Cytochrome P450 Activity Research Articles

Evaluation of stabilizing additives to protect activities of cytochrome P450 enzymes for in vitro drug testing and pharmacogenetic studies: Focus on CYP2D6.

In vitro and ex vivo studies on drug metabolism and stability are vital for drug development and pre-clinical safety assessment. Traditional in vitro models, such as liver enzyme (S9) fractions and microsomes, often fail to account for individual variability. Personalized models, including 3D cell models and organoids, offer promising alternatives but may not fully replicate physiological processes, especially for Cytochrome P450 (CYP) families involved in extrahepatic metabolism. A major challenge in these studies is the low stability and expression of CYP enzymes. This study aimed to stabilize native CYP activity in vitro by developing an optimized buffer formulation. Initial experiments using recombinant CYP supersomes and liver microsomes identified 45 μM cysteine, 4 mM dithiothreitol (DTT), and 300 μM phosphocholine (PC) as the most effective stabilizers. The applicability of these stabilizers was subsequently confirmed in primary human brain tissue, where they enabled the successful determination of CYP2D6 activity. This highlights the stabilizing buffer's utility for enhancing CYP functionality in diverse tissue types, including the brain, which plays a critical role in cerebral detoxification and drug metabolism. These findings suggest that specific enzyme stabilization can enable comprehensive evaluations of CYP function in ex vivo tissue samples, advancing the development of organoid human tissue models and supporting drug metabolism research.

Long Non-Coding RNA LOC113219358 Regulates Immune Responses in Apis mellifera Through Protein Interactions.

Long non-coding RNAs (lncRNAs) are emerging as critical regulators in honeybee physiology, influencing development, behavior, and stress responses. This study investigates the role of lncRNA LOC113219358 in the immune response and neurophysiological regulation of Apis mellifera brains. Using RNA interference (RNAi) and RNA sequencing (RNA-seq), we demonstrate that silencing lncLOC113219358 significantly alters the expression of 162 mRNA transcripts, including genes associated with detoxification, energy metabolism, and neuronal signaling. Functional enrichment analysis revealed involvement in neuropeptide signaling, ATP synthesis, and oxidative phosphorylation pathways. Acetylcholinesterase (AChE), Glutathione-S-transferase (GST) and cytochrome P450 (CYP450) activities were significantly downregulated with 48 h of RNAi treatment. Additionally, RNA pull-down assays identified 113 proteins interacting with lncLOC113219358, including ATP synthase subunits, heat shock proteins, and major royal jelly proteins, suggesting its role in cellular stress responses and neural activity modulation. These findings provide mechanistic insights into how lncLOC113219358 mediates honeybee responses to environmental stressors, contributing to our understanding of lncRNA-regulated neural and immune functions in pollinators.

Dichlormid protect wheat from fomesafen residual injury by increasing PPO expression and the photosynthesis characterize.

Fomesafen is a herbicide with long persistence in soil, causing damage to succeeding crops. Dichlormid is a widely used safener protecting maize from chloroacetanilide and thiocarbamate injury. We found that dichlormid treatment could restore the growth of wheat seedlings exposed to fomesafen stress. To explore its molecular mechanism, RNA-Seq was conducted to analysis transcript profiles between fomesafen and fomesafen+dichlormid treated wheat seedlings. The gene expression level was determined by qRT-PCR. Results showed that the up-regulated genes by dichlormid treatment were significantly enriched in pathways related to photosynthesis. The expression level of glutamyl-tRNA reductase (GTR), protoporphyrinogen IX oxidase (PPO, target of fomesafen), and magnesium chelatase (MAG) involved in chlorophyll biosynthesis was significantly up-regulated by dichlormid. And the expression level of genes in chlorophyll binding, energy biosynthesis, gibberellin biosynthesis and salicylic acid signal pathway was also validated to be significantly up-regulated by dichlormid. The detoxification enzyme activity of cytochrome P450 or glutathione S-transferase (GSTs), and their gene expression level was found to show no significant difference between fomesafen and fomesafen+dichlormid treatment. The antioxidant enzyme activity of peroxidase, superoxide, and the content malondialdehyde content was decreased by dichlormid, while the reduced glutathione content was increased by dichlormid significantly. The metabolism of fomesafen was further validated to be not influenced by dichlormid. These results suggested that dichlormid acted by increasing the expression of fomesafen target and photosynthesis related genes to alleviate fomesafen injury to wheat, but not accelerating fomesafen metabolism.

Molecular insights into developmental toxicity induced by PCB77 exposure on zebrafish via integrating transcriptomics with adverse outcome pathway.

Polychlorinated biphenyls (PCBs), a typical type of persistent organic pollutants (POPs), were previously widely employed as insulating and heat exchange fluids in transformers and capacitors. Despite knowledge of its adverse effects, the precise mechanism underlying PCB77 toxicity remains enigmatic. In this study, we utilized zebrafish as a model organism to explore the toxic effects of various concentrations of PCB77 (10, 200, and 1000μg/L) and its molecular toxicity mechanisms. Upon exposure to dosages of PCB77 throughout embryonic and larval stages, the zebrafish exhibited adverse phenotypic manifestations, including deformities, decreased heart rates, increased distances between the bulbus arteriosus (BA) and sinus venosus (SV) and reduced locomotor ability. Transcriptome analysis revealed the common enriched pathways across all PCB77 concentration groups, such as retinol metabolism, steroid hormone biosynthesis, and metabolism of xenobiotics by cytochrome P450, which are closely related to the activity of cytochrome P450 (cyp1a) enzymes. Furthermore, Adverse Outcome Pathway (AOP) framework which integrates AOPs and dose-dependent transcriptomics to predict PCB77-induced adverse outcomes (AOs) revealed that aryl hydrocarbon receptor (AhR) associated AOPs triggered by PCB77 exposure may increase early-life stage mortality and decrease cardiac development, indicating that the primary toxic pathways of PCB77 in zebrafish may involve AhR-mediated signaling. Besides, molecular docking modeling demonstrated that PCB77 could bind to the groove within the AhR domain, suggesting that PCB77 induces embryotoxicity in zebrafish through its interaction with AhR. Collectively, these findings not only deliver a thorough examination of PCB77-induced developmental toxicity as well as the underlying mechanisms, but also validate the efficacy of the analytical approach leveraging AOP framework in unraveling toxicity mechanisms of environmental contaminants, which holds promise for risk assessment associated with novel environmental pollutants.

The effect of synergists on the inhibition of detoxification enzyme activities and acaricide sensitivity in Rhizoglyphus robini.

The saffron bulb mite, Rhizoglyphus robini Claparede (Acari: Acaridae), is the most important pest of the saffron crop in Iran. This pest attacks and feeds on saffron corms. For this reason, the corms are treated with acaricides before planting. The high activity of detoxification enzymes in arthropods may reduce their pesticide sensitivity. Diethyl maleate (DEM) is an inhibitor of glutathione S-transferases (GSTs), piperonyl butoxide (PBO) is an inhibitor of cytochrome P450 monooxygenases, and triphenyl phosphate (TPP) is an inhibitor of esterase activity. A filter paper method was used to determine the efficiency of these synergists in inhibiting the activity of detoxifying enzymes of R. robini. Adult mites were treated with these three synergists for 6, 12, 24, and 48 h, respectively. The activity of each detoxifying enzyme was measured and compared to the control treatment, and the inhibition percentage was calculated each time. The results showed that DEM reduced GST activity by 59.9% after 48 h, PBO inhibited cytochrome P450 activity by 30%, and TPP suppressed esterase activity by 38.5%. The most statistically significant inhibition occurred 24 h after pretreatment with each synergist. Bioassays with 24 h pretreatment showed that the sensitivity of R. robini to propargite increased by 1.6 times with PBO, 1.7 times with TPP, and 2.5 times with DEM. In conclusion, synergists and efficient inhibition of detoxifying enzymes can play a significant role in increasing the sensitivity of agricultural pests to pesticides and can be considered in managing pesticide resistance.

The Octadecanoids: Synthesis and Bioactivity of 18-Carbon Oxygenated Fatty Acids in Mammals, Bacteria, and Fungi.

The octadecanoids are a broad class of lipids consisting of the oxygenated products of 18-carbon fatty acids. Originally referring to production of the phytohormone jasmonic acid, the octadecanoid pathway has been expanded to include products of all 18-carbon fatty acids. Octadecanoids are formed biosynthetically in mammals via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) activity, as well as nonenzymatically by photo- and autoxidation mechanisms. While octadecanoids are well-known mediators in plants, their role in the regulation of mammalian biological processes has been generally neglected. However, there have been significant advancements in recognizing the importance of these compounds in mammals and their involvement in the mediation of inflammation, nociception, and cell proliferation, as well as in immuno- and tissue modulation, coagulation processes, hormone regulation, and skin barrier formation. More recently, the gut microbiome has been shown to be a significant source of octadecanoid biosynthesis, providing additional biosynthetic routes including hydratase activity (e.g., CLA-HY, FA-HY1, FA-HY2). In this review, we summarize the current field of octadecanoids, propose standardized nomenclature, provide details of octadecanoid preparation and measurement, summarize the phase-I metabolic pathway of octadecanoid formation in mammals, bacteria, and fungi, and describe their biological activity in relation to mammalian pathophysiology as well as their potential use as biomarkers of health and disease.

Effects of mixed exposure to PFAS on adolescent non-alcoholic fatty liver disease: Integrating evidence from human cohorts, toxicogenomics, and animal models to uncover mechanisms and potential target sites.

Extensive evidence suggests a correlation between environmental pollutants, specifically perfluoroalkyl and polyfluoroalkyl substances (PFAS) and non-alcoholic fatty liver disease (NAFLD). This study aims to investigate the association and underlying mechanisms of PFAS-induced NAFLD in adolescents by employing a comprehensive approach of population-based studies, toxicogenomics, and animal models. A total of 2014 freshmen from Dali University were recruited for this study, with 1694 participants undergoing serum testing for PFAS exposure. Additionally, Comparative Toxicogenomics Database analysis and PFAS exposure experiments were conducted by orally administering PFAS to 8-week-old adult C57/6 J mice for 28 days. Epidemiological analysis of the adolescent cohort revealed that perfluorohexanesulfonic acid and perfluorooctanoic acid are significant risk factors for NAFLD in adolescents. Toxicogenomic analysis revealed that the negative regulation of gap junction assembly and glutathione derivative biosynthesis contributes to NAFLD development. Animal model studies further demonstrated that combined PFAS exposure led to pathological changes in hepatocytes, including inflammation and steatosis, elevated liver enzymes, cholestasis, and bile canalicular blockage. This study reveals that PFAS exposure serves as a significant risk factor for hepatic steatosis/NAFLD in adolescents. The activation of cytochrome P4502E1 and glutathione S-transferase A1 signaling highlights new molecular targets for PFAS-induced disruptions in hepatic lipid metabolism.

Heterotropic Activation of Cytochrome P450 3A4 by Perillyl Alcohol.

Background/Objectives: Perillyl alcohol (POH), a monoterpene natural product derived from the essential oils of plants such as perilla (Perilla frutescens), is currently in phase I and II clinical trials as a chemotherapeutic agent. In this study, we investigated the effect of POH on cytochrome P450 (CYP) activity for evaluating POH-drug interaction potential. Methods: The investigation was conducted using pooled human liver microsomes (HLMs), recombinant CYP3A4 (rCYP3A4) enzymes, and human pluripotent stem cell-derived hepatic organoids (hHOs) employing liquid chromatography-tandem mass spectrometry. Results: POH inhibited the activities of CYP2A6 and CYP2B6 with Ki of 6.35 and 3.78 μM, respectively, whereas it stimulated CYP3A4 activity in pooled HLMs incubated with midazolam (MDZ). In a direct CYP inhibition assay using HLMs, activities of CYP2C9, CYP2C19, and CYP2E1 were also inhibited by POH, with IC50 values greater than 50 μM, but those of CYP1A2, CYP2C8, CYP2D6, and CYP3A4 (testosterone) were not significantly inhibited. In pooled HLMs, the Vmax/Km value of 1'-hydroxy MDZ, but not that of 4-hydroxy MDZ, was increased 2.7-fold by 100 μM POH compared with that in the absence of POH. Moreover, stimulation of MDZ 1'-hydroxylation by CYP3A4 was observed in hHOs and rCYP3A4 with cytochrome b5 but not rCYP3A4 without cytochrome b5. Furthermore, activation of CYP3A4-mediated metabolism by POH was observed in HLMs incubated with fimasartan but not atorvastatin, buspirone, donepezil, nifedipine, or tadalafil, suggesting a substrate-dependent activation of CYP3A4 by POH. Conclusions: POH inhibits CYP2A6 and CYP2B6, but it activates CYP3A4. These findings underscore the need for further evaluation of the interactions of clinical drugs with POH.

Assessment of detoxification enzymes in pyrethroid‐resistant Listronotus maculicollis Kirby suggests additional mechanisms

AbstractInsecticide resistance is a pressing issue in urban pest management. The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby, is a highly destructive turfgrass pest in northeastern North America. The overuse of pyrethroids has led to resistance in some ABW populations in the northeastern United States. Understanding ABW resistance mechanisms is pivotal for sustaining turf quality and minimizing environmental impact. This study investigated the potential for enzymatic detoxification, a common resistance mechanism in insects, to be the mechanism behind ABW tolerance to pyrethroids. Different ABW populations’ resistance levels to bifenthrin were determined through dose–response Petri dish assays. Cytochrome P450 activity and protein concentrations were measured following bifenthrin exposure. Results indicated golf course‐specific effects on enzyme and protein levels but found no direct correlation between these levels and bifenthrin dosage. P450 activity varied among different golf course populations, showing no dependence on bifenthrin dosage. These findings question prior studies suggesting P450 involvement in bifenthrin detoxification in populations of all resistance levels. This study establishes a foundation for comprehending ABW resistance mechanisms and devising effective management strategies. However, further research is needed to investigate additional detoxification enzymes and other resistance mechanisms.

Glutathione's Role in Liver Metabolism and Hangover Symptom Relief: Dysregulation of Protein S-Glutathionylation and Antioxidant Enzymes.

Hangovers from alcohol consumption cause symptoms like headaches, nausea, and fatigue, disrupting daily activities and overall well-being. Over time, they can also lead to inflammation and oxidative stress. Effective hangover relief alleviates symptoms, prevents dehydration, and replenishes energy needed for daily tasks. Natural foods considered high in antioxidants and antiinflammatory properties may aid in the hepatic breakdown of alcohol. The study aims to investigate the impact of glutathione or its enriched yeast extract, which is recognized for its antioxidant characteristics, on alcohol metabolism and alleviating hangovers in a rat model exposed to binge drinking. In this study, glutathione and its enriched yeast extract controlled hangover behaviour patterns, including locomotor activity. Additionally, it enhanced the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) following ethanol ingestion (3 g/kg). Further, the incorporation of glutathione led to an increase in the expression of antioxidant enzymes, such as SOD and catalase, by activating the nuclear erythroid 2-related factor 2 (Nrf2) signaling pathway. This activation reduced the excessive production of reactive oxygen species (ROS) and malondialdehyde. Next, glutathione modulated the activity of cytochrome P450 2E1 (CYP2E1) and the protein expressions of Bax and Bcl2. Besides, in vitro and in vivo investigations with glutathione demonstrated a regulating effect on the pan-s-glutathionylation and its associated protein expression, glutaredoxin 1 (Grx1), glutathione-S-transferase Pi (GST-π), and glutathione reductase (GR). Together, these findings suggest that glutathione or its enriched yeast extract as a beneficial dietary supplement for alleviating hangover symptoms by enhancing alcohol metabolism and its associated Nrf2/Keap1 signalings.

Insecticide Resistance of Xenopsylla cheopis in Madagascar: Revision of Diagnostic Doses for Bioassay and Exploration of Biochemical Mechanisms.

The Oriental rat flea, Xenopsylla cheopis, is known worldwide as an efficient plague vector, including in Madagascar, where the disease remains a public health concern. Chemical control is the primary response method against X. cheopis in Madagascar. Previous bioassays focusing on different flea populations from Madagascar showed phenotypic resistance to various insecticides, including deltamethrin and fenitrothion, which, respectively, represent the previous and current chemicals for flea vector control. Despite apparent insecticide resistance, the associated mechanisms of this resistance remain poorly known. The aims of this study were to adjust diagnostic doses of deltamethrin and fenitrothion and to investigate the metabolism-based insecticide resistance of X. cheopis in Madagascar. Five available laboratory-reared flea strains of X. cheopis were selected, and their susceptibility statuses to deltamethrin and fenitrothion were determined using the WHO standard bioassay. Diagnostic doses of each insecticide were determined by the probit method, in accordance with concentration gradients. Biochemical microplate-based assays were performed to detect overproduction of cytochrome P450, alpha-/beta-esterases, and glutathione S-transferase (GST), which are signatures of metabolic resistance. The five tested strains showed different susceptibility statuses against deltamethrin and fenitrothion. The diagnostic doses were estimated to be 0.07% for deltamethrin and 1.56% for fenitrothion. Increased activities of cytochrome P450, beta-esterase, and GST enzymes in the resistant strains were revealed in comparison with those of the susceptible strain. In conclusion, readjusted diagnostic doses will help to better understand the susceptibility status of X. cheopis to deltamethrin and fenitrothion. The overproduction of cytochrome P450, beta-esterase, and GST observed on deltamethrin-resistant flea strains suggests metabolic resistance.

Genetic analyses reveal wildfire particulates as environmental pollutants rather than nutrient sources for corals.

Heterotrophic nutrients are crucial for coral growth and recovery from bleaching events. Although wildfire emissions are a potential source of these nutrients, their impact on corals was minimally investigated. In this microcosm experiment, Acropora formosa corals exhibited rapid tissue detachment upon exposure to wildfire fine particulate matter (PM2.5). Physiological and genetic analyses revealed mechanisms associated with oxidation-reduction homeostasis and nutrient metabolism. Excessive hydrogen peroxide was generated as corals activated cytochrome P450 enzymes and the respiratory burst in phagocytic cells to metabolize PM2.5, leading to oxidative damage, mitochondrial dysfunction, and cell apoptosis due to reduced superoxide dismutase activity and compromised glutathione antioxidant function. Subsequently, corals upregulated the transcriptions of genes which are related to tyrosine receptor proteins to regulate multicellular development for self-repair, increasing energy consumption. However, Symbiodiniaceae upregulated their metabolism and retained photosynthates, reducing nutrient supply to the coral host. Therefore, the host temporarily utilized lipid reserves via the glyoxylate cycle, but excessive consumption disrupted lipid and carbohydrate metabolism, ultimately weakening cell adhesion and causing coral tissue detachment. Additionally, the downregulation of HSP70 expression, potentially linked to decreased sacsin and mitochondrial apoptosis, accelerated coral heat bleaching. This study elucidates the mechanisms by which wildfire PM2.5 at environmental concentrations poses risks to corals, particularly in a warming climate.

Revealing the Immune Response of Sitona callosus Gyllenhal to Entomopathogenic Fungi Beauveria bassiana Infection Through Integrative Analyses of Transcriptomics and Metabolomics.

In this study, we selected Sitona callosus, one of the primary insect pests of alfalfa, as the experimental insect and infected it with Beauveria bassiana. Transcriptomic and metabolomic analyses were conducted to explore alterations in gene expression and metabolic processes in S. callosus at 48, 96, and 144 h post infection with B. bassiana. The transcriptomic analysis unveiled that B. bassiana infection boosted immune responses in tubercula, affecting carbohydrate metabolism, cytochrome P450 activity, lysosome function, apoptosis regulation, phagosome formation, glutathione metabolism, amino acid metabolism, and pathogen response pathways. Subsequent metabolomics analysis confirmed that glycerophospholipids, carboxylic acids and derivatives, organooxygen compounds, keto acids and derivatives, and azane immune metabolites were significantly upregulated in response to B. bassiana infection. Additionally, we utilized the Pearson correlation coefficient method to examine the relationships between differentially expressed immune-related genes and metabolites, revealing notably strong correlations between these two sets of variables. By leveraging the WGCNA method to analyze immune metabolite data for immune-related genes, we identified hub genes crucial at various stages of immune activation. These central genes predominantly included C-type lectin receptors for pattern recognition, cytochrome P450 enzymes linked to detoxification processes, and cathepsin proteases. By combining transcriptome and metabolome analyses, it was determined that autophagy and arachidonic acid metabolism play significant roles in the response of S. callosus to infection by B. bassiana. This research will facilitate the understanding of the immune response to B. bassiana infection in adult S. callosus, laying a theoretical groundwork for future biological control strategies targeting S. callosus.

Human Cytochrome P450 Cancer-Related Metabolic Activities and Gene Polymorphisms: A Review.

Cytochromes P450 (CYPs) are heme-containing oxidoreductase enzymes with mono-oxygenase activity. Human CYPs catalyze the oxidation of a great variety of chemicals, including xenobiotics, steroid hormones, vitamins, bile acids, procarcinogens, and drugs. In our review article, we discuss recent data evidencing that the same CYP isoform can be involved in both bioactivation and detoxification reactions and convert the same substrate to different products. Conversely, different CYP isoforms can convert the same substrate, xenobiotic or procarcinogen, into either a more or less toxic product. These phenomena depend on the type of catalyzed reaction, substrate, tissue type, and biological species. Since the CYPs involved in bioactivation (CYP3A4, CYP1A1, CYP2D6, and CYP2C8) are primarily expressed in the liver, their metabolites can induce hepatotoxicity and hepatocarcinogenesis. Additionally, we discuss the role of drugs as CYP substrates, inducers, and inhibitors as well as the implication of nuclear receptors, efflux transporters, and drug-drug interactions in anticancer drug resistance. We highlight the molecular mechanisms underlying the development of hormone-sensitive cancers, including breast, ovarian, endometrial, and prostate cancers. Key players in these mechanisms are the 2,3- and 3,4-catechols of estrogens, which are formed by CYP1A1, CYP1A2, and CYP1B1. The catechols can also produce quinones, leading to the formation of toxic protein and DNA adducts that contribute to cancer progression. However, 2-hydroxy- and 4-hydroxy-estrogens and their O-methylated derivatives along with conjugated metabolites play cancer-protective roles. CYP17A1 and CYP11A1, which are involved in the biosynthesis of testosterone precursors, contribute to prostate cancer, whereas conversion of testosterone to 5α-dihydrotestosterone as well as sustained activation and mutation of the androgen receptor are implicated in metastatic castration-resistant prostate cancer (CRPC). CYP enzymatic activities are influenced by CYP gene polymorphisms, although a significant portion of them have no effects. However, CYP polymorphisms can determine poor, intermediate, rapid, and ultrarapid metabolizer genotypes, which can affect cancer and drug susceptibility. Despite limited statistically significant data, associations between CYP polymorphisms and cancer risk, tumor size, and metastatic status among various populations have been demonstrated. The metabolic diversity and dual character of biological effects of CYPs underlie their implications in, preliminarily, hormone-sensitive cancers. Variations in CYP activities and CYP gene polymorphisms are implicated in the interindividual variability in cancer and drug susceptibility. The development of CYP inhibitors provides options for personalized anticancer therapy.

Combining azole antifungals with venetoclax plus azacitidine in patients with newly diagnosed acute myeloid leukemia

ABSTRACT The combination of venetoclax (VEN) with hypomethylating agents (HMAs) improves survival in patients with acute myeloid leukemia (AML) and may cause neutropenia requiring combined antifungal therapy or prophylaxis. The inhibition of cytochrome P450 activity by azole antifungal agents leads to elevated blood concentrations of VEN. This study aimed to evaluate the efficacy and safety of venetoclax plus azacitidine (AZA) with azoles in newly diagnosed AML patients. The primary endpoints included complete remission (CR), complete remission with incomplete blood cell recovery (CRi), composite CR (CRc, CR + CRi), blood cell recovery time and incidence of infections. The CRc was 50.0% in the azole group and 56% in the nonazole group (p > 0.05). In the azole group, the median recovery times for patients with ANC >500 cells/mm3 and ANC >1,000 cells/mm3 were 19 and 25 days, respectively. For the nonazole group, the corresponding times were 16 and 19 days (p < 0.05). In the azole group, the median durations for patients with a PLT >50,000/mm3 and >100,000/mm3 were 18 and 20 days, respectively. For the nonazole group, the corresponding times were 16 and 19 days (p > 0.05). The incidences of fungal and bacterial infections were not significantly different (30.8% vs 26.1% and 50.0% vs 56.0%) (p > 0.05). The cost-effectiveness ratio of the azole group is lower. There was no significant difference between VEN + AZA with or without azole in terms of efficacy, infection, or partial hematological toxicity. However, the combination of azoles may prolong the neutrophil recovery time. Azole combination could reduce the amount of venetoclax and improve health economics.

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.