Cytochrome P450 Activity Research Articles

Implantable 3D printed hydrogels with intrinsic channels for liver tissue engineering

This study presents the design, fabrication, and evaluation of a general platform for the creation of three-dimensional printed devices (3DPDs) for tissue engineering applications. As a demonstration, we modeled the liver with 3DPDs consisting of a pair of parallel millifluidic channels that function as portal-venous (PV) and hepatobiliary (HB) structures. Perfusion of medium or whole blood through the PV channel supports the hepatocyte-containing HB channel. Device computer-aided design was optimized for structural stability, after which 3DPDs were 3D printed in a polyethylene(glycol) diacrylate photoink by digital light processing and evaluated in vitro. The HB channels were subsequently seeded with hepatic cells suspended in a collagen hydrogel. Perfusion of 3DPDs in bioreactors enhanced the viability and function of rat hepatoma cells and were maintained over time, along with improved liver-specific functions. Similar results were observed with primary rat hepatocytes, including significant upregulation of cytochrome p450 activity. Additionally, coculture experiments involving primary rat hepatocytes, endothelial cells, and mesenchymal stem cells in 3DPDs showed enhanced viability, broad liver-specific gene expression, and histological features indicative of liver tissue architecture. In vivo implantation of 3DPDs in a rat renal shunt model demonstrated successful blood flow through the devices without clot formation and maintenance of cell viability. 3D printed designs can be scaled in 3D space, allowing for larger devices with increased cell mass. Overall, these findings highlight the potential of 3DPDs for clinical translation in hepatic support applications.

The Mediator complex subunit MoMed15 plays an important role in conferring sensitivity to isoprothiolane by modulating xenobiotic metabolism in M. oryzae.

Rice blast caused by Magnaporthe oryzae is one of the most economically important rice diseases. Fungicides such as isoprothiolane (IPT) have been used extensively for rice blast control, but resistance to IPT in M. oryzae is an emerging threat. In this study, molecular mechanisms of resistance in IPT-resistant mutants were identified. Through whole-genome sequencing and genetic transformation, we identified the gene MoMed15, encoding a transcriptional glutamine-rich co-activator Mediator complex subunit, in which mutations or deletion resulted in moderate IPT resistance. Further research found that MoMed15 physically interacted with the IPT resistance regulatory factor MoIRR to simultaneously regulate both MoIRR expression and the expression of multiple xenobiotic-metabolizing enzymes in response to IPT stress. We hypothesize that some xenobiotic-metabolizing enzymes enhance IPT toxicity by modifying the IPT structure. Variation of MoMed15 affected the recruitment of the transcriptional Mediator complex and decreased the expression of these xenobiotic-metabolizing enzymes, resulting in moderate IPT resistance. We also found that MoPGR1, encoding a protein that activates cytochrome P450 enzymes, was essential to confer IPT sensitivity, and its expression was directly regulated by MoIRR.IMPORTANCEIsoprothiolane (IPT) has been used extensively for the management of rice blast disease and IPT-resistant subpopulations have emerged in Chinese rice fields. The emergence of resistant pathogen populations has led to a steep increase in fungicide use, increasing pesticide risk for the applicator and the environment. The molecular mechanisms of IPT resistance in M. oryzae remain elusive. In this study, we demonstrated that transcriptional co-activator MoMed15 interacts with IPT resistance regulator MoIRR to recruit the Mediator complex, which promotes the expression of xenobiotic-metabolizing enzymes, leading to exacerbated IPT toxicity. The MoMed15 could be used for IPT resistance detection in rice fields.

Valifenalate-induced non-adverse thyroid changes via adaptive induction of uridine 5′-diphospho-glucuronosyltransferase (UGT) in the liver of dogs and rats but not humans

Some rat and dog toxicology studies with the fungicide valifenalate showed minimal, non-adverse thyroid changes, mostly above the maximum tolerated dose, and concomitantly with liver effects. This publication describes their mode of action (MOA), combining in vivo and new approach methodologies (NAMs), in a weight of evidence approach.Data demonstrate a MOA of liver enzyme induction via nuclear receptor CAR/PXR activation, increased thyroxine (T4) metabolism and elevated thyroid stimulating hormone (TSH) level, leading to thyroid follicular cell hypertrophy and increased thyroid weight. Non-human relevance of the MOA was demonstrated in in vitro cross species assays in rat, dog and human hepatocytes. Increased gene expression and activity of cytochrome P450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs) were observed in rat and dog hepatocytes exposed to valifenalate, with increased T4 clearance and/or T4 glucuronidation/T4-UGT activity. Therefore, a causal relationship between increased liver enzyme induction and thyroid effects in dogs and rats is concluded. Rat hepatocytes were most sensitive, while valifenalate did not increase T4-UGT activity above 2-fold of vehicle control or T4 glucuronidation and T4 clearance in human hepatocytes. Consequently, valifenalate exposure in humans is unlikely to lead to decreased T4 levels, and subsequent thyroid and developmental neurotoxicity effects. Alternative human-relevant thyroid MOAs were excluded, i.e. inhibition of deiodinases (DIO), thyroperoxidase (TPO) or the sodium iodide symporter (NIS).Due to known species differences in thyroid homeostasis between humans and laboratory animals and, importantly, based on the presented data, this liver enzyme mediated MOA is considered not relevant for human hazard assessment.

CYP5122A1 encodes an essential sterol C4-methyl oxidase in Leishmania donovani and determines the antileishmanial activity of antifungal azoles

Visceral leishmaniasis is a life-threatening parasitic disease, but current antileishmanial drugs have severe drawbacks. Antifungal azoles inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. CYP5122A1 overexpression results in growth delay, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1 possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes.

Toxicity and Sublethal Effect of Chlorantraniliprole on Multiple Generations of Aedes aegypti L. (Diptera: Culicidae)

Due to the quick development of insecticide resistance, it is crucial to optimize management programs by understanding the sublethal effects of effective insecticides like chlorantraniliprole on Aedes aegypti L. populations. Using age-stage and two-sex life tables, we investigated the sublethal impacts of chlorantraniliprole on Ae. aegypti. Larval duration in the progeny of exposed parents was reduced by 0.33–0.42 days, whereas, the longevity of male and female adults was decreased by 1.43–3.05 days. Similarly, the egg-laying capacity of F1 and F2 progeny of the exposed parents was significantly reduced from 27.3% to 41.2%. The mean generation time (T) increased up to 11.8% in exposed populations, and the net reproduction rate (Ro) decreased by 51.50–55.60%. After 24 h of chlorantraniliprole treatment, there was a significant increase in cytochrome P450 activity. Contrarily, the activity of glutathione S-transferase (GST) initially declined but started increasing after 48 h of treatment. This research highlights the importance of chlorantraniliprole in mosquito management, as well as the importance of considering sublethal effects when developing strategies to handle them. Having a thorough understanding of the harmful effects of insecticides on mosquito populations can greatly enhance the effectiveness of insecticide-based interventions, while also minimizing the risk of pest resurgence.

Detection and Quantification of Drug-Protein Adducts in Human Liver.

Covalent protein adducts formed by drugs or their reactive metabolites are risk factors for adverse reactions, and inactivation of cytochrome P450 (CYP) enzymes. Characterization of drug-protein adducts is limited due to lack of methods identifying and quantifying covalent adducts in complex matrices. This study presents a workflow that combines data-dependent and data-independent acquisition (DDA and DIA) based liquid chromatography with tandem mass spectrometry (LC-MS/MS) to detect very low abundance adducts resulting from CYP mediated drug metabolism in human liver microsomes (HLMs). HLMs were incubated with raloxifene as a model compound and adducts were detected in 78 proteins, including CYP3A and CYP2C family enzymes. Experiments with recombinant CYP3A and CYP2C enzymes confirmed adduct formation in all CYPs tested, including CYPs not subject to time-dependent inhibition by raloxifene. These data suggest adducts can be benign. DIA analysis showed variable adduct abundance in many peptides between livers, but no concomitant decrease of unadducted peptides. This study sets a new standard for adduct detection in complex samples, offering insights into the human adductome resulting from reactive metabolite exposure. The methodology presented will aid mechanistic studies to identify, quantify and differentiate between adducts that result in adverse drug reactions and those that are benign.

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

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

Oxygen control in bioreactor drives high yield production of functional hiPSC-like hepatocytes for advanced liver disease modelling

Hepatocytes-like cells (HLC) derived from human induced pluripotent stem cells show great promise for cell-based liver therapies and disease modelling. However, their application is currently hindered by the low production yields of existing protocols. We aim to develop a bioprocess able to generate high numbers of HLC. We used stirred-tank bioreactors with a rational control of dissolved oxygen concentration (DO) for the optimization of HLC production as 3D aggregates. We evaluated the impact of controlling DO at physiological levels (4%O2) during hepatic progenitors’ stage on cell proliferation and differentiation efficiency. Whole transcriptome analysis and biochemical assays were performed to provide a detailed characterization of HLC quality attributes. When DO was controlled at 4%O2 during the hepatic progenitors’ stage, cells presented an upregulation of genes associated with hypoxia-inducible factor pathway and a downregulation of oxidative stress genes. This condition promoted higher HLC production (maximum cell concentration: 2 × 106 cell/mL) and improved differentiation efficiencies (80% Albumin-positive cells) when compared to the bioreactor operated under atmospheric oxygen levels (21%O2, 0.6 × 106 cell/mL, 43% Albumin positive cells). These HLC exhibited functional characteristics of hepatocytes: capacity to metabolize drugs, ability to synthesize hepatic metabolites, and inducible cytochrome P450 activity. Bioprocess robustness was confirmed with HLC derived from different donors, including a primary hyperoxaluria type 1 (PH1) patient. The generated PH1.HLC showed metabolic features of PH1 disease with higher secretion of oxalate compared with HLC generated from healthy individuals. This work reports a reproducible bioprocess, that shows the importance of controlling DO at physiological levels to increase HLC production, and the HLC capability to display PH1 disease features.

Upregulated DNMT3a coupling with inhibiting p62-dependent autophagy contributes to NNK tumorigenicity in human bronchial epithelial cells

NNK, formally known as 4-(methyl nitrosamine)-1-(3-pyridyl)-1-butanoe, is a potent chemical carcinogen prevalent in cigarette smoke and is a key contributor to the development of human lung adenocarcinomas. On the other hand, autophagy plays a complex role in cancer development, acting as a "double-edged sword" whose impact varies depending on the cancer type and stage. Despite this, the relationship between autophagy and NNK-induced lung carcinogenesis remains largely unexplored. Our current study uncovers a marked reduction in p62 protein expression in both lung adenocarcinomas and lung tissues of mice exposed to cigarette smoke. Interestingly, this reduction appears to be contingent upon the activity of extrahepatic cytochrome P450 (CYP450), revealing that NNK metabolic activation by CYP450 enzyme escalates its potential to induce p62 downregulation. Further mechanistic investigations reveal that NNK suppresses autophagy by accelerating the degradation of p62 mRNA, thereby promoting the malignant transformation of human bronchial epithelial cells. This degradation process is facilitated by the hypermethylation of the Human antigen R (HuR) promoter, resulting in the transcriptional repression of HuR - a key regulator responsible for stabilizing p62 mRNA through direct binding. This hypermethylation is triggered by the activation of ribosomal protein S6, which is influenced by NNK exposure and subsequently amplifies the translation of DNA methyltransferase 3 alpha (DNMT3a). These findings provide crucial insights into the nature of p62 in both the development and potential treatment of tobacco-related lung cancer.

Deciphering the biodegradation of thiamethoxam by Phanerochaete chrysosporium with natural siderite: Synergistic mechanisms, transcriptomics characterization, and molecular simulation

Fungi play vital roles in the fate of organic pollutants, particularly when interacting with minerals in aquatic and soil environments. Mechanisms by which fungi may mitigate pollutions in fungus-mineral interactions are still unclear. Inspired by biogeochemical cycling, we constructed a range of co-culture systems to investigate synergistic effects of the white-rot fungus Phanerochaete chrysosporium and the iron-bearing mineral siderite on thiamethoxam (THX) transformation, a common neonicotinoid pesticide. Co-culturing with siderite significantly enhanced THX transformation during the initial 10 days with a dose effect, achieving 86 % removal within 25 days. Fungi could affect siderite’s dissolution, transformation, and precipitation through their biological activities. These interactions triggered physiological adaptation and resilience in fungi. Siderite could enhance the activity of fungal ligninolytic enzymes and cytochrome P450, facilitating biotransformation. Genes expression related to growth, energy metabolism, and oxidative stress response upregulated, enhancing fungal resilience to THX. The primary THX degradation pathways included nitro-reduction, C-N cleavage, and de-chlorination. Molecular dynamics simulations provided insights into catalytic mechanisms of enzyme-THX interactions. Together, siderite could act as natural enhancers that endowed fungi to resist physical and chemical stresses in environments, providing insights into contaminants attenuation, fungal biomineralization, and the coevolution of the Earth's lithosphere and biosphere.

The Biological Effects and Mechanisms of Fisetin on Hepatotoxicity, Liver Injury, and Liver Fibrosis: A Systematic Review

Background: Liver disease is a common cause of death worldwide. Objectives: This study aims to investigate the effects and mechanisms of Fisetin on hepatotoxicity, liver injury, and liver fibrosis. Methods: We adhered to the PRISMA 2020 guidelines in this systematic review. Our search used MeSH keywords encompassed Embase, PubMed, Web of Science, Scopus, and Cochrane Library for articles published before March 2, 2024. Relevant data was extracted from the publications, meticulously recorded in a standard form, and subsequently reviewed for outcomes and mechanisms. Results: Fisetin protects hepatocytes from oxidative stress by neutralizing free radicals (O2 −and H2O2), reduces oxidative stress, prevents lipid peroxidation, and increases endogenous antioxidants. It also reduces inflammation via lowering the production of tumor necrosis factor α (TNF-α), interleukins (IL)1α, IL-6, IL-18, IL-1β suppressing nuclear factor kappa B (NF-κB) activation, and cyclooxygenase- 2 (COX-2), inducible nitric oxide synthase (iNOS) inhibition, reducing monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), NLR family pyrin domain-containing 3 (NLRP3) inflammasome and interferon-gamma (IFN‐γ). Moreover, it inhibited apoptosis-modulated enzyme activity and detoxification enzymes via modulating the activity of cytochrome P450 and Phase II detoxification enzymes. Fisetin prevented fibrosis by inhibiting the activation of hepatic stellate cells (HSCs), attenuating extracellular matrix (ECM) remodeling-associated genes, and suppressing transforming growth factor-β (TGF-β) signaling pathway and attenuating collagen production. It decreased lipid accumulation and liver function tests. Conclusion: In vivo and in vitro studies indicated that Fisetin can enhance detoxification, attenuate liver injury, and reduce fibrosis, which helps maintain liver health.

Effects of diphenhydramine on crayfish cytochrome P450 activity and antioxidant defence mechanisms: First evidence of CYP2C- and CYP3A-like activity in marbled crayfish

Growing evidence has reported that diphenhydramine (DPH), an ionisable antihistamine, is widely present in surface waters across the world. Relative to vertebrates studied, its impact on invertebrates, particularly concerning cytochrome P450 (CYP) metabolism and oxidative stress, remains poorly understood. In this study, we aimed to investigate the effects of 2, 20, and 200 µg/L DPH on marbled crayfish (Procambarus virginalis) after 96-h exposure. Specifically, we assessed CYP activity, antioxidant enzyme responses, and acetylcholinesterase (AChE) activity in gills, muscle, and hepatopancreas. The crayfish CYP metabolised fluorogenic CYP-metabolic substrates of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) and dibenzylfluorescein (DBF), which evidenced the activity of CYP2C and CYP3A isoforms, well known in mammalian detoxification metabolism. Both BFC and DBF dealkylations showed a positive correlation with each other but were negatively correlated to water and haemolymph DPH concentrations. Exposure to 200 µg/L DPH elicited an apparent inhibition trend, albeit not significant, in BFC- and DBF-transformation activities in crayfish. Other tested 7-benzyloxyresorufin and 7-pentoxyresorufin substrates were poorly metabolised, suggesting their relatively low activity or the lack of mammalian-like CYP1A and CYP2B isoforms in marbled crayfish. The significant modulation of antioxidant enzymes was demonstrated in gills and hepatopancreas. The exposure to DPH did not alter the activity of AChE. Integrated biomarker response version 2 showed the highest cumulative effect of DPH exposure on gills, implying that gill tissue is the most reliable matrix for evaluating DPH toxicity. Activities of glutathione peroxidase and glutathione-S-transferase were the most deviated determinants among the investigated biomarkers, providing insights into the DPH toxicity in crayfish. This study brought the first insight into utilising the fluorogenically active substrates BFC and DBF to demonstrate the CYP involvement in the detoxification metabolism in marbled crayfish. Further, our results provided information on valuable antioxidant defence mechanisms and biomarker responses for a future DPH toxicity assessment in aquatic organisms.

Different ethanol exposure durations affect cytochrome P450 2E1-mediated sevoflurane metabolism in rat liver

BackgroundChronic alcohol users often exhibit an increased minimum alveolar concentration (MAC) of sevoflurane, yet the specific mechanism remains unclear. It has been reported that ethanol exposure can upregulate the protein expression and enzyme activity of cytochrome P450 2E1 (CYP2E1). CYP2E1 is a key enzyme that converts 2–5% of sevoflurane into equimolar amounts of hexafluoroisopropanol (HFIP) and F−. This study aims to explore whether ethanol exposure could alter sevoflurane metabolism through CYP2E1 modulation, potentially explaining the increased MAC observed in alcohol users.MethodsEighty adult male Sprague-Dawley (SD) rats were randomly divided into two groups and received either 50% ethanol (dose: 3 g/kg) or 0.9% saline twice daily by gavage. After 1, 2, 3, and 4 weeks of gavage, ten rats were randomly selected from each group to undergo 1-hour anesthesia with 2.3% sevoflurane. Blood samples were collected after anesthesia to measure the concentration of free HFIP using gas chromatography. Additionally, the left lobe tissue of the liver was collected for the analysis of CYP2E1 protein expression by Western blot and CYP2E1 enzyme activity by colorimetric assay. Correlations between these parameters were analyzed using Pearson’s correlation.ResultsIn the ethanol group, CYP2E1 expression, activity, and the concentration of free HFIP were significantly higher at all time points compared to the control group (P < 0.05), except for protein expression in the first week (P > 0.05). Within-group comparisons indicated no significant changes in any of the parameters for the control group (P > 0.05). In the ethanol group, there was no difference in free HFIP concentration between the first and second weeks (P > 0.05), but a significant increase was observed in the third and fourth weeks (P < 0.01); protein expression and enzyme activity significantly varied over time, especially showing a notable increase from the first to the third and fourth weeks (P < 0.05). Correlation analysis revealed strong positive correlations between free HFIP concentration and CYP2E1 activity (r = 0.7898), free HFIP concentration and CYP2E1 expression (r = 0.8418), and CYP2E1 activity and expression (r = 0.8740), all with P < 0.001.ConclusionsEthanol exposure increased both the expression and enzymatic activity of CYP2E1, consequently enhancing the metabolism of sevoflurane.

Advanced Liver-on-a-Chip Model for Evaluating Drug Metabolism and Hepatotoxicity.

The liver has many important functions, including the biotransformation of drugs and detoxification of the human organism. As such, it is also exposed to many harmful substances, which leads to disorders and diseases such as cirrhosis. For these reasons, it seems important to consider liver metabolism and the direct effects on the liver when evaluating the efficacy of new drugs. Accordingly, we have developed an advanced in vitro liver model using an organ-on-a-chip approach that replicates many of the morphological and functional features of the liver in vivo. The model we created can metabolize drugs, which we demonstrated using two widely used anticancer drugs, 5-fluorouracil (5FU) and capecitabine (CAP). In addition, to the best of our knowledge, we are the first who evaluate the direct effects of these drugs not only on the viability of liver model-building cells but on their functions, such as cytochrome P450 activity and albumin production. Our study brings new hope to properly evaluating drug efficacy at the in vitro level.

Effect of cannabidiol and hemp extract on viability and function of hepatocytes derived from human induced pluripotent stem cells

Since the passage of the 2018 Agriculture Improvement Act (2018 Farm Bill), the number of products containing cannabis-derived compounds available to consumers have rapidly increased. Potential effects on liver function as a result from consumption of products containing cannabidiol (CBD), including hemp extracts, have been observed but the mechanisms for the effects are not fully understood. In this study, hepatocytes derived from human induced pluripotent stem cells (iPSCs) were used to evaluate potential hepatic effects of CBD and hemp extract at exposure concentrations ranging from 0.1 to 30 μM. Despite that a significant reduction in cell viability occurred only in the 30 μM group for both CBD and hemp extract, significant changes to cytochrome P450 activity, mitochondrial membrane potential, and lipid accumulation occurred within the concentration range of 0.1–3 μM for both CBD and hemp extract. Albumin and urea production, caspase 3/7 activity, and intracellular glutathione were significantly affected within the concentration range of 3–30 μM by CBD or hemp extract. These findings indicate that CBD and hemp extract can alter hepatic function and metabolism. The current study contributes data to help inform the evaluation of potential hepatotoxic effects of products containing cannabis-derived compounds.

The tyrosine kinase inhibitor lenvatinib is oxidized by rat cytochromes P450 and affects their expression in rat liver.

Lenvatinib is an orally effective tyrosine kinase inhibitor used to treat several types of tumors, including progressive, radioiodine-refractory differentiated thyroid cancer and advanced renal cell carcinoma. Although this drug is increasingly used in therapy, its metabolism and effects on the organism are still not described in detail. Using the rat as an experimental animal model, this study aimed to investigate the metabolism of lenvatinib by rat microsomal enzymes and cytochrome P450 (CYPs) enzymes recombinantly expressed in SupersomesTM in vitro and to assess the effect of lenvatinib on rat CYP expression in vivo. Two metabolites, O-desmethyl lenvatinib, and lenvatinib N-oxide, were produced by rat CYPs in vitro. CYP2A1 and 2C12 were found to be the most effective in forming O-desmethyl lenvatinib, while CYP3A2 was found to primarily form lenvatinib N-oxide. The administration of lenvatinib to rats caused changes in the expression of mRNA and protein, as well as the activity of various CYPs, particularly in an increase in CYP1A1. Thus, the administration of lenvatinib to rats has an impact on the level of CYPs.

Maternal obesogenic diet during pregnancy and its impact on fetal hepatic function in baboons.

Maternal obesity (MO) increases the risk of later-life liver disease in offspring, especially in males. This may be due to impaired cytochrome P450 (CYP) enzyme activity driven by an altered maternal-fetal hormonal milieu. MO increases fetal cortisol concentrations that may increase CYP activity; however, glucocorticoid receptor (GR)-mediated signaling can be modulated by alternative GR isoform expression. We hypothesized that MO induces sex-specific changes in GR isoform expression and localization that contribute to reduced hepatic CYP activity. Nonpregnant, nulliparous female baboons were assigned to either an ad libitum control diet or a high-fat, high-energy diet (HF-HED) at 9 months pre pregnancy. At 165 days' gestation (term = 180 days), fetal liver samples were collected (n = 6/sex/group). CYP activity was quantified using functional assays, and GR was measured using quantitative RT-PCR and Western blot. CYP3A activity was reduced in the HF-HED group, whereas CYP2B6 activity was reduced in HF-HED males only. Total GR expression was increased in the HF-HED group. Relative nuclear expression of the antagonistic GR isoform GRβ was increased in HF-HED males only. Reduced CYP activity in HF-HED males may be driven in part by dampened hepatic-specific glucocorticoid signaling via altered GR isoform expression. These findings highlight targetable mechanisms that may reduce later-life sex-specific disease risk.

Characterization of Neonicotinoid Metabolites by Cytochrome P450-Mediated Metabolism in Poultry.

Neonicotinoids, a neuro-effective class of insecticides, are heavily applied in agricultural activities worldwide. Poultry can be exposed to neonicotinoids by several routes, but the knowledge of neonicotinoid's metabolism in poultry and its associated interspecies differences is highly limited. Hence, this study aims to investigate the species differences in metabolite formations, as well as cytochrome P450 (CYP)-dependent metabolism of four major neonicotinoid compounds, acetamiprid, imidacloprid, clothianidin, and thiamethoxam, in poultry. In vitro biotransformation assays using hepatic microsomes of chicken, ducks, geese, quails, and rats were conducted. Metabolites of neonicotinoids were then screened by LC/Q-TOF and quantified by LC/MS/MS. The results revealed an existence of interspecies differences in the formations of N-[(6-chloro-3-pyridyl) methyl] -N-methyl acetamidine (IM-1-5) of acetamiprid and dm-clothianidin of clothianidin between chicken and other species. In addition, the greatest CYP activities in the metabolism of most neonicotinoid substrates, such as acetamiprid to dm-acetamiprid, imidacloprid to hydroxylated-imidacloprid and imidacloprid-olefin, clothianidin to dm-clothianidin, and thiamethoxam to clothianidin, were found in chicken. These results suggested that the CYPs in chicken may have a greater capacity for metabolism of neonicotinoids compared to other poultry. This study further revealed that the maximum intrinsic clearance of dn-imidacloprid and dn-clothianidin in ducks may be superintended by CYP-mediated nitro-reductions of imidacloprid and clothianidin. Further studies employing CYP recombinant enzymes may be required to elucidate the specific CYP isoforms that may be involved in neonicotinoid metabolism in avian species.

Gal-1-mediated cytochrome p450 activation promotes fibroblast into myofibroblast differentiation in pulmonary fibrosis

Pulmonary fibrosis (PF) results from excessive extracellular matrix (ECM) deposition and tissue remodeling after activation of fibroblasts into myofibroblasts. Abnormally deposited fibrotic ECM, in turn, promotes fibroblast activation and accelerates loss of lung structure and function. However, the molecular mediators and exact mechanisms by which fibrotic ECM promotes fibroblast activation are unclear. In a bleomycin-induced PF mouse model, we found Galectin-1 (Gal-1) expression was significantly increased in lung tissue, and overexpression of Gal-1 plasmid-transfected fibroblasts were activated into myofibroblasts. Using the decellularization technique to prepare decellularized fibrotic ECM and constructing a 3D in vitro co-culture system with fibroblasts, we found that decellularized fibrotic ECM induced a high expression of Gal-1 and promoted the activation of fibroblasts into myofibroblasts. Therefore, Gal-1 has been identified as a pivotal mediator in PF. Further, we found that decellularized fibrotic ECM delivered mechanical signals to cells through the Gal-1-mediated FAK-Src-P130Cas mechanical signalling pathway, while the CYP450 enzymes (mainly involved in CYP1A1, CYP24A1, CYP3A4, and CYP2D6 isoforms) acted as a chemical signalling pathway to receive mechanical signals transmitted from upstream Gal-1, thereby promoting fibroblast activation. The Gal-1 inhibitor OTX008 or the CYP1A1 inhibitor 7-Hydroxyflavone prevented PF in mice and inhibited the role of fibrotic ECM in promoting fibroblast activation into myofibroblasts, preventing PF. These results reveal novel molecular mechanisms of lung fibrosis formation and identify Gal-1 and its downstream CYP1A1 as potential therapeutic targets for PF disease treatmnts.

Qualitative and quantitative status of cytochrome P450s after the administration of a liposomal platelet substitute in rat liver

In the process of the drug development, studies on the cytochrome P450 (CYP) profiles after its administration provided fundamental information regarding drug interactions with concomitantly administered drugs. Here, we evaluated the influence of the administration of H12-(ADP)-liposomes, a platelet substitute, on the mRNA and protein expression, and metabolic activity of CYPs, with focus on the CYP1A2, CYP2C11 and CYP3A2, in rat liver. At 24 h after administering saline or H12-(ADP)-liposomes (10 mg of lipids/kg), a quantitative RT-PCR and western blot analysis revealed that the mRNA and proteins expression of all of the target hepatic CYP isoforms were not different between the saline and H12-(ADP)-liposome groups. Furthermore, an ex vivo CYP metabolic activity assay showed that hepatic CYP metabolic activities in the H12-(ADP)-liposome group were comparable to the corresponding saline group. On the other hand, the area under the blood concentration-time curve for substitutes for CYP1A2 and CYP2C11 was higher in H12-(ADP)-liposome group than in saline group, but the degree of elevations was negligible levels. At a minimum, based on these results, we conclude that H12-(ADP)-liposomes have no quantitative and qualitative effect on the hepatic CYP isoforms, indicating that the drug interactions of H12-(ADP)-liposomes with CYP-metabolizing drugs would be negligible.