Cytochrome P450 1A1 Expression Research Articles

Establishment and Characterization of Novel Human Intestinal In Vitro Models for Absorption and First-Pass Metabolism Studies.

Commonly used intestinal in vitro models are limited in their potential to predict oral drug absorption. They either lack the capability to form a tight cellular monolayer mimicking the intestinal epithelial barrier or the expression of cytochrome P450 3A4 (CYP3A4). The aim of this study was to establish a platform of colorectal cancer patient-derived cell lines for evaluation of human intestinal drug absorption and metabolism. We characterized ten 2D cell lines out of our collection with confluent outgrowth and long-lasting barrier forming potential as well as suitability for high throughput applications with special emphasis on expression and inducibility of CYP3A4. By assessment of the transepithelial electrical resistance (TEER) the cells barrier function capacity can be quantified. Very high TEER levels were detected for HROC60. A high basal CYP3A4 expression and function was found for HROC32. Eight cell lines showed higher CYP3A4 induction by stimulation via the vitamin D receptor compared to Caco-2 cells (5.1- to 16.8-fold change). Stimulation of the pregnane X receptor led to higher CYP3A4 induction in two cell lines. In sum, we identified the two cell lines HROC183 T0 M2 and HROC217 T1 M2 as useful tools for in vitro drug absorption studies. Due to their high TEER values and inducibility by drug receptor ligands, they may be superior to Caco-2 cells to analyze oral drug absorption and intestinal drug–drug interactions. Significance statement: Selecting appropriate candidates is important in preclinical drug development. Therefore, cell models to predict absorption from the human intestine are of the utmost importance. This study revealed that the human cell lines HROC183 T0 M2 and HROC217 T1 M2 may be better suited models and possess higher predictive power of pregnane X receptor- and vitamin D-mediated drug metabolism than Caco-2 cells. Consequently, they represent useful tools for predicting intestinal absorption and simultaneously enable assessment of membrane permeability and first-pass metabolism.

Homeostatic regulation of the aryl hydrocarbon receptor-cytochrome P450 1a axis by Scutellaria baicalensis-Coptis chinensis herb pair and its main constituents

Ethnopharmacological relevanceScutellaria baicalensis (SB) and Coptis chinensis (CC) are widely used traditional Chinese medicine (TCM) for “heat-clearing and damp-drying” and “purging fire and detoxifying”. SB-CC are commonly used as a herbal pair for synergistic treatment of various diseases such as bacteria-related infections, metabolic syndromes, and some inflammatory disorders. This herbal pair is commonly used in many famous TCM formula, like Huang-Lian-Jie-Du, Gegen-Qinlian, Banxia Xiexin decoction. Aryl hydrocarbon receptor (AHR) plays an essential role in the disposition of both xenobiotics and endogenous substances through the induction of cytochrome P450 1A (CYP1A) enzymes. Regulation of the AHR-CYP1A axis is increasingly implicated in drug-drug and drug-herb interactions. Research on SB-CC for regulatory effect on the AHR-CYP1A axis is only limited to few compounds. Aim of the studyThis study aimed to systematically investigate the regulatory effect of SB-CC and its main constitutes on the AHR-CYP1A axis in vitro and in vivo. Materials and methodsThe livers of mice treated with SB-CC extract were subjected to RNA-sequencing (RNA-seq). The key target genes related to drug metabolism were screened, and the differential expression genes (DEGs) were validated by qRT-PCR, Western blot, and enzyme activity assay. Luciferase reporter gene, qRT-PCR, and Western blot assays were used to determine whether SB-CC and their main constituents could activate AHR and regulate CYP1A expression in HepG2 cells. The effect of SB-CC on the pharmacokinetics of phenacetin, a CYP1A substrate, were further observed in mice to test the net effect of SB-CC on CYP1A functions. The potential CYP1A inhibitors in SB-CC were screened and their inhibitory mechanisms were also studied using human liver microsomes. ResultsAHR and drug metabolism system, especially CYP1A1 and CYP1A2, were strongly affected in the liver of SB-CC-treated mice. These results were further validated by the findings that SB-CC increased CYP1A's mRNA, protein expression and activity in mouse liver. In HepG2 cells, SB, CC, baicalin, baicalein, chrysin, oroxylin A, berberine, coptisine and epiberberine increased CYP1A1 mRNA expression in an AHR-dependent way. Interestingly, SB-CC treatment for 14 days only slightly increased the systemic exposure of paracetamol in mice. In the CYP1A inhibition assay, SB, CC, baicalin, baicalein, wogonoside, wogonin, chrysin, oroxylin A, scutellarein, columbamine, coptisine, palmatine, epiberberine, and berberrubine inhibited CYP1A activity in different degree. ConclusionsThese results suggested that SB-CC exerted dual regulatory effect on the AHR-CYP1A axis by increasing CYP1A expression but simultaneously inhibiting CYP1A activity, which may contribute to a tight modulation of AHR signaling for homeostatic control.

Aryl hydrocarbon receptor activation-mediated vascular toxicity of ambient fine particulate matter: contribution of polycyclic aromatic hydrocarbons and osteopontin as a biomarker

BackgroundExposure to ambient fine particulate matter (PM2.5) is associated with vascular diseases. Polycyclic aromatic hydrocarbons (PAHs) in PM2.5 are highly hazardous; however, the contribution of PM2.5-bound PAHs to PM2.5-associated vascular diseases remains unclear. The ToxCast high-throughput in vitro screening database indicates that some PM2.5-bound PAHs activate the aryl hydrocarbon receptor (AhR). The present study investigated whether the AhR pathway is involved in the mechanism of PM2.5-induced vascular toxicity, identified the PAH in PM2.5 that was the major contributor of AhR activation, and identified a biomarker for vascular toxicity of PM2.5-bound PAHs.ResultsTreatment of vascular smooth muscle cells (VMSCs) with an AhR antagonist inhibited the PM2.5-induced increase in the cell migration ability; NF-κB activity; and expression of cytochrome P450 1A1 (CYP1A1), 1B1 (CYP1B1), interleukin-6 (IL-6), and osteopontin (OPN). Most PM2.5-bound PAHs were extracted into the organic fraction, which drastically enhanced VSMC migration and increased mRNA levels of CYP1A1, CYP1B1, IL-6, and OPN. However, the inorganic fraction of PM2.5 moderately enhanced VSMC migration and only increased IL-6 mRNA levels. PM2.5 increased IL-6 secretion through NF-κB activation; however, PM2.5 and its organic extract increased OPN secretion in a CYP1B1-dependent manner. Inhibiting CYP1B1 activity and silencing OPN expression prevented the increase in VSMC migration ability caused by PM2.5 and its organic extract. The AhR activation potencies of seven PM2.5-bound PAHs, reported in the ToxCast database, were strongly correlated with their capabilities of enhancing the migration ability of VSMCs. Benzo(k)fluoranthene (BkF) contributed the most to the AhR agonistic activity of ambient PM2.5-bound PAHs. The association between PM2.5-induced vascular toxicity, AhR activity, and OPN secretion was further verified in mice; PM2.5-induced intimal hyperplasia in pulmonary small arteries and OPN secretion were alleviated in mice with low AhR affinity. Finally, urinary concentrations of 1-hydroxypyrene, a major PAH metabolite, were positively correlated with plasma OPN levels in healthy humans.ConclusionsThe present study offers in vitro, animal, and human evidences supporting the importance of AhR activation for PM2.5-induced vascular toxicities and that BkF was the major contributor of AhR activation. OPN is an AhR-dependent biomarker of PM2.5-induced vascular toxicity. The AhR activation potency may be applied in the risk assessment of vascular toxicity in PAH mixtures.

Long Noncoding RNAs Hepatocyte Nuclear Factor 4A Antisense RNA 1 and Hepatocyte Nuclear Factor 1A Antisense RNA 1 are Involved in Ritonavir‐induced Cytotoxicity in Hepatoma Cells

Ritonavir (RTV), a pharmacoenhancer used in anti‐HIV regimens, can induce liver damage. RTV is mainly metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A‐AS1) and HNF1A antisense RNA 1 (HNF1A‐AS1) are long noncoding RNAs (lncRNAs) that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and molecular mechanisms of HNF4A‐AS1 and HNF1A‐AS1 in RTV‐induced hepatotoxicity. Small hairpin RNAs were used to knock down HNF4A‐AS1 and HNF1A‐AS1 in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV‐induced hepatotoxicity. Chromatin immunoprecipitation qPCR was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A‐AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV‐induced cytotoxicity, whereas HNF1A‐AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A‐AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A‐AS1 knockdown. Alterations in RTV‐induced hepatotoxicity caused by decreasing HNF4A‐AS1 or HNF1A‐AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV‐induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A‐AS1 overexpression or HNF1A‐AS1 knockdown. Taken together, these results revealed that HNF4A‐AS1 and HNF1A‐AS1 modulated RTV‐induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter.

Intermittent Hypoxia Inhibits Hepatic CYP1a2 Expression and Delays Aminophylline Metabolism.

Purpose In this study, we aimed to determine the effects of intermittent hypoxia (IH) on hepatic cytochrome P450 1A2 (CYP1A2) expression and the pharmacokinetics of CYP1A2-mediated aminophylline and warfarin in vitro and in a rabbit model of obstructive sleep apnea. Materials Human normal liver (LO-2) cells were exposed to 30 min each of 1%, 1–21%, 21%, and 21–1% O2, and then, CYP1A2 expression and drug concentrations were analyzed. We compared the pharmacokinetic parameters of drugs administered to normoxic rabbits and those exposed to 10 min of IH during which the oxygen level fluctuated from 21% to 8%–10% (n = 10 per group). Results. The expression of CYP1A2 protein in vitro was significantly reduced in the IH compared with the normoxic cells (0.56 ± 0.11 vs. 1.27 ± 0.17, p < 0.001). Aminophylline was more abundant in cell culture supernatants after 48 h of IH than in those under normoxia. The T1/2, AUC0–24 h, and Ke values for aminophylline were significantly higher in the IH group. Conclusion Intermittent hypoxia inhibits hepatic CYP1A2 expression and delays aminophylline metabolism, suggesting that the impact of IH on the expression of CYP enzymes should be closely monitored in clinical practice.

The Host CYP1A1-Microbiota Metabolic Axis Promotes Gut Barrier Disruption in Methicillin-Resistant Staphylococcus aureus-Induced Abdominal Sepsis.

BackgroundHost-microbiota crosstalk has been implicated in multiple host metabolic pathway axes that regulate intestinal barrier function. Although constitutive cytochrome P4501A1 (CYP1A1) expression perturbs the microbiome-derived autoregulatory loop following enteric infection, little is known about the role of host CYP1A1 in modulating gut microbiome-mediated signaling during methicillin-resistant Staphylococcus aureus (MRSA)-induced abdominal sepsis and its effects on intestinal barrier integrity.MethodsAbdominal sepsis was induced by the intraperitoneal injection of MRSA in mice. The effect of CYP1A1 deficiency on gut barrier integrity was investigated using RNA sequencing, microbiome analyses, and targeted metabolomics. The microbiota-produced metabolites were validated in patients with sepsis and persistent MRSA infection.ResultsMice lacking CYP1A1 exhibited an altered gut microbiome, a reduced metabolic shift from lysine to cadaverine in the caecal contents and antimicrobial molecule production (Retnlb, Gbp7, and Gbp3), and they were protected against gut barrier disruption when subjected to MRSA challenge. These beneficial effects were validated in aryl hydrocarbon receptor (AHR) knockout (KO) mice by cohousing with CYP1A1 KO mice and abrogated after supplementation with cadaverine or Enterococcus faecalis, the primary microbiota genus for cadaverine synthesis. Antibiotic-driven gut dysbacteriosis impaired the survival benefit and disrupted the intestinal barrier integrity in CYP1A1 KO mice after MRSA infection. Furthermore, increased cadaverine levels in feces and serum were detected in critically ill patients with gut leakiness during persistent MRSA infection, whereas cadaverine was not detected in healthy controls. Additionally, microbiota-derived cadaverine induced enterocyte junction disruption by activating the histamine H4 receptor/nuclear factor-κB/myosin light-chain kinase signaling pathway.ConclusionThis study revealed the unexpected function of host CYP1A1 in microbiota-mediated cadaverine metabolism, with crucial consequences for dysbacteriosis following MRSA-induced abdominal sepsis, indicating that inhibiting CYP1A1 or blocking cadaverine-histamine H4 receptor signaling could be a potential therapeutic target against abdominal sepsis.Clinical Trial Registration[http://www.chictr.org.cn/index.aspx], identifier [ChiCTR1800018646].

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

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

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

Increasing the bioactivity of kynurenine by ultraviolet irradiation via resonance energy transfer in vitro

Kynurenine (Kyn) is involved in a variety of physiological/pathological reactions via activating aryl hydrocarbon receptor (Ahr). However, how to activate Ahr by Kyn under physiological/pathological conditions is still unclear. Here, we presented that Kyn (8 μM, a concentration less than the dose of Kyn-induced Ahr activation) significantly induced the nuclear transfer of Ahr and the expression of cytochrome P450 1A1 (CYP1A1, a classic biomarker for Ahr activation) when co-administered with ultraviolet (UV) irradiation in 95D cells, which were transfected transiently with siRNA against indoleamine 2,3-dioxygenase 1 (IDO 1) and cultured in cell medium supplemented with bovine serum containing bovine serum albumin (BSA), in vitro. Additionally, we found that the fluorescence intensity of BSA was attenuated by Kyn (2, 4, 6, 8, 10, 12 and 14 μM) mainly through quenching the fluorescence of tryptophan (Trp) residues in the pattern of dynamic quenching related to molecular diffusion. More important, resonance energy transfer from excited-state BSA to Kyn was confirmed, leading to the generation “energetic” Kyn that might be ability of hyperactivity according to the theory of photochemical reaction. These data indicate that UV irradiation is contributable for Kyn to function, and present a novel pattern of altering the activity of biomolecules to some degree.

PLIN2 IS AN ARYL HYDROCARBON RECEPTOR TRANSCRIPTIONAL TARGET AND MAY CONTRIBUTE TO MUCOSAL RESTITUTION

Abstract INTRODUCTION Dysregulated barrier function contributes to the pathogenesis in inflammatory bowel diseases (Crohn’s (CD) and ulcerative colitis (UC)). The cytosolic transcription factor aryl hydrocarbon receptor (AHR) is activated by both endogenous and exogenous ligands, and functionally contributes to maintaining mucosal homeostasis. Recently, we demonstrated that AHR activity promotes a pro-restitutive program in small intestine, but this pathway is disrupted in Crohn’s disease. In this study, we sought to identify genetic loci associated with AHR pathway dysfunction and to identify candidate downstream effectors linking AHR activity to mucosal healing. METHODS Organoid lines were established with biopsies from healthy patients (N=4) and patients with IBD (CD=4, UC=5) and from mice with intact Ahr (WT) or Ahr null (KO). Genomic DNA was isolated and genotyped with a custom GSA SNP chip. Organoids were differentiated and treated with AHR agonist 6-Formylindolo[3,2-b]carbazole (FICZ) or vehicle. Expression of AHR transcriptional target cytochrome P450 1A1 (CYP1A1) was determined by quantitative realtime (qRT) PCR and was used to measure AHR activity. Genomic association analysis was performed using PLINK with cell lines with high (&amp;gt;3 fold) CYP1A1 expression as controls and cells with low CYP1A1 expression as cases. RESULTS Using human organoids, nine genetic variants were associated with low AHR activity (p&amp;lt;5x10-8). One variant, rs12338361, has been identified as an expression quantitative trait locus (eQTL) for Perilipin 2 (PLIN2) in other tissues. PLIN2 is involved in lipid droplet formation and trafficking in the liver, but has also been shown to be a PPARγ effector and reduce inflammation by lowering the release of TNFa in the intestine. In WT mouse organoids, expression of Plin2 was increased in response to FICZ treatment (∼50% increase). This upregulation was dependent on Ahr, as Plin2 expression did not increase in KO mouse organoids (∼7% increase). Expression of PLIN2 was assessed in human organoids treated with FICZ and was found to correlate with AHR activity (r=0.953, p=4.896 x 10-7). CONCLUSIONS PLIN2 is a transcriptional target of AHR and may contribute to AHR-mediated epithelial restitution through activation of the PPARγ pathway. This may represent a novel method of controlling inflammation in the intestinal epithelium.

Reduced Colonic Mucosal Injury in 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Poly ADP-Ribose Polymerase (TIPARP/PARP7)-Deficient Mice.

Poly-ADP-ribose polymerases (PARPs) are important regulators of the immune system, including TCDD-inducible poly-ADP-ribose polymerase (TIPARP), also known as poly-ADP-ribose polymerase 7 (PARP7). PARP7 negatively regulates aryl hydrocarbon receptor (AHR) and type I interferon (IFN-I) signaling, both of which have been implicated in intestinal homeostasis and immunity. Since the loss of PARP7 expression increases AHR and IFN-I signaling, we used a murine dextran sulfate sodium (DSS)-induced colitis model to investigate the effect of PARP7 loss on DSS-induced intestinal inflammation. DSS-exposed Parp7−/− mice had less body weight loss, lower disease index scores, and reduced expression of several inflammation genes, including interleukin IL-6, C-x-c motif chemokine ligand 1 (Cxcl1), and lipocalin-2, when compared with wild-type mice. However, no significant difference was observed between genotypes in the colonic expression of the AHR target gene cytochrome P450 1A1 (Cyp1a1). Moreover, no significant differences in microbial composition were observed between the genotypes. Our findings demonstrate that the absence of PARP7 protein results in an impaired immune response to colonic inflammation and suggests that PARP7 may participate in the recruitment of immune cells to the inflammation site, which may be due to its role in IFN-I signaling rather than AHR signaling.

Puromycin-based purification of cells with high expression of the cytochrome P450 CYP3A4 gene from a patient with drug-induced liver injury (DILI)

BackgroundMany drugs have the potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in hepatocytes. Hepatocytes can be accurately evaluated for drug-mediated CYP3A4 induction; this is the gold standard for in vitro hepatic toxicology testing. However, the variation from lot to lot is an issue that needs to be addressed. Only a limited number of immortalized hepatocyte cell lines have been reported. In this study, immortalized cells expressing CYP3A4 were generated from a patient with drug-induced liver injury (DILI).MethodsTo generate DILI-derived cells with high expression of CYP3A4, a three-step approach was employed: (1) Differentiation of DILI-induced pluripotent stem cells (DILI-iPSCs); (2) Immortalization of the differentiated cells; (3) Selection of the cells by puromycin. It was hypothesized that cells with high cytochrome P450 gene expression would be able to survive exposure to cytotoxic antibiotics because of their increased drug-metabolizing activity. Puromycin, a cytotoxic antibiotic, was used in this study because of its rapid cytocidal effect at low concentrations.ResultsThe hepatocyte-like cells differentiated from DILI-iPSCs were purified by exposure to puromycin. The puromycin-selected cells (HepaSM or SI cells) constitutively expressed the CYP3A4 gene at extremely high levels and exhibited hepatocytic features over time. However, unlike primary hepatocytes, the established cells did not produce bile or accumulate glycogen.ConclusionsiPSC-derived hepatocyte-like cells with intrinsic drug-metabolizing enzymes can be purified from non-hepatocytes and undifferentiated iPSCs using the cytocidal antibiotic puromycin. The puromycin-selected hepatocyte-like cells exhibited characteristics of hepatocytes after immortalization and may serve as another useful source for in vitro hepatotoxicity testing of low molecular weight drugs.

Long Noncoding RNAs Hepatocyte Nuclear Factor 4A Antisense RNA 1 and Hepatocyte Nuclear Factor 1A Antisense RNA 1 Are Involved in Ritonavir-Induced Cytotoxicity in Hepatoma Cells.

Ritonavir (RTV), a pharmacoenhancer used in anti-HIV regimens, can induce liver damage. RTV is primarily metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A-AS1) and HNF1A antisense RNA 1 (HNF1A-AS1) are long noncoding RNAs that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and underlying mechanisms of HNF4A-AS1 and HNF1A-AS1 in RTV-induced hepatotoxicity. HNF4A-AS1 and HNF1A-AS1 were knocked down by small hairpin RNAs in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV-induced hepatotoxicity. Chromatin immunoprecipitation quantitative real-time polymerase chain reaction was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A-AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV-induced cytotoxicity, whereas HNF1A-AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A-AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A-AS1 knockdown. Alterations in RTV-induced hepatotoxicity caused by decreasing HNF4A-AS1 or HNF1A-AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV-induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A-AS1 overexpression or HNF1A-AS1 knockdown. Taken together, these results revealed that HNF4A-AS1 and HNF1A-AS1 modulated RTV-induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter. SIGNIFICANCE STATEMENT: HNF4A-AS1 and HNF1A-AS1, transcribed separately from neighboring antisense genes of the human transcription factor genes HNF4A and HNF1A, were identified as long noncoding RNAs that can affect RTV-induced hepatotoxicity and susceptibility to RTV-induced hepatotoxicity caused by rifampicin exposure, mainly by affecting the expression of CY3A4 via alterations in PXR enrichment and histone modification status in the CYP3A4 promoter. This discovery provides directions for further research on the mechanisms of RTV-induced liver injury.

Inhibition of 6-formylindolo[3,2-b]carbazole metabolism sensitizes keratinocytes to UVA-induced apoptosis: Implications for vemurafenib-induced phototoxicity

Ultraviolet (UV) B irradiation of keratinocytes results in the formation of the tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) which is a high-affinity ligand for the aryl hydrocarbon receptor (AHR). The resulting activation of AHR signaling induces the expression of cytochrome P450 (CYP) 1A1 which subsequently metabolizes FICZ. Importantly, FICZ is also a nanomolar photosensitizer for UVA radiation. Here, we assess whether a manipulation of the AHR-CYP1A1 axis in human epidermal keratinocytes affects FICZ/UVA-induced phototoxic effects and whether this interaction might be mechanistically relevant for the phototoxicity of the BRAF inhibitor vemurafenib. Treatment of keratinocytes with an AHR agonist enhanced the CYP1A1-catalyzed metabolism of FICZ and thus prevented UVA photosensitization, whereas an inhibition of either AHR signaling or CYP1A1 enzyme activity resulted in an accumulation of FICZ and a sensitization to UVA-induced oxidative stress and apoptosis. Exposure of keratinocytes to vemurafenib resulted in the same outcome. Specifically, CYP phenotyping revealed that vemurafenib is primarily metabolized by CYP1A1 and to a lesser degree by CYP2J2 and CYP3A4. Hence, vemurafenib sensitized keratinocytes to UVA-induced apoptosis by interfering with the CYP1A1-mediated oxidative metabolism of FICZ. In contrast to this pro-apoptotic effect, a treatment of UVB-damaged keratinocytes with vemurafenib suppressed apoptosis, a process which might contribute to the skin carcinogenicity of the drug. Our results provide insight into the mechanisms responsible for the photosensitizing properties of vemurafenib and deliver novel information about its metabolism which might be relevant regarding potential drug-drug interactions. The data emphasize that the AHR-CYP1A1 axis contributes to the pathogenesis of cutaneous adverse drug reactions.

Regulation of CYP3A4 and CYP3A5 by a lncRNA: a potential underlying mechanism explaining the association between CYP3A4*1G and CYP3A metabolism.

The cytochrome P450 3A4 (CYP3A4) enzyme is the most abundant drug-metabolizing enzyme in the liver, displaying large inter-person variability with unknown causes. In this study, we found that the expression of CYP3A4 is negatively correlated with AC069294.1 (ENSG00000273407, ENST00000608397.1), a lncRNA generated antisense to CYP3A4. Knockdown of AC069294.1 in Huh7 cells increased CYP3A4 mRNA ~3-fold, whereas overexpression of AC069294.1 decreased CYP3A4 mRNA by 89%. We also observed changes in CYP3A5 expression when AC069294.1 was knocked down or overexpressed, indicating dual effects of AC069294.1 on both CYP3A4 and CYP3A5 expression. Consistently, the expression level of CYP3A5 is also negatively correlated with AC069294.1. Previous studies have shown associations between an intronic single nucleotide polymorphism CYP3A4*1G (rs2242480) and CYP3A metabolism, but the results are inconsistent and the underlying mechanism is unclear. We show here that CYP3A4*1G (rs2242480) is associated with 1.26-fold increased expression of AC069294.1 (P < 0.0001), and decreased expression of CYP3A4 by 31% (P = 0.008) and CYP3A5 by 39% (P = 0.004). CYP3A4*1G is located ~2.7 kb upstream of AC069294.1 and has been previously reported to have increased transcriptional activity in reporter gene assays. Taken together, our results demonstrate the regulation of CYP3A4 and CYP3A5 by a novel lncRNA AC069294.1. Our results also indicate that the clinically observed CYP3A4*1G associations may be caused by its effect on the expression of AC069294.1, and thereby altered expression of both CYP3A4 and CYP3A5. Furthermore, because CYP3A4*1G is in high linkage disequilibrium with CYP3A5*1, increased AC069294.1 expression caused by CYP3A4*1G may decrease expression of the normal-functioning CYP3A5*1, explaining additional inter-person variability of CYP3A5.

Protective Effects of Maclurin against Benzo[a]pyrene via Aryl Hydrocarbon Receptor and Nuclear Factor Erythroid 2-Related Factor 2 Targeting.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon formed during the incomplete combustion of organic matter, has harmful effects. Therefore, much research is ongoing to develop agents that can mitigate the effects of B[a]P. The aim of this study was to examine the effect of maclurin, one component of the branches of Morus alba L., on the B[a]P-induced effects in HaCaT cells, a human keratinocyte cell line. Maclurin treatment inhibited aryl hydrocarbon receptor (AHR) signaling as evidenced by reduced xenobiotic response element (XRE) reporter activity, decreased expression of cytochrome P450 1A1 (CYP1A1), and reduced nuclear translocation of AHR. The B[a]P-induced dissociation of AHR from AHR-interacting protein (AIP) was suppressed by maclurin. Maclurin also inhibited the production of intracellular reactive oxygen species (ROS) induced by B[a]P. In addition, the antioxidant property of maclurin itself was demonstrated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. Furthermore, maclurin activated antioxidant response element (ARE) signaling through enhancement of ARE luciferase reporter activity and the expression of ARE-dependent genes including nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). Nrf2 activation and its nuclear translocation were promoted by maclurin through p38 MAPK activation. These data indicate that maclurin had antagonistic activity against B[a]P effects through activation of Nrf2-mediated signaling and inhibition of AHR signaling and, suggesting its potential in protecting from harmful B[a]P-containing pollutants.

Tumor Microenvironment Alters Chemoresistance of Hepatocellular Carcinoma Through CYP3A4 Metabolic Activity.

Variations in tumor biology from patient to patient combined with the low overall survival rate of hepatocellular carcinoma (HCC) present significant clinical challenges. During the progression of chronic liver diseases from inflammation to the development of HCC, microenvironmental properties, including tissue stiffness and oxygen concentration, change over time. This can potentially impact drug metabolism and subsequent therapy response to commonly utilized therapeutics, such as doxorubicin, multi-kinase inhibitors (e.g., sorafenib), and other drugs, including immunotherapies. In this study, we utilized four common HCC cell lines embedded in 3D collagen type-I gels of varying stiffnesses to mimic normal and cirrhotic livers with environmental oxygen regulation to quantify the impact of these microenvironmental factors on HCC chemoresistance. In general, we found that HCC cells with higher baseline levels of cytochrome p450-3A4 (CYP3A4) enzyme expression, HepG2 and C3Asub28, exhibited a cirrhosis-dependent increase in doxorubicin chemoresistance. Under the same conditions, HCC cell lines with lower CYP3A4 expression, HuH-7 and Hep3B2, showed a decrease in doxorubicin chemoresistance in response to an increase in microenvironmental stiffness. This differential therapeutic response was correlated with the regulation of CYP3A4 expression levels under the influence of stiffness and oxygen variation. In all tested HCC cell lines, the addition of sorafenib lowered the required doxorubicin dose to induce significant levels of cell death, demonstrating its potential to help reduce systemic doxorubicin toxicity when used in combination. These results suggest that patient-specific tumor microenvironmental factors, including tissue stiffness, hypoxia, and CYP3A4 activity levels, may need to be considered for more effective use of chemotherapeutics in HCC patients.

Nicotine stimulates CYP1A1 expression in human hepatocellular carcinoma cells via AP-1, NF-κB, and AhR

Cytochrome P450 1A1 (CYP1A1) is a member of a subfamily of enzymes involved in the metabolism of both endogenous and exogenous substrates and the chemical activation of xenobiotics to carcinogenic derivatives. Here, the effects of nicotine, a major psychoactive compound present in cigarette smoke, on CYP1A1 expression and human hepatocellular carcinoma (HepG2) cell proliferation were investigated. Nicotine stimulated CYP1A1 expression via the transcription factors, activator protein 1, nuclear factor-kappa B, and the aryl hydrocarbon receptor (AhR) signaling pathway. Pharmacological inhibition and mutagenesis studies indicated that p38 mitogen-activated protein kinase, as well as RelA (or p65), mediated the upregulation of CYP1A1 of nicotine in HepG2 cells. The antioxidant compound, N-acetyl-cysteine, abrogated nicotine-activated production of reactive oxygen species and inhibited CYP1A1 expression by nicotine. Furthermore, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was inhibited by diphenyleneiodonium (an NADPH oxidase inhibitor). Thus, these results demonstrated that AhR played an important role in nicotine-induced CYP1A1 expression. Additionally, liver hepatocellular carcinoma HepG2 cells treated with nicotine exhibited markedly enhanced proliferation via CYP1A1 expression and Akt activation.

Yinzhihuang Oral Liquid Ameliorates Hyperbilirubinemia Induced by δ-Aminolevulinic Acid and Novobiocin in Neonatal Rats.

Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200 mg/kg) or novobiocin (NOVO, 200 mg/kg). Oral treatment of YZH (80, 160 and 320 mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated protein 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.

Furmonertinib (Alflutinib, AST2818) is a potential positive control drug comparable to rifampin for evaluation of CYP3A4 induction in sandwich-cultured primary human hepatocytes.

Furmonertinib (Alflutinib, AST2818), as a third-generation epidermal growth factor receptor inhibitor with an advanced efficacy and a relatively wide safety window, has been commercially launched in China recently. However, previous clinical studies demonstrated its time- and dose-dependent clearance in a multiple-dose regimen. In vitro drug metabolism and pharmacokinetic studies have suggested that furmonertinib is mainly metabolized by cytochrome P450 3A4 (CYP3A4) and can induce these enzymes via an increased mRNA expression. This study investigated two important evaluation criteria of CYP3A4 induction by furmonertinib through quantitative proteomics and probe metabolite formation: simultaneous (1) protein expression and (2) enzyme activity with sandwich-cultured primary human hepatocytes in the same well of cell culture plates. Results confirmed that furmonertinib was a potent CYP3A4 inducer comparable with rifampin and could be used as a positive model drug in in vitro studies to evaluate the induction potential of other drug candidates in preclinical studies. In addition, inconsistencies were observed between the protein expression and enzyme activities of CYP3A4 in cells induced by rifampin but not in groups treated with furmonertinib. As such, furmonertinib could be an ideal positive control in the evaluation of CYP3A4 induction. The cells treated with 10 µM rifampin expressed 20.16 ± 5.78 pmol/mg total protein, whereas the cells induced with 0.5 µM furmonertinib expressed 4.8 ± 0.66 pmol/mg protein compared with the vehicle (0.1% dimethyl sulfoxide), which contained 0.65 ± 0.45 pmol/mg protein. The fold change in the CYP3A4 enzyme activity in the cells treated with rifampin was 5.22 ± 1.13, which was similar to that of 0.5 µM furmonertinib (3.79 ± 0.52).