Drug-metabolizing Enzyme Genes Research Articles

Pan-cancer single-cell landscape of drug-metabolizing enzyme genes.

Varied expression of drug-metabolizing enzymes (DME) genes dictates the intensity and duration of drug response in cancer treatment. This study aimed to investigate the transcriptional profile of DMEs in tumor microenvironment (TME) at single-cell level and their impact on individual responses to anticancer therapy. Over 1.3 million cells from 481 normal/tumor samples across 9 solid cancer types were integrated to profile changes in the expression of DME genes. A ridge regression model based on the PRISM database was constructed to predict the influence of DME gene expression on drug sensitivity. Distinct expression patterns of DME genes were revealed at single-cell resolution across different cancer types. Several DME genes were highly enriched in epithelial cells (e.g. GPX2, TST and CYP3A5) or different TME components (e.g. CYP4F3 in monocytes). Particularly, GPX2 and TST were differentially expressed in epithelial cells from tumor samples compared to those from normal samples. Utilizing the PRISM database, we found that elevated expression of GPX2, CYP3A5 and reduced expression of TST was linked to enhanced sensitivity of particular chemo-drugs (e.g. gemcitabine, daunorubicin, dasatinib, vincristine, paclitaxel and oxaliplatin). Our findings underscore the varied expression pattern of DME genes in cancer cells and TME components, highlighting their potential as biomarkers for selecting appropriate chemotherapy agents.

Pharmacogenetics and pharmacogenomics in glaucoma therapeutics: the way to personalized therapy.

Pharmacogenetics and pharmacogenomics in glaucoma therapeutics: the way to personalized therapy.

Effects of intermittent fasting on the histology and mRNA expression of major drug-metabolizing cyp450s in the liver of diabetic mice

ABSTRACT Introduction:There is a variation in drug response among patients who practice intermittent fasting. Alteration in the expression of drug-metabolizing enzymes (DMEs) can affect the pharmacokinetics and drug response. Aims: This research aimed to determine the effect of intermittent fasting on the mRNA expression of major drug-metabolizing cyp450s in the liver of diabetic mice. Methods: Thirty-two male Balb/c mice were divided into four groups; control, nonfasting diabetic, non-diabetic fasting, and diabetic fasting mice. Insulin-dependent diabetes was induced in mice by a single high-dose (250 mg/kg) streptozocin. Mice of non-diabetic and diabetic fasting groups were subjected to 10-day intermittent fasting for 17 hours daily. Then, the mRNA expression of mouse phase I DMEs cyp1a1, cyp2c29, cyp2d9, and cyp3a11 was analyzed using real-time polymerase chain reaction. In addition, the liver of mice in all groups was examined for pathohistological alterations. Results: Diabetes downregulated the mRNA expression of hepatic drug-metabolizing cyp450s in diabetic mice, while intermittent fasting significantly (P < 0.05) increased it. Also, cyp2d9 and cyp3a11 were upregulated in the liver of diabetic fasting mice. These alterations in the gene expression were correlated with the pathohistological alterations, where livers of diabetic mice showed dilatation in the blood sinusoids and inflammatory cells leukocyte infiltrations. Whereas livers of diabetic fasting mice showed almost comparable histological findings to control mice. Conclusions: Intermittent fasting can protect the liver against diabetes-induced hepatotoxicity and the down-regulation of DME genes in the diabetic liver. These results can explain, at least partly, the inter-individual variation in the drug response during practicing fasting.

An extraction-free method for rapid detection of CYP2C19 * 2/3/17 polymorphisms in one tube using melting curve analysis.

Drug-metabolizing enzymes play an important role in the metabolism of drugs in vivo. Their activity is an important factor affecting the rate of drug metabolism, which directly determines the intensity and persistence of drug action. Patients taking medication can be divided into different metabolic types through detection of CYP2C19 drug-metabolizing enzyme gene polymorphisms, which can then be used for medication guidance for clopidogrel. Here, we describe a detection method based on real-time polymerase chain reaction. This method uses multicolor melting curve analysis to accurately identify different mutation sites and genotypes of CYP2C19 * 2, CYP2C19 * 3, and CYP2C19 * 17. The detection limit of plasmid samples was 1 copies/μl; that of genomic samples was 0.1ng/μl. The system can detect nine types of CYP2C19 * 2/3/17 at three sites in one tube, quickly achieving detection within 1h. Combined with the sample release agent, sample extraction was completed in 5 s, achieving rapid diagnosis without extraction for timely diagnosis and treatment. Furthermore, the system is not limited to blood samples and can also be applied to oropharyngeal and saliva samples, increasing sampling diversity and convenience. When using clinical blood samples (n=93), the detection system we established was able to quickly and accurately identify different genotypes, and the accuracy and effectiveness of the detection were confirmed by Sanger sequencing. Due to its accuracy, rapidity, simple operation and low cost, detection technology based on real-time polymerase amplification combined with melting curve analysis is expected to become a powerful tool for detecting and guiding clopidogrel use in countries with limited resources. This article is protected by copyright. All rights reserved.

NAT2 phenotype alters pharmacokinetics of rivaroxaban in healthy volunteers

Rivaroxaban is a direct inhibitor of factor Xa, a member of direct oral anticoagulant group of drugs (DOACs). Despite being a widely extended alternative to vitamin K antagonists (i.e., acenocoumarol, warfarin) the interindividual variability of DOACs is significant, and may be related to adverse drug reaction occurrence or drug inefficacy, namely hemorrhagic or thromboembolic events. Since there is not a consistent analytic practice to monitor the anticoagulant activity of DOACs, previously reported polymorphisms in genes coding for proteins responsible for the activation, transport, or metabolism of DOACs were studied. The study population comprised 60 healthy volunteers, who completed two randomized, crossover bioequivalence clinical trials between two different rivaroxaban formulations. The effect of food, sex, biogeographical origin and 55 variants (8 phenotypes and 47 single nucleotide polymorphisms) in drug metabolizing enzyme genes (such as CYP2D6, CYP2C9, NAT2) and transporters (namely, ABCB1, ABCG2) on rivaroxaban pharmacokinetics was tested. Individuals dosed under fasting conditions presented lower tmax (2.21 h vs 2.88 h, β = 1.19, R2 =0.342, p = 0.012) compared to fed volunteers. NAT2 slow acetylators presented higher AUC∞ corrected by dose/weight (AUC∞/DW; 8243.90 vs 7698.20 and 7161.25 h*ng*mg /ml*kg, β = 0.154, R2 =0.250, p = 0.044), higher Cmax/DW (1070.99 vs 834.81 and 803.36 ng*mg /ml*kg, β = 0.245, R2 =0.320, p = 0.002), and lower tmax (2.63 vs 3.19 and 4.15 h, β = −0.346, R2 =0.282, p = 0.047) than NAT2 rapid and intermediate acetylators. No other association was statistically significant. Thus, slow NAT2 appear to have altered rivaroxaban pharmacokinetics, increasing AUC∞ and Cmax. Nonetheless, further research should be conducted to verify NAT2 involvement on rivaroxaban pharmacokinetics and to determine its clinical significance.

Regional Transcriptomics and Proteomics of Pharmacokinetics-Related Genes in Human Intestine.

The intestine is an organ responsible for the absorption and metabolism of orally administered drugs. To predict pharmacokinetics behavior in the small intestine, it is necessary to examine the human intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). In this study, to obtain more accurate expression profiles in various regions of the human intestine, biopsy samples were collected from endoscopically noninflamed mucosa of the duodenum, jejunum, ileum, colon, and rectum from Japanese including Crohn's disease or ulcerative colitis patients, and both RNA-seq and quantitative proteomics analyses were performed. We also analyzed the expression of drug-metabolizing enzymes (cytochromes P450 (CYPs) and non-CYP enzymes), drug transporters, and nuclear receptors. Overall, the mRNA expression levels of these ADME-related genes correlated highly with the protein expression levels. The characteristics of the expression of ADME-related genes differed significantly between the small and large intestines, including the expression levels of CYP enzymes, which were higher and lower in the small and large intestines, respectively. Most CYPs were expressed dominantly in the small intestine, especially the jejunum, but were rarely expressed in the large intestine. On the other hand, non-CYP enzymes were expressed in the large intestine but at lower expression levels than in the small intestine. Moreover, the expression levels of drug metabolizing enzyme genes differed even between the proximal and distal small intestine. Transporters were expressed most highly in the ileum. The data in the present study will enhance understanding of the intestinal ADME of drug candidates and would be useful for drug discovery research.

HepaSH cells: Experimental human hepatocytes with lesser inter-individual variation and more sustainable availability than primary human hepatocytes

Primary human hepatocytes (PHHs) have been commonly used as the gold standard in many drug metabolism studies, regardless of having large inter-individual variation. These inter-individual variations in PHHs arise primarily from genetic polymorphisms, as well as from donor health conditions and storage conditions prior to cell processing. To equalize the effects of the latter two factors, PHHs were transplanted to quality-controlled mice providing human hepatocyte proliferation niches, and engrafted livers were generated. Cells that were harvested from engrafted livers, call this as experimental human hepatocytes (EHH; termed HepaSH cells), were stably and reproducibly produced from 1014 chimeric mice produced by using 17 different PHHs. Expression levels of acute phase reactant (APR) genes as indicators of a systemic reaction to the environmental/inflammatory insults of liver donors varied widely among PHHs. In contrast to PHHs, the expression of APR genes in HepaSH cells was found to converge within a narrower range than in donor PHHs. Further, large individual differences in the expression levels of drug metabolism-related genes (28 genes) observed in PHHs were greatly reduced among HepaSH cells produced in a unified in vivo environment, and none deviated from the range of gene expression levels in the PHHs. The HepaSH cells displayed a similar level of drug-metabolizing enzyme activity and gene expression as the average PHHs but retained their characteristics for drug-metabolizing enzyme gene polymorphisms. Furthermore, long-term 2D culture was possible and HBV infection was confirmed. These results suggest that the stably and reproducibly providable HepaSH cells with lesser inter-individual differences in drug-metabolizing properties, may have a potential to substitution for PHH as practical standardized human hepatocytes in drug discovery research.

Polymorphism of genes encoding drug-metabolizing and inflammation-related enzymes for susceptibility to cholangiocarcinoma in Thailand.

Cholangiocarcinoma (CCA) is an intractable cancer, and its incidence in northeastern Thailand is the highest worldwide. Infection with the liver fluke Opisthorchis viverrini (OV) has been associated with CCA risk. However, animal experiments have suggested that OV alone does not induce CCA, but its combination with a chemical carcinogen like nitrosamine can cause experimentally induced CCA in hamsters. Therefore, in humans, other environmental and genetic factors may also be involved. To examine relations between risk for CCA and genetic polymorphisms in carcinogen-metabolizing and inflammation-related genes. This hospital-based case-control study enrolled 95 case-control pairs matched by age (± 5 years) and sex. We examined relations between risk for CCA and genetic polymorphisms in carcinogen-metabolizing and inflammation-related genes, serum anti-OV, alcohol consumption, and smoking. Polymorphisms of CYP2E1, IL-6 (-174 and -634), IL-10 (-819), and NF-κB (-94) and their co-occurrence with polymorphisms in the drug-metabolizing enzyme gene GSTT1 or GSTM1 were also analyzed. Although CCA risk was not significantly associated with any single polymorphism, persons with the GSTT1 wild-type and CYP2E1 c1/c2 + c2/c2 genotype had an increased risk (OR = 3.33, 95%CI: 1.23-9.00) as compared with persons having the GSTT1 wild-type and CYP2E1 c1/c1 wild genotype. The presence of anti-OV in serum was associated with a 7- to 11-fold increased risk, and smoking level was related to an OR of 1.5-1.8 in multivariable analyses adjusted for each of the seven genetic polymorphisms. In addition to infection with OV, gene-gene interactions may be considered as one of the risk factors for CCA development.

DNMT1/PKR double knockdowned HepG2 (HepG2-DP) cells have high hepatic function and differentiation ability

HepG2 cells are widely used as a human hepatocytes model, but their functions, including drug metabolism, are inferior to primary hepatocytes. We previously reported that the hepatic gene expressions in HepG2 cells were upregulated by treatment with zebularine, which is an inhibitor of DNA methylation, through the inhibition of both DNA methyltransferase 1 (DNMT1) and double-stranded RNA-dependent protein kinase (PKR). In this study, we established a new HepG2 cell subline, HepG2-DP cells, by stable double knockdown of DNMT1 and PKR and evaluated its function. Albumin production, expression of CYP1A2 genes, and accumulation of lipid droplets were increased in HepG2-DP cells compared with the original HepG2 cells. Comprehensive gene expression analysis of transcription factors revealed that the expression of important genes for hepatic function, such as HNF1β, HNF4α, ONECUT1, FOXA1, FOXA2, FOXA3, and various nuclear receptors, was upregulated in HepG2-DP cells. These results indicate that the newly established HepG2-DP cells are a highly functional hepatocyte cell line. In addition, we investigated whether HepG2-DP cells are able to mature by differentiation induction, since HepG2 cells are derived from hepatoblastoma. The gene expression of major CYPs and Phase II, III drug-metabolizing enzyme genes was significantly increased in HepG2-DP cells cultured in differentiation induction medium. These results suggest that HepG2-DP cells can be further matured by the induction of differentiation and could therefore be applied to studies of drug metabolism and pharmacokinetics.

Comparison of genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma in Japanese patients using a novel panel for cancer-related drug-metabolizing enzyme genes

The differences in genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma remain unclear. We developed a customized, targeted gene sequencing panel for efficient and sensitive identification of germline variants, including whole-gene deletion types for cancer-related drug-metabolizing enzyme genes in lung adenocarcinoma and squamous cell carcinoma. The minor allele frequencies of the variants, confirmed as clinically significant in the Japanese population, did not differ significantly from those of normal participants listed in the public database. Genotype analysis comparing lung adenocarcinoma (n = 559) and squamous cell carcinoma (n = 151) indicated that the variants of DPYD (rs190771411, Fisher’s exact test, P = 0.045; rs200562975, P = 0.045) and ALDH2 (rs568781254, P = 0.032) were associated with an increased risk of squamous cell carcinoma compared to adenocarcinoma. Conversely, whole-gene deletion of CYP2A6 was associated with adenocarcinoma but not squamous cell carcinoma. Notably, whole-gene deletion of CYP2A6 was confirmed in 22 patients with lung adenocarcinoma but not in any patients with squamous cell carcinoma. Most patients with whole-gene deletion of CYP2A6 were female non-smokers. The discovery of a whole-gene deletion of CYP2A6 in patients with lung adenocarcinoma may have an important role in clinical practice and advance our understanding of CYP2A6 germline variants and their association with carcinogenesis or their susceptibility to lung adenocarcinoma.

Associations of HLA and drug-metabolizing enzyme genes in co-trimoxazole-induced severe cutaneous adverse reactions

Co-trimoxazole is mainly used as a first-line drug for treatment and prophylaxis against Pneumocystis jiroveci pneumonia. This drug, however, has been reported as the most common causative drug for severe cutaneous adverse reactions (SCARs). This study aimed to extensively elucidate the associations between genetic polymorphisms of HLA class I and genes involved in bioactivation and detoxification of co-trimoxazole on co-trimoxazole-induced SCARS in a large sample size and well-defined Thai SCARs patients. A total of 67 patients with co-trimoxazole-induced SCARs, consisting of 51 SJS/TEN patients and 16 DRESS patients, and 91 co-trimoxazole tolerant controls were enrolled in the study. The results clearly demonstrated that the HLA-B∗13:01 allele was significantly associated with co-trimoxazole-induced SCARs, especially with DRESS (OR = 8.44, 95% CI = 2.66–26.77, P = 2.94 × 10−4, Pc = 0.0126). Moreover, the HLA-C∗08:01 allele was significantly associated with co-trimoxazole-induced SJS/TEN in the HIV/AIDS patients with an OR of 8.51 (95% CI = 2.18–33.14, P = 8.60 × 10−4, Pc = 0.0241). None of the genes involved in the bioactivation and detoxification of co-trimoxazole investigated in this study play any major role in the development of all phenotypes of SCARs.

Efek Polimorfisme Gen Enzim Pemetabolisme Obat terhadap Kadar Rivaroxaban: Sebuah Kajian Naratif

Rivaroxaban is an anticoagulant agent that often experience biotransformation in the liver through CYP3A4/5 (18%); CYP2J2 (14%) and CYP independent hydrolytic cleavage (14%). Several studies revealed that Polymorphism in drug-metabolizing enzyme genes can affect the rivaroxaban levels in the body. Therefore, this study aims to review the effect of gene variations of metabolizing enzymes on rivaroxaban concentration in patient’s body. A search using keyword rivaroxaban, polymorphism, CYP3A4, CYP3A5 and CYP2J2 was carried out in Google Scholar. Several studies showed that polymorphisms of metabolizing genes do not have a significant effect on rivaroxaban concentration. There is also a need for comprehensive studies involving all single nucleotide polymorphisms (SNPs) that play a role in the drug’s pharmacokinetics. This is because rivaroxaban is metabolized by several CYP isoenzymes and excreted by different transporters. This still opens the opportunity for further studies on its pharmacogenomic.

Impact of CYP3A7, CYP2D6 and ABCC2/ABCC3 polymorphisms on tacrolimus steady state concentrations in Bulgarian kidney transplant recipients

Polymorphisms in the genes of drug-metabolizing enzymes have the potential to contribute to inter-individual differences in drug pharmacokinetics and toxicity. A custom next-generation sequencing (NGS) panel was used in 71 kidney transplanted patients to study the polymorphisms of 11 genes relevant to the metabolism of immunosuppressive drugs. Cyclosporine A and tacrolimus concentrations were determined by a validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method. More than 1000 polymorphisms were found in the studied 11 genes, and 45% of them were different non-synonymous variants. Eleven missense mutations were observed in the CYP3A7 gene, resulting in increased metabolism of tacrolimus at day 21 post-transplantation (6.7 µg/L vs. 10.3 µg/L; p = 0.048). Two alleles encoding a cytochrome P450 2D6 enzyme with impaired function—CYP2D6*4 (non-functional) and CYP2D6*10 (decreased function), were found in the studied group and both of them were associated with higher levels of tacrolimus at day 14 (10.1 µg/L; p = 0.021 and 9.7 µg/L; p = 0.036, vs. 7.7 µg/L respectively). Altered function of ABC transporters C3 and C2 was also associated with increased TAC concentration. ABCC3 significantly influenced TAC metabolism by itself, but polymorphisms affecting both ABCC3 and ABCC2 resulted in higher changes: 13.6 µg/L vs. 7.9 µg/L, day 14 (p = 0.003) and 20 µg/L versus 9.3 µg/L, day 21 (p = 0.019). All associations were also checked for variants affecting the activity of CYP3A4 and CYP3A5. Despite its small size, the study points out that the pharmacogenetics of calcineurin inhibitors may also be influenced by other genes besides CYP3A4 and CYP3A5.

Association study identifies genetic determinants and non-genetic factors on steady-state plasma and therapeutic outcome of galantamine in mixed dementia.

This study aimed to evaluate the influence of genetic polymorphisms of drug-metabolizing enzyme genes, transporter gene, pathological gene (APOE), and non-genetic factors on therapeutic outcomes as well as steady-state plasma concentrations (Cpss) of galantamine in Thai patients with mixed dementia. Fifty-one Thai patients with mixed dementia who received galantamine for at least 6months were recruited. CYP2D6, CYP3A5, and ABCB1 polymorphisms were detected by TaqMan® Genotyping Assay. UGT1A1 and APOE polymorphism was detected by direct Sanger sequencing technique and restriction fragment length polymorphism technique. Cpss of galantamine was measured by ultra-performance liquid chromatography. Associations of genetic and non-genetic factors with Cpss and clinical outcomes (change in cognitive function as measured by the Thai Mental State Examination (ΔTMSE) scores) were determined by using univariate and multivariate analysis. The multivariate regression model revealed that patients who carried one or more detrimental allelic variant (CYP2D6, CYP3A5, and UGT1A1) showed a tendency toward a higher galantamine adjusted Cpss (B = 34.559, 95% CI = 0.741-68.377, p value = 0.045). Logistic regression analysis also revealed CYP2D6*10 carriers were significantly associated with higher ΔTMSE (B = 5.227, 95% CI = 2.395-8.060, p value = 0.001). UGT1A1 mutant alleles and non-genetic factors including concomitant use of statin drugs and higher education level can attenuate therapeutic outcomes of galantamine. Pharmacokinetic-related genes including CYP2D6*10 and UGT1A1 mutant alleles were significantly associated with galantamine adjusted Cpss and cognitive function. Determination of Cpss and genotype could be an adjunct examination to provide further explanation in interindividual variability of galantamine therapeutic outcome.

Pharmacogenomics: Relevance and opportunities for clinical pharmacology

Pharmacogenomics: Relevance and opportunities for clinical pharmacology

Use of a Baculovirus-Mammalian Cell Expression-System for Expression of Drug-Metabolizing Enzymes: Optimization of Infection With a Focus on Cytochrome P450 3A4.

Heterologous expression systems are important for analyzing the effects of genetic factors including single nucleotide polymorphisms on the functions of drug-metabolizing enzymes. In this study, we focused on a baculovirus-mammalian cell (Bac-Mam) expression system as a safer and more efficient approach for this purpose. The baculovirus-insect cell expression system is widely utilized in large-scale protein expression. Baculovirus has been shown to also infect certain mammalian cells, although the virus only replicates in insect cells. With this knowledge, baculovirus is now being applied in a mammalian expression system called the Bac-Mam system wherein a gene-modified baculovirus is used whose promotor is replaced with one that can function in mammalian cells. We subcloned open-reading frames of cytochrome P450 3A4 (CYP3A4), UDP-glucuronosyltransferase (UGT) 1A1, and UGT2B7 into a transfer plasmid for the Bac-Mam system, and prepared recombinant Bac-Mam virus. The obtained virus was amplified in insect Sf9 cells and used to infect mammalian COS-1 cells. Expression of CYP3A4, UGT1A1, and UGT2B7 in COS-1 cell homogenates were confirmed by immunoblotting. Optimum infection conditions including the amount of Bac-Mam virus, culture days before collection, and concentration of sodium butyrate, an enhancer of viral-transduction were determined by monitoring CYP3A4 expression. Expressed CYP3A4 showed appropriate activity without supplying hemin/5-aminolevulinic acid or co-expressing with NADPH-cytochrome P450 reductase. Further, we compared gene transfer efficiency between the Bac-Mam system and an established method using recombinant plasmid and transfection reagent. Our results indicate that the Bac-Mam system can be applied to introduce drug-metabolizing enzyme genes into mammalian cells that are widely used in drug metabolism research. The expressed enzymes are expected to undergo appropriate post-translational modification as they are in mammalian bodies. The Bac-Mam system may thus accelerate pharmacogenetics and pharmacogenomics research.

Association of Drug-Metabolizing Enzyme and Transporter Gene Polymorphisms and Lipid-Lowering Response to Statins in Thai Patients with Dyslipidemia.

PurposeStatins are increasingly widely used in the primary and secondary prevention of cardiovascular disease. However, there is an inter-individual variation in statin response among patients. The study aims to determine the association between genetic variations in drug-metabolizing enzyme and transporter (DMET) genes and lipid-lowering response to a statin in Thai patients with hyperlipidemia.Patients and MethodsSeventy-nine patients who received statin at steady-state concentrations were recruited. Serum lipid profile was measured at baseline and repeated after 4-month on a statin regimen. The genotype profile of 1936 DMET markers was obtained using Affymetrix DMET Plus genotyping microarrays.ResultsIn this DMET microarray platform, five variants; SLCO1B3 (rs4149117, rs7311358, and rs2053098), QPRT (rs13331798), and SLC10A2 (rs188096) showed a suggestive association with LDL-cholesterol-lowering response. HDL-cholesterol-lowering responses were found to be related to CYP7A1 gene variant (rs12542233). Seven variants, SLCO1B3 (rs4149117, rs7311358, and rs2053098); SULT1E1 (rs3736599 and rs3822172); and ABCB11 (rs4148768 and rs3770603), were associated with the total cholesterol-lowering response. One variant of the ABCB4 gene (rs2109505) was significantly associated with triglyceride-lowering response.ConclusionThis pharmacogenomic study identifies new genetic variants of DMET genes that are associated with the lipid-lowering response to statins. Genetic polymorphisms in DMET genes may impact the pharmacokinetics and lipid-lowering response to statin. The validation studies confirmations are needed in future pharmacogenomic studies.

Dill Shows Potential for Herb-Drug Interactions via Up-Regulation of CYP1A2, CYP2C19, SULT1A1, NAT2 and ABCB1 in Caco-2 Cells.

&lt;b&gt;Background and Objective:&lt;/b&gt; Dill&lt;i&gt; &lt;/i&gt;(&lt;i&gt;Anethum graveolens&lt;/i&gt; L.) has the potential to develop as a new alternative medicine due to its pharmacological activities. However, studies into its safety regarding herb-drug interactions have been neglected. This study investigated the risk of dill-induced herb-drug interactions (HDI) by examining its effect on the expression of phase I and II drug-metabolizing enzyme and transporter genes in Caco-2 cells. &lt;b&gt;Materials and Methods:&lt;/b&gt; Caco-2 cells (5×10&lt;sup&gt;5&lt;/sup&gt; cells/well) were treated with 10 μM ketoconazole, 20 μM rifampicin or dill extract (60-240 μg mL&lt;sup&gt;1&lt;/sup&gt;) for 72 hrs. Cell viability was assessed using the resazurin assay and reactive oxygen species (ROS) content was determined with 2 ,7 -dichlorofluorescein diacetate. Aspartate (AST) and alanine aminotransferase (ALT) levels were measured using L-aspartate and L-alanine with α-ketoglutarate as substrate. Expression of phase I (&lt;i&gt;CYP1A2&lt;/i&gt;, &lt;i&gt;CYP2C19&lt;/i&gt;, &lt;i&gt;CYP2D6&lt;/i&gt;, &lt;i&gt;CYP2E1 &lt;/i&gt;and &lt;i&gt;CYP3A4&lt;/i&gt;) and II (&lt;i&gt;UGT1A6&lt;/i&gt;,&lt;i&gt; SULT1A1&lt;/i&gt;,&lt;i&gt; NAT1&lt;/i&gt;,&lt;i&gt; NAT2 &lt;/i&gt;and&lt;i&gt; GSTA1/2&lt;/i&gt;) metabolizing genes and transporters (&lt;i&gt;ABCB1&lt;/i&gt;,&lt;i&gt; ABCC2&lt;/i&gt;,&lt;i&gt; ABCG2 &lt;/i&gt;and &lt;i&gt;SLCO1B1&lt;/i&gt;) were determined by RT/qPCR. &lt;b&gt;Results:&lt;/b&gt; All tested concentrations of dill did not affect cell viability or AST and ALT levels. The highest concentration of dill extract (240 μg mL&lt;sup&gt;1&lt;/sup&gt;) significantly lowered the ROS level. Expression of &lt;i&gt;CYP1A2&lt;/i&gt;, &lt;i&gt;CYP2C19&lt;/i&gt;, &lt;i&gt;SULT1A1&lt;/i&gt;, &lt;i&gt;NAT2 &lt;/i&gt;and &lt;i&gt;ABCB1 &lt;/i&gt;mRNA was significantly up-regulated by dill extract. &lt;b&gt;Conclusion:&lt;/b&gt; Dill extract did not directly damage Caco-2 cells but prolonged use of dill may increase the risk of HDI via the up-regulation of the drug-metabolizing genes &lt;i&gt;CYP1A2&lt;/i&gt;, &lt;i&gt;CYP2C19&lt;/i&gt;, &lt;i&gt;SULT1A1&lt;/i&gt;, &lt;i&gt;NAT2 &lt;/i&gt;and the transporter &lt;i&gt;ABCB1&lt;/i&gt;.

CYP2C9 Variations and Their Pharmacogenetic Implications Among Diverse South Asian Populations.

IntroductionAllelic frequency distribution of drug metabolizing enzyme genes among populations is important to identify risk groups for adverse drug reaction and to select representative populations for clinical trials. Although India emerged as an important hub for clinical trials, information about the pharmacogenetic diversity for this region is still lacking. Here, we investigated genetic diversity of cytochrome-P450-2C9 (CYP2C9) gene which metabolizes wide range of drugs and is highly expressed in the human liver.MethodsIn total, 1278 individuals from 36 diverse Indian populations, 210 individuals from in-house data-repository and 489 other South Asian samples from the 1000 Genomes Project were selected. Variants observed in CYP2C9 gene were subjected to various statistical analyses.ResultsHigh frequency of CYP2C9*3 (~13%) and CYP2C9*3/*3 (~1%) was observed among South Asians, compared to 21 populations living outside the Indian subcontinent. The allelic/genotypic frequency does not correlate with geographical location or linguistic affiliation, except populations speaking Tibeto-Burmans language, who have lower frequency of CYP2C9*3 and CYP2C9*3/*3. Since, South Asians practice strict endogamy, presence of unique mutation and high frequency of homozygous genotypes not surprising. CYP2C9*3 has been associated with therapeutic response.The effect of CYP2C9*3/*3 is more pronounced compared to heterozygous and wild type homozygous genotypes as evident in many in vitro studies. As South Asians have high frequency, it would be interesting to explore potential of CYP2C9*3 as a marker for personalized therapy. Our study revealed several rare functional variants, which form eight novel and rare haplotypes of CYP2C9 (CYP2C9*63–*70). Of which, CYP2C9*64, *65, *66, *68, *69 and *70 haplotypes are South Asian-specific.ConclusionOverall, we find high genetic heterogeneity within South Asians and identified South Asian-specific putative functional CYP2C9 haplotypes. High frequency of CYP2C9*3 and CYP2C9*3/*3 was observed in South Asian populations. Taken together, current study greatly enriches the knowledge of naturally occurring CYP2C9 variants and its diversity in South Asia, which are relevant to further CYP2C9-related functional research and for personalized medicine.

Association of ABCB1 and CYP450 Gene Polymorphisms and their DNA Methylation Status with Steroid-Induced Osteonecrosis of the Femoral Head in the Chinese Population.

Objective: Osteonecrosis of the femoral head (ONFH) is a severe pathological state with multiple etiologies. Steroid hormone metabolism-related genes play an important role in ONFH. The aim of this study was to investigate the relationships between polymorphisms of the drug-metabolizing enzyme gene, cytochrome P450 (CYP450), and the drug transporter gene, ATP-binding cassette subfamily B member 1 (ABCB1), as well as their DNA methylation status with the pathogenesis of steroid-induced ONFH. Methods: In this case-control study, we evaluated five single nucleotide polymorphisms (SNPs) in two genes in a Han Chinese population, including 79 patients with steroid-induced ONFH and 114 persons who took steroids but did not develop steroid-induced ONFH. SNPs were genotyped by the improved multiplex ligation detection reaction. MethylTarget technology was used to ascertain the methylation status at two CpG islands in the ABCB1 gene for statistical analysis. Finally, interactions between the SNPs and the CpG site's methylation levels were statistically analyzed by methylation quantitative trait locus. Results: We found that the T allele of the CYP450 rs2242480 locus was associated with steroid-induced ONFH risk reduction (odds ratio [OR] = 0.598, 95% confidence interval [CI]: 0.360-0.992, p = 0.046). In the genetic model analysis, the T allele of the rs2032582 locus in the ABCB1 gene was associated with a reduced risk of steroid-induced ONFH under the dominant model (OR = 0.465, 95% CI: 0.223-0.972, p = 0.042). The CpG sites with significant differences (p < 0.05) in methylation levels between the cases and controls were ABCB1_1_192…ABCB1_2_43. A total of 14 pairs of linear regression tests between SNPs and methylation sites demonstrated statistical significance (p < 0.05). Conclusions: This study provides evidence for two steroid-induced ONFH susceptibility genes (ABCB1, CYP450) in the Han Chinese population.