CYP3A4 Expression Research Articles

Abstract C008: Pharmacokinetic analysis of vincristine and its m1 metabolite in Kenyan pediatric cancer patients through co-modeling

Abstract Objectives: The aim of this study was to develop a pharmacokinetic (PK) model that can determine the PK of vincristine (VCR) and its M1 metabolite using concentration data from Kenyan pediatric cancer patients. VCR is one of the most widely used anticancer agents in treating a variety of malignancies in pediatric oncology. Regardless of its extensive pediatric use, dosing strategies for VCR are largely empirical as there is little information about its disposition and optimal therapeutic dosing. Our collaborator, Dr. Jamie Renbarger and her group have reported that CYP3A5 enzyme generates the major VCR metabolite, M1, more efficiently than CYP3A4 enzyme. This finding maybe clinically significant because CYP3A5 expression varies with up to 90%, 70% and 10-20% of expressions in Kenyans, African Americans and Caucasians, respectively. Therefore, it is crucial to monitor M1 and characterize its disposition in humans to provide an insight of inter-ethnic variability in VCR metabolism and clearance. Methods: Kenyan pediatric cancer patients (8 males:8 females, age range: 1-13 years, weight range: 7.0 - 36.6 kg, body surface area: 0.36 - 1.24 m2) were intravenously administered with VCR (delivered dose, 0.4 - 2.5 mg). Blood samples were collected using human dried blood spot (DBS) collection paper via finger prick at various time points depending on the feasibility and duration of patient stay for care in the hospital. Using a developed LC-MS-MS assay, the concentrations of VCR and M1 were measured from patient DBS samples. A PK compartmental model was developed through co-modeling of VCR and M1 using Phoenix (version 8.0). The model was selected based on comparison of quality of fit plots and on the likelihood ratio test on the difference of criteria (-2LL). Results: The best fit structural model for VCR and its M1 metabolite was established. The model consists of a one-way metabolite formation transfer from VCR to M1 compartments. Good correlations were observed between observed and predicted values of VCR and M1 in the subjects used. The best-fit PK parameter estimates were derived from the PK compartmental model. In one subject, values estimated for PK parameters that only our study could derive include: conversion rate constant from VCR to M1 of 0.04 1/hr, M1 metabolite volume of distribution of 51.49 L, and M1 metabolite elimination rate constant of 0.44 1/hr. Conclusion: For the first time, a compartmental PK model that could determine the PK parameters of VCR and its M1 metabolite from DBS samples through co-modeling was developed. The model resulted in a good fit for the subjects used. The model will be validated after further adjustments and could potentially be used to rationally modify future VCR dosing regimen for Kenyan pediatric patients. Further, this modification could be possibly extrapolated for African American pediatric patients. Additionally, using our validated model, we will characterize and predict the population PK of VCR and its M1 metabolite among different ethnic groups. Citation Format: Lorita Agu, Jamie Renbarger, Diana S-L. Chow. Pharmacokinetic analysis of vincristine and its m1 metabolite in Kenyan pediatric cancer patients through co-modeling [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr C008.

Abstract B017: Role of CYP3A5 in modulating androgen receptor signaling and its relevance in African American prostate cancer patients

Abstract Introduction: African-American men (AA) often present with aggressive castration-resistant prostate cancer (CRPC), due to highly active androgen receptor (AR). AR is a ligand-activated transcription factor that promotes expression of genes responsible for cell proliferation and growth. Previously, we have shown that CYP3A5 promotes AR nuclear translocation and activation leading to increased prostate cancer (PC) growth. 73% of AAs carrying wild-type CYP3A5 (*1/*1) express full-length functional CYP3A5, whereas 95% of Caucasians carry mutant (*3/*3) variant producing truncated nonfunctional protein. Difference in CYP3A5 expression leads to highly active AR and aggressive disease in AAs. Additionally, most of the PC patients are prescribed concomitant medications to manage age-related comorbidities. Many of these drugs are known modulators of CYP3A5; modulation of CYP3A5 may alter efficacy of ADT in these patients. Our work is focused on characterizing the effect of CYP3A5 modulating drugs on AR signaling in AAs. Methods: q-RT-PCR based profiler assay was used to study effect of CYP3A5 modulation on AR-regulated genes using cDNA from nontarget (NT) and CYP3A5 siRNA treated cells. Confocal microscopy and cell fractionation assays were performed to evaluate and confirm the effect of CYP3A5 modulating drugs on AR nuclear translocation. Genes shortlisted from q-PCR based profiler assays were analyzed for change of expression in response to CYP3A5 modulating drugs. MDAPCA2b (AA origin, *1/*3) and LNCaP (Caucasian, *3/*3) cell lines were used for above experiments. Results: CYP3A5 siRNA pool treatment downregulates AR regulated genes identified using q-PCR based profiler assay, performed with cDNA from CYP3A5 siRNA pool and NT treated MDAPCA2b cells. These downregulated genes include SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1 and SPDEF with p-value of ≤0.005. These genes are known to regulate AR nuclear translocation, cell cycle progression, and cell growth. CYP3A5 siRNA treated MDACPA2b/ LNCaP cells showed decreased AR nuclear translocation and PSA production. Commonly prescribed drugs that are either CYP inhibitors (amiodarone, ritonavir) or inducers (phenytoin, rifampicin) were tested for their ability to alter AR signaling. The results show that the CYP inducers promoted AR nuclear migration and downstream signaling whereas CYP3A5 inhibitors blocked it. Further, CYP3A5 siRNA treated MDAPCa2b cells do not show any increase in AR nuclear migration with phenytoin treatment (CYP3A inducer), confirming that the activation of AR activity is specific to changes in CYP3A5 activity. Conclusions: Concomitantly prescribed CYP3A5 modulating drugs can alter downstream AR signaling, cell growth, and ADT efficacy in men, more so in AAs expressing wild-type CYP3A5. We propose to further characterize drug-induced CYP3A5 modulation of AR signaling to develop a guideline for physicians coprescribing CYP3A5 modulating drugs to treat comorbidities in elderly patients undergoing ADT. Citation Format: Priyatham Gorjala, Oscar B. Goodman Jr, Ranjana Mitra. Role of CYP3A5 in modulating androgen receptor signaling and its relevance in African American prostate cancer patients [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B017.

Cytochrome P450 expression-associated multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD) in HepG2 cells

Purpose: To establish a free fatty acid (FFA)-induced non-alcoholic fatty liver disease (NAFLD) model in HepG2 cells.Methods: HepG2 cells were incubated with 0.1, 1, or 5 mM oleic acid (OA) or palmitic acid (PA) for 24 h. Histological features were examined by oil-red-O staining. Expression levels of metabolic genes (peroxisome proliferator activated receptors α/γ, sterol regulatory element binding proteins 1a/1c, acetyl-CoA carboxylase, acyl-CoA oxidase, and fatty acid synthase), antioxidative genes (catalase and superoxide dismutases 1/2), and cytochrome P450 genes (CYP1A2, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP4A11) were determined by reverse transcription-real time polymerase chain reaction (RT-qPCR).Results: Intracellular lipid storage was observed in cells treated with 1 mM OA or PA while cell shrinkage was present at 5 mM concentrations of both. Expression of all metabolic genes were elevated by 1 mM PA and 5 mM OA and PA. Expression of all antioxidative genes were diminished by 5 mM OA whereas 5 mM PA only reduced superoxide dismutase-2 expression. Expression of CYP1A2, CYP2D6, and CYP3A4 genes were down-regulated by both FFAs, CYP2C19 was induced by PA, while CYP2E1 and CYP4A11 were up-regulated in a concentration-dependent manner.Conclusion: PA was the more potent steatogenic agent in an OA- or PA- induced NAFLD model in HepG2 cells. Increase in intracellular hepatic lipid and expression of metabolic genes, suppression of antioxidative genes, suppression of CYP1A2, CYP2D6, and CYP3A4, and induction of CYP2E1 andCYP4A11 correlated with the multiple-hit pathogenesis model of NAFLD. These findings suggest that PA-induced NAFLD model in HepG2 cells is a suitable in vitro model for studying novel therapeutic approaches to NAFLD treatment.
 Keywords: NAFLD, Multiple-hit pathogenesis, Free fatty acid, Oleic acid, Palmitic acid

Placenta-derived mesenchymal stem cells enhance human liver organoid maturation: translational implications for liver regeneration

Background & Aim The global prevalence of end-stage liver disease (ESLD) has reached very high levels, and, to date, the best treatment option is orthotopic liver transplantation (1). However, due to the complexity of this procedure and the limited numbers of healthy organ donors, new therapeutic options are being sought. Liver progenitor and multipotent stem cells offer potential cell sources that could be used clinically (2). Different studies have reported therapeutic effects of transplanted mesenchymal stem cells (MSCs) on ESLD (3). Indeed, an improvement of liver function has been achieved in patients with liver cirrhosis after autologous MSCs injection (4). Based on our recent work where we showed that a three-dimensional (3D) culture method is effective to improve MSCs paracrine activity (5), in this work, we used a human liver organoid system to study if the paracrine signal secreted by human amnion-derived MSCs (hAMSCs) affect liver stem/progenitor cells to differentiate (Fig. 1). Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ function to model and treat human disease (6). Methods, Results & Conclusion Our data show that the expanded human liver organoids had distinct traits in the ALB production, Krt19 and HNF1β expression when compared to the reference cell line HepG2 cells. On liver organoids, we tested conventional differentiation medium (DM) conditioned by both 2D (2D CM) and 3D (3D CM) hAMSCs cultures observing that ALB, NTCP (markers of mature hepatocytes), and LGR5 (stem cell marker) expression were significantly upregulated by 3D CM compared with both 2D CM and conventional DM. Furthermore, CYP3A4 expression (marker of mature hepatocytes) was upregulated in both 2D and 3D CM. These data were also confirmed by the over-production/activity of both ALB and CYP3A4 observed in organoids grown with 3D CM. Liver epithelial dysfunctional repair play a role in the development of liver disease, and, effective repair likely requires the normal functioning of liver stem/progenitor cells. Herein, we showed that hAMSCs, principally grown in 3D cultures, has the potential to increase hepatic stem/progenitor cell differentiation, demonstrating that soluble factors secreted by hAMSCs are likely responsible for the response. This work revealing a potential approach to improve cell transplantation with the ultimate goal being efficient liver regeneration.

Silybin enhances the blood concentration of brexpiprazole in rats by downregulating expression of CYP3A4 and CYP2D6.

In this study, we investigated the effect of silybin on the pharmacokinetics of brexpiprazole and the underlying mechanism in rats. Two groups of animals received silybin at different doses (50 mg/kg, 25 mg/kg) for 2 weeks, while another group was given vehicle alone. After that, rats were intragastrically administrated with 2 mg/kg brexpiprazole. Then, the tail blood and liver tissues were collected from each rat at different time points. Brexpiprazole in serum was determined by an established UPLC-MS/MS assay. Finally, pharmacokinetic parameters of animals in each group were figured out. The results show that silybin remarkably changed the pharmacokinetic properties of brexpiprazole, especially at the highest dose. AUC and Cmax in the combination group with 50 mg/kg silybin were enhanced approximately 4 times as much as after a single dose of brexpiprazole, p < 0.05. Meanwhile, total liver protein of each sample was extracted, and was subjected to immunoblotting assay for probing CYP3A4 and CYP2D6. Therein CYP3A4 was significantly downregulated compared to the control group. Overall, silybin can increase blood concentration of brexpiprazole in rat by downregulating its main metabolic enzyme CYP3A4. Therefore, the maintenance dose of brexpiprazole should be decreased while co-treating with silybin.

Effects of supplementing yeast fermentate in the feed or drinking water on stress susceptibility, plasma chemistry, cytokine levels, antioxidant status, and stress- and immune-related gene expression of broiler chickens

This study aimed to elucidate the mechanism by which adding Saccharomyces cerevisiae–derived yeast fermentate to the feed (XPC) or drinking water reduces stress in poultry. Day-old male Cobb 500 broiler chicks were assigned to 1 of 3 treatments: stressed control (CS), stressed + XPC (1.25 kg/metric ton feed, day 0–43; XPC), or stressed + AviCare (160 mL/100 L drinking water, day 0–43; AVI). All birds were spray-vaccinated for coccidiosis (day 0), raised on reused litter, spray-vaccinated for Newcastle/Bronchitis (day 18), and exposed to heat stress (32°C–34°C) and feed/water withdrawal for 12 h (day 18). Blood samples were collected to assess plasma corticosterone (CORT) and heterophil/lymphocyte (H/L) ratio (60 birds/treatment; day 40); plasma biochemistry and growth hormone (12 birds/treatment; day 38); and serum serotonin and plasma prolactin, thyroid hormones, antioxidant capacity, and selected cytokines (12 birds/treatment; day 39). Composite asymmetry scores were obtained from 60 birds/treatment on day 41. Organs were collected from 20 birds/treatment on day 43 to measure gene expression of CYP1A2 and melanocortin 2 receptor (MC2R) in the adrenal glands and IL10 and AvBD1 in the spleen. Serotonin was lower in CS than XPC (P = 0.049), whereas AVI was intermediate. Plasma interleukin (IL)-1β was higher in AVI than CS (P = 0.009) and XPC (P = 0.009). The CS treatment had higher CORT than AVI (P = 0.013) and XPC (P = 0.037) and higher H/L ratios than AVI (P = 0.026) and XPC (P = 0.034). Expression of CYP1A2, MC2R, and IL10 was lower (P < 0.05) in XPC and AVI compared with CS. Furthermore, IL10 expression was lower in XPC than AVI (P < 0.05). Adding yeast fermentate to the feed or drinking water reduced measures of stress and MC2R gene expression in birds exposed to acute and rearing stressors. However, differences in IL10 gene expression and circulating serotonin and IL-1β suggest that supplementing yeast fermentate in the feed is slightly more effective than supplementation via the drinking water in mitigating the physiological effects associated with the stress response in broilers.

Cis-acting regulatory elements regulating CYP3A4 transcription in human liver.

The CYP3A4 enzyme is the most abundant drug-metabolizing enzyme in the liver, metabolizing ~50% of commonly used medications. CYP3A4 displays large interperson variability in expression and enzyme activity with unknown causes. This study aims to identify cis-acting regulatory elements controlling the transcription of CYP3A4, using chromatin conformation capture (4C and 3C assays), chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR), clustered regularly interspaced short palindromic repeats (CRISPR)-mediated deletions of genomic regions and reporter gene assays in primary culture human hepatocytes and hepatic cell lines. 4C assays identified four regions (R1-R4) interacting with the CYP3A4 promoter, one of which overlaps with the previously identified upstream enhancers CLEM4/XREM (R2) while the other three are novel. ChIP-qPCR, reporter gene assays and CRISPR-mediated deletion experiments indicate regulatory roles for both R2 and R4. Interestingly, the deletion of R4 increased CYP3A4 while decreasing CYP3A43 expression, possibly due to competitive domain-domain interactions within the CYP3A cluster, supported by deletion of R4 increasing interaction between the CYP3A4 promoter and R2. We also identified a single nucleotide polymorphism rs62471956 within R4, with the variant allele A having increased transcriptional activity in a reporter gene assay. The rs62471956 A allele is associated with higher CYP3A43 expression and lower CYP3A4 expression in a cohort of 136 liver samples, further supporting the opposing effects of R4 on CYP3A4 and CYP3A43. rs62471956 is in complete linkage disequilibrium with CYP3A4*22, potentially contributing to reduced expression of CYP3A4*22. These results validate previously identified enhancers (CLEM4 and XREM) of CYP3A4 and demonstrate additional regulatory mechanisms underlying CYP3A4 transcriptional control via competitive domain-domain interactions within the CYP3A cluster.

Trigonella stellata reduced the deleterious effects of diabetes mellitus through alleviation of oxidative stress, antioxidant- and drug-metabolizing enzymes activities

Ethnopharmacological relevanceGenus Trigonella has a history of folkloric medicinal uses in China, Japan, Egypt and India. There are a variety of therapeutic actions of Trigonella including hypocholesterolemia, hypoglycemia, antibacterial, antiviral, anti-inflammatory activities, antioxidants and appetite stimulant. Aim of the studyThe prevalence of diabetes mellitus is increasing annually. The present study aims at investigating the protective effects of Trigonella stellata against the adverse effects of diabetes mellitus through investigation of the changes in phase I & II drug-metabolizing enzyme activities, protein expression of cytochrome P450 isoenzymes [CYP2E1 & 3A4], oxidative stress, antioxidant enzymes as well as histopathology of both liver and kidney tissues. MethodsGC-MS and MALDI-TOF were used to analyze the main constituents of the aqueous and the ethanolic extract of Trigonella stellata. Western immunoblotting technique used to show the protein expression of CYP450 isozymes in different groups. Spectrophotometric- and fluorophotometric techniques were also used for assessment of different hepatic integrity enzymes. Histopathological techniques used to illustrate the changes in the tissues of both livers and kidneys after different treatments. ResultsTrigonelline was found to be the main constituent of both aqueous and ethanolic extract of Trigonella stellata. Administration of the aqueous and/or the ethanolic extracts of Trigonella stellata to the diabetic rats was found to decrease the blood glucose level, the biochemical markers of both liver (transaminases activities, Lactate dehydrogenase, gamma-glutamyl transferase) and the renal functions (urea, creatinine and bilirubin) which were increased in diabetic-treated rats relative to their normal levels. Diabetes mellitus potentially induced the oxidative stress, and also activities of dimethylnitrosamine N-demethylase I, cytochrome c-reductase, ethoxyresourfin O-deethylase, and the total hepatic content of cytochrome P450. On the other hand, the activity of catalase [CAT], superoxide dismutase [SOD], glutathione S-transferase [GST], glutathione reductase [GR], glutathione peroxidase [GPx] and levels of reduced glutathione [GSH] were potentially inhibited in diabetic rats compared to the control rats. However, treatments of diabetic rats with either aqueous and/or ethanolic extracts of Trigonella stellata restored such changes caused by diabetes almost nearly to their normal levels compared to the control group. Supporting the activity of dimethyl nitrosamine N-demethylase I activity, the protein expression of CYP2E1 was also induced in diabetic rats. However, the aqueous extract of Trigonella stellata was more effective than ethanolic extract in restoring the changes in the protein expression of CYP2E. On the other hand, the protein expression of CYP3A4 was markedly decreased in diabetic rats, and this decrease was partially restored to its normal level after treatment of diabetic rats with aqueous and/or ethanolic extracts. In addition, Trigonella stellata extracts alleviated the histopathological changes in livers and kidneys caused by diabetes mellitus. ConclusionIt is concluded that diabetes mellitus induced changes in oxidative stress, phase I & II drug-metabolizing enzymes, and antioxidant enzymes activities, whereas both extracts of Trigonella stellata alleviated such changes. Alterations in cytochrome P450 system should be considered when therapeutic drugs are administered to diabetic patients since most of xenobiotic are mainly metabolized by this system.

Establishment of a novel CYP3A4-transduced human hepatic sinusoidal endothelial cell model and its application in screening hepatotoxicity of pyrrolizidine alkaloids

Pyrrolizidine alkaloids (PAs) are extensively distributed in plants and are known to damage hepatic sinusoidal endothelial cells (HSECs) via metabolic activation mediated by hepatic cytochrome P450 enzymes (CYPs), particularly the CYP3A4 isozyme. Different PAs have distinct toxic potencies and their toxic effects on HSECs are difficult to be determined in cultured cells, because HSECs lack the key CYP3A4 isozyme for metabolic activation. This study aims to establish a novel, convenient and reliable CYP3A4-expressing HSEC model using human HSECs transduced with lentivirus carrying CYP3A4-ires-eGFP, for evaluating the hepatotoxicity of different PAs on their target HSECs. The developed CYP3A4-expressing HSEC (HSEC-CYP3A4) model was verified by the expression of GFP and CYP3A4 and by the ability to metabolize nifedipine, a classic CYP3A4 substrate. Treated with retrorsine, a representative toxic PA, HSEC-CYP3A4 cells showed significantly reduced cell viability, depletion of GSH, and increased formation of pyrrole-protein adducts. Furthermore, this newly developed cell model successfully discriminated the cytotoxic potency of different PAs evidenced by their IC40 values. In conclusion, the established HSEC-CYP3A4 cell model can be used as a rapid screening platform for assessing the relative potencies of individual PAs on their target HSECs and for investigating the mechanisms underlying PA-induced hepatic sinusoidal damage.

Novel Contributions of Neutrophil‐derived Myeloperoxidase and Hypochlorous Acid to 20‐HETE Production that drives Post‐Ischemic Angiogenesis

Objective20‐hydroxy‐eicosatetraenoic acid (20‐HETE), an arachidonic acid metabolite produced by cytochrome P450 (CYP) 4A/F ω‐hydroxylases, was recently uncovered as a novel contributor to ischemia‐induced angiogenesis. The current study aims to determine whether neutrophil‐derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) mediate post‐ischemic 20‐HETE increases that drive angiogenesis and its underlying mechanism.MethodsMice were subjected to a femoral artery ligation with the contralateral hindlimb acting as the non‐ischemic control. Hindlimb angiogenesis was assessed by laser doppler blood perfusion imaging (LDPI) as well as quantifying the microvascular density in the gracilis muscles where angiogenesis is taking place. Endothelial cells (EC) in culture were subjected to physiological concentrations of MPO and HOCl. Gracilis muscles and EC were analyzed for 20‐HETE production by LC/MS/MS. The mRNA expression of the 20‐HETE synthesizing enzyme, CYP4A11 was measured by real‐time PCR. Protein expression of CYP4A11 and HIF1a were analyzed using western blotting. The rate and mechanism of HOCl entry into EC was evaluated using live‐cell fluorescence microscopy with a HOCl‐specific indicator dye (FDOCl‐1) in the presence/absence of chloride channel blocker 9‐anthracenecarboxylic acid (9‐Ac) and/or gap junction inhibitor 18‐beta‐glycyrrhetinic acid (18β‐GA).ResultsIn animals that were selectively depleted of neutrophils, ischemia failed to induce 20‐HETE increases in the gracilis muscle compared to the controls (13 ± 1.5 vs 35 ± 5 pg/mg of protein). Additionally, MPO‐null mice failed to show increased post‐ischemic 20‐HETE (~2 ± .25 vs 35 ± 5 pg/mg of protein) as well. Both animals display a decreased post‐ischemic angiogenenic phenotype. In EC, the addition of MPO or HOCl markedly stimulated the expression of CYP4A11 and production of 20‐HETE (40 ± 12 vs 8 ± 5 pg/mg of protein). Upregulation of CYP4A11 transcript/protein (&gt;2.5‐fold) and HIF1a protein expression (2‐fold) was observed as early as 15 minutes post‐HOCl exposure. The mechanism of HOCl entry was also exmained by FDOCl‐1 fluorescence quantification, which was markedly decreased in EC pre‐treated with 9‐Ac or 18β‐GA by approximately 40% and 60%, respectively.ConclusionOur study demonstrates for the first time the contribution of MPO and HOCl in promoting 20‐HETE production that drives post‐ischemic angiogenesis. Moreover, this study aims to further expand on the known mechanisms involved in regulating ischemia‐induced angiogenesis. These new findings have high clinical relevancy in studying vascular pathologies by identifing novel mechanisms that contribute to pathological angiogenesis.Support or Funding InformationThis study was supported by grants from AHA[11SDG6870004 (AMG) and 17GRNT33430003 (AMG)], the NIH [HL34300 (M.L.S.) and grant DK38226 (J.R.F.)]; &amp; the R.A. Welch Foundation [GL625910 (J.R.F.)].

Long noncoding RNA LINC00844‐mediated molecular network regulates expression of drug metabolizing enzymes and nuclear receptors in HepaRG cells

Noncoding RNAs, such as long noncoding RNAs (lncRNAs) and microRNAs (miRNAs), regulate gene expression in many physiological and pathological processes, including drug metabolism. Drug metabolizing enzymes (DMES) are critical components in drug‐induced liver toxicity. In this study, we used human hepatic HepaRG cells treated with 5 or 10 mM acetaminophen (APAP) as our model system and identified LINC00844 as a toxicity‐associated lncRNA. We analyzed the expression profiles and the correlations between the expression of long noncoding RNAs and key enzymes for acetaminophen metabolism in humans. Our results showed that lncRNA LINC00844 is enriched in the liver and its expression correlates positively with that of CYP3A4, CYP2E1, SULT2A1, NR1I2, and HNF4A mRNA transcripts. We demonstrated that LINC00844 regulates the expression of these five genes in HepaRG cells using gain‐ and loss‐of‐function assays. Further, we discovered that LINC00844 was predominantly localized in the cytoplasm and acted as a hsa‐miR‐486‐5p sponge, via direct binding, to protect SULT2A1 from miRNA‐mediated gene silencing. Our data also showed that hsa‐miR‐486‐5p upregulated the expression of CYP3A4, SULT2A1, NR1I2, and HNF4A by positively regulating LINC00844. We depict a LINC00844‐mediated regulatory network that involves miRNA and NRs and influences the expression of DMEs.Support or Funding InformationFDA E0753201

Janus kinase-dependent regulation of drug detoxifying protein expression by interleukin-22 in human hepatic cells

Interleukin (IL)-22 is a cytokine up-regulated in inflammatory situations and known to exert various hepatic effects. The potential impact of IL-22 towards liver drug detoxifying proteins remains nevertheless unknown, but may be important to determine owing to the well-established alterations of liver detoxification occuring during inflammation. The present study was therefore designed to analyze the effects of IL-22 towards drug metabolizing enzyme and drug transporter expression and activity in cultured human hepatic cells. Exposure of differentiated hepatoma HepaRG cells or primary human hepatocytes to 10 ng/mL IL-22 was found to repress mRNA expression of cytochrome P-450 (CYP) 1A2, CYP3A4, CYP2B6 and CYP2C9 and of the sinusoidal sodium-taurocholate co-transporting polypeptide (NTCP); such IL-22 effects were concentration-dependent for CYP3A4 (IC50 = 1.7 ng/mL), CYP2B6 (IC50 = 0.9 ng/mL) and NTCP (IC50 = 1.8 ng/mL). Activity of CYP1A2 (phenacetin O-deethylation), CYP3A4 (midazolam hydroxylation) and CYP2B6 (bupropion hydroxylation), as well as that of NTCP (taurocholate uptake) were concomitantly decreased in IL-22-treated HepaRG cells; by contrast, activity of organic anion transporter polypeptides (OATPs) (estrone-3-sulfate uptake) and of organic cation transporter (OCT) 1 (tetra-ethylammonium uptake) remained unchanged. IL-22 was next found to activate the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) 3 pathway, whose inhibition by the JAK inhibitor ruxolitinib fully prevented the IL-22-mediated CYP3A4, CYP2B6 and NTCP repression in HepaRG cells. This JAK-dependent down-regulation of hepatic drug detoxifying proteins, notably of CYPs, by IL-22 may contribute to alteration of pharmacokinetics in patients suffering from acute and chronic inflammatory diseases and may be the source of drug-drug interactions.

Reused palm oil from frying pork or potato induced expression of cytochrome P450s and the SLCO1B1 transporter in HepG2 cells.

Deep frying degrades the oil and generates harmful products. This study evaluated effects of reused palm oil (from frying pork or potato) on expression of cytochrome P450s (CYPs), the transporter (SLCO1B1), and lipid metabolism regulators; proliferator-activated receptors (PPAR) and sterol regulatory element binding protein (SREBP). Human hepatic carcinoma cell line (HepG2) cells were incubated with oleic acid (OA), new palm oil, or reused palm oils for 24hr. Fatty acid accumulation was examined by Nile red staining. Total RNA was extracted, followed by RT/qPCR of the target genes. Fatty acid accumulation was significantly different between the new and the reused oils. Expression of CYP1A2, CYP2C19, CYP2E1, CYP3A4, CYP4A11, and SLCO1B1 was induced by reused oils. Expression of PPAR-α was strongly increased in all treatments while SREBP-1a and SREBP-1c were suppressed. Modification of CYPs, PPAR-α, and SLCO1B1 by palm oil might increase the risk of fatty acid accumulation with associated oxidative stress. Therefore, consumption of palm oil or reused oil should be limited. PRACTICAL APPLICATIONS: Deep frying degrades the oil and generates harmful products. This study evaluated effects of reused palm oil (from frying pork or potato) on expression of cytochrome P450s (CYPs), the transporter (SLCO1B1), and lipid metabolism regulators; PPAR and SREBP in HepG2 cells. Both of the reused oils-induced profiles of all CYP and SLCO1B1, but the new oil upregulated CYP2E1, CYP3A4, and CYP4A11. PPAR-α was induced while SREBP-1a and SREBP-1c were suppressed by all treatments. Inductions of CYPs with suppression of SREBP-1a and SREBP-1c might contribute to an increased risk of fatty acid accumulation. These findings revealed the impacts of reused palm oil on metabolism via CYPs which related to oxidative stress for further study. Hence, consumption of palm oil or reused cooking oil should be of concern.

Identification of rhythmic human CYPs and their circadian regulators using synchronized hepatoma cells

Cytochromes P450 (CYPs) catalyze a great number of metabolic reactions that have profound effects on the biological activities of xenobiotics and endobiotics. In this study, we aimed to characterize rhythmic expressions of drug-metabolizing CYPs using synchronized hepatoma cells, and to investigate the potential roles of cis-elements of circadian clock system (E-box, D-box and RevRE or RORE) in generating the rhythms.Serum was used to synchronize circadian cycles and to induce circadian gene expression in cultured hepatoma cells (HepRG and HepG2 cells). Regulation of CYP genes by circadian clock components was investigated by performing luciferase reporter, overexpression and knockdown experiments. mRNA and protein expression were determined by qPCR and Western blotting assays, respectively.Of ten major drug-metabolizing CYP genes, six are rhythmically expressed (CYP1A2, 2B6, 2C8, 2D6, 2E1 and 3A4), whereas other four are non-rhythmic (CYP1B1, 2A6, 2C9 and 2C19).The E-box binding protein BMAL1 directly controls the rhythmic expression of CYP1A2. Rhythmic expressions of CYP2E1 and CYP3A4 are generated via both E-box and D-box elements. The RevRE binding protein REV-ERBα contributes to rhythmic oscillations in CYP2B6 and CYP2C8.In conclusion, rhythmic expressions of five human CYPs (CYP1A2, 2B6, 2C8, 2E1 and 3A4) are generated and regulated by E-box-, D-box-, and/or RevRE-acting clock components. Our findings may have implications for understanding chronopharmacokinetic events in humans.

Disruption of cytochrome P450 enzymes in the liver and small intestine in chicken embryos in ovo exposed to glyphosate.

Glyphosate is the active component of several commercial formulations as in Roundup®. The present study was investigated the toxic effects of pure glyphosate or Roundup® on the liver and small intestine of chick embryos. On day 6, a total of 180 fertile eggs injected with deionized water (control group), 10mg pure glyphosate, or 10mg of the active ingredient glyphosate in Roundup®/kg egg mass. The results showed an increase in relative weights of the liver in embryos that treated with Roundup®. Furthermore, oxidative stress was observed in the embryos treated with glyphosate or Roundup®, increased total superoxide dismutase, and content of malondialdehyde in the liver and intestine; moreover, decrease of glutathione peroxidase in the liver with increased in the intestine compared with the control. Besides, glutamic-pyruvic transaminase was increased in Roundup® group compared with other groups. Moreover, histopathological alterations in the liver and intestine tissues were observed in treated groups. Suppression of hepatic CYP1A2, CYP1A4, CYP1B1, and MDR1 mRNA expression after exposed to Roundup®. Furthermore, inhibition of CYP1A4 in the duodenum, CYP1A4, and MRP2 in the jejunum in embryos exposed to glyphosate or Roundup®. In addition, glyphosate treatment caused an increase of CYP3A5, CYP1C1, and IFNY mRNA expression in the jejunum and CYP1A2 expression in the ileum, while IFN-Y gene increase in embryos treated with Roundup®. In conclusion, in ovo exposure to glyphosate caused histopathological alterations and induced oxidative stress in the liver and small intestines. Moreover, the expression of cytochrome P450, MDR1, and MRP2 transporters was also modulated in the liver and small intestines for chick embryos.

Severity of coeliac disease and clinical management study when using a CYP3A4 metabolised medication: a phase I pharmacokinetic study

ObjectiveSeverity of coeliac disease depends in part on the extent of small intestinal mucosa injury. Patients with the most abnormal pathology have loss of duodenal villi CYP3A4, a drug-metabolising enzyme...

Effects of plasma concentration of micro-RNA Mir-27b and CYP3A4*22 on equilibrium concentration of alprazolam in patients with anxiety disorders comorbid with alcohol use disorder.

The objective of our study was to investigate the effect of 99366316G>A polymorphism of the CYP3A4 gene on the concentration/dose indicator of alprazolam in patients with anxiety disorders comorbid with alcohol use disorder, using findings on enzymatic activity of CYP3A (as evaluated by the 6-beta-hydroxy-cortisol/cortisol ratio measurement) and on CYP3A4 expression level obtained by measuring the miR-27b plasma concentration levels in patients with anxiety disorders comorbid with alcoholism. Our study enrolled 105 patients with anxiety disorders comorbid with alcohol use disorder (age - 37.8±14.6 years). Therapy included alprazolam in an average daily dose of 5.6±2.4 mg per day. Treatment efficacy was evaluated using the psychometric scales. Therapy safety was assessed using the UKU Side-Effect Rating Scale. For genotyping and estimation of the microRNA (miRNA) plasma levels, we performed the real-time polymerase chain reaction. The activity of CYP3A was evaluated using the HPLC-MS/MS method by the content of the endogenous substrate of the given isoenzyme and its metabolite in urine (6- beta-hydroxy-cortisol/cortisol). Therapeutic drug monitoring (TDM) has been performed using HPLC-MS/MS. Our study revealed the statistically significant results in terms of the treatment efficacy evaluation (HAMA scores at the end of the treatment course): (GG) 3.0 [2.0; 5.0] and (GA) 4.0 [4.0; 5.0], p=0.007; at the same time, the statistical significance in the safety profile was not obtained (the UKU scores): (GG) 3.0 [2.0; 3.8] and (GA) 3.0 [1.5; 4.0], p=0.650. We revealed a statistical significance for concentration/dose indicator of alprazolam in patients with different genotypes: (GG) 1.583 [0.941; 2.301] and (GA) 2.888 [2.305; 4.394], p=0.001). Analysis of the results of the pharmacotranscriptomic part of the study didn't show the statistically significant difference in the miR-27b plasma levels in patients with different genotypes: (GG) 25.6 [20.4; 28.8], (GA) 25.7 [19.7; 33.1], p=0.423. At the same time, correlation analysis revealed a statistically significant relationship between the alprazolam efficacy profile evaluated by changes in HAMA scale scores and the miR-27b plasma concentration: rs=0.20, p=0.042. Also, we didn't reveal the correlation between the miRNA concentration and safety profile: rs=0.15, p=0.127. In addition, we revealed the relationship between the CYP3A enzymatic activity (as evaluated by 6-beta-hydroxycortisol/ cortisol ratio measurement) and the miR-27b plasma concentration: rs=-0.27, p=0.006. At the same time, correlation analysis revealed a statistically significant relationship between the alprazolam concentration and the miR-27b plasma concentration: rs=0.28, p=0.003. The effect of genetic polymorphism of the CYP3A4 gene on the efficacy and safety profiles of alprazolam was demonstrated in a group of 105 patients with anxiety disorders comorbid with alcohol use disorder. At the same time, miR-27b remains a promising biomarker for assessing the level of CYP3A4 expression, because it correlates with the encoded isoenzyme's activity.

Knockdown of Long Noncoding RNAs Hepatocyte Nuclear Factor 1α Antisense RNA 1 and Hepatocyte Nuclear Factor 4α Antisense RNA 1 Alters Susceptibility of Acetaminophen-Induced Cytotoxicity in HepaRG Cells

Acetaminophen (APAP) is a commonly used over-the-counter drug for its analgesic and antipyretic effects. However, APAP overdose leads to severe APAP-induced liver injury (AILI) and even death as a result of the accumulation of N-acetyl-p-benzoquinone imine, the toxic metabolite of APAP generated by cytochrome P450s (P450s). Long noncoding RNAs HNF1α antisense RNA 1 (HNF1α-AS1) and HNF4α antisense RNA 1 (HNF4α-AS1) are regulatory RNAs involved in the regulation of P450 expression in both mRNA and protein levels. This study aims to determine the impact of HNF1α-AS1 and HNF4α-AS1 on AILI. Small hairpin RNAs were used to knock down HNF1α-AS1 and HNF4α-AS1 in HepaRG cells. Knockdown of these lncRNAs altered APAP-induced cytotoxicity, indicated by MTT and LDH assays. Specifically, HNF1α-AS1 knockdown decreased APAP toxicity with increased cell viability and decreased LDH release, whereas HNF4α-AS1 knockdown exacerbated APAP toxicity, with opposite effects in the MTT and LDH assays. Alterations on gene expression by knockdown of HNF1α-AS1 and HNF4α-AS1 were examined in several APAP metabolic pathways, including CYP1A2, CYP2E1, CYP3A4, UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Knockdown of HNF1α-AS1 decreased mRNA expression of CYP1A2, 2E1, and 3A4 by 0.71-fold, 0.35-fold, and 0.31-fold, respectively, whereas knockdown of HNF4α-AS1 induced mRNAs of CYP1A2, 2E1, and 3A4 by 1.3-fold, 1.95-fold, and 1.9-fold, respectively. These changes were also observed in protein levels. Knockdown of HNF1α-AS1 and HNF4α-AS1 had limited effects on the mRNA expression of UGT1A1, UGT1A9, SULT1A1, GSTP1, and GSTT1. Altogether, our study suggests that HNF1α-AS1 and HNF4α-AS1 affected AILI mainly through alterations of P450-mediated APAP biotransformation in HepaRG cells, indicating an important role of the lncRNAs in AILI. SIGNIFICANCE STATEMENT: The current research identified two lncRNAs, hepatocyte nuclear factor 1α antisense RNA 1 and hepatocyte nuclear factor 4α antisense RNA 1, which were able to affect susceptibility of acetaminophen (APAP)-induced liver injury in HepaRG cells, possibly through regulating the expression of APAP-metabolizing cytochrome P450 enzymes. This discovery added new factors, lncRNAs, which can be used to predict cytochrome P450-mediated drug metabolism and drug-induced toxicity.

Effect of genetic polymorphisms in CYP3A4, CYP3A5, and m-TOR on everolimus blood exposure and clinical outcomes in cancer patients.

Genetic variations in CYP3A4, CYP3A5, and m-TOR could contribute to interpatient variability regarding m-TOR inhibitors pharmacokinetics or cellular effects. The purpose of this study was to evaluate the influence of selected candidate variations in these genes on everolimus pharmacokinetics, efficacy, and toxicity in cancer patients. Thirty-four patients receiving everolimus for breast (n = 22) or renal (n = 10) cancers, or neuroendocrine tumors of pancreatic origin (n = 2) were included in the study. Six variants in genes related to everolimus pharmacokinetics (CYP3A4*22 and CYP3A5*3) or pharmacodynamics (m-TOR rs2295079, rs2295080, rs2024627 and rs1057079) were genotyped. Associations with trough concentrations (C0), dose reductions, or treatment interruptions due to toxicity and progression-free survival were investigated using generalized estimating equations and Cox models. CYP3A5 nonexpressers had significantly higher C0 as compared with expressers (βGG vs AG = + 6.32 ± 2.22 ng/mL, p = 0.004). m-TOR rs2024627 was significantly associated with an increased risk of cancer progression studied alone or as part of an haplotype (T vs C: HR = 2.60, 95% CI [1.16-5.80], p = 0.020; CTCG vs other haplotypes HR = 2.29, 95% CI [1.06-4.95], p = 0.035, respectively). This study showed that CYP3A5 expression impacts everolimus pharmacokinetics in cancer patients and identified a genetic variation in m-TOR associated with the risk of cancer progression.

Expression Patterns of Xenobiotic-Metabolizing Enzymes in Tumor and Adjacent Normal Mucosa Tissues among Patients with Colorectal Cancer: The ColoCare Study.

Xenobiotic-metabolizing enzymes (XME) play a critical role in the activation and detoxification of several carcinogens. However, the role of XMEs in colorectal carcinogenesis is unclear. We investigated the expression of XMEs in human colorectal tissues among patients with stage I-IV colorectal cancer (n = 71) from the ColoCare Study. Transcriptomic profiling using paired colorectal tumor and adjacent normal mucosa tissues of XMEs (GSTM1, GSTA1, UGT1A8, UGT1A10, CYP3A4, CYP2C9, GSTP1, and CYP2W1) by RNA microarray was compared using Wilcoxon rank-sum tests. We assessed associations between clinicopathologic, dietary, and lifestyle factors and XME expression with linear regression models. GSTM1, GSTA1, UGT1A8, UGT1A10, and CYP3A4 were all statistically significantly downregulated in colorectal tumor relative to normal mucosa tissues (all P ≤ 0.03). Women had significantly higher expression of GSTM1 in normal tissues compared with men (β = 0.37, P = 0.02). By tumor site, CYP2C9 expression was lower in normal mucosa among patients with rectal cancer versus colon cancer cases (β = -0.21, P = 0.0005). Smokers demonstrated higher CYP2C9 expression levels in normal mucosa (β = 0.17, P = 0.02) when compared with nonsmokers. Individuals who used NSAIDs had higher GSTP1 tumor expression compared with non-NSAID users (β = 0.17, P = 0.03). Higher consumption of cooked vegetables (>1×/week) was associated with higher CYP3A4 expression in colorectal tumor tissues (β = 0.14, P = 0.007). XMEs have lower expression in colorectal tumor relative to normal mucosa tissues and may modify colorectal carcinogenesis via associations with clinicopathologic, lifestyle, and dietary factors. Better understanding into the role of drug-metabolizing enzymes in colorectal cancer may reveal biological differences that contribute to cancer development, as well as treatment response, leading to clinical implications in colorectal cancer prevention and management.