Expression Of Cytochrome P450 Enzymes Research Articles

Involvement of P450s and nuclear receptors in the hepatoprotective effect of quercetin on liver injury by bacterial lipopolysaccharide

Objective: Quercetin (Que), a flavonoid, possesses anti-inflammatory and antioxidant properties. It has been shown to protect against liver injury induced by various factors. This study was designed to investigate the underlying mechanism of its protective effect against lipopolysaccharide (LPS)- induced liver damage.Methods: Mice were pretreated with Que for 7 consecutive days and then exposed to LPS. To study the hepatoprotective effect of Que, oxidative stress parameters, inflammatory cytokine levels in liver and serum liver function indexes were examined. Protein and mRNA expression of nuclear orphanreceptors and cytochrome P450 enzymes were measured by Western Blotting and qPCR, respectively.Results: Que significantly reduced circulating ALT, AST, ALP, and ameliorated LPS-induced histological alterations. In addition, Que obviously decreased markers of oxidative stress and pro-inflammatory cytokines. Furthermore, Que carried out the hepatoprotective effect via regulation of the expression of nuclear orphan receptors (CAR, PXR) and cytochrome P450 enzymes (CYP1A2, CYP2E1, CYP2D22, CYP3A11).Conclusions: Our findings suggested that Que pretreatment could ameliorate LPS-induced liver injury.

Resident bacteria in the small intestine can shape host drug metabolism through innate lymphoid cell‐derived IL‐22

The ability of the intestinal microbiota to influence drug metabolism has been recognized, however the mechanisms through which this occurs remain relatively unexplored. Recent work in germ‐free mice has demonstrated that conventionalization with specific pathogen free (SPF) microbiota influences the expression of cytochrome P450 (CYP) enzymes in the liver and small intestine (SI), two important sites for drug metabolism. Given that CYP enzymes, including CYP3A11 in mice, account for roughly 75% of total drug metabolism, we sought to investigate the role of specific gut‐residing microbes in shaping the expression and activity of these enzymes. Utilizing PCR arrays, we found that colonization of SPF mice with the immunomodulatory bacteria segmented filamentous bacteria (SFB) altered the expression of various CYP enzymes in the SI (but not liver), with Cyp3a11 being one of the most downregulated genes. Further analysis showed that SFB‐induced IL‐22 production by type 3 innate lymphoid cells (ILC3) correlated with reduced SI Cyp3a11 expression. Additionally, SFB colonization had no effect on the expression of Cyp3a11 in the SI of IL‐22−/− mice or RAG1−/− mice in which ILCs were depleted with anti‐Thy1 antibody. Both SFB colonization and administration of IL‐23, to activate IL‐22 from ILC3, increased the ability of the CYP3A11‐metabolized drug glibenclamide to decrease basal blood glucose levels when administered orally. In mouse SI enteroid cultures, IL‐22 dose‐dependently reduced the expression of Cyp3a11 and decreased the ability of SI enteroids to metabolize CYP3A11 specific substrates, effects that were blocked by the STAT3 inhibitor Stattic. Finally, when applied to human ileal organoids, IL‐22 induced robust changes in the transcriptome, with substantial effects on drug metabolism pathways centred around CYP3A. Our data suggest that specific gut‐resident microbes can influence the expression and activity of the drug metabolizing enzyme CYP3A11. This involves the production of IL‐22 by small intestinal ILC3, which down regulates CYP3A11 in the SI epithelium. These findings provide an understanding of how the intestinal microbiota modulates host drug metabolism, and how microbiota‐immune cell crosstalk may influence the efficacy and toxicity of various drugs.Support or Funding InformationAlberta Innovates, Canadian Association of Gastroenterology, Canadian Institute of Health Research

Combined Maternal Exposure to Cypermethrin and Stress Affect Embryonic Brain and Placental Outcomes in Mice.

Prenatal exposure to cypermethrin is a risk factor for adverse neurodevelopmental outcomes in children. In addition, maternal psychological stress during pregnancy has significant effects on fetal neurodevelopment and may influence end-stage toxicity to offspring by altering maternal xenobiotic metabolism. As such, this study examined effects of maternal exposure to alpha-cypermethrin and stress, alone and in combination, on offspring development, with a focus on fetal neurotoxicity. CD1 mouse dams were administered 10 mg/kg alpha-cypermethrin or corn oil vehicle via oral gavage from embryonic day 11 (E11) to E14. In addition, dams from each treatment were subjected to a standard model of restraint stress from E12 to E14. Cypermethrin treatment impaired fetal growth, reduced fetal forebrain volume, and increased ventral forebrain proliferative zone volume, the latter effects driven by combined exposure with stress. Cypermethrin also impaired migration of GABAergic progenitors, with different transcriptional changes alone and in combination with stress. Stress and cypermethrin also interacted in effects on embryonic microglia morphology. In addition, levels of cypermethrin were elevated in the serum of stressed dams, which was accompanied by interacting effects of cypermethrin and stress on hepatic expression of cytochrome P450 enzymes. Levels of cypermethrin in amniotic fluid were below the limit of quantification, suggesting minimal transfer to fetal circulation. Despite this, cypermethrin increased placental malondialdehyde levels and increased placental expression of genes responsive to oxidative stress, effects significantly modified by stress exposure. These findings suggest a role for interaction between maternal exposures to cypermethrin and stress on offspring neurodevelopment, involving indirect mechanisms in the placenta and maternal liver.

AK-1, a Sirt2 inhibitor, alleviates carbon tetrachloride-induced hepatotoxicity in vivo and in vitro

Background/Aim: Acute liver injury (ALI) is a life-threatening clinical syndrome that is usually caused by toxic chemicals, drugs, or pathogen infections. Sirtuin2 (Sirt2), an NAD+-dependent deacetylase, appears to play detrimental roles in liver injury. Here, we evaluated the therapeutic application targeting Sirt2 in carbon tetrachloride (CCl4)-induced ALI, by using AK-1 (a Sirt2 inhibitor).Methods: For in vivo experiments, a single injection of CCl4 was used to induce ALI. One hour later, mice were intraperitoneally injected with AK-1 and were sacrificed 24 h after CCl4 administration. For in vitro experiments, primary mouse hepatocytes were used to determine the effects of AK-1 on oxidative stress and hepatocellular death induced by CCl4.Results: AK-1 alleviated CCl4-induced ALI as confirmed by histopathologic analysis, and decreased levels of serum biochemicals and inflammatory cytokines. Although it barely affected the expression of hepatic cytochrome P450 enzymes, AK-1 attenuated CCl4-induced oxidative stress and its related cell death. Mechanistically, Sirt2 inhibition significantly increased the nuclear protein level of nuclear factor erythroid 2-related factor 2 (Nrf2), and meanwhile decreased phosphorylation of c-Jun N-terminal kinases (JNK), in normal and injured livers. Similar results were observed in vitro. AK-1 significantly attenuated CCl4-induced cytotoxicity and oxidative stress by up-regulating the activity of Nrf2, and down-regulating JNK signaling in hepatocytes.Conclusions: Our results suggest that AK-1 treatment attenuated oxidative stress and cell death in the ALI model, at least partially, via activating Nrf2 and inhibiting JNK signaling, and that Sirt2 inhibition might be a potential approach to cure ALI.

A14 GUT-RESIDING BACTERIA CAN SHAPE HOST DRUG METABOLISM IN THE SMALL INTESTINE THROUGH AN INNATE LYMPHOID CELL-IL-22 DRIVEN AXIS

Abstract Background The ability of the intestinal microbiota to influence drug metabolism has been recognized, however the mechanisms through which this occurs remain unexplored. Recent work in germ-free mice showed that conventionalization with specific pathogen free (SPF) microbiota influences the expression of cytochrome P450 (CYP) enzymes in the liver and small intestine (SI), two important sites of drug metabolism. Given that CYP enzymes, including CYP3A11 in mice, account for roughly 70% of total drug metabolism, we hypothesized that commensal gut bacteria can shape the CYP landscape to influence drug metabolism and therapeutic outcomes. Aims To investigate the role of specific gut-residing microbes in shaping the expression and activity of host drug metabolism enzymes. Methods Segmented filamentous bacteria (SFB)-free mice were obtained from Jackson Lab (Jax) and colonized with feces from SFB-mono-associated mice via oral gavage. 14 days later, expression of drug metabolism enzymes in the SI were probed using PCR arrays, and lamina propria cells isolated for flow cytometry. A monoclonal antibody for Thy1.2 was used to deplete innate lymphoid cells (ILCs) in RAG1-/- mice (lacking T- and B-cells). CYP3A11 activity was determined through the colorimetric breakdown of the CYP3A11-specific substrate 7-benzyloxyresorufin. SI organoids were generated from mice and humans, and treated with IL-22 to further assess the dynamics of CYP3A11/CYP3A4 expression and activity. Results Colonization of Jax mice with immunomodulatory SFB altered the expression of various CYP enzymes in the SI (but not liver), with Cyp3a11 being the most downregulated gene. Further analysis showed that SFB-induced IL-22 production by type 3 ILCs (ILC3) correlated with reduced SI Cyp3a11 expression. Additionally, SFB colonization had no effect on the expression of Cyp3a11 in the SI of mice in which ILCs were depleted. Both SFB colonization and administration of IL-23, to induce IL-22 from ILC3, increased the ability of the CYP3A11-metabolized drug glyburide to decrease blood glucose levels when given orally. In mouse SI enteroid cultures, IL-22 dose-dependently reduced the expression of Cyp3a11 and decreased the ability of enteroids to metabolize CYP3A11-specific substrates. Finally, IL-22 induced wide changes in the transcriptome of human SI enteroids, with substantial effects on a drug metabolism pathway centred around CYP3A. Conclusions Our data suggest that a gut-resident microbe (SFB) can influence the expression and activity of the drug metabolising enzyme CYP3A11 in the SI through an ILC3-IL-22 dependent mechanism. These findings provide an understanding of how the intestinal microbiota may modulate host drug metabolism to influence the efficacy and toxicity of various pharmaceutical compounds. Funding Agencies CAG, CIHRAbbvie, Lloyd Sutherland Investigatorship

Acetaminophen-Induced Liver Injury Alters Expression and Activities of Cytochrome P450 Enzymes in an Age-Dependent Manner in Mouse Liver.

Drug-induced liver injury (DILI) is a global medical problem. The risk of DILI is often related to expression and activities of drug-metabolizing enzymes, especially cytochrome P450s (P450s). However, changes on expression and activities of P450s after DILI have not been determined. The aim of this study is to fill this knowledge gap. Acetaminophen (APAP) was used as a model drug to induce DILI in C57BL/6J mice at different ages of days 10 (infant), 22 (child), and 60 (adult). DILI was assessed by levels of alanine aminotransferase and aspartate aminotransferase in plasma with a confirmation by H&E staining on liver tissue sections. The expression of selected P450s at mRNA and protein levels was measured by real-time polymerase chain reaction and liquid chromatography-tandem mass spectrometry, respectively. The activities of these P450s were determined by the formation of metabolites from probe drugs for each P450 using ultraperformance liquid chromatography-quadrupole time of flight mass spectrometry. DILI was induced at mild to severe levels in a dose-dependent manner in 200, 300, and 400 mg/kg APAP-treated groups at child and adult ages, but not at the infant age. Significantly decreased expression at mRNA and protein levels as well as enzymatic activities of CYP2E1, 3A11, 1A2, and 2C29 were found at child and adult ages. Adult male mice were more susceptible to APAP-induced liver injury than female mice with more decreased expression of P450s. These results suggest that altered levels of P450s in livers severely injured by drugs may affect the therapeutic efficacy of drugs, which are metabolized by P450s, more particularly for males. SIGNIFICANCE STATEMENT: The current study in an animal model demonstrates that acetaminophen-induced liver injury results in decreased expression and enzyme activities of several examined drug-metabolizing cytochrome P450s (P450s). The extent of such decreases is correlated to the degree of liver injury severity. The generated data may be translated to human health for patients who have drug-induced liver injury with decreased capability to metabolize drugs by certain P450s.

Sexual Dimorphism in Doxorubicin-induced Systemic Inflammation: Implications for Hepatic Cytochrome P450 Regulation.

Doxorubicin (DOX) is an effective chemotherapeutic agent used to treat a wide variety of malignancies. In addition to its multi-organ toxicity, DOX treatment has been shown to induce systemic inflammation in patients and experimental animals. Inflammation alters the expression of hepatic cytochrome P450 (CYP) enzymes, which play important roles in drug metabolism and DOX-induced toxicity. Significant sex differences have been reported in DOX-induced toxicity; however, sex differences in DOX-induced systemic inflammation and the potential effects on hepatic CYP expression have not been determined. In the current work, male and female C57Bl/6 mice were administered DOX (20 mg/kg by intraperitoneal injection), and groups of mice were sacrificed 24 and 72 h after DOX administration. DOX elicited a systemic inflammatory response in both male and female mice, but the inflammatory response was stronger in male mice. DOX altered the expression of hepatic CYP isoforms in a sex-dependent manner. Most notably, inhibition of Cyp2c29 and Cyp2e1 was stronger in male than in female mice, which paralleled the sex differences in systemic inflammation. Therefore, sex differences in DOX-induced systemic inflammation may lead to sexually dimorphic drug interactions, in addition to contributing to the previously reported sexual dimorphism in specific DOX-induced organ toxicity.

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

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

Burmese grape, common lime, pomelo, salak, Southern langsat, star fruit, and orange modulate the expression of cytochrome P450 enzymes in mouse livers

Burmese grape, common lime, pomelo, salak, Southern langsat, star fruit, and orange modulate the expression of cytochrome P450 enzymes in mouse livers

日本人表皮組織における薬物代謝酵素Cytochrome P450（CYP）の発現量解析

日本人表皮組織における薬物代謝酵素Cytochrome P450（CYP）の発現量解析

P16 - Drug-induced liver injury alters expression and activities of drug-metabolizing cytochrome P450 enzymes in mouse liver at different ages

P16 - Drug-induced liver injury alters expression and activities of drug-metabolizing cytochrome P450 enzymes in mouse liver at different ages

Influence of Selected Carbon Nanostructures on the CYP2C9 Enzyme of the P450 Cytochrome.

Carbon nanostructures have recently gained significant interest from scientists due to their unique physicochemical properties and low toxicity. They can accumulate in the liver, which is the main expression site of cytochrome P450 (CYP450) enzymes. These enzymes play an important role in the metabolism of exogenous compounds, such as drugs and xenobiotics. Altered activity or expression of CYP450 enzymes may lead to adverse drug effects and toxicity. The objective of this study was to evaluate the influence of three carbon nanostructures on the activity and expression at the mRNA and protein levels of CYP2C9 isoenzyme from the CYP2C subfamily: Diamond nanoparticles, graphite nanoparticles, and graphene oxide platelets. The experiments were conducted using two in vitro models. A microsome model was used to assess the influence of the three-carbon nanostructures on the activity of the CYP2C9 isoenzyme. The CYP2C9 gene expression at the mRNA and protein levels was determined using a hepatoma-derived cell line HepG2. The experiments have shown that all examined nanostructures inhibit the enzymatic activity of the studied isoenzymes. Moreover, a decrease in the expression at the mRNA and protein levels was also observed. This indicates that despite low toxicity, the nanostructures can alter the enzymatic function of CYP450 enzymes, and the molecular pathways involved in their expression.

NADPH-cytochrome P450 reductase expression and enzymatic activity in primary-like human hepatocytes and HepG2 cells for in vitro biotransformation studies.

BACKGROUND:Human hepatocyte in vitro cell culture systems are important models for drug development and toxicology studies in the context of liver xenobiotic metabolism. Often, such culture systems are used to elucidate the biotransformation of xenobiotics or drugs and further investigate drug and drug metabolite effects on biological systems in terms of potential therapeutic benefit or toxicity. Human hepatocytes currently used for such in vitro studies are mostly primary cells or cell lines derived from liver cancers. Both approaches have limitations such as low proliferation capacity and progressive dedifferentiation found in primary cells or lack of liver functions in cell lines, which makes it difficult to reliably predict biotransformation of xenobiotics in patients. In order to overcome these limitations, HepaFH3 cells and Upcyte® hepatocytes representing primary-like hepatocytes of the first and second generation are increasingly used. Based on primary human hepatocyte cells transduced for stable expression of Upcyte® proliferation genes, they are mitotically active and exhibit liver functions over an extended period, making them comparable to primary human hepatocytes. These hepatocyte models show active liver metabolism such as urea and glycogen formation as well as biotransformation of xenobiotics. The latter is based on the expression, activity and inducibility of cytochrome P450 enzymes (CYP) as essential phase I reaction components. However, for further characterisation in terms of performance and existing limitations, additional studies are needed to elucidate the mechanisms involved in phase I reactions. One prerequisite is sufficient activity of microsomal NADPH-cytochrome P450 reductase (POR) functionally connected as electron donor to those CYP enzymes.OBJECTIVE:For Upcyte® hepatocytes and HepaFH3 cells, it is so far unknown to what extent POR is expressed, active, and may exert CYP-modulating effects. Here we studied POR expression and corresponding enzyme activity in human hepatoblastoma cell line HepG2 and compared this with HepaFH3 and Upcyte® hepatocytes representing proliferating primary-like hepatocytes.METHODS:POR expression of those hepatocyte models was determined at mRNA and protein level using qRT-PCR, Western Blot and immunofluorescence staining. Kinetic studies on POR activity in isolated microsomes were performed by a colorimetric method.RESULTS:The investigated hepatocyte models showed remarkable differences at the level of POR expression. Compared to primary-like hepatocytes, POR expression of HepG2 cells was 4-fold higher at mRNA and 2-fold higher at protein level. However, this higher expression did not correlate with corresponding enzyme activity levels in isolated microsomes, which were comparable between all cell systems tested. A tendency of higher POR activity in HepG2 cells compared to HepaFH3(p = 0.0829) might be present. Compared to primary human hepatocyte microsomes, POR activity was considerably lower in all hepatocyte models.CONCLUSION:In summary, our study revealed that POR expression and activity were clearly detectable in all in vitro hepatocyte models with the highest POR expression in cancer cell line HepG2. However, POR activity was lower in tested hepatocyte models when compared to human primary hepatocyte microsomes. Whether this was caused by e.g. polymorphisms or metabolic differences of investigated hepatocyte models will be target for future studies.

Molecular mechanisms underlying zinc oxide nanoparticle induced insulin resistance in mice

Zinc oxide nanoparticles (ZnO NPs) represent an important class of commercially applied materials. Recently, adverse effects of ZnO NPs were found in humans and animals following ingestion, although the effects on endocrine system disease remain unclear. In this study, ZnO NPs were orally administered to mice, and at doses of 25 mg/kg bw (body weight) ZnO NPs and above, plasma glucose increased significantly. The genome-wide effects of ZnO NPs were then investigated using RNA-sequencing technology. In the cluster analysis, the most significantly enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways concerned membranes and their close association with endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) generation. Biochemical and gene and protein expression analyses revealed that ZnO NPs activated a xenobiotic biodegradation response and increased the expression of cytochrome P450 (CYP) enzymes in mice livers, leading to ER stress. The ER stress increased ROS generation. The high levels of ROS activated the MAPK and NF-κB pathways and induced an inflammation response, resulting in the phosphorylation of insulin receptor substrate 1. Thus, the insulin resistance that developed was the primary mechanism for the increase in the plasma glucose of mice treated orally with ZnO NPs.

Ligand-Free Estrogen Receptor α (ESR1) as Master Regulator for the Expression of CYP3A4 and Other Cytochrome P450 Enzymes in the Human Liver.

Cytochrome P450 3A4 isoform (CYP3A4) transcription is controlled by hepatic transcription factors (TFs), but how TFs dynamically interact remains uncertain. We hypothesize that several TFs form a regulatory network with nonlinear, dynamic, and hierarchical interactions. To resolve complex interactions, we have applied a computational approach for estimating Sobol's sensitivity indices (SSI) under generalized linear models to existing liver RNA expression microarray data (GSE9588) and RNA-seq data from genotype-tissue expression (GTEx), generating robust importance ranking of TF effects and interactions. The SSI-based analysis identified TFs and interacting TF pairs, triplets, and quadruplets involved in CYP3A4 expression. In addition to known CYP3A4 TFs, estrogen receptor α (ESR1) emerges as key TF with the strongest main effect and as the most frequently included TF interacting partner. Model predictions were validated using small interfering RNA (siRNA)/short hairpin RNA (shRNA) gene knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)-mediated transcriptional activation of ESR1 in biliary epithelial Huh7 cells and human hepatocytes in the absence of estrogen. Moreover, ESR1 and known CYP3A4 TFs mutually regulate each other. Detectable in both male and female hepatocytes without added estrogen, the results demonstrate a role for unliganded ESR1 in CYP3A4 expression consistent with unliganded ESR1 signaling reported in other cell types. Added estrogen further enhances ESR1 effects. We propose a hierarchical regulatory network for CYP3A4 expression directed by ESR1 through self-regulation, cross regulation, and TF-TF interactions. We also demonstrate that ESR1 regulates the expression of other P450 enzymes, suggesting broad influence of ESR1 on xenobiotics metabolism in human liver. Further studies are required to understand the mechanisms underlying role of ESR1 in P450 regulation. SIGNIFICANCE STATEMENT: This study focuses on identifying key transcription factors and regulatory networks for CYP3A4, the main drug metabolizing enzymes in liver. We applied a new computational approach (Sobol's sensitivity analysis) to existing hepatic gene expression data to determine the role of transcription factors in regulating CYP3A4 expression, and used molecular genetics methods (siRNA/shRNA gene knockdown and CRISPR-mediated transcriptional activation) to test these interactions in life cells. This approach reveals a robust network of TFs, including their putative interactions and the relative impact of each interaction. We find that ESR1 serves as a key transcription factor function in regulating CYP3A4, and it appears to be acting at least in part in a ligand-free fashion.

AHR and GPER mediate the stimulatory effects induced by 3-methylcholanthrene in breast cancer cells and cancer-associated fibroblasts (CAFs)

BackgroundThe chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity.MethodsMolecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA.ResultsWe first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC.ConclusionsIn the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.

Functional expression and activity screening of all human cytochrome P450 enzymes in fission yeast.

Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2 , and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result.

Effect of "Jiaji" (EX-B2)-electroacupuncture preconditioning on expression of myocardial cytochrome P450 enzyme in rats with acute myocardial ischemia-reperfusion injury

To investigate the effect of "Jiaji"(EX-B2)-electroacupuncture(EA) preconditioning on structural changes of myocardium and expression of cytochrome P450 (CYP450) enzymes in acute myocardial ischemia-reperfusion injury (MIRI) rats. Sixty male Wistar rats were randomly divided into sham operation, model, EX-B2, Neiguan (PC6), Yanglingquan(GB34) and Quchi (LI11) groups (n＝10 in each group). The MIRI model was established by occlusion of the descending anterior branch of the left coronary artery for 30 min, followed by reperfusion for 30 min. EA preconditioning (2 Hz/100 Hz, 1 mA) was respectively applied to EX-B2, PC6, GB34, and LI11 for 30 min, once daily for 7 days. Morphological changes of myocardium were observed by H．E. staining. The ultrastructure of myocardial cells was observed by transmission electron microscope (TEM). The protein expression levels of myocardial CYP450 (CYP1A1, CYP2B2, CYP2C11 and CYP4A2) were detected by Western blot. In MIRI rats, myocardial pathological changes as swollen, disorganized arrangement, and partially ruptured myocardial fibers with unclear limit and necrosis and infiltration of lots of inflammatory cells under microscope, and focal myofilament degeneration, dissolution and necrosis with interstitial edema, vague mitochondria with swelling and vacuolation, ruptured and disorganized arrangement of crests under TEM were found in the ischemic area, which was obviously milder in rats of both EX-B2 and PC6 groups. After modeling, the expression of myocardial CYP1A1 and CYP2B2 protein were significantly up-regulated in the model group relevant to the sham operation group (P<0.05). In EA preconditioning groups, the expression levels of CYP1A1 and CYP4A2 proteins in the EX-B2, PC6 and GB34 groups were obviously down-regulated (P<0.01), and those of CYP2B2 and CYP2C11 proteins in both EX-B2 and PC6 groups were markedly up-regulated relevant to the model group (P<0.01). Compared with the EX-B2 group, the expression levels of CYP1A1, CYP4A2 proteins in the LI11 group were significantly up-regulated (P<0.05), and CYP2B2, CYP2C11 proteins were significantly down-regulated (P<0.05). No significant differences were found between the model and sham operation groups in the expression levels of myocardial CYP2C11 and CYP4A2 proteins, between the LI11 and model groups in the expression of CYP1A1, CYP2B2, CYP2C11 and CYP4A2, between the GB34 and model groups in the expression of CYP2B2, CYP2C11 (all P>0.05)．. EA preconditioning at EX-B2 and PC6 can suppress MIRI induced up-regulation of myocardial CYP450 ω-hydroxylase CYP1A1 and CYP4A2 expression and further up-regulate the expression of myocardial epoxygenase CYP2B2 and CYP2C11 proteins in acute MIRI rats, thus playing a role in myocardial protection.

Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver.

Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24hours rocked culture technique. Hepatocyte cell density was 5×106 /mL in 1000mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK+1mM deferoxamine (DEF); HTK+5mM N-acetyl-L-cysteine (NAC); and HTK+1mM DEF+5mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK+DEF group hepatocyte spheroids was increased compared with other cold storage groups (P<0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72hours (P<0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK+DEF group at both 48- and 72-hour time points (P<0.05). The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.

Role of CYP3A4 in bone marrow microenvironment–mediated protection of FLT3/ITD AML from tyrosine kinase inhibitors

AbstractJones et al address a critical issue in the field of FMS-like tyrosine kinase 3/internal tandem duplication–positive (FLT3/ITD+) leukemias, the fact that FLT3 inhibitors demonstrate superior activity against peripheral compared to bone marrow disease. They demonstrate that expression of cytochrome P450 enzymes (CYPs), in particular CYP3A4, play a major role in bone marrow stromal cell–mediated inhibition in the activity of several FLT3 inhibitors. Their findings suggest combining CYP inhibitors with FLT3 inhibitors may enhance clearance of bone marrow disease. These findings may have major implications for the treatment of FLT3+ acute myeloid leukemia.