Expression Of Drug-metabolizing Enzymes Research Articles

Hypertensive Nephropathy Changes the Expression of Drug-Metabolizing Enzymes and Transporters in Spontaneously Hypertensive Rat Liver and Kidney

Hypertensive Nephropathy Changes the Expression of Drug-Metabolizing Enzymes and Transporters in Spontaneously Hypertensive Rat Liver and Kidney

Novel Regulatory Mechanisms for Expression of Drug Metabolism-related Factors

Interindividual differences in the expression and activity of drug-metabolizing enzymes, including cytochrome P450, UDP-glucuronosyltransferase, and esterases, cause variability of therapeutic effectiveness and side effects during drug treatment. Conventional research has focused on transcriptional regulation by transcription factors and nuclear receptors such as aryl hydrocarbon receptor, pregnane X receptor (PXR), constitutive androstane receptor, and hepatocyte nuclear factor 4α, as the major mechanisms causing the differences in the expression of drug-metabolizing enzymes. Recently, we have revealed that adenosine-to-inosine RNA editing and methylation of adenosine at the N6 position on RNA, two major types of posttranscriptional modification, play a pivotal role in the regulation of drug metabolism. In addition, switch/sucrose non-fermentable complex, a chromatin remodeler, is required for PXR-mediated transcriptional regulation of drug-metabolizing enzymes. This review article introduces the significance of these epitranscriptomic and epigenetic regulations as factors in determining drug metabolism potency. Further research on this link is expected to lead to a deeper understanding of interindividual differences in the therapeutic effectiveness and side effects of medicines.

Isoform-level expression of the constitutive androstane receptor (CAR or NR1I3) transcription factor better predicts the mRNA expression of the cytochrome P450s in human liver samples.

Many factors cause inter-person variability in the activity and expression of liver cytochrome P450 (CYP) drug-metabolizing enzymes, leading to variable drug exposure and treatment outcomes. Several liver-enriched transcription factors (TFs) are associated with CYP expression, with estrogen receptor alpha (ESR1) and constitutive androstane receptor (CAR or NR1I3) being the two top factors. ESR1 and NR1I3 undergo extensive alternative splicing that results in numerous splice isoforms, but how these splice isoforms associate with CYP expression is unknown. Here, we quantified 18 NR1I3 splice isoforms and the three most abundant ESR1 isoforms in 260 liver samples derived from African Americans (AA, n=125) and European Americans (EA, n=135). Our results showed variable splice isoform populations in the liver for both NR1I3 and ESR1. Multiple linear regression analyses revealed that, compared to gene-level NR1I3, isoform-level NR1I3 expression better predicted the mRNA expression of most CYPs and three UDP-glucuronosyltransferases (UGTs), while ESR1 isoforms improved predictive models for the UGTs and CYP2D6, but not for most CYPs. Also, different NR1I3 isoforms were associated with different CYPs, and the associations varied depending on sample ancestry. Surprisingly, non-canonical NR1I3 isoforms having retained introns (introns 2 or 6) were abundantly expressed and associated with the expression of most CYPs and UGTs, whereas the reference isoform (NR1I3-205) only associated with CYP2D6. Moreover, NR1I3 isoform diversity increased during the differentiation of induced pluripotent stem cells to hepatocytes, paralleling increasing CYP expression. These results suggest that isoform-level TF expression may help to explain variation in CYP or UGT expression between individuals. Significance Statement We quantified 18 NR1I3 splice isoforms and three ESR1 splice isoforms in 260 liver samples derived from AA and EA donors and found variable NR1I3 and ESR1 splice isoform expression in the liver. Multiple linear regression analysis showed that, compared to gene-level expression, isoform-level expression of NR1I3 and ESR1 better predicted the mRNA expression of some CYPs and UGTs, highlighting the importance of isoform-level analyses to enhance our understanding of gene transcriptional regulatory networks controlling the expression of drug-metabolizing enzymes.

Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture.

Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.

OCT1 (SLC22A1) transporter kinetics and regulation in primary human hepatocyte 3D spheroids

3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a model to study both transport kinetics and the long-term regulation of transporter activity via relevant signalling pathways. The kinetics of the OCT1 transporter was studied using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, 3D PHH were treated with xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomic analysis was used to track hepatic phenotypes as well as prototypical changes in other regulated proteins, such as P-glycoprotein and Cytochrome P450 3A4. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14 ± 4.0µM as the mean from three donors. Co-incubation with known OCT1 inhibitors decreased the uptake of ASP+ in the 3D PHH spheroids by 35–52%. The long-term exposure studies showed that OCT1 is relatively stable upon activation of nuclear receptor signalling or exposure to compounds that could induce inflammation, steatosis or liver injury. Our results demonstrate that 3D PHH spheroids express physiologically relevant levels of fully active OCT1 and that its transporter kinetics can be accurately studied in the 3D PHH configuration. We also confirm that OCT1 remains stable and functional during the activation of key metabolic pathways that alter the expression and function of other drug transporters and drug-metabolizing enzymes. These results will expand the range of studies that can be performed using 3D PHH.

Enhanced hepatotoxicity assessment through encapsulated HepG2 spheroids in gelatin hydrogel matrices: Bridging the gap from 2D to 3D culture

Conventional 2D drug screening often fails to accurately predict clinical outcomes. We present an innovative approach to improve hepatotoxicity assessment by encapsulating HepG2 spheroids in gelatin hydrogel matrices with different mechanical properties. Encapsulated spheroids exhibit sustained liver-specific functionality, enhanced expression of drug-metabolizing enzymes, and increased drug sensitivity compared to 2D cultures. The platform detects critical variations in drug response, with significant differences in IC50 values between 2D and spheroid cultures ranging from 1.3-fold to > 13-fold, particularly for acetaminophen. Furthermore, drug-metabolizing enzyme expression varies across hydrogel concentrations, suggesting a role for matrix mechanical properties in modulating hepatocyte function. This novel spheroid-hydrogel platform offers a transformative approach to hepatotoxicity assessment, providing increased sensitivity, improved prediction, and a more physiologically relevant environment. The use of such advanced in vitro models can accelerate drug development, reduce animal testing, and contribute to improved patient safety and clinical outcomes.

Hydrogen Peroxide Induces Ethanol-inducible CYP2E1 via the NFkB-classical Pathway: CYP2E1 mRNA Levels are not High in Alcoholic Hepatitis.

The aim of the present study is to elucidate the mechanism of CYP2E1 induction as a causative factor of alcoholic hepatitis (AH) and its relationship with inflammation. Chronic alcohol consumption induces CYP2E1, which is involved in the development of alcoholic hepatitis (AH). However, the mechanisms underlying the induction of CYP2E1 by alcohol remain unclear. Therefore, we herein investigated the induction of drug-metabolizing enzymes, particularly CYP2E1, by hydrogen peroxide (H2O2), the concentration of which is elevated under inflammatory conditions. The mechanisms underlying the induction of CYP2E1 by H2O2 were examined with a focus on Keap1, a target factor of H2O2. We assessed changes in the expression of drug-metabolizing enzymes in the human hepatoma cell line, Hep3B, following treatment with H2O2, and evaluated changes in the expression of the NFkB-related factor RelA(p65) after the knockdown of Keap1, a regulator of Nrf2 expression by reactive oxygen species. We also performed a promoter analysis using the upstream region of the CYP2E1 gene. We herein used the GSE89632 series for non-alcoholic hepatitis (NASH) and the GSE28619 series for AH. The induction of CYP2E1 by H2O2 was significantly stronger than that of other drugmetabolizing enzymes. On the other hand, the knockdown of Keap1, a target of H2O2, markedly increased RelA(p65), an NFkB factor. Furthermore, the overexpression of RelA(p65) strongly induced the expression of CYP2E1. Four candidate p65-binding sequences were identified upstream of the CYP2E1 gene, and promoter activity assays showed that the third sequence was responsive to the overexpression of RelA(p65). We used the GSE89632 series for NASH and the GSE28619 series for AH in the present study. The expression of CYP2E1 mRNA in the liver was significantly lower in AH patients than in HC patients, but was similar in HC patients and NASH patients. We herein demonstrated that the expression of CYP2E1 was induced by H2O2. The overexpression of RelA(p65) also induced CYP2E1 mRNA expression, whereas H2O2 did not after the knockdown of RelA. These results suggest that H2O2 acts on Keap1 to upregulate RelA (p65) in the NFkB system. One of the mechanisms underlying the induction of CYP2E1 was dependent on the H2O2-Keap1-RelA axis. The results of the database analysis revealed that the expression of CYP2E1 in the liver was significantly lower in AHH patients than in NASH patients, suggesting that CYP2E1 is not the main cause of AH; however, CYP2E1 may exacerbate the pathogenesis of AH.

Oral Drug Dosing After Gastric Bypass and Diet-Induced Weight Loss: Simpler Than We Think? Lessons Learned From the COCKTAIL Study.

This article summarizes the lessons learned from the COCKTAIL study: an open, three-armed, single-center study including patients with obesity scheduled for treatment with Roux-en-Y gastric bypass (RYGB) or nonsurgical calorie restriction, and a normal- to overweight control group. The clinical implications of the results from multiple peer-reviewed articles describing the effects of RYGB, severe caloric restriction, weight loss, and type 2 diabetes on the in vivo activity and protein expression of drug-metabolizing enzymes (cytochrome P450 (CYP) 1A2, 2C9, 2C19, and 3A) and transporters (DMETs; organic anion-transporting polypeptide (OATP) 1B1 and P-glycoprotein (P-gp)) are discussed in the perspective of three clinically relevant questions: (1) How should clinicians get the dose right in patients after RYGB? (2) Will drug disposition in patients with obesity be normalized after successful weight loss? (3) Are dose adjustments needed according to obesity and diabetes status? Overall, RYGB seems to have a lower impact on drug disposition than previously assumed, but clinicians should pay close attention to drugs with a narrow therapeutic range or where a high maximum drug concentration may be problematic. Whether obesity-related alterations of DMETs normalize with substantial weight loss depends on the DMET in question. Obesity and diabetes downregulate the in vivo activity of CYP2C19 and CYP3A (only obesity) but whether substrate drugs should be dose adjusted is also dependent on other factors that influence clearance, that is, liver blood flow and protein binding. Finally, we recommend frequent and individualized follow-up due to high inter- and intraindividual variability in these patients, particularly following RYGB.

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.

Non-alcoholic fatty liver disease changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats

Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, which can cause serious complications and gradually increase the mortality rate. However, the effects of NAFLD on drug-metabolizing enzymes and transporters remain unclear, which may cause some confusion regarding patient medication. In this study, a NAFLD rat model was constructed by feeding rats with methionine and choline deficiency diets for 6 weeks, and the mRNA and protein levels of drug-metabolizing enzymes and transporter were analyzed by real-time fluorescent quantitative PCR and Western blot, respectively. The activity of drug-metabolizing enzymes was detected by cocktail methods. In the NAFLD rat model, the mRNA expression of phase I enzymes, phase II enzymes, and transporters decreased. At the protein level, only CYP1A1, CYP1B1, CYP2C11, and CYP2J3 presented a decrease. In addition, the activities of CYP1A2, CYP2B1, CYP2C11, CYP2D1, CYP3A2, UGT1A1, UGT1A3, UGT1A6, and UGT1A9 decreased. These changes may be caused by the alteration of FXR, HNF4α, LXRα, LXRβ, PXR, and RXR. In conclusion, NAFLD changes the expression and activity of hepatic drug-metabolizing enzymes and transporters in rats, which may affect drug metabolism and pharmacokinetics. In clinical medication, drug monitoring should be strengthened to avoid potential risks.

PHARMACOGENETICS OF ANESTHESIOLOGICAL SUPPORT

Pharmacogenetics studies the relationship between a person’s individual genetic characteristics and the human body’sresponse to the action of various drugs, particularly the occurrence of undesirable side eff ects. Thanks to the development of the latest technologies and methods, this branch of medical genetics and clinical pharmacology is developing very actively. Data are being accumulated, special databases are being created with the aim of creating individual genetic passports in the future, which will allow the selection of personalized treatment schemes.Anesthesiology is a special area of pharmacogenetic research because, more than any other medical specialty, it ischaracterized by polypharmacy-the simultaneous or sequential administration of many drugs. The same dose of a drug may be inadequate for some patients and may be life-threatening or cause unwanted side eff ects for others. Today, information about genetic factors is being used by clinicians to prescribe drugs to tailor drug therapy to a patient’s genome. In anesthesiology, the principles of pharmacogenetics have been explained for neuromuscular blocking agents, opioid metabolism, different types of anesthetics, and postoperative nausea and vomiting. On the other hand, a large number of anesthetics have a narrow therapeutic index.This review summarizes the most recent data from the scientifi c literature on the pharmacogenetics of diff erent types ofanesthetics. Inhalational anesthetics are halogenated derivatives of methyl ethyl ether, the exact mechanism of action of which is not yet fully understood. One of the rare but very serious side eff ects of all halogenated anesthetics is malignant hyperthermia, a genetically determined autosomal dominant disorder that manifests as a hypermetabolic response to drug administration. The dosage of intravenous anesthetics should also be carefully determined, taking into account the patient’s age, cardiovascular, hepatic, and renal status, concomitant drug therapy, and genetic factors. Ontogeny and genetic variability of drug-metabolizing enzymes are interrelated because genetic variability in drug-metabolizing enzyme expression cannot be assessed until the required protein is suffi ciently expressed. Pharmacogenetic variants may contribute to unpredictable drug exposure at the same weight- based drug dose.There are a number of potentially clinically applicable pharmacogenetic data in newborns, but more research is needed toconfi rm these fi ndings and understand how to incorporate them into clinical care.The selection of drugs and dosing regimens based on a patient’s pharmacogenomic profi le may be an important part of thefuture of medicine. Personalized treatment based on the specifi c variants in the genome will ultimately reduce the incidence of side eff ects and length of hospital stay for patients and save healthcare costs. Although pharmacogenomics and its application in clinical practice are still in their infancy, different variants and their implications for many clinical areas, including anesthesiology, are emerging every day.

Design and engineering of logic genetic-enzymatic gates based on the activity of the human CYP2C9 enzyme in permeabilized Saccharomyces cerevisiae cells

Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes. In this study, we combined, in the yeast S. cerevisiae, genetic regulatory elements with the enzymatic reactions of the human CYP2C9 and its redox partner CPR on luciferin substrates and diclofenac. S. cerevisiae cells were permeabilized and used as enzyme bags in order to host these metabolic reactions. We engineered three different (genetic)-enzymatic basic Boolean gates (YES, NOT, and N-IMPLY). In the YES and N-IMPLY gates, human CYP2C9 was expressed under the galactose-inducible GAL1 promoter. The carbon monoxide releasing molecule CORM-401 was used as an input in the NOT and N-IMPLY gates to impair CYP2C9 activity through inhibition of the Fe+2- heme prosthetic group in the active site of the human enzyme. Our study provides a new approach in designing synthetic bio-circuits and optimizing experimental conditions to favor the heterologous expression of human drug metabolic enzymes over their endogenous counterparts. This new approach will help study precise metabolic attributes of human P450s.

Effects of Calamintha incana (Sm.) Helder Ethanolic Extract on the mRNA Expression of Drug-metabolizing cyp450s in the Mouse Livers.

Alteration in the expression and activity of drug-metabolizing enzymes (DMEs) can alter the pharmacokinetics and hence the response of the drug. Some chemicals found in herbs and fruits affect the expression of DMEs. Calamintha incana is commonly used in Middle Eastern Arabic countries. There is no report regarding the influence of Calamintha incana on the hepatic expression of DMEs. The current investigation aimed to investigate the effect of Calamintha incana consumption on the mRNA expression of major hepatic drug-metabolizing cytochrome (cyp) P450 genes in mice. The chemical composition of the ethanoic extract was analyzed using liquid chromatography/ mass spectrometry. Then, 21 BALB/c mice were used for the in vivo experiment. The mice were divided into three groups, each consisting of seven mice. The first group (low-dose group) was treated with 41.6 mg/kg of Calamintha incana extract and the second group was administered the high-dose (125 mg/kg) of the extract for one month. The mice in the third "control" group administrated the vehicle 20% polyethylene glycol 200. Then, the expression of cyp3a11, cyp2c29, cyp2d9, and cyp1a1 was analyzed using the real-time polymerase chain reaction. The relative liver weights of the mice and the hepatic pathohistological alterations were assessed. The ethanolic extract of Calamintha incana contained 27 phytochemical compounds. The most abundant compounds were linolenic acid, myristic acid, and p-cymene. It was found that the low dose of Calamintha incana extract upregulated significantly (P < 0.05) the expression of cyp3a11 by more than ten folds in the liver of treated mice. Furthermore, the histological analysis showed that low- and high-dose administration of the C. incana did not cause pathological alterations. It can be concluded from these findings that consumption of low doses of Calamintha incana upregulated the mRNA expression of mouse cyp3a11 without causing histopathological alterations in the livers. Further studies are needed to determine the influence of Calamintha incana on the pharmacokinetics and response of drugs metabolized by cyp3a11.

Expression of intestinal drug transporter proteins and metabolic enzymes in neonatal and pediatric patients

The development of pediatric oral drugs is hampered by a lack of predictive simulation tools. These tools, in turn, require data on the physiological variables that influence oral drug absorption, including the expression of drug transporter proteins (DTPs) and drug-metabolizing enzymes (DMEs) in the intestinal tract. The expression of hepatic DTPs and DMEs shows age-related changes, but there are few data on protein levels in the intestine of children. In this study, tissue was collected from different regions of the small and large intestine from neonates (i.e., surgically removed tissue) and from pediatric patients (i.e., gastroscopic duodenal biopsies). The protein expression of clinically relevant DTPs and DMEs was determined using a targeted mass spectrometry approach. The regional distribution of DTPs and DMEs was similar to adults. Most DTPs, with the exception of MRP3, MCT1, and OCT3, and all DMEs showed the highest protein expression in the proximal small intestine. Several proteins (i.e., P-gp, ASBT, CYP3A4, CYP3A5, CYP2C9, CYP2C19, and UGT1A1) showed an increase with age. Such increase appeared to be even more pronounced for DMEs. This exploratory study highlights the developmental changes in DTPs and DMEs in the intestinal tract of the pediatric population. Additional evaluation of protein function in this population would elucidate the implications of the presented changes in protein expression on absorption of orally administered drugs in neonates and pediatric patients.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

Human intestinal organoid-derived PDGFRα + mesenchymal stroma enables proliferation and maintenance of LGR4 + epithelial stem cells

BackgroundIntestinal epithelial cells derived from human pluripotent stem cells (hPSCs) are generally maintained and cultured as organoids in vitro because they do not exhibit adhesion when cultured. However, the three-dimensional structure of organoids makes their use in regenerative medicine and drug discovery difficult. Mesenchymal stromal cells are found near intestinal stem cells in vivo and provide trophic factors to regulate stem cell maintenance and proliferation, such as BMP inhibitors, WNT, and R-spondin. In this study, we aimed to use mesenchymal stromal cells isolated from hPSC-derived intestinal organoids to establish an in vitro culture system that enables stable proliferation and maintenance of hPSC-derived intestinal epithelial cells in adhesion culture.MethodsWe established an isolation protocol for intestinal epithelial cells and mesenchymal stromal cells from hPSCs-derived intestinal organoids and a co-culture system for these cells. We then evaluated the intestinal epithelial cells and mesenchymal stromal cells' morphology, proliferative capacity, chromosomal stability, tumorigenicity, and gene expression profiles. We also evaluated the usefulness of the cells for pharmacokinetic and toxicity studies.ResultsThe proliferating intestinal epithelial cells exhibited a columnar form, microvilli and glycocalyx formation, cell polarity, and expression of drug-metabolizing enzymes and transporters. The intestinal epithelial cells also showed barrier function, transporter activity, and drug-metabolizing capacity. Notably, small intestinal epithelial stem cells cannot be cultured in adherent culture without mesenchymal stromal cells and cannot replaced by other feeder cells. Organoid-derived mesenchymal stromal cells resemble the trophocytes essential for maintaining small intestinal epithelial stem cells and play a crucial role in adherent culture.ConclusionsThe high proliferative expansion, productivity, and functionality of hPSC-derived intestinal epithelial cells may have potential applications in pharmacokinetic and toxicity studies and regenerative medicine.

Drug regulation of microRNA

The scientific review provides the mechanisms of drug regulation of microRNA in the human body. To write the article, information was searched using Scopus, Web of Science, MEDLINE, PubMed, Google Scholar, Embase, Global Health, The Cochrane Library databases. To restore the reduced functional activity of microRNAs, replacement therapy is used, with modified synthetic analogs of endogenous microRNAs, and drugs that enhance the production of the body’s own microRNAs. The authors state that numerous studies have confirmed the effectiveness of miRNA replacement therapy. It is known that there are several groups of drugs among miRNA inhibitors: anti-miRNA oligonucleotides, miRNA traps, miRNA mimics that prevent miRNA binding; peptide nucleic acids, small-molecule inhibitors. The authors suggest that the expression of drug-metabolizing enzymes is controlled by nuclear receptors and transcription factors, epigenetic regulation such as DNA methylation and histone acetylation, and post-translational modification. It is emphasized that ursodeoxycholic acid modulates the expression of some miRNAs. It is known that probiotic bacteria can modulate the expression level of miRNA genes. The use of probiotics is accompanied by a change in the expression of nume­rous genes of the body involved in the regulation of the inflammatory response, allergic reactions, metabolism and other biological processes. Thus, modern science is intensively studying the potential of using drugs that restore miRNA content or inhibit miRNA acti­vity for the therapy of miRNA-dependent conditions. The results of scientific research confirmed the therapeutic effect of ursodeoxycholic acid and probiotic preparations due to the effect on the acti­vity of miRNA generation in hepatobiliary diseases. Therefore, the introduction into clinical practice of drugs than can modulate the content and expression of specific miRNAs will certainly open new perspectives in the treatment of patients with hepatobiliary diseases.

Cyclosporine-A induced cytotoxicity within HepG2 cells by inhibiting PXR mediated CYP3A4/CYP3A5/MRP2 pathway

Cyclosporine-A (CsA) is currently used to treat immune rejection after organ transplantation as a commonly used immunosuppressant. Liver injury is one of the most common adverse effects of CsA, whose precise mechanism has not been fully elucidated. Pregnane X receptor (PXR) plays a critical role in mediating drug-induced liver injury as a key regulator of drug and xenobiotic clearance. As a nuclear receptor, PXR transcriptionally upregulates the expression of drug-metabolizing enzymes and drug transporters, including cytochrome P4503A (CPY3A) and multidrug resistance-associated protein 2 (MRP2). Our study established CsA-induced cytotoxic hepatocytes in an in vitro model, demonstrating that CsA dose-dependently increased the aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) level secreted in the HepG2 cell supernatant, as well as viability and oxidative stress of HepG2 cells. CsA also dose-dependently decreased the PXR, CYP3A4, CPY3A5, and MRP2 levels of HepG2 cells. Mechanistically, altering the expression of PXR, CYP3A4, CYP3A5, and MRP2 affected the impact of CsA on AST and LDH levels. Moreover, altering the expression of PXR also changed the level of CYP3A4, CPY3A5, and MRP2 of HepG2 cells treated by CsA. Our presented findings provide experimental evidence that CsA-induced liver injury is PXR tightly related. We suggest that PXR represents an attractive target for therapy of liver injury due to its central role in the regulation of the metabolizing enzymes CYP3A and MRP2-mediated bile acid transport and detoxification.

Acceleration of benzo(a)pyrene-induced colon carcinogenesis by Western diet in a rat model of colon cancer

Colorectal cancer (CRC) is the third leading cause of cancer-related mortalities in the USA and around 52,550 people were expected to die from this disease by December 2023. The objective of this study was to investigate the effect of diet type on benzo(a)pyrene [B(a)P]-induced colon cancer in an adult male rat model, the Polyposis In the Rat Colon (PIRC) kindred type. Groups of PIRC rats (n = 10) were fed with AIN-76A regular diet (RD) or Western diet (WD) and received 25, 50 and 100 µg B(a)P/kg body wt. via oral gavage for 60 days. Rats fed diets alone, but no B(a)P, served as controls. After exposure, rats were euthanized; colon and liver samples were analyzed for activation of drug metabolizing enzymes (DMEs) CYP1A1, CYP1B1, SULT and GST. Plasma and tissue samples were analyzed by reverse phase-HPLC for B(a)P metabolites. In addition to these studies, DNA isolated from colon and liver tissues was analyzed for B(a)P-induced DNA adducts by the 32P-postlabeling method using a thin-layer chromatography system. Western diet consumption resulted in a marked increase in DME expression and B(a)P metabolite concentrations in rats that were administered 100 µg/kg B(a)P + WD (p < 0.05) compared to other treatment groups. Our findings demonstrate that WD accelerates the development of colon tumors induced by B(a)P through enhanced biotransformation, and the products of this process (metabolites) were found to bind with DNA and form B(a)P-DNA adducts, which may have given rise to colon polyps characterized by gain in tumor number, sizes, and dysplasia.

A Literature Review of Changes in Phase II Drug-Metabolizing Enzyme and Drug Transporter Expression during Pregnancy.

The purpose of this literature review is to comprehensively summarize changes in the expression of phase II drug-metabolizing enzymes and drug transporters in both the pregnant woman and the placenta. Using PubMed®, a systematic search was conducted to identify literature relevant to drug metabolism and transport in pregnancy. PubMed was searched with pre-specified terms during the period of 26 May 2023 to 10 July 2023. The final dataset of 142 manuscripts was evaluated for evidence regarding the effect of gestational age and hormonal regulation on the expression of phase II enzymes (n = 16) and drug transporters (n = 38) in the pregnant woman and in the placenta. This comprehensive review exposes gaps in current knowledge of phase II enzyme and drug transporter localization, expression, and regulation during pregnancy, which emphasizes the need for further research. Moreover, the information collected in this review regarding phase II drug-metabolizing enzyme and drug transporter changes will aid in optimizing pregnancy physiologically based pharmacokinetic (PBPK) models to inform dose selection in the pregnant population.