbal medicines. In this study, the pharmacokinetic profiles of four effective lignans from Schisandra chinensis (SC), schisandrin, schisantherin A, deoxyshisandrin and γ-schisandrin, were investigated in carbon tetrachloride (CCl4)-intoxicated rats. The metabolism of the four lignans was also studied using microsomes from patients with advanced hepatocellular carcinoma. In situ intestinal and hepatic perfusions were conducted to clarify the contributions from impairments of gut and liver on the pharmacokinetics of the four schisandra lignans in CCl4-intoxicated rats. The metabolism in rat and human liver microsomes and transport in Caco-2 monolayer cell model were studied to reveal the key factors for the in vivo disposition of the four lignans. When SC alcoholic extract was orally administrated to CCl4-intoxicated rat for a short term (4 days), the pharmacokinetics of four active SC lignans was significantly changed while its hepatotherapeutic effect was not obviously observed. The plasma concentrations of the four schisandra lignans were dramatically elevated compared with the control. The Cmax, AUC and MRT were all increased or prolonged significantly while parameter CLz/F was obviously reduced in rat pretreated with CCl4. In hepatic perfusion study and liver microsomes incubation, it was found that the hepatic metabolism of the four lignans was markedly decreased mainly due to the activity reduction of multiple CYP450 isoenzymes involved the metabolism, which, eventually, might lead to the alternation of their pharmacokinetic profiles in CCl4-intoxicated rats or patients with advanced hepatocellular carcinoma. The pharmacokinetic studies of SC components in pathological situation of liver dysfunction are expected to provide useful data for rational and safe application of SC preparations in clinic or further pharmacological and toxicological research.

Effects of Herbal Formulas Bojungikgi-tang and Palmijihwang-hwan on Inflammation in RAW 264.7 Cells and the Activities of Drug-Metabolizing Enzymes in Human Hepatic Microsomes.

In the present study, Bojungikgi-tang (BJIKT: Buzhongyiqi-tang, Hochuekki-to) and Palmijihwang-hwan (PMJHH: Baweidìhuang-wan, Hachimijio-gan), traditional herbal formulas, investigated anti-inflammatory efficacies in murine macrophage cell line and the influence on the activities of drug-metabolizing enzymes (DMEs). The anti-inflammatory potentials of the herbal formulas were evaluated to inhibit the production of the inflammatory mediators and cytokines and the protein expression of inducible nitric oxide and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-treated RAW 264.7 cells. The activities of the major human DMEs, cytochrome P450 isozymes (CYP450s) and UDP-glucuronosyltransferase isozymes (UGTs), were measured by in vitro enzyme assay systems. BJIKT and PMJHH significantly suppressed the prostaglandin E2 (PGE2) production (IC50 = 317.3 and 282.2 µg/mL, respectively) and the protein expression of COX-2 in LPS-treated RAW264.7 cells. On the human microsomal DMEs, BJIKT inhibited the activities of CYP1A2 (IC50 = 535.05 µg/mL), CYP2B6 (IC50 > 1000 µg/mL), CYP2C9 (IC50 = 800.78 µg/mL), CYP2C19 (IC50 = 563.11 µg/mL), CYP2D6 (IC50 > 1000 µg/mL), CYP2E1 (IC50 > 1000 µg/mL), CYP3A4 (IC50 = 879.60 µg/mL), UGT1A1 (IC50 > 1000 µg/mL), and UGT1A4 (IC50 > 1000 µg/mL), but it showed no inhibition of the UGT2B7 activity at doses less than 1000 µg/mL. PMJHH inhibited the CYP2D6 activity (IC50 = 280.89 µg/mL), but IC50 values of PMJHH exceeded 1000 µg/mL on the activities of CYP1A2, CYP2C19, CYP2E1, and CYP3A4. At concentrations less than 1000 µg/mL, PMJHH did not affect the activities of CYP2B6, CYP2C9, UGT1A1, UGT1A4, and UGT2B7. The results indicate that both BJIKT and PMJHH may be potential candidates to prevent and treat PGE2- and COX-2-mediated inflammatory diseases. In addition, this study will expand current knowledge about herb-drug interactions by BJIKT and PMJHH.

Interaction between 3,4­dichlorophenyl­propenoyl­sec.­butylamine (3,4­DCPB), an antiepileptic drug, and cytochrome P450 in rat liver microsomes and recombinant human enzymes in vitro.

The compound 3,4­dichlorophenyl­propenoyl­sec.­butylamine (3,4­DCPB) is an antiepileptic drug. The purpose of the present research was to identify cytochrome P450 (CYP450) responsible for the metabolism of 3,4­DCPB and evaluate the effects of 3,4­DCPB on the activities of CYP450 enzymes. 3,4­DCPB was incubated with rat liver microsomes (RLMs) plus six CYP450 enzyme-specific inhibitors, or six recombinant human CYP450 enzymes (rhCYP450s). The concentrations of 3,4­DCPB and six CYP450 enzyme-activities probe drugs were detected by high-performance liquid chromatographic (HPLC). The results showed that the prototype of 3,4­DCPB was metabolized by multiple CYP450 enzymes into three metabolites, and the predominant isoforms were CYP2D6 (metabolite M1), CYP1A2 (M2), CYP2C19 and CYP3A4 (M3), respectively., in the presence of ß-NADPH (1 mM) in RLMs or rhCYP450s. Compared with the control (PB-), phenobarbital pre-treatment (PB+) significantly enhanced levels (all of p < 0.01) of hydroxylmethytobutamide (CYP2C9), 4­hydroxy­mephenytoin (CYP2C19), acetaminophen (CYP1A2), 6­hydroxychlorzoxazone (CYP2E1) and oxidized nifedipine (CYP3A4), respectively, in spite of dextrophan (CYP2D6) was not markedly enhanced in RLMs. Conversely, the inhibitory ratios of 3,4­DCPB (16 µg/mL, 59 µM) on the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6 were 97.6%, 59.0%, 53.5% and 36.5%, respectively. However, CYP2E1 (both of PB- and PB+) and CYP3A4 (PB+) were not inhibited by 3,4­DCPB in RLMs. In conclusion, the present study showed that 3,4­DCPB was metabolized by multiple CYP450 enzymes. 3,4­DCPB inhibited the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6, rather than that CYP2E1 and CYP3A4 enzymes, suggesting that the different effects of 3,4­DCPB on the CYP450 enzymes might influence metabolic drug-drug interaction in antiepileptics therapy.

The relevance of cytochrome P450 polymorphism in forensic medicine and akathisia-related violence and suicide.

Adverse drug reactions and interactions are among the major causes of death in the United States. Antidepressants have been reported as causing suicide and homicide and share the class attribute of frequently producing akathisia, a state of severe restlessness associated with thoughts of death and violence. Medical examiners can now identify some pharmacogenetic interactions that cause drugs, deemed safe for most, to be lethal to others. Such deaths do not yet include medication-induced, akathisia-related suicides and homicides. An extrapyramidal side effect, akathisia is a manifestation of drug toxicity whose causes lie, inter alia, in drugs, doses, and co-prescribed medications that inhibit and compete for metabolizing enzymes, which may themselves be defective. In this paper, we report our investigation into adverse drug reactions/interactions in three persons who committed homicide, two also intending suicide, while on antidepressants prescribed for stressful life events. Their histories of medication use, adverse reactions and reasons for changes in medications are presented. DNA samples were screened for variants in the cytochrome P450 gene family; that produce drug metabolizing enzymes. All three cases exhibit genotype-based diminished metabolic capability that, in combination with their enzyme inhibiting/competing medications, decreased metabolism further and are the likely cause of these catastrophic events.

Skatole metabolites in urine as a biological marker of pigs with enhanced hepatic metabolism.

Boar taint is a quality defect in meat, related to accumulation of skatole and androstenone in male pigs. The levels of skatole and its main metabolites in plasma and urine samples were measured with a validated liquid chromatography-MS method and related to activity of hepatic cytochrome P450 (CYP450) in order to identify 'fast metabolizing' pigs. Urine (n=46), blood (n=12), liver (n=25) and adipose tissue (n=46) were sampled from a total of 46 entire male pigs. Skatole levels in fat were negatively correlated to CYP2E1 activity and positively to 3-hydroxy-3-methyloxindole (HMOI), indole-3-carboxylic acid (ICA) and 2-aminoacetophenone in urine. HMOI and ICA levels in urine were the best predictors of high skatole levels in fat. In summary, the present study provided further evidence for the key role of CYP2E1 in skatole metabolism and suggested that measurement of HMOI and/or ICA in urine might provide information about skatole levels in live pigs.

Mutagenic and genotoxic potential of native air borne particulate matter from industrial area of Rourkela city, Odisha, India.

In this study, we examined potential adverse health effect of particulate matter (PM) collected from industrial areas of Rourkela, Odisha, India. Results indicate that PM in these areas contains benzo[a]pyrene in addition to other unidentified molecules. Ames test revealed the above PM to be highly mutagenic. Further studies of PM in HaCaT cells suggest its DNA damaging potential which may lead to apoptosis. Generation of reactive oxygen and nitrogen species following PM exposure may be an early event in the PM induced apoptosis. In addition, the activity of cytochrome P450 (CYP450), the key xenobiotic metabolism enzyme, was found to be increased following PM exposure indicating its role in PM induced toxicity. To confirm this, we used genetic and pharmacological inhibitors of CYP450 like CYP1B1 siRNA and Clotrimazole. Interestingly, we found that the use of these inhibitors significantly suppressed the PM induced apoptosis in HaCaT cells, which confirm the crucial role of CYP1B1 in the toxic manifestation of PM. For further analysis, blood samples were collected from the volunteer donor and analyzed for immunophenotypes and comet assay to survey any change in immune cells and DNA damage in blood cells respectively. The study was performed with 55 blood samples including 32 from industrial areas and 23 people from non-industrial zone of Rourkela city. Samples had a mean±SD age of 35±6.2years (35 men and 20 women). Our investigation did not observe any significant alteration in lymphocytes (P=0.671), B cell (P=0.104), cytotoxic T cell (P=0.512), helper T cell (P=0.396), NK cell (P=0.675) and monocytes (P=0.170) of blood cells from these two groups. Taken together; this study first time reports the possible health hazards of PM from industrial areas of Odisha, India.