As part of toxicity assessment in the development of pharmaceuticals and pesticides, ex vivo method using testicular tissue slices is suggested for evaluating the impact and underlying mechanisms of action of compounds on androgen synthesis. However, relevant comprehensive assessments, such as comprehensive profiling of androgens and related hormones and histological examination of tissue slices, remain limited. We evaluated an ex vivo method using testicular tissue slices by determining whether the amounts of each hormone synthesized in successive metabolic steps could be measured to reflect compound-induced effects. First, rat testicular tissue slices were incubated with ketoconazole (Ket) solutions (a representative azole fungicide that inhibits androgen synthesis) for 3 h; hormone assays of the supernatant and histological examination of the slices, including hematoxylin and eosin staining, ultrastructural examination, and immunohistochemistry, were conducted. Changes in androgen and precursor hormone levels were consistent with cytochrome P450 (CYP) 17A1 inhibition by Ket, together with morphological observations in Leydig cells that generally reflected the altered hormone profiles. Subsequently, eight azoles (hexaconazole, flusilazole, triticonazole, imazalil, penconazole, myclobutanil, triadimefon, and fluconazole) with inhibitory effects on CYP17A1 were examined. Hormone assays revealed changes suggestive of CYP17A1 inhibition for all the compounds, along with apparent differences in their inhibitory potency. Histological examination provided supportive morphological observations that were generally consistent with differences in hormone synthesis among the compounds. These results demonstrate that this ex vivo method can detect azole-induced disruptions in androgen synthesis and associated histopathological changes with fewer resources, thus offering a valuable tool for mechanistic toxicity studies.
Toxicology has long depended on animal studies and static computational models to evaluate the safety of chemical substances and pharmaceutical compounds. These conventional approaches are increasingly critiqued for their limited translational accuracy, ethical concerns, and inability to capture real-time, individual-level physiological variation. Digital twin technology, virtual replicas of biological systems that are continuously updated with real-world data, has emerged as a promising paradigm that addresses many of these shortcomings. This review examines the current and potential applications of digital twin technology in toxicology, highlighting its utility across drug safety evaluation, environmental toxicology, occupational health, and personalised medicine. A narrative review framework was employed. Peer-reviewed literature published between 2015 and 2026 was retrieved from PubMed, Scopus, and Web of Science. Studies were selected based on relevance to the review objectives, methodological rigour, and contribution to the conceptual synthesis. Evidence indicates that digital twins are being applied in toxicology to simulate organ-level and whole-body responses to toxic exposures, predict dose-response relationships, and model individual susceptibility. Integration with omics data, wearable biosensors, and artificial intelligence further enhances their predictive fidelity. Regulatory bodies including the United States Environmental Protection Agency and the European Medicines Agency are beginning to acknowledge in silico approaches as complementary evidence in safety assessments. Digital twin technology offers a promising basis for advancing toxicological research and risk assessment, although much of this potential remains to be demonstrated. Challenges around data standardisation, model validation, and regulatory acceptance require concerted interdisciplinary efforts to overcome.
Polystyrene nanoplastics (PS-NPs) are emerging food safety contaminants. Ferroptosis is iron-dependent cell death, but its role in PS-NPs hepatotoxicity is unclear. Mice received tail-vein injection of PS-NPs (2-8 mg/kg). PS-NPs caused liver injury (elevated transaminases) and possible renal impairment (increased uric acid/creatinine/urea). Hepatic GSH and SOD decreased, IL-1β and TNF-α increased. Mitochondrial shrinkage and cristae loss (ferroptotic features) were observed. PS-NPs upregulated ACSL4, MDA, 4-HNE and TfR, but suppressed FTH1, FPN1, SLC7A11 and GPx4. Thus, ferroptosis mediates PS-NPs liver injury with oxidative stress and inflammation, and PS-NPs may exert multi-organ toxicity.
Phytolacca acinosa Roxb. (PR) is a saponin-rich medicinal plant associated with gastrointestinal toxicity, but the mechanisms underlying PR-induced intestinal barrier injury remain unclear. In this study, raw PR extract was analytically characterized by UPLC-ZenoTOF-MS/MS, confirming triterpenoid saponins as the predominant constituents. C57BL/6 J mice were orally exposed to characterized PR extract (1.20 or 12.0 g/kg for 5 h), and Caco-2 cells and mouse intestinal organoids were used to assess epithelial toxicity and barrier disruption. Histopathology, ELISA, FITC-dextran permeability assays, immunofluorescence, CCK-8, LDH release, western blotting, DIA-based proteomics and untargeted metabolomics were integrated to define toxicological mechanisms. PR induced dose-dependent intestinal inflammation and barrier dysfunction, with the ileum as the most sensitive target. PR increased serum DAO and D-lactate and intestinal TNF-α and IL-1β, disrupted organoid morphology, enhanced epithelial permeability, and reduced ZO-1 expression. Proteomics revealed changes in inflammatory, lipid-metabolic, cytoskeletal and tight-junction pathways, including upregulation of MLCK3 and phospholipase-related proteins and downregulation of ZO-1 and ZO-2. Metabolomics identified histidine metabolism disturbance and histamine accumulation. Integrated multi-omics and pharmacological validation indicated that histamine activated the PLC/IP₃/Ca²⁺/CaM/MLCK cascade, promoting MLC phosphorylation, tight-junction disassembly and epithelial leakiness. MLCK inhibition partially restored ZO-1/ZO-2 expression and attenuated PR-induced epithelial injury. These findings identify the histamine-MLCK-tight junction axis as a key mechanism of PR-induced intestinal toxicity and support hazard identification of saponin-rich PR exposure.
BACKGROUND:Benzene exposure is a recognized environmental risk factor for acute myeloid leukemia (AML). This study aimed to identify key drivers of benzene-associated AML and elucidate its pathogenic mechanisms. METHODS:Candidate genes were screened by integrating two-sample Mendelian randomization (MR), transcriptomics, and single-cell sequencing data. Their biological functions were validated using functional experiments and metabolomics, and the efficacy of targeted interventions was assessed using in vivo and in vitro models. RESULTS:Integrated analysis identified 138 genes associated with AML, among which ATF7IP2 was a reliable prognostic biomarker and independent risk factor. Benzene significantly upregulated ATF7IP2 expression in a dose-dependent manner. Mechanistically, ATF7IP2 is a key regulator of glutamate metabolism; knockdown of ATF7IP2 reduces intracellular glutamate/glutamine levels, thereby impairing cell viability and inducing cell cycle arrest, while exogenous glutamate can prevent these effects. scRNA-seq and trajectory analysis showed that in a benzene-associated microenvironment, ATF7IP2 drives the malignant differentiation of hematopoietic stem cells (HSCs) into a leukemia precursor stem cell (pre-LSC) subset. The peptidomimetic inhibitor TCMCB07, targeting this axis, inhibited leukemia cell proliferation, delayed disease progression, and prolonged mouse survival. CONCLUSION:Our results indicate that ATF7IP2 is a key transcriptional metabolic hub that mediates benzene-induced AML by reprogramming glutamate metabolism and driving the conversion of HSCs to pre-LSCs.
Lead (Pb) exposure is a well-established environmental risk factor for cognitive impairment. Emerging evidence indicates that the combined effects of co-occurring metals may modify their neurotoxicity, but the underlying mechanisms remain elusive. The present study investigated how high copper (Cu) intake exacerbates Pb-induced synaptic loss, with a specific focus on the critical mediating role of N6-methyladenosine (m6A) RNA methylation in this process. We established a mouse model divided into four groups: control, Pb exposure, high Cu, and lead-copper co-exposure (Pb+Cu, PC). Cognitive function was assessed using the Morris water maze test and novel object recognition test. Golgi-Cox and immunofluorescence staining for postsynaptic density protein 95 (PSD95) were utilized to quantify dendritic spine density and synaptic number. Additionally, quantitative real-time PCR (RT-qPCR), Western blot, and dot blot assays were performed to analyze global m6A methylation levels and METTL3 expression, the core catalytic subunit of the m6A methyltransferase complex. Results showed that lead-copper co-exposure significantly exacerbated spatial learning and memory impairments in mice, accompanied by more severe loss of hippocampal dendritic spines and synapses. Mechanistically, heavy metal co-exposure induced an aberrant elevation of PSD95 mean methylation in the hippocampus, a dysregulation of epitranscriptomic modification that serves as a core driver of synaptic impairment. This elevated m⁶A level was due to the specific upregulation and enhanced activity of METTL3, the core catalytic subunit of m⁶A methyltransferase. Specific knockdown of METTL3 in vitro in primary hippocampal neurons and HT22 cells attenuated co-exposure-induced m6A hyper-methylation and partially restored synaptic structure and function, supporting the mediating role of aberrant m6A modification in synergistic heavy metal neurotoxicity. This study first identifies that high Cu intake synergistically exacerbates Pb neurotoxicity by activating the METTL3-driven m6A epitranscriptomic pathway, offering a potential target for intervening in cognitive impairment related to combined heavy metal exposure.
2,4-Di-tert-butylphenol (DTBP) is a degradation product of the antioxidant and plastic additive tris(2,4-di-tert-butylphenyl)phosphite. DTBP can migrate into food or drinking water from high production volume polyolefins containing this additive, leading to potential human exposures, confirmed by findings of DTBP in blood and urine. Natural sources of DTBP have also been described. We have recently developed a sensitive analytical method for the quantification of urinary DTBP and its postulated oxidised metabolite 2-tert-butyl-4-(2-hydroxy-1,1-dimethylethyl)phenol (DTBP-4-OH) after enzymatic cleavage of glucuronides. This method has now been employed in a human metabolism study. Five adult volunteers were orally dosed with 9.715 mg of DTBP and their urine voids were quantitatively and separately collected for 48 h. DTBP and DTBP-4-OH were quantifiable in all post-dose samples and biphasic excretion kinetics were observed. Urinary peak concentrations of the oxidised metabolite DTBP-4-OH were around six times higher than those of DTBP and occurred around the same time (1.5-3.8 h). Overall, we recovered 62.2% of the oral dose as DTBP-4-OH and 9.3% as parent DTBP within 48 h. Over 95% of the excretion occurred within the first 24 h. External contamination with DTBP e.g. from commonly used polypropylene tubes was observed, potentially interfering with the determination of parent DTBP. Hence, we strongly recommend using DTBP-4-OH as the most robust and most sensitive urinary exposure biomarker in human biomonitoring studies. With the toxicokinetic information provided here, reverse dosimetry of DTBP-4-OH enables an informed exposure and risk assessment for DTBP both in environmentally and occupationally exposed populations.
Perfluorooctane sulfonate (PFOS), a persistent organic pollutant, is associated with male reproductive disorders, yet its mechanisms remain poorly understood. Using in vivo (ICR mice exposed to 0.5, 5, and 10 mg/kg/d PFOS for 28 days) and in vitro models, we investigated the role of the transcription factor 2 (ATF2) / Wilms' tumor 1 (WT1) axis in PFOS-induced Sertoli cells (SCs) injury. Firstly, male ICR mice were administered PFOS (0.5, 5, and 10 mg/kg/d) for 28 days. PFOS exposure caused decreased sperm counts, disrupted blood-testis barrier (BTB) integrity, and ultrastructural damage to the SC. Integrated transcriptomic-metabolomic analyses revealed mitochondrial metabolic reprogramming as a core alteration: Transcriptomics: 271 differentially expressed genes (DEGs) were identified, with significant downregulation of Wt1 (a master SC differentiation regulator) and Cyp21a1 (mitochondrial regulatory gene). Pathway enrichment confirmed dysregulation in oxidative phosphorylation, TNF signaling, and steroid hormone biosynthesis. Metabolomics: Accumulated TCA intermediates (succinate) and glycolytic metabolites (glucose, phosphoenolpyruvate) indicated energy metabolic reprogramming; elevated lipid peroxidation markers (13-HODE) confirmed oxidative stress. These changes aligned with WT1 suppression, mitochondrial impairment (swelling, cristae loss, reduced ATP), and SC vacuolization. Pathologically, PFOS elevated testicular transforming growth factor-β3 (TGF-β3), p-ATF2, and p-p38 while suppressing WT1, Connexin43, and Occludin-corroborated in vitro with concurrent mitochondrial ROS (mtROS) overproduction. Crucially, ATF2 knockdown or WT1 overexpression rescued BTB junction proteins, restored transepithelial electrical resistance (TEER) in primary SCs, and normalized mtROS. We conclude that PFOS hijacks the ATF2/WT1 axis to drive SC injury, converging on mitochondrial metabolic reprogramming and dysfunction as the central mechanism.
Higenamine is a catecholic benzyltetrahydroisoquinoline alkaloid occurring in plant-derived foods and food supplements, the latter potentially resulting in substantially higher exposure levels than conventional foods. Because of its catechol and alkylphenol structures, oxidative conversion to electrophilic quinone or quinone methide intermediates is plausible, raising concern about a potential genotoxic hazard. However, experimental data on the genotoxic potential of higenamine are lacking. The present study, therefore, investigated its potential to induce gene mutations and chromosomal damage in cultured V79 cells. Gene mutations were examined at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus after 24 h treatment without metabolic activation and after 4 h in the absence or presence of rat liver S9-mix. Clastogenicity and aneugenicity were assessed using in vitro micronucleus assay with CREST staining after 4 h of treatment, followed by compound-free postincubation periods of 16 and 24 h. Proliferation and mitotic activity were monitored, and higenamine stability was analyzed by HPLC-UV. Higenamine caused a statistically significant increase in the frequency of micronuclei at 300-510 µM, reaching maxima of 51 and 73 micronucleated cells per 1000 cells without and with metabolic activation, respectively. Micronuclei mainly contained chromosomal fragments, indicating a prevalent clastogenic mode of action, although a smaller increase in micronuclei containing whole chromosomes was also observed. Mutant frequencies in the HPRT assay were not significantly increased under any condition tested, despite concentration-dependent cytotoxicity. During incubation, higenamine concentrations decreased while only minor formation of O-methylated metabolites was detected, suggesting conversion into additional reactive products. Overall, these findings provide the first experimental evidence that higenamine exhibits clastogenic potential in vitro and support the hypothesis that oxidative activation of its catechol and/or alkylphenol moiety to electrophilic intermediates capable of reacting with cellular nucleophiles might contribute to its genotoxic activity.
Immune-mediated drug-induced liver injury (DILI) is triggered or exacerbated by the immune system mounting an attack against the drug or its metabolites. The array of in vitro assays for evaluating drug immune liability is limited, highlighting a significant gap in effectively predicting and understanding immune-mediated hepatotoxicity. We aimed to investigate whether monocytes differentiated with the Metaheps (MH) protocol could provide insights into the molecular mechanisms of immune-mediated DILI. MH were generated from monocytes of healthy volunteers (HV) and DILI patients. MH phenotypic characterization was performed by proteomics and qPCR. MH sensitivity to drugs associated with immune-mediated DILI was assessed by lactate dehydrogenase (LDH) assay. Drug-induced LDH release by DILI-derived MH was compared to the upper limit of the 95% CI calculated from HV-derived MH cells treated with the same drug. The 95% CI determined in HV-derived MH was set as the sensitivity threshold for the specific drug. MH cells retain the expression of several immune-related proteins of the parental monocytes and activate a pro-inflammatory response upon exposure to lipopolysaccharide. For all MH (6 out of 6) generated from patients with penicillin-induced DILI, the LDH release upon re-challenge was above the threshold. The sensitivity of MH generated from seven patients with immune checkpoint inhibitor (ICI)-induced hepatotoxicity was ICI-dependent, responding to nivolumab and/or ipilimumab (4 out of 5), but not to pembrolizumab (0 out of 2). Additionally, DILI-derived MH were not sensitive to non-DILI drugs. In conclusion, monocyte-derived cells may serve as an additional tool for drug-specific mechanistic studies of immune-mediated DILI.
Heated tobacco products (HTPs) operate without combustion and are designed to reduce exposure to harmful constituents compared to cigarettes. Within the HTP category, rapid consumer-led technological development has resulted in a growing diversity of heating approaches across products, including the incorporation of multiple heating modes within individual devices. In this study, to gain deeper insight into the molecular mechanisms underlying biological responses to HTP aerosols, high-throughput transcriptomics was used to evaluate acute transcriptomic responses in human bronchial epithelial cells exposed to the extract of aerosols from multiple HTPs across all operable heating modes. Eleven emission constituents, including nine cigarette smoke constituents proposed for mandated reduction by the World Health Organization and combustion markers defined by British Standards Institution, were analyzed. HTP aerosols contained fewer detectable constituents and lower levels of measurable analytes than smoke from the reference cigarette (1R6F), confirming reduced combustion-related toxicants and non-combustion operation. Consistent with these chemical differences, HTP aerosols elicited weaker cytotoxic and transcriptomic responses than 1R6F smoke at comparable concentrations. While concentration-response modeling identified comparable transcriptomic perturbation patterns between HTP aerosols and 1R6F smoke, transcriptomic amplitudes were uniformly lower in response to HTP aerosols and no product-specific pathways were detected under the tested conditions. Transcriptomic points of departure (PoD) demonstrated greater sensitivity than cytotoxicity-based PoD, supporting their utility for evaluating the potential toxicological effects of HTPs at lower exposure levels than those identified by conventional cytotoxicity endpoints.
Sulfur mustard (SM), a highly toxic vesicant chemical warfare agent, can induce severe acute lung injury and poses a substantial threat to human health. Oxidative stress and inflammatory responses play central roles in the pathogenesis of SM-induced lung injury, and these two processes interact in complex and mutually reinforcing ways. This review systematically summarizes recent research advances and elucidates the role of oxidative stress in SM-induced pulmonary injury from three key aspects: oxidative stress-mediated DNA damage, the stimulation of inflammatory cytokine production, and cellular apoptosis. In addition, the major components of the inflammatory response are examined, including inflammatory cell activation and infiltration, the release of inflammatory mediators, and the ability of inflammation to further amplify oxidative stress. Building on this foundation, the mechanisms underlying the interaction between oxidative stress and inflammatory responses are discussed in depth from three perspectives: ROS/RNS-mediated crosstalk, regulation of signal transduction pathways, and interactions within apoptotic signaling pathways. This review aims to provide a conceptual framework for advancing the mechanistic understanding of SM-induced lung injury and to support the development of effective therapeutic strategies.
The increasing presence of microplastics (MPs) and nanoplastics (NPs) in food and water has raised concerns about their potential effects on gut microbiota. This study provides a comprehensive synthesis through a systematic review and meta-analysis evaluating the impact of micro and nanoplastics (MNPs) on gut microbiota diversity in rodent experimental models. Following PRISMA guidelines, eligible studies were identified from PubMed, Scopus, and Web of Science, and risk of bias was assessed using the SYRCLE tool. A quantitative meta-analysis was conducted on three commonly reported α-diversity indices (Chao1, Shannon, Simpson), while β-diversity and taxonomic changes were qualitatively synthesized. MNPs exposure showed no statistically significant effect on α-diversity (g = 0.17,p = 0.259), with substantial heterogeneity across studies. Subgroup analyses confirmed the absence of significant effects across particle size, animal model, polymer type, dose, and exposure duration. β-diversity was consistently and significantly altered in the vast majority of studies, indicating consistent microbial community restructuring. Taxonomic shifts were variable at the phylum level, particularly for Firmicutes and Bacteroidota, while decreases in Lactobacillaceae/Lactobacillus and increases in Ruminococcaceae, Lachnospiraceae, and Desulfobacterota were frequently observed. These findings indicate that MNPs primarily reshape microbial composition. The high heterogeneity highlights the need for standardized, environmentally relevant experimental designs to better assess microbiome-related alterations associated with MNPs exposure and their potential implications for host health.
Bisphenol-A (BPA) is a widely used chemical used in plastic and an endocrine disruptor ubiquitous in most developed settings. Exposure, particularly in early life, is implicated in a range of negative health outcomes. This study assessed whether various sources of BPA exposure in pregnant women, including diet, food preparation practices and personal care product use, are relevant predictors of measured BPA concentrations. Exposures were assessed via questionnaires. Maternal urine samples from 842 mothers were collected in trimester 3 and analysed for five bisphenols from the Barwon Infant Study, (infants born 2010-2013). Measurable BPA concentrations were detected in 54% of women. Positive associations with urinary BPA concentrations were observed for food-related factors, including a contemporaneous whole-foods dietary pattern and the proportion of raw foods introduced to the infant at 12 months, as well as renovation activities and use of dermal or spray products such as insect repellents. In contrast, cleaning frequency was negatively associated with BPA concentrations. Notably, women who reported attempting to reduce or avoid BPA exposure during pregnancy did not have lower urinary BPA concentrations. Findings show that BPA exposure goes beyond dietary and other ingestion-related factors, also occur via inhalation and skin absorption from the home environment and personal care products, respectively. Household cleaning practices offer a potential opportunity to reduce population-level exposure. The knowledge generated here will inform the design of bisphenol-reducing interventions for pregnant women or women of reproductive age.
Fenazaquin (FEN) is an emerging low-dose acaricide used for mite control in vegetable crops. It is potential to enter aquatic environments via runoff, yet studies on its toxicity to aquatic organisms remain limited. This study aims to assess the harmful impact of FEN on the liver, with zebrafish serving as the animal model. FEN triggered developmental toxicity and impaired liver development in zebrafish embryos, along with liver injury in adults. Remarkably, F1 offspring that were never directly exposed to FEN still exhibited deficits in growth and development. In zebrafish embryos, FEN exposure led to reduced hepatic lipid accumulation, whereas in adult zebrafish liver, FEN promoted lipid deposition. FEN also increased the enzymatic activities of alanine transaminase and aspartate aminotransferase, alter total cholesterol and triglycerides contents in zebrafish embryos and adult zebrafish livers, and disrupt lipid and carbohydrate metabolism in adult zebrafish livers. Furthermore, qRT-PCR analysis revealed that FEN altered the expressions of genes related to growth and development, fatty acid, sterol, steroid, glyceride, carbohydrate metabolism. Transcriptomic analysis of embryos revealed extensive remodeling of lipid and carbohydrate metabolic pathways in embryos. Therefore, FEN may impair liver development and metabolic homeostasis by disrupting glucose and lipid metabolism in zebrafish.
Silicosis is an incurable fibrotic lung disease caused by crystalline silica exposure. Aberrant activation of lung fibroblasts into pathological fibroblasts is a hallmark of silicosis. CTHRC1 has been considered a novel fibroblast activation biomarker. This study sought to explore CTHRC1's role in silicosis and determine the signaling pathways it modulates. A combination of transcriptomic analysis of clinical samples, LC-MS/MS, Co-IP, and cationic nanoliposome-based techniques was utilized. In vitro models of lung fibroblast activation induced by TGF-β1, PDGF or recombinant CTHRC1 (rCTHRC1), as well as in vivo models of pulmonary fibrosis induced by silica or bleomycin in mice were established. RNA sequencing, Western blot, RT-qPCR, AlphaFold2 analyses and rescue experiments using siCTHRC1, siCD44, CTHRC1 plasmids, and the PI3K activator 740 Y-P were performed to explore the underlying mechanisms. CTHRC1 is a significantly dysregulated gene implicated in myogenesis and ECM-related pathways. Mechanistically, lymphoid enhancer-binding factor 1 (LEF1) exerts its transcriptional regulator role by binding to the CTHRC1 promoter region in TGF-β1-activated fibroblasts. Further, CTHRC1 mediates its pro-fibroblast-to-myofibroblast transition function by directly binding to the CD44 receptor. AlphaFold2 analyses revealed the binding between CTHRC1 and CD44 is accomplished by three critical CD44 residues: Arg41, Asn172, and Trp650. Rescue experiments demonstrated that the CTHRC1-CD44 complex exerts its pro-fibrotic effects through AKT signaling. In vivo, liposomal Cthrc1 siRNA mitigated fibrogenesis in both silica- and bleomycin-induced mouse fibrosis models. Our results uncover a previously unrecognized role of the LEF1-CTHRC1-CD44 axis in silicosis and highlight the therapeutic target potential of CTHRC1 in fibrotic lung diseases.
Pharmaceuticals and antibiotics occur in the environment as complex, time-varying mixtures, but their toxicological interpretation remains limited by targeted chemical lists, parent-compound monitoring, and single-compound testing. This narrative review synthesizes representative peer-reviewed evidence and integrates established exposomics, HRMS, EDA, AEP, and AOP concepts into a framework for mechanistic interpretation of environmental pharmaceutical and antibiotic mixtures. This integration connects target, suspect, and non-target HRMS screening with internal exposure verification, effect-directed analysis, aggregate exposure pathways, and AOP-informed mechanistic prioritization. The synthesis focuses on peer-reviewed studies that illustrate chemical screening, internal and tissue-resolved exposure, bioactivity anchoring, antibiotic transformation products, antimicrobial-resistance-relevant endpoints, and AEP/AOP-based mechanistic interpretation. Internal and tissue-resolved exposure data are emphasized as important for identifying biologically plausible drivers, particularly for neuroactive pharmaceuticals with conserved molecular targets and antibiotics that act through microbial, microbiome, immune, and resistance-selection pathways. Effect-directed analysis and mode-of-action-relevant bioassays provide a bridge between feature-rich exposome datasets and bioactivity-informed mechanistic interpretation. For antibiotics, inclusion of transformation products and antimicrobial resistance-related endpoints is significant because parent-only workflows can underestimate both chemical burden and biological relevance. AOP-network mapping offers a structured method for prioritizing key events, convergence points, and follow-up assays while separating confirmed evidence from tentative HRMS annotations. Finally, we summarize practical reporting and study-design considerations covering exposure verification, annotation confidence, QA/QC, bioactivity anchoring, omics interpretation, and qualitative evidence evaluation. This integrated approach can improve reproducibility, comparability, and decision relevance in the mechanistic toxicology of environmental pharmaceutical and antibiotic mixtures.
