CYP3A4 Expression Research Articles

Hepatotoxicity of cyanotoxin microcystin-LR in human: Insights into mechanisms of action in the 3D culture model Hepoid-HepaRG

Microcystin-LR (MC-LR) is a potent hepatotoxin produced by harmful cyanobacterial blooms (CyanoHABs). MC-LR targets highly differentiated hepatocytes expressing organic anion transporting polypeptides OATP1B1 and OATP1B3 that are responsible for hepatocellular uptake of the toxin. The present study utilized an advanced 3D in vitro human liver model Hepoid-HepaRG based on the cultivation of collagen-matrix embedded multicellular spheroids composed of highly differentiated and polarized hepatocyte-like cells. 14-d-old Hepoid-HepaRG cultures showed increased expression of OATP1B1/1B3 and sensitivity to MC-LR cytotoxicity at concentrations >10 nM (48 h exposure, EC20 = 26 nM). MC-LR induced neither caspase 3/7 activity nor expression of the endoplasmic reticulum stress marker gene BiP/GRP78, but increased release of pro-inflammatory cytokine IL-8, indicating a necrotic type of cell death. Subcytotoxic (10 nM) and cytotoxic (≥100 nM) MC-LR concentrations disrupted hepatocyte functions, such as xenobiotic metabolism phase-I enzyme activities (cytochrome P450 1A/1B) and albumin secretion, along with reduced expression of CYP1A2 and ALB genes. MC-LR also decreased expression of HNF4A gene, a critical regulator of hepatocyte differentiation and function. Genes encoding hepatobiliary membrane transporters (OATP1B1, BSEP, NTCP), hepatocyte gap junctional gene connexin 32 and the epithelial cell marker E-cadherin were also downregulated. Simultaneous upregulation of connexin 43 gene, primarily expressed by liver progenitor and non-parenchymal cells, indicated a disruption of tissue homeostasis. This was associated with a shift in the expression ratio of E-cadherin to N-cadherin towards the mesenchymal cell marker, a process linked to epithelial-mesenchymal transition (EMT) and hepatocarcinogenesis. The effects observed in the human liver cell in vitro model revealed mechanisms that can potentially contribute to the MC-LR-induced promotion and progression of hepatocellular carcinoma (HCC). Hepoid-HepaRG cultures provide a robust, accessible and versatile in vitro model, capable of sensitively detecting hepatotoxic effects at toxicologically relevant concentrations, allowing for assessing hepatotoxicity mechanisms, human health hazards and impacts of environmental hepatotoxins, such as MC-LR.

Alterations in the gene expression of SARS-COV-2 entry receptors and enzymes in lungs and hearts of controlled and uncontrolled diabetic mice.

The entry of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into the host cell is carried out by specific receptors and enzymes, including human angiotensin-converting enzyme 2 receptor (ACE2), transmembrane serine protease 2 (TMPRSS2), and cathepsin-L (CTSL). COVID-19 patients with comorbidities, such as diabetes mellitus (DM), are more prone to severe symptoms and have a higher risk of mortality. The present study aimed to investigate the impact of controlled and uncontrolled type 1 DM (T1DM) on the gene expression of mouse Ace2, Tmprss2, and Ctsl and correlate it with the pathological alterations in the lungs and the heart of DM mice. Balb/c mice were administered a single dose of 240 mg/kg streptozocin to induce T1DM. The blood glucose level was measured to confirm the induction of DM. Normalization of blood glucose levels in T1DM mice was achieved using 0.1mL/kg Mixtard® insulin therapy. The mice's lungs and hearts were harvested, and the mRNA was extracted and converted to cDNA. The gene expression of Ace2, Tmprss2, Ctsl, Cyp4a11, and Adrb1 genes, which play a role in the homeostasis of lungs and hearts, were measured using quantitative real-time polymerase chain reaction (RT-PCR). The pathological alterations in the hearts and lungs induced by T1DM were evaluated using the relative heart and lung weights, in addition to the pathohistological examination. After inducing T1DM for 14 days, we observed a significant reduction in the total weight of uncontrolled DM (UDM) mice (P<0.05). Pathohistological examination of UDM lung tissues revealed thickening of the alveolar walls with narrowing of the surface of the alveolar sacs. Additionally, we found that UDM mice exhibited downregulation of Ace2 gene expression (P< 0.05) in their lungs, while both UDM and control DM (CDM) mice showed upregulation of Ctsl gene expression in their hearts (P<0.05). Notably, Cyp4a12 gene expression was significantly downregulated (P<0.05) in UDM mice but returned to normal levels in CDM mice. We conclude from this study that T1DM downregulates Ace2 receptor and Cyp4a12 gene expression, which is correlated with the thickening of alveolar walls and narrowing of the surface of alveolar sacs in the lungs. Insulin administration for controlling T1DM ameliorated these pathological alterations. These results can help increase our understanding of the impact of controlled and uncontrolled T1DM on the lungs and may explain, at least in part, why DM patients with COVID-19 experience exacerbation of symptoms.

Quercetin inhibits the metabolism of arachidonic acid by inhibiting the activity of CYP3A4, thereby inhibiting the progression of breast cancer

BackgroundRecent years have witnessed impressive growth in applying natural medicine in tumor treatment. Saffron is reported to elicit an inhibitory property against BC. Herein, we sought to explore the specific components and mechanistic basis of saffron’s anti-breast carcinoma (BC) function.MethodsBioinformatics analysis was employed to analyze saffron components' anti-BC activity and screen the corresponding target genes involved in BC. Then, the roles of the main saffron ingredient quercetin in the activity of BC cells were examined using CCK-8, MTS, flow cytometry, colony formation, Transwell, and Gelatin zymogram assays. Additionally, the interactions among Quercetin, EET, and Stat3 were assessed by immunofluorescence and Western blot, and LC–MS/MS determined the levels of AA, EETs, and CYP3A. Finally, BC xenograft mouse models were established to verify the anti-BC function of Quercetin in vivo.ResultsQuercetin, the main active component of saffron, inhibited BC progression. Quercetin suppressed BC cell growth, migration, and invasion and inhibited CYP3A4 expression and activity in BC. Mechanistically, Quercetin down-regulated CYP3A4 to block the nuclear translocation of Stat3 by decreasing the metabolization of AA to EETs, thereby alleviating BC. Moreover, exogenously added EETs counteracted the anti-tumor effect of Quercetin on BC. Quercetin also inhibited the tumor growth of tumor-bearing nude mice.ConclusionQuercetin could inhibit the activity of CYP3A to down-regulate AA metabolites EETs, consequently hampering p-Stat3 and nuclear translocation, thus impeding BC development.

Scutellarin Mediates Cytochrome P450 3A4 and 2C19 Expression via Pregnane X Receptor and Constitutive Androstane Receptor.

Breviscapine is a flavonoid extracted from Erigeron breviscapus (Vant.) Hand.-Mazz., and mainly contains scutellarin. Nuclear receptors play important roles in regulating transporter and drug metabolic enzymes. To investigate the regulatory effects of scutellarin on CYP3A4 and 2C19 in HepG2 and Caco-2 cells based on nuclear receptors PXR and CAR. The proteins and mRNA levels of CYP3A4 and CYP2C19 treated with scutellarin were detected by Western Blot and RT-qPCR. Using assays of the dual-luciferase reporter, promoter sequences containing hPXR and hCAR protein recognition and binding regulatory elements CYP3A4 and CYP2C19 were inserted upstream of the reporter gene, and the expression vector and the reporter vector were cotransfected into HepG2 and Caco-2 cells. Scutellarin inhibited mRNA of CYP3A4 and PXR, and promoted mRNA expression of CYP2C19 and CAR in RT-qPCR results. Western-blot results showed scutellarin inhibited the expression of CYP3A4 and promoted the expression of CYP2C19. The dual-luciferase reporter genes showed that scutellarin enhanced the expression level of CYP2C19, and when its concentration was 40 and 80μmol/L, CYP3A4 was significantly increased. Scutellarin down-regulates CYP3A4 through PXR, and its mechanism may work by up-regulating CAR, binding to PXR to inhibit PXR-mediated expression of CYP3A4. Scutellarin up-regulates CYP2C19 through CAR.

Coding RNA expression profile and transcription factor analysis of H.pylori-associated chronic atrophic gastritis

PurposeAtrophic gastritis, one of the processes leading to gastric cancer (GC), is closely related to Helicobacter pylori (HP) infection. This study aimed to understand how HP causes chronic inflammation that leads to ulcers and stomach problems. MethodsTwenty-eight CAG patients were included in the study (9 HP-infected and 19 HP-uninfected). Endoscopy, histopathology, and high-throughput mRNA sequencing were performed. Differentially expressed genes (DEGs) were validated via qRT-PCR. ResultsPrincipal component analysis (PCA) results showed that more than 88.9 ​% of the samples were classified into the HP (+) group. A total of 157 DEGs were identified, of which 38 were up-regulated and 119 were down-regulated. The DEGs were mainly enriched in the biological process (BP) terms associated with immune system process, adaptive immune response, G protein-coupled receptor signaling pathway, as well as point to numerous key pathways, including fat digestion and absorption, retinol metabolism, steroid hormone biosynthesis, ascorbate and aldarate metabolism, and chemical carcinogenesis. APOA1, APOA4, FOXP3, NR1H4, ABCG5, ACTA1, CCL19, CCR7, CYP3A4, and PDCD had the highest degrees in protein–protein interaction network as the hub genes; they were also included into the transcription factor (TF)-target network except for PDCD. APOA1 and CYP3A4 were extremely significantly up-regulated in HP (+) CAG patients compared with the HP (−) CAG patients, while FOXP3, CCR7 and CCL19 were significantly down-regulated. ConclusionThe expression of APOA1, CYP3A4, FOXP3, CCR7, and CCL19 are the potential indicators for CAG to GC development, being the biomarkers to predict progression of CAG and poor prognosis of GC.

Study on the protein expression of CYP3A4 and CYP2C18

Background As a drug enzyme, CYP450 has important significance in clinical treatment and the combined use of drugs, but the current research has not yet achieved high efficiency and large amount of expression in vitro. Therefore, the expression of CYP450 in vitro has become a hot spot.Objective Study the in vitro high-efficiency expression and time optimization of CYP3A4 and CYP2C18. Methods (1) CYP3A4 and CYP2C18 yeast recombinants were induced, and 24h, 48h and 72h bacterial liquid were collected respectively, to obtain the crude enzyme solution at different time periods, and then the enzyme solution at different time periods was subjected to SDS-PAGE protein electrophoresis to obtain its target bands. The results of comparative analysis between the protein bands and the Marker were observed by observing the gel running results. Finally, the enzyme activity was determined by liquid chromatography. Results The expressed proteins of CYP3A4 and CYP2C18 yeast recombinants were successfully induced. The target bands were obtained by SDS-PAGE protein electrophoresis and the optimal expression time was analyzed. Finally, the activity of the products was verified by liquid chromatography. Conclusion CYP3A4 and CYP2C18 yeast recombinants were successfully expressed in this experiment, and the most abundant expression of CYP3A4 and CYP2C18 was found at 72h by comparing the position of electrophoresis bands. At the same time, it was also verified that the expressed products were active, and CYP3A4 had the highest activity at 48h, which could be used for the study of CYP450 metabolism in vitr.

NcBAF enhances PXR-mediated transcriptional activation in the human and mouse liver

Pregnane X receptor (PXR) is one of the key regulators of drug metabolism, gluconeogenesis, and lipid synthesis in the human liver. Activation of PXR by drugs such as rifampicin, simvastatin, and efavirenz causes adverse reactions such as drug-drug interaction, hyperglycemia, and dyslipidemia. The inhibition of PXR activation has merit in preventing such adverse events. Here, we demonstrated that bromodomain containing protein 9 (BRD9), a component of non-canonical brahma-related gene 1-associated factor (ncBAF), one of the chromatin remodelers, interacts with PXR. Rifampicin-mediated induction of CYP3A4 expression was attenuated by iBRD9, an inhibitor of BRD9, in human primary hepatocytes and CYP3A/PXR-humanized mice, indicating that BRD9 enhances the transcriptional activation of PXR in vitro and in vivo. Chromatin immunoprecipitation assay reveled that iBRD9 treatment resulted in attenuation of the rifampicin-mediated binding of PXR to the CYP3A4 promoter region, suggesting that ncBAF functions to facilitate the binding of PXR to its response elements. Efavirenz-induced hepatic lipid accumulation was attenuated by iBRD9 in C57BL/6J mice, suggesting that the inhibition of BRD9 would be useful to reduce the risk of efavirenz-induced hepatic steatosis. Collectively, we found that inhibitors of BRD9, a component of ncBAF that plays a role in assisting transactivation by PXR, would be useful to reduce the risk of PXR-mediated adverse reactions.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate toxicity of the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine in human placental villous trophoblast BeWo cells

Trichloroethylene (TCE) is a known human carcinogen with toxicity attributed to its metabolism. S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is a metabolite of TCE formed downstream in TCE glutathione (GSH) conjugation and is upstream of several toxic metabolites. Despite knowledge that DCVC stimulates reactive oxygen species (ROS) generation and apoptosis in placental cells, the extent to which these outcomes are attributable to DCVC metabolism is unknown. The current study used N-acetyl-L-cysteine (NAC) at 5 mM and aminooxyacetic acid (AOAA) at 1 mM as pharmacological modifiers of DCVC metabolism to investigate DCVC toxicity at concentrations of 5–50 µM in the human placental trophoblast BeWo cell model capable of forskolin-stimulated syncytialization. Exposures of unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells were studied. NAC pre/co-treatment with DCVC either failed to inhibit or exacerbated DCVC-induced H2O2 abundance, PRDX2 mRNA expression, and BCL2 mRNA expression. Although NAC increased mRNA expression of CYP3A4, which would be consistent with increased generation of the toxic metabolite N-acetyl-DCVC sulfoxide (NAcDCVCS), a CYP3A4 inhibitor ketoconazole did not significantly alter BeWo cell responses. Moreover, AOAA failed to inhibit cysteine conjugate β-lyase (CCBL), which bioactivates DCVC, and did not affect the percentage of nuclei condensed or fragmented, a measure of apoptosis, in all BeWo cell models. However, syncytialized cells had higher CCBL activity compared to unsyncytialized cells, suggesting that the former may be more sensitive to DCVC toxicity. Together, although neither NAC nor AOAA mitigated DCVC toxicity, differences in CCBL activity and potentially CYP3A4 expression dictated the differential toxicity derived from DCVC.

Identification of Key Biomarkers in Hepatocellular Carcinoma Induced by Non-alcoholic steatohepatitis or Metabolic Syndrome via Integrated Bioinformatics Analysis.

The burden of hepatocellular carcinoma (HCC) is steadily growing because obesity, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD) are replacing viral- and alcohol-related liver disease as major pathogenic promoters. The current study attempted to identify the key genes and pathways in the non-alcoholic steatohepatitis (NASH) induced development of HCC using integrated bioinformatics analyses. Two gene expression profiling datasets, GSE102079 and GSE164760 were downloaded. Differentially expressed genes (DEGs) from HCC and healthy control samples were screened. Functional enrichment analyses based on Gene Ontology (GO) resource, Kyoto Encyclopedia of Genes and Genomes (KEGG) resource. Then protein-protein interaction (PPI) of these DEGs was visualized by Cytoscape with Search Tool for the Retrieval of Interacting Genes (STRING). Expression and survival analysis of hub genes, methylation and genetic mutation analysis were explored withGEPIA2, UALCAN, GSCA, and TIMER2.0 databases. We identified 158 overlapping genes from the 2 datasets. Up-regulated genes were mainly related to the proliferation, adhesion and metastasis of tumors, while down-regulated genes were mainly related to oxidative stress and energy metabolism. CDKN2A, SPP1, CYP2C9 and CYP4A11 were associated with prognostic performance and were considered the potential crucial genes, which SPP1, CYP2C9 and CYP4A11 were identified as the DNA methylation-driven genes. In different mutation statuses of HCC, gene expression of CDKN2A, SPP1, CYP2C9 and CYP4A11 showed significant differences. CDKN2A and SPP1 were identified as risk genes, while CYP2C9 and CYP4A11 were identified as protective genes, which may affect the transformation of NASH into HCC.

Anti-Cancer Prodrug Cyclophosphamide Exerts Thrombogenic Effects on Human Venous Endothelial Cells Independent of CYP450 Activation-Relevance to Thrombosis.

Cancer patients are at a very high risk of serious thrombotic events, often fatal. The causes discussed include the detachment of thrombogenic particles from tumor cells or the adverse effects of chemotherapeutic agents. Cytostatic agents can either act directly on their targets or, in the case of a prodrug approach, require metabolization for their action. Cyclophosphamide (CPA) is a widely used cytostatic drug that requires prodrug activation by cytochrome P450 enzymes (CYP) in the liver. We hypothesize that CPA could induce thrombosis in one of the following ways: (1) damage to endothelial cells (EC) after intra-endothelial metabolization; or (2) direct damage to EC without prior metabolization. In order to investigate this hypothesis, endothelial cells (HUVEC) were treated with CPA in clinically relevant concentrations for up to 8 days. HUVECs were chosen as a model representing the first place of action after intravenous CPA administration. No expression of CYP2B6, CYP3A4, CYP2C9 and CYP2C19 was found in HUVEC, but a weak expression of CYP2C18 was observed. CPA treatment of HUVEC induced DNA damage and a reduced formation of an EC monolayer and caused an increased release of prostacyclin (PGI2) and thromboxane (TXA) associated with a shift of the PGI2/TXA balance to a prothrombotic state. In an in vivo scenario, such processes would promote the risk of thrombus formation.

Role of circadian clock in the chronoefficacy and chronotoxicity of clopidogrel.

The role of circadian locomotor output cycles kaput (CLOCK) in regulating drug chronoefficacy and chronotoxicity remains elusive. Here, we aimed to uncover the impact of CLOCK and dosing time on clopidogrel efficacy and toxicity. The antiplatelet effect, toxicity and pharmacokinetics experiments were conducted with Clock-/- mice and wild-type mice, after gavage administration of clopidogrel at different circadian time points. The expression levels of drug-metabolizing enzymes were determined by quantitative polymerase chain reaction (qPCR) and western blotting. Transcriptional gene regulation was investigated using luciferase reporter and chromatin immunoprecipitation assays. The antiplatelet effect and toxicity of clopidogrel in wild-type mice showed a dosing time-dependent variation. Clock ablation reduced the antiplatelet effect of clopidogrel, but increased clopidogrel-induced hepatotoxicity, with attenuated rhythms of clopidogrel active metabolite (Clop-AM) and clopidogrel, respectively. We found that Clock regulated the diurnal variation of Clop-AM formation by modulating the rhythmic expression of CYP1A2 and CYP3A1, and altered clopidogrel chronopharmacokinetics by regulation of CES1D expression. Mechanistic studies revealed that CLOCK activated Cyp1a2 and Ces1d transcription by directly binding to the enhancer box (E-box) elements in their promoters, and promoted Cyp3a11 transcription through enhancing the transactivation activity of albumin D-site-binding protein (DBP) and thyrotroph embryonic factor (TEF). CLOCK regulates the diurnal rhythmicity in clopidogrel efficacy and toxicity through regulation of CYP1A2, CYP3A11 and CES1D expression. These findings may contribute to optimizing dosing schedules for clopidogrel and may deepen understanding of the circadian clock and chronopharmacology.

Pregnancy related hormones increase CYP3A mediated buprenorphine metabolism in human hepatocytes: a comparison to CYP3A substrates nifedipine and midazolam.

Introduction: Pregnancy increases the clearance of CYP3A4 substrate drugs and pregnancy-related hormones (PRHs) induce hepatic CYP3A4 expression and metabolism. However, it remains unclear to what extent the magnitude of PRH-evoked changes in hepatic CYP3A metabolism varies across multiple substrates. This study quantified the impact of PRHs on CYP3A protein concentrations and buprenorphine metabolism in human hepatocytes, and compared the magnitude of these effects to nifedipine and midazolam metabolism. Methods: Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRHs, administered in combination across a range of physiologically relevant concentrations, for 72h. Absolute protein concentrations of CYP3A4, CYP3A5, and CYP3A7 in SCHH membrane fractions were quantified by nanoLC-MS/MS, and norbuprenorphine (nor-BUP), dehydro-nifedipine (dehydro-NIF), and 1-hydroxy-midazolam (1-OH-MDZ) formation was evaluated. Results: Compared to control, PRH exposure increased CYP3A4, CYP3A7, and total CYP3A protein concentrations, but not CYP3A5 concentrations, and increased nor-BUP, dehydro-NIF, and 1-OH-MDZ formation in a concentration-dependent manner. The formation of nor-BUP, dehydro-NIF, and 1-OH-MDZ each positively correlated with PRH-mediated changes in total CYP3A protein concentrations. The PRH-evoked increase in nor-BUP formation was evident in all donors; however, the PRH induction of dehydro-NIF and 1-OH-MDZ formation was diminished in a hepatocyte donor with high basal CYP3A5 expression. Discussion: These findings demonstrate that PRHs increase buprenorphine, nifedipine, and midazolam metabolism in SCHH via induction of CYP3A4 and total CYP3A protein concentrations, and the magnitude of these effects vary across hepatocyte donors in a substrate-specific manner. These data provide insight into the contribution of PRH induction of CYP3A4 metabolism to increased buprenorphine clearance during pregnancy.

Mechanistic, functional and clinical aspects of pro-inflammatory cytokine mediated regulation of ADME gene expression in 3D human liver spheroids.

During systemic inflammation, pro-inflammatory cytokines alter metabolism and transport of drugs affecting the clincal outcome. We used an in vivo like human 3D liver spheroid model to study the effects and mechanisms of pro-inflammatory cytokines on the expression of nine different genes encoding enzymes responsible for the metabolism of > 90% of clinically used drugs. Treatment of spheroids with pathophysiologically relevant concentrations of IL-1β, IL-6 or TNFα resulted in a pronounced decrease in mRNA expression of CYP3A4 and UGT2B10 within 5 h. The reduction of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 mRNA expression was less pronounced, whereas the pro-inflammatory cytokines caused increased CYP2E1 and UGT1A3 mRNA expression. The cytokines did not influence expression of key nuclear proteins, nor the activities of specific kinases involved in the regulation of genes encoding drug metabolizing enzymes. However, ruxolitinib, a JAK1/2 inhibitor, inhibited the IL-6 dependent increase in CYP2E1 and the decrease in CYP3A4 and UGT2B10 mRNA expression. We evaluated the effect of TNFα in hepatocytes in 2D plates and found a rapid decrease in DME mRNA both in the absence or presence of the cytokines. Taken together, these data suggest that pro-inflammatory cytokines regulate multiple gene- and cytokine-specific events seen in in vivo and in 3D but not in 2D liver models. We propose that the 3D spheroid system is suitable for the prediction of drug metabolism under conditions of inflammation and constitutes a versatile system for short- and long-term pre-clinical and mechanistic studies of cytokine-induced changes in drug metabolism.

Exploring the Mechanism of Hepatotoxicity Induced by Dictamnus dasycarpus Based on Network Pharmacology, Molecular Docking and Experimental Pharmacology.

The root bark of Dictamnus dasycarpus Turcz is a traditional Chinese medicine, Dictamni Cortex (DC), which is mainly used in the clinical treatment of skin inflammation, eczema, rubella, rheumatism, and gynecological inflammation. Unexpectedly, there are some cases of liver injury after the administration of DC. However, the mechanism of hepatotoxicity remains ambiguous. The aim of this study was to explore the mechanism and substance bases of DC hepatotoxicity based on network pharmacology and molecular docking, verified through pharmacological experiments. Partial prototype components and metabolites in vivo of quinoline alkaloids from DC were selected as candidate compounds, whose targets were collected from databases. Network pharmacology was applied to study the potential hepatotoxic mechanism after correlating the targets of candidate compounds with the targets of hepatotoxicity. Molecular docking was simulated to uncover the molecular mechanism. Furthermore, the hepatotoxicity of the extract and its constituents from DC was evaluated in vivo and in vitro. We constructed the "potential toxic components-toxic target-toxic pathway" network. Our results showed that the targets of DC included CYP1A2 and GSR, participating in heterologous steroid metabolism, REDOX metabolism, drug metabolism, heterocyclic metabolic processes, the synthesis of steroid hormone, cytochrome P450 metabolism, chemical carcinogens and bile secretion pathways. In vitro and in vivo experiments displayed that DC could result in a decrease in GSH-Px and oxidative stress, simultaneously inhibiting the expression of CYP1A2 and inducing hepatotoxicity. These results further indicated the mechanism of hepatotoxicity induced by Dictamnus dasycarpus, providing a basic theory to explore and prevent hepatotoxicity in the clinical usage of Dictamnus dasycarpus.

Expression of CYP2B6 Enzyme in Human Liver Tissue of HIV and HCV Patients.

Background and Objectives: Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections present significant public health challenges worldwide. The management of these infections is complicated by the need for antiviral and antiretroviral therapies, which are influenced by drug metabolism mediated by metabolic enzymes and transporters. This study focuses on the gene expression of CYP2B6, CYP3A4, and ABCB1 transporters in patients with HIV, HCV, and HIV/HCV co-infection, aiming to assess their potential association with the choice of therapy, patohistological and clinical parameters of liver damage such as the stage of liver fibrosis, serum levels of ALT and AST, as well as the grade of liver inflammation and other available biochemical parameters. Materials and Methods: The study included 54 patients who underwent liver biopsy, divided into HIV-infected, HCV-infected, and co-infected groups. The mRNA levels of CYP2B6, CYP3A4, and ABCB1 was quantified and compared between the groups, along with the analysis of liver fibrosis and inflammation levels. Results: The results indicated a significant increase in CYP2B6 mRNA levels in co-infected patients, a significant association with the presence of HIV infection with an increase in CYP3A4 mRNA levels. A trend towards downregulation of ABCB1 expression was observed in patients using lamivudine. Conclusions: This study provides insight into gene expression of CYP2B6 CYP3A4, and ABCB1 in HIV, HCV, and HIV/HCV co-infected patients. The absence of correlation with liver damage, inflammation, and specific treatment interventions emphasises the need for additional research to elucidate the complex interplay between gene expression, viral co-infection, liver pathology, and therapeutic responses in these particular patients population.

Pharmacokinetic Drug-Drug Interactions: A Systematic Review of the Cytochrome P450 (CYP) Isoenzyme 3A4

The cytochrome P450 (CYP) isoenzyme 3A4 or CYP3A4 is a major drug-metabolizing enzyme that has the potential to cause pharmacokinetic drug-drug interactions. Primary research studies have demonstrated CYP3A4-mediated drug-drug interactions through a variety of mechanisms. However, there has been no review during the last 10 years of pharmacokinetic drug-drug interactions mediated by CYP3A4 isoenzymes. It is necessary to systematically review the pharmacokinetic drug-drug interactions mediated by CYP3A4. Source review of articles were retrieved from the PubMed and Scopus databases. The preparation of keywords through the population, intervention, comparison, and outcomes (PICO) method written based on the Boolean operator. Reporting the results of the paper search is presented in the Prisma version 1 2020 flowchart. The risk of bias assessment used COHORT tools and Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) tools. Data was analyzed narratively. Pharmacokinetic drug interactions are mediated by CYP3A4 through the mechanism of induction, activation, inhibition, and inactivation. Induction or activation of CYP3A4 can cause an increase in CYP3A4 expression, so that the drug is metabolized more quickly and has the potential to lose drug efficacy. Inhibition and inactivation of CYP3A4 causes plasma drug levels to increase and drug elimination time to last longer. CYP3A4 plays a major role in the bioactivation of drugs that cause hepatotoxicity through the formation of reactive metabolites. The use of drugs needs to be monitored to avoid pharmacokinetic drug interactions.

Protocatechuic acid inhibits LPS-induced mastitis in mice through activating the pregnane X receptor.

Mastitis refers to the inflammation in the mammary gland caused by various reasons. Protocatechuic acid (PCA) exerts anti-inflammatory effect. However, no studies have shown the protective role of PCA on mastitis. We investigated the protective effect of PCA on LPS-induced mastitis in mice and elucidated its possible mechanism. LPS-induced mastitis model was established by injection of LPS into the mammary gland. The pathology of mammary gland, MPO activity and inflammatory cytokine production were detected to evaluate the effects of PCA on mastitis. In vivo, PCA significantly attenuated LPS-induced mammary pathological changes, MPO activity, TNF-α and IL-1β production. In vitro, the production of inflammatory cytokines TNF-α and IL-1β was significantly reduced by PCA. Furthermore, LPS-induced NF-κB activation was also inhibited by PCA. In addition, PCA was found to activate pregnane X receptor (PXR) transactivation and PCA dose-dependently increased the expression of PXR downstream molecule CYP3A4. In addition, the inhibitory effect of PCA on inflammatory cytokine production was also reversed when PXR was knocked down. In conclusion, the protective effects of PCA on LPS-induced mastitis in mice through regulating PXR.

Development of an Induced Pluripotent Stem Cell-Based Liver-on-a-Chip Assessed with an Alzheimer's Disease Drug.

Liver-related drug metabolism is a key aspect of pharmacokinetics and possible toxicity. From this perspective, the availability of advanced in vitro models for drug testing is still an open need, also to the end of reducing the burden of in vivo experiments. In this scenario, organ-on-a-chip is gaining attention as it couples a state-of-the art in vitro approach to the recapitulation of key in vivo physiological features such as fluidodynamics and a tri-dimensional cytoarchitecture. We implemented a novel liver-on-a-chip (LoC) device based on an innovative dynamic device (MINERVA 2.0) where functional hepatocytes (iHep) have been encapsulated into a 3D hydrogel matrix interfaced through a porous membrane with endothelial cells (iEndo)]. Both lines were derived from human-induced pluripotent stem cells (iPSCs), and the LoC was functionally assessed with donepezil, a drug approved for Alzheimer's disease therapy. The presence of iEndo and a 3D microenvironment enhanced the expression of liver-specific physiologic functions as in iHep, after 7 day perfusion, we noticed an increase of albumin, urea production, and cytochrome CYP3A4 expression compared to the iHep static culture. In particular, for donepezil kinetics, a computational fluid dynamic study conducted to assess the amount of donepezil diffused into the LoC indicated that the molecule should be able to pass through the iEndo and reach the target iHep construct. Then, we performed experiments of donepezil kinetics that confirmed the numerical simulations. Overall, our iPSC-based LoC reproduced the in vivo physiological microenvironment of the liver and was suitable for potential hepatotoxic screening studies.

Interaction between Changan Granule and its main components in the plasma and CYP450 enzymes.

Changan Granule (CAG) is a Chinese patent drug developed based on an empirical prescription in accordance with the formulation theory of Traditional Chinese Medicine. The prescription is composed of eight herbal drugs which have been traditionally used by Chinese people for a long history. It has effects of invigorating spleen and supplementing qi, as well as regulating liver and ceasing diarrhea, and is indicated for the treatment of irritable bowel syndrome (IBS). This study was aimed to investigate the interaction between CAG and its main components and cytochrome P450 (CYP450) enzymes so as to characterize the major metabolites and metabolic enzymes and evaluate the safety concerns to its clinical use. Both in vivo and in vitro experiments using such as diarrhea-predominant IBS (IBS-D) rat model, HepG2 cells, and human liver microsomes (HLM) were carried out to investigate the interaction between CAG and its main components and CYP450 enzymes. Real-time quantitative PCR (qPCR), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and cocktail probes were employed to qualitatively or quantitatively measure the metabolites and metabolic enzymes. CAG inhibited the enzyme activities of CYP1A2, CYP2E1, CYP2D6, CYP2C9, and CYP3A4 and the mRNA expressions of CYP2E1, CYP2C9, CYP3A4, and CYP2D6 in vitro. CAG down-regulated the increased expression of CYP1A2 and up-regulated the decreased expression of CYP3A1 in vivo. Twenty-two metabolites were characterized from the main components of CAG after incubation with HLM in vitro. CYP2D6, CYP2E1, CYP3A4 and CYP2C9 were identified as the characteristic metabolic enzymes. This study provides a reference for clinical application of CAG in safety. CAG and CYP450 enzymes are interacted. CAG is mainly metabolized by CYP2E1 and CYP2D6. The expression of CYP2E1 and CYP2D6 are more susceptible to be influenced by CAG in comparison with that of CYP3A4, CYP2C9 and CYP1A2. It implies the potential risk of interaction when CAG is taken together with the drugs metabolized by CYP2E1 and CYP2D6.

Impairment of aryl hydrocarbon receptor signalling promotes hepatic disorders in cancer cachexia.

The aryl hydrocarbon receptor (AHR) is expressed in the intestine and liver, where it has pleiotropic functions and target genes. This study aims to explore the potential implication of AHR in cancer cachexia, an inflammatory and metabolic syndrome contributing to cancer death. Specifically, we tested the hypothesis that targeting AHR can alleviate cachectic features, particularly through the gut-liver axis. AHR pathways were explored in multiple tissues from four experimental mouse models of cancer cachexia (C26, BaF3, MC38 and APCMin/+ ) and from non-cachectic mice (sham-injected mice and non-cachexia-inducing [NC26] tumour-bearing mice), as well as in liver biopsies from cancer patients. Cachectic mice were treated with an AHR agonist (6-formylindolo(3,2-b)carbazole [FICZ]) or an antibody neutralizing interleukin-6 (IL-6). Key mechanisms were validated in vitro on HepG2 cells. AHR activation, reflected by the expression of Cyp1a1 and Cyp1a2, two major AHR target genes, was deeply reduced in all models (C26 and BaF3, P<0.001; MC38 and APCMin/+ , P<0.05) independently of anorexia. This reduction occurred early in the liver (P<0.001; before the onset of cachexia), compared to the ileum and skeletal muscle (P<0.01; pre-cachexia stage), and was intrinsically related to cachexia (C26 vs. NC26, P<0.001). We demonstrate a differential modulation of AHR activation in the liver (through the IL-6/hypoxia-inducing factor 1α pathway) compared to the ileum (attributed to the decreased levels of indolic AHR ligands, P<0.001), and the muscle. In cachectic mice, FICZ treatment reduced hepatic inflammation: expression of cytokines (Ccl2, P=0.005; Cxcl2, P=0.018; Il1b, P=0.088) with similar trends at the protein levels, expression of genes involved in the acute-phase response (Apcs, P=0.040; Saa1, P=0.002; Saa2, P=0.039; Alb, P=0.003), macrophage activation (Cd68, P=0.038) and extracellular matrix remodelling (Fga, P=0.008; Pcolce, P=0.025; Timp1, P=0.003). We observed a decrease in blood glucose in cachectic mice (P<0.0001), which was also improved by FICZ treatment (P=0.026) through hepatic transcriptional promotion of a key marker of gluconeogenesis, namely, G6pc (C26 vs. C26+FICZ, P=0.029). Strikingly, these benefits on glycaemic disorders occurred independently of an amelioration of the gut barrier dysfunction. In cancer patients, the hepatic expression of G6pc was correlated to Cyp1a1 (Spearman's ρ=0.52, P=0.089) and Cyp1a2 (Spearman's ρ=0.67, P=0.020). With this set of studies, we demonstrate that impairment of AHR signalling contributes to hepatic inflammatory and metabolic disorders characterizing cancer cachexia, paving the way for innovative therapeutic strategies in this context.