Search Results (1,409)

Triterpenoids are the primary bioactive constituents responsible for the medicinal efficacy of Ziziphus jujuba Mill. cv. ‘Junzao’ (Junzao) fruits, and there are significant differences in their functional composition depending on the harvest period. However, the metabolic characteristics and dynamic accumulation patterns of triterpenoids during fruit development remain poorly understood. This study represented the first systematic integration of transcriptomic and metabolomic analysis combined with weighted gene co-expression network analysis (WGCNA) to elucidate the biosynthetic pathways of triterpenoids during the harvest times of Junzao fruits, and provide a scientific basis for the future development of functional foods. Total triterpenoid content exhibited a stage-specific accumulation pattern, peaking at the YG (young fruit) stage of fruit development, declining sharply by 37% at the PD (expansion) stage, rebounding at the BS (white-ripe) stage, and gradually decreasing through the CS (crispy-ripe) stage and WS (full-ripe) stages. A total of 347 terpenoid differentially accumulated metabolites (DAMs) and 18,925 differentially expressed genes (DEGs) were identified, among which 224 triterpenoids were predominant. A total of 347 terpenoid differentially accumulated metabolites (DAMs) and 18,925 differentially expressed genes (DEGs) were identified, among which 224 triterpenoids were predominant. WGCNA identified six key modules (salmon, midnightblue, black, blue, yellow, and brown modules) strongly correlated with the accumulation of methyl oleanolate, 3-oxopomolic acid, hederagenin, and other triterpenoids. Furthermore, integrated correlation analysis revealed that cytochrome P450 family genes, particularly CYP716, CYP72A and CYP88, were likely the hub genes governing triterpenoid biosynthesis and RT-qPCR validation of eight key genes confirmed the transcriptome expression trends. These findings provide a comprehensive framework for understanding triterpenoid biosynthesis and offer theoretical foundations for optimizing harvest timing and advancing metabolic engineering in Junzao fruits. Full article

Metabolic dysfunction-associated steatohepatitis (MASH; formerly non-alcoholic steatohepatitis, NASH) is characterized by oxidative stress, inflammatory activation, hepatocellular injury, and progressive liver dysfunction. However, the global transcriptomic landscape underlying stress-induced hepatic injury remains incompletely understood. In this study, we employed a methionine–choline-deficient (MCD) diet-induced murine model to characterize the phenotypic and transcriptomic alterations associated with liver injury. Male C57BL/6J mice were fed either a control or MCD diet, and hepatotoxicity was assessed by survival analysis, body and liver weight measurements, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, histopathological examination, RNA sequencing, quantitative real-time PCR (qRT-PCR), and tumor necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay (ELISA). MCD feeding markedly reduced survival and body weight while inducing hepatomegaly and significant elevations in serum ALT and AST, indicating severe hepatocellular injury. Histopathological analysis demonstrated hepatic steatosis, hepatocellular ballooning, and lobular inflammation without histological evidence of fibrosis. Transcriptomic profiling revealed extensive gene expression remodeling, characterized by activation of inflammatory pathways, enrichment of MAPK-related signaling, dysregulation of lipid metabolism, suppression of antioxidant defense systems, impairment of cytochrome P450-mediated detoxification, and upregulation of apoptosis-associated genes. qRT-PCR further validated the differential expression of representative genes involved in inflammatory signaling (Tlr4, Nfkb1, Nlrp3, and Casp1), MAPK signaling (Fos), xenobiotic metabolism (Cyp4f18), lipid metabolism (Apoa4 and Lpl), extracellular matrix remodeling (Mmp12), and oxidative stress responses (Sod1 and Gstp1). In addition, elevated serum TNF-α levels provided protein-level evidence supporting activation of the TLR4/NF-κB/TNF-α/NLRP3 inflammatory axis. Although fibrosis-associated transcriptional responses were detected, the absence of histological fibrosis suggests transcriptional priming of fibrogenic pathways rather than established fibrogenesis. Collectively, these findings provide a transcriptomic framework linking oxidative stress, impaired detoxification, inflammatory activation, and stress-responsive signaling to MCD-induced hepatic injury. The MCD model provides a valuable experimental platform for characterizing hepatic stress-response transcriptomes and for generating hypotheses that can subsequently be evaluated in environmentally relevant toxicological models. Nevertheless, caution should be exercised when extrapolating these findings to obesity-associated human MASLD, as the MCD model lacks key metabolic features of the human disease, including obesity and insulin resistance. Therefore, the present findings should be interpreted primarily as transcriptomic signatures of stress-induced hepatic injury rather than as a direct representation of the pathophysiological processes underlying human obesity-associated MASLD. Full article

Background/Objectives: Antidepressant treatment response is highly variable, and CYP2D6 metabolizer phenotype has been proposed as a contributor to this variability. It was examined whether CYP2D6 metabolizer phenotype is associated with real-world antidepressant treatment outcomes in a large population-based cohort. Methods: Using genetic and longitudinal primary care prescription data from the UK Biobank, we evaluated associations between CYP2D6 metabolizer phenotype and prescription-based proxies of treatment outcomes, including discontinuation, switching, and side effects. Analyses were stratified by antidepressant and adjusted for demographic covariates. Results: Among 26,957 individuals of European ancestry prescribed CYP2D6-metabolized antidepressants, reduced metabolic capacity was significantly associated with early discontinuation of paroxetine (N = 5718), venlafaxine (N = 2327), and mirtazapine (N = 3340). For paroxetine, poor metabolizers had higher odds of discontinuation compared with normal metabolizers and, among discontinuers, were more likely to stop immediately rather than later. Similar early discontinuation signals were observed for venlafaxine, with intermediate metabolizers showing increased risk. Mirtazapine also demonstrated increased odds of early discontinuation among poor metabolizers. No significant association was observed for fluoxetine. Associations with switching were limited, and no significant associations were detected for side effects. Conclusions: CYP2D6 variation appears to primarily influence early antidepressant discontinuation within the first 30 days of treatment, particularly for paroxetine, venlafaxine, and mirtazapine, rather than treatment switching or side effects. These findings provide observational support relevant to drug-specific gene interactions and suggest a role for CYP2D6-guided prescribing in clinical practice, notably in the first 30 days of antidepressant treatment. Full article

The Cytochrome P450 (CYP) superfamily plays an important role in the regulation of plant growth and development. However, the composition, evolutionary characteristics, and potential functions of CYPs in celery remain largely unexplored. Therefore, the objective of this study was to perform a genome-wide characterization of the Apium graveolens Cytochrome P450 (AgCYP) gene family and investigate its potential roles in seed development. In this study, a total of 227 AgCYPs were identified, and phylogenetic analysis classified them into six clades. Conserved motif and domain evaluations indicated that most AgCYP proteins possess conserved P450 domains. Chromosomal localization revealed an unequal distribution of AgCYPs across the 11 celery chromosomes. Duplicated AgCYP gene pairs were identified by synteny and Ka/Ks analyses, indicating that the duplicated AgCYPs have undergone strong purifying selection. Inter-genomic synteny analysis further reflects the closer relationship within Apiaceae. Analysis of cis-acting elements in the promoter regions identified an abundance of elements associated with light, hormone, and environmental stress. Moreover, AgCYPs showed stage-specific expression patterns and were correlated with monoterpene and phthalide accumulation during celery seed development, suggesting their potential functions in secondary metabolism in seed development. Treatment with exogenous auxin and its transport and biosynthesis inhibitors differentially induced distinct expression responses among AgCYPs, indicating their possible participation in auxin-related regulatory pathways. Moreover, candidate genes were selected. They exhibited diverse tissue-specific expression patterns and were potentially localized to the endoplasmic reticulum and interacted with some auxin-related proteins. In conclusion, this study provides the first comprehensive framework for understanding the functional diversification of AgCYPs in celery seeds, providing new insights into the evolutionary features and biological functions of the AgCYP gene family and establishing a foundation for future functional studies and molecular breeding applications. Full article

Plant cytochrome P450 monooxygenases (CYPs) are valuable biocatalysts for the regioselective hydroxylation of aromatic compounds. However, their expression in bacterial hosts is hampered by poor solubility, membrane anchoring and the requirement for redox partners. In this work, we report the design and characterization of modular expression systems that enable the functional production of SbCYP82D1.1 from Scutellaria baicalensis (SbF6H) in Escherichia coli. Both independent expression and synthetic fusion systems were evaluated by combining a CYP with a compatible reductase (ATR2_tr from Arabidopsis thaliana) to catalyze the conversion of chrysin into baicalein. A combinatorial library of N-terminal variants, host strains, media, and induction strategies was constructed and screened. Among the tested host, E. coli DH 10-beta provided the highest product titers, particularly when cultures were supplemented with 5-aminolevulinic acid. Truncation of the native transmembrane anchor significantly improved catalytic performance, whereas the addition of the heterologous MALLLAVF tag decreased activity. Fusion systems outperformed separate expression formats, showing approximately two-fold higher activity, with the flexible glycine–serine linker (L_GS) supporting the highest hydroxylation product formation. The corresponding fusion construct showed an apparent conversion of 0.1 mM chrysin to baicalein of up to 90% under the applied whole-cell reaction and analytical conditions, although this value should be interpreted with caution due to the concurrent instability of baicalein observed in all reactions and culture conditions. This result nevertheless indicates a marked improvement in whole-cell baicalein formation compared with previously reported bacterial systems. Together, these results demonstrate that rational N-terminal engineering combined with fusion protein design can enable efficient bacterial expression of plant CYPs, representing a promising step toward scalable production of hydroxylated flavonoids. Full article

Background and Objectives: Ticagrelor reduces ischemic events in acute coronary syndrome (ACS) but increases bleeding risk. Clinical predictors of bleeding are well established; the contribution of cytochrome P450 polymorphisms involved in ticagrelor metabolism remains uncertain, with conflicting reports in the literature. We examined the association of CYP3A4* 22 (rs 35599367), CYP3A5* 3 (rs 776746), and CYP4F2 (rs3093135) with bleeding in a Serbian ACS cohort. Materials and Methods: This prospective, single- center observational study enrolled 105 consecutive ACS patients undergoing percutaneous coronary intervention (PCI) or medical management after coronary angiography and receiving dual antiplatelet therapy (DAPT) with acetylsalicylic acid and ticagrelor at the University Clinical Center Niš between January 2024 and the end of May 2025. Bleeding events occurring during the index hospitalization and the six-month follow-up were classified according to the Bleeding Academic Research Consortium (BARC) criteria. Genotyping used TaqMan assays. Associations with bleeding were assessed using Firth’s penalized logistic regression, with multivariable adjustment for age and renal function. Severity-stratified analyses and gradient-boosted machine learning (XGBoost with SHAP) were performed as exploratory analyses. Results: Thirteen patients (12.4%) experienced bleeding (nine minor [BARC 1/2], four major [BARC 3/5]). Age ≥ 75 years (univariable OR 7.62, p = 0.001) and eGFR < 60 mL/min/1. 73 m ² (OR 3.68, p = 0.006) were the strongest predictors. CYP3A5 *1 carrier status was univariably associated with bleeding (OR 4.16, p = 0.043) but did not remain significant after adjustment for age and renal function, and *1 carriers were significantly older and more likely to have impaired renal function. No genotype was associated with major (BARC 3/5) bleeding. The apparent effect was concentrated in minor bleeding (BARC 1/2 rate: 30.8% versus 5.5%), with no major events among *1 carriers. CYP 3 A 4* 22 (OR 1.37, p = 0.109) and CYP 4 F 2 (OR 1.17, p = 0.111) showed no association. Machine-learning analyses confirmed eGFR and age as the dominant predictors. Conclusions: In this Serbian ACS cohort, clinical factors—particularly advanced age and impaired renal function—dominated the prediction of bleeding risk. The CYP3A5 signal was largely explained by baseline imbalances in age and renal function. CYP 3 A 4* 22 and CYP 4 F 2 polymorphisms did not contribute additional predictive information. Preemptive genotyping for these variants is unlikely to materially improve bleeding-risk assessment beyond standard clinical evaluation in patients of this type. Full article

Thiamethoxam is the main neonicotinoid insecticide used for controlling Riptortus pedestris (Fabricius) (Hemiptera: Alydidae). However, sublethal concentration stress may induce intergenerational transcriptional memory, leading to transcriptional patterns that may contribute to the intergenerational accumulation of metabolic tolerance, and evaluating only the toxicity of the current generation would underestimate the long-term risk. Therefore, this study investigated the effect of parental exposure on the expression of detoxification enzyme genes in offspring. Using transcriptome sequencing, we systematically identified three detoxification enzyme gene families (cytochrome P450 monooxygenases (CYPs), carboxylesterases (CCEs), and glutathione S-transferases (GSTs)) in R. pedestris and compared their differential expression patterns between the parental and filial generations after thiamethoxam treatment at three sublethal concentrations (LC₁₀, LC₃₀, and LC₅₀). In the parental generation, a Theta family GST was consistently upregulated, while in the filial generation, detoxification genes were predominantly downregulated, and the genes upregulated in the parents were not also upregulated in the offspring. Comparisons of parents and offspring at the same concentration revealed that the medium concentration induced the highest number of intergenerationally upregulated genes, exhibiting a non-linear response pattern. These results indicate that parental exposure to sublethal thiamethoxam leaves an intergenerational transcriptional imprint in the offspring, and the transmission pattern involves transcriptional reprogramming rather than simple replication of the parental response, the mechanism of which remains to be determined. This study provides transcriptomic evidence for understanding the metabolic adaptation and intergenerational resistance evolution of R. pedestris to thiamethoxam, offering important reference value for field resistance monitoring and rational insecticide application. Full article

Introduction: Temperature is a key environmental factor influencing the physiological and biochemical processes of aquatic organisms, including xenobiotic metabolism. Understanding how temperature modulates the toxicological effects of pollutants such as polycyclic aromatic hydrocarbons (PAHs) is crucial in the context of climate change. Among these compounds, benzo[a]pyrene (BaP) and benzo[a]anthracene (BaA) are priority pollutants in aquatic environments, resulting from incomplete combustion. Their relevance is attributed to persistence and metabolic bioactivation potential. Fish primary hepatocyte cultures represent a relevant in vitro model for studying combined effects of thermal stress and chemical exposures, while supporting the 3Rs principles (Replacement, Reduction, and Refinement). Objective: This study aims to assess temperature-dependent effects of BaP and BaA, and their mixtures in brown trout hepatocytes using cytochrome P450 1A (CYP1A) immunohistochemistry as an indicator of xenobiotic metabolism. Methodology: Primary hepatocytes were isolated using a two-step collagenase perfusion method and cultured in 24-well plates at 18 °C and 22 °C. Cells were exposed for 72 h to supplemented L-15 medium (control) or to 0.1% dimethyl sulfoxide in supplemented L-15 medium (solvent control), as well as to single exposures of 1 and 10 µM of BaP and BaA and to equimolar mixtures of both compounds (1 and 10 µM). Viability was assessed using the lactate dehydrogenase (LDH) assay. CYP1A immunostaining was quantified based on cytoplasmic staining intensity relative to background area. Results: No significant effects on cell viability were observed under any condition. Temperature significantly reduced CYP1A expression in single exposures at 22 °C compared to 18 °C. BaP induced a significant dose-dependent increase, while BaA differed from controls only at 10 µM. In mixtures, only treatment- and dose-dependent effects were observed, with no temperature influence detected. Conclusions: Overall, the data highlight temperature as a key modulator of biochemical responses to PAHs, with single and mixed exposures eliciting distinct effects and suggesting potential synergism in mixtures. Full article

Introduction: The temperature of rivers in the Iberian Peninsula has increased due to global warming. In addition, these rivers are polluted by contaminants of emerging concern, such as polycyclic aromatic hydrocarbons (PAHs). Higher temperatures and pollution concurrently impose threats to the Iberian Peninsula’s endemic species, including the brown trout (Salmo trutta), a cold-water species widely used in ecotoxicological studies. Because the liver is the main biotransformation organ, and is particularly sensitive to both chemical and temperature changes, in vitro liver models may represent valuable alternatives for assessing combined stressor effects, complying with the 3Rs principle. Objective: In line with the above, the present study aimed to evaluate the combined effects of a 4 °C temperature increase and the model PAH benzo[k]fluoranthene (B[k]F) on fish liver cells using a primary brown trout hepatocyte culture as a model. Methodology: Primary hepatocytes were seeded in 6-well plates at a density of 1.0 × 10⁶ cells/mL and exposed for 48 h to 1, 10, and 20 µM B[k]F at 18 °C (normothermia) and 22 °C (warming scenario). Cell viability was assessed using trypan blue, alamarBlue, and lactate dehydrogenase (LDH) assays. Cytochrome P450 (CYP)1A was evaluated in terms of its gene expression by RT-qPCR and its protein expression through immunocytochemistry (ICC). The immunostaining was quantified using a score system which considered five intensity staining levels. Results: Exposure to B[k]F and to the higher temperature increased LDH leakage without interaction effects. In contrast, the other viability assays did not show significant differences across conditions. Regarding CYP1A, both gene and protein expression increased with all B[k]F concentrations in relation to the controls, but were not influenced by temperature. Notably, the lowest B[k]F concentration (1 µM) elicited the highest CYP1A gene expression, suggesting a non-monotonic response. Conclusions: Overall, the model was responsive to both temperature (4 °C) increase and to B[k]F, validating its usefulness for assessing liver pollutant effects in the context of global warming. These findings support the application of fish primary hepatocyte models as relevant tools in ecotoxicology under environmentally realistic multi-stressor scenarios. Full article

Plagiodera versicolora (Coleoptera: Chrysomelidae), the willow leaf beetle, is a leaf-eating pest that generally occurs on salicaceous trees. However, there is a blank of identification and phylogenetic relationship of the detoxification genes in P. versicolora. Here, we identified four detoxification gene families (glutathione S-transferases: GSTs, UDP-glycosyltransferases: UGTs, cytochrome P450 monooxygenases: CYPs and carboxylesterases: COEs) from the adult antennal transcriptome data. In all, 146 candidate detoxification genes including 22 GSTs, 20 UGTs, 60 CYPs, and 44 COEs were identified. We used quantitative real-time PCR technology to explore the tissue expression patterns of 12 PvGSTs in P. versicolora. The results showed that 7 PvGSTs have significantly high expression in antennae, indicating these PvGSTs may play an important role in degrade and/or inactivate the sex pheromones and host volatiles. The identification and phylogenetics of the detoxification genes in P. versicolora extended the database in Coleoptera and contributed to the subsequent in-depth research for function about detoxification genes. Full article

Kadsura heteroclita (Roxb.) Craib, commonly known as “Xuetong”, is a traditional Tujia ethnomedicine with anti-rheumatoid arthritis (RA) activity, and schizanlactone E (Xuetongsu) is its major bioactive component whose biosynthetic pathway remains uncharacterized. As a cycloartane-type tetracyclic triterpenoid, Xuetongsu’s biosynthesis is likely to involve multiple oxidation steps. Cytochrome P450 (CYP450) is a versatile monooxygenase encoded by a large and diverse gene superfamily and plays a critical role in various oxidation reactions in plants’ secondary metabolism. In this study, 367 KhCYP450s were identified and systematically analyzed for their physicochemical properties, phylogenetic analysis, conserved motifs, gene structures, collinearity, and cis-acting elements. Weighted gene co-expression network analysis (WGCNA) revealed a turquoise module strongly associated with Xuetong root tissue, which had the highest Xuetongsu accumulation; 32 candidate KhCYP450s within this module were screened via correlation analysis between gene expression and xuetongsu content and partially validated by qRT-PCR. Five of these candidates showed significant homology with known triterpenoid biosynthetic genes via protein structure analyses. This study deepened our comprehension of the CYP450 superfamily in Xuetong and provided a valuable reference for further research on the biosynthesis of Xuetongsu. Full article

Introduction: The 3 Rs principle advocates developing alternative, biologically relevant models. Thus, 3D fish liver in vitro models have been increasingly used for ecotoxicological studies. We previously optimized spheroids from the rainbow trout non-tumoral liver cell line RTL-W1 and employed them to assess the effects of aquatic pollutants. Although they demonstrated potential for assessing ecotoxicological effects, further optimization is warranted to enhance their physiological relevance. Incorporating an extracellular matrix (ECM), such as collagen, has been shown to be a promising strategy to improve spheroids’ structural organization and functionality. Objective: This study aimed to optimize 3D culturing conditions of RTL-W1 spheroids by evaluating the effects of collagen supplementation on viability, morphology, and functional response. Methodology: Spheroids from the RTL-W1 cell line (60,000 cells per well) were cultured in 96-well ultra-low attachment (ULA) plates at 18 °C. After spheroids’ formation, rat tail collagen was supplemented at concentrations of 15 (C15), 30 (C30), and 60 (C60) µg/mL at culture days 7, 8, and 9. Spheroids were collected at two sampling days (10 and 14). Viability was assessed using alamarBlue and lactate dehydrogenase (LDH) assays, while morphology was assessed by optical microscopy. Collagen penetration was evaluated using Masson’s trichrome staining technique. Protein expression of cytochrome P450(CYP)1A was assessed by quantifying immunocytochemistry staining using an anti-CYP1A antibody. Results: On day 10, LDH leakage decreased in C15 and C60, compared with the control, whilst C15 spheroids showed lower absorbance levels in the alamarBlue assay. On day 14, LDH showed no significant differences; however, C30 and C60 had higher alamarBlue absorbance, indicating greater metabolic capacity. Spheroid morphology appeared intact in all conditions. Masson trichrome revealed collagen fibrils at the periphery of the spheroids, especially in C30 and C60, indicating that spheroids incorporated collagen. CYP1A immunostain was present in all conditions, localized in the spheroids’ border, and tended to be higher when supplementation occurred in earlier days. Conclusions: Our results suggest that RTL-W1 spheroids interacted with the collagen matrix and appeared to functionally improve. Data suggest that incorporating ECM may increase the complexity and physiological relevance of RTL-W1 spheroids, thereby better supporting mechanistic and ecotoxicological applications. Full article

Oxytetracycline (OTC) is widely used in aquaculture to treat bacterial diseases, but its effects on drug metabolism and immune responses remain poorly understood in olive flounder (Paralichthys olivaceus). This study investigated the effects of OTC immersion for 1 h at different concentrations on the expression profile of drug metabolism- and immune-related genes and the histopathological changes in the gills and liver of olive flounder. The expression of Paralichthys olivaceus cytochrome P450 (PoCYP), Paralichthys olivaceus diphosphate (UDP)-glucuronosyltransferases (PoUGTs), and immune-related genes significantly increased at 12 and 24 h and then decreased at 72 h in the 100 and 200 ppm groups. By contrast, the 400 ppm group exhibited variable expression patterns depending on the gene and tissue. The highest expression levels were observed for PoIL-1β in the gills (22.7-fold) and PoCYP27B1 in the liver (58.0-fold) in the 400 ppm group. Histopathological analysis revealed macrophage activation in the liver at 72 h in the 200 ppm group, whereas epithelial edema and mild aneurysmal changes in the gills and macrophage activation in the liver were observed in the 400 ppm group. These findings provide insights into the effects of OTC exposure on drug metabolism and immune responses in olive flounder. Full article

Spermatogenesis is a highly coordinated biological process in which diploid spermatogonia undergo mitotic expansion, meiotic division, and terminal differentiation into haploid spermatozoa. This process is tightly regulated by intrinsic germ cell programs and extrinsic signals from Sertoli cells within the seminiferous epithelium. Among the signaling pathways governing male germ cell development, all-trans retinoic acid (RA), a bioactive metabolite of vitamin A, has emerged as a master regulator of meiotic initiation and spermatogonial differentiation in mammals. RA functions through nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which regulate transcriptional networks essential for germ cell progression, including the activation of Stimulated by Retinoic Acid 8 (STRA8), a key determinant of meiotic entry. Intratesticular RA homeostasis is maintained by a balance between synthesis via aldehyde dehydrogenase (ALDH) enzymes and degradation by cytochrome P450 family 26 (CYP26) enzymes, ensuring precise temporal and spatial control of germ cell development. While rodent models have defined core mechanisms of RA signaling, the canine testis provides a valuable comparative and translational system due to its physiological similarity to human spermatogenesis and relevance to reproductive management. Recent studies highlight conserved RA signaling pathways in dogs, including receptor-mediated transcriptional regulation, feedback control of RA metabolism, and post-transcriptional modulation via microRNAs. Importantly, pharmacological manipulation of RA signaling can reversibly disrupt spermatogenesis, supporting its potential applications in non-hormonal male contraception. This review integrates molecular, developmental, pharmacological, and comparative evidence and presents RA signaling as a central regulatory axis of spermatogenesis with important translational applications. Full article

Background/Objectives: Atrasentan is a selective endothelin A receptor antagonist (SERA) developed as a potential therapy for chronic renal diseases, including diabetic nephropathy and immunoglobulin A nephropathy. Despite this potential, understanding its metabolic bioactivation is essential for assessing the risks of drug-induced liver injury (DILI). However, the metabolic profile of atrasentan remains poorly characterized, and the mechanisms underlying its potential hepatotoxicity remain underexplored. Therefore, this study aims to investigate the metabolic pathways of atrasentan in human liver microsomes (HLMs) in the presence of nicotinamide adenine dinucleotide phosphate (NADP⁺), uridine diphosphate glucuronic acid (UDPGA), or glutathione (GSH). Methods: A liquid chromatography–high resolution mass spectrometry (LC-HRMS) coupled with a feature-based molecular networking approach was used to characterize metabolites. Characterization of the major metabolites was achieved through cytochrome P450 (P450) phenotyping with human recombinant P450 isoforms. Results: A total of eighteen metabolites were characterized through phase I and II metabolic reactions, including demethylenation, N-dealkylation, O-demethylation, hydroxylation, dehydrogenation, and glucuronidation. Atrasentan acyl glucuronide (M8) was confirmed as the predominant metabolite, and we also putatively annotated a catechol intermediate (M5) and its corresponding GSH conjugate (M15). Characterizing the GSH conjugate (M15) indicates that catechol intermediate (M5) can be further oxidized to a reactive ortho-quinone intermediate, which is subsequently trapped by GSH, suggesting the potential for a bioactivation mechanism. Reaction phenotyping demonstrated that the formation of M5 is catalyzed almost exclusively by the CYP3A subfamily. However, its direct translation to in vivo oxidative stress or covalent protein binding requires further studies. Conclusions: These findings demonstrate that feature-based molecular networking is a valuable strategy for metabolite characterization, underscoring the urgent need for further in vivo metabolism studies to definitively assess hepatotoxic risks associated with these reactive metabolites. Full article