Archives of Toxicology最新文献

Inhalation administration of therapeutics is a crucial method for treatment of respiratory diseases, offering direct access to the target organ. However, the progression of candidate drugs is frequently impacted by clinical dose level limitations due to lung histopathological findings or functional effects identified in in vivo studies. Addressing these safety concerns is crucial in advancing compounds with the right safety profile. To that end, there is a need for predictive in vitro model systems to evaluate lung toxicities, including inflammatory responses across various modalities. This study aimed to assess the predictive capability of the AlveoliX Lung-on-Chip (^AXLung-on-Chip) model in determining respiratory toxicity of eight inhaled substances of varying modalities. Experiments using a two-dimensional (2D) culture were conducted to assess cellular responses, optimize dose settings and study design. Differentiation between compounds with lower and higher inflammatory potential was not possible in the 2D model. In contrast however, the response following treatment in the ^AXLung-on-Chip model was more pronounced, and the use of multiple endpoints enabled differentiation based on their inflammatory potential. Our study also indicated a potential increased sensitivity in cytokine response following treatment when mechanical stretch was incorporated in the ^AXLung-on-Chip. Comparison to in vivo toxicology studies demonstrated that the ^AXLung-on-Chip model predicted drug-induced inflammatory responses, capturing a spectrum of lung pathologies from mild toxicity to severe inflammatory damage, and illustrates the potential of the ^AXLung-on-Chip to identify inhaled compound toxicity across various modalities.

Fentanyl analogs, a major subclass of synthetic opioids, have emerged worldwide and pose serious public health threats owing to their widespread misuse. This study investigated the comparative in vitro metabolism of five fentanyl analogs with varying N-acyl side chain lengths (acetylfentanyl, fentanyl, butyrylfentanyl, valerylfentanyl, and crotonylfentanyl) to elucidate the relationship between drug structure and metabolism. Each analog (5 μmol/L) was incubated with human liver microsomes for 1 h, and after deproteinization, the samples were analyzed using liquid chromatography-high-resolution mass spectrometry. Elongation of the N-acyl side chain resulted in shorter half-lives and higher clearance values. Among the four analogs with N-acyl alkyl side chains, the formation of the nor-metabolites, the metabolites hydroxylated at the ethyl linker, and the metabolites hydroxylated at the piperidine ring increased with increasing side chain length, peaking with fentanyl or butyrylfentanyl, and then decreasing with valerylfentanyl, thereby exhibiting an overall inverted U-shaped trend. In contrast, the metabolites hydroxylated at the phenyl ring of the phenethyl group declined as the side chain lengthened, whereas the metabolites hydroxylated at the acyl side chain and the carboxylated metabolites increased. Crotonylfentanyl, featuring an N-acyl alkenyl side chain, deviated from the structure-metabolism relationship observed among the other analogs. These findings highlight distinct structure-dependent metabolic stabilities and biotransformation pathways among fentanyl analogs, providing valuable information for identifying diagnostic metabolites to confirm fentanyl analog use in humans.

Drug-induced liver injury remains a major obstacle in pharmaceutical development and a leading cause of acute liver failure, underscoring the need for predictive and human-relevant in vitro models. Acetaminophen, a widely used analgesic with well-characterized dose-dependent hepatotoxicity, serves as a benchmark compound for evaluating liver toxicity mechanisms. Here, we applied single cell RNA sequencing to characterize cellular responses to acetaminophen exposure in two distinct liver cell culture formats: two-dimensional monolayers (2D) and three-dimensional (3D) spheroids composed of primary human hepatocytes, Kupffer cells, and liver endothelial cells. Cultures were exposed for 24 h to low (350 µM) and high (2687 µM) acetaminophen concentrations, with 2D cultures receiving only the low dose. Compared to 2D monolayers, 3D spheroids exhibited greater transcriptional diversity and elevated expression of ribosomal genes, indicative of enhanced metabolic and biosynthetic activity. Reactome pathway analysis revealed pronounced hypoxia-associated signaling in 3D cultures even under baseline conditions, likely due to restricted oxygen diffusion within spheroids. These hypoxia signatures were most prominent in endothelial cells. Upon acetaminophen exposure, hypoxic hepatocytes displayed elevated expression of cytochrome P450 enzymes, while conjugation enzymes involved in detoxification declined with increasing dose, suggesting compromised phase II metabolism under oxygen-limited conditions. Normoxic hepatocytes showed minimal transcriptional response. These results suggest a dynamic interplay between oxygen availability and acetaminophen metabolism, where oxygen tension influences metabolic activity, and drug metabolism in turn alters the hypoxic landscape. Our findings underscore the physiological relevance of 3D liver models and highlight the importance of spatial microenvironmental context for improving mechanistic insights into hepatotoxicity.

Cytochrome P450 enzymes (CYPs) are central to metabolism and stress adaptation. In Caenorhabditis elegans, the CYP-13 family performs diverse and conserved functions beyond xenobiotic detoxification. cyp-13 links lifespan regulation to the APP ortholog apl-1 and the heterochronic factor lin-14, integrating with DAF-16/FOXO, HSF-1, and DAF-12 pathways. In apoptosis, cyp-13 contributes to the degradosome complex with CPS-6/EndoG and WAH-1, facilitating DNA degradation. Several isoforms are inducible by aflatoxin B1 and PCB1254, underscoring roles in toxicant metabolism. Notably, cyp-13A12 regulates behavioral responses to reoxygenation via the EGL-9-HIF-1-PUFA-eicosanoid pathway, paralleling mammalian ischemia-reperfusion responses. Epigenetic regulation adds another layer, as BRCA1/BARD1 homologs brc-1 and brd-1 repress distinct subsets of cyp-13A genes through H2A ubiquitylation. Collectively, CYP-13 emerges as a multifunctional hub linking developmental, apoptotic, metabolic, stress, and chromatin-level processes, with clear parallels to human CYPs, highlighting its translational relevance to aging, cancer, and toxicology.

Inferior efficacy of antibiotics (e.g. rifampicin) in M2 macrophage-dominant tuberculosis granuloma might be related to poor drug uptake into these cells, potentially mediated by altered expression and activity of drug transporters such as P-glycoprotein (P-gp, encoded by ABCB1). Consequently, THP-1 cells were differentiated (200 nM phorbol 12-myristate 13-acetate; 72 h) and polarized to M1 (50 ng/mL lipopolysaccharide, 20 ng/mL interferon-gamma; 48 h) or M2 (20 ng/mL interleukin-4, interleukin-13; 48 h) macrophages. Then, quantitative polymerase-chain reaction array-based transcriptional analysis, flow cytometry, and ultra-performance liquid chromatography coupled to tandem mass spectrometry were used to evaluate the impact of differentiation and polarization on the expression levels of 84 drug transporter genes, the efflux activity of P-gp, and the cellular rifampicin uptake kinetics. ABCB1 was enhanced 166-fold during M1 polarization and 55-fold during M2 polarization. P-gp efflux activity in M2 cells was 1.55-fold higher than in M1 cells (P < 0.05). Rifampicin uptake into M2 cells was lower than into M1 cells after 2 h (P < 0.01) or 4 h exposure (P = 0.06) to 0.05 µM rifampicin. Together, when compared to M1 macrophages, M2 cells exhibit a considerably altered fingerprint of drug transporter expression levels, including an enhanced expression and activity of P-gp, being accompanied by lowered rifampicin uptake.

Translational research utilizing mass spectrometry to identify and validate biomarkers requires high-quality samples to maintain the integrity of research results. Key steps, including study design and sample/data quality control, are often overlooked or undervalued, which can compromise the consistency, accuracy, and verifiability of scientific and clinical outcomes. This is a prospective, longitudinal, multicenter cohort clinical trial that is recruiting participants who are victims of snakebite in Brazil and healthy individuals. Previously published studies on clinical trials and clinical-epidemiological research related to snakebites have identified universal measures and their categories, which were used to develop the study design and prepare the Case Report Forms. The biorepository presents a collection of clinical and epidemiological data, images of clinical manifestations, results of laboratory tests, and biological samples (serum and plasma in the presence of EDTA and sodium citrate). This article presents recommendations for essential steps in designing and implementing a mass spectrometry-based biomarker and mechanistic study focused on snakebite envenomation. We outline critical reporting models for conducting each phase of clinical data acquisition, such as collecting biological materials (serum and blood plasma) and processing samples to minimize the study's pre-analytical variables. The guidelines provided, covering everything from study design to implementation and data interpretation, serve as a reference to increasing the rigor and reproducibility of clinical proteomics studies. The results of this study can transcend basic research, connecting technological development to real clinical application, optimizing diagnosis and monitoring, but also contributing to reducing the economic burden, morbidity and mortality associated with these accidents.

Under the European Cosmetic Regulation, safety assessments of cosmetics and their ingredients must be conducted without the use of animals. This regulatory requirement poses a number of challenges, as validated alternative methods are only available for some of the toxicological endpoints that are typically considered in standard human health risk assessments. Despite significant progress since the ban in 2013, particularly in the development of New Approach Methodologies (NAMs) for local and acute toxicity, and for mutagenicity/genotoxicity, there remains an urgent need for non-animal test methods to assess systemic toxicity, which often becomes evident after repeated or long-term exposure. Currently, no validated animal-free alternatives are available for assessing sub-acute, sub-chronic and chronic toxicity, carcinogenicity, developmental/reproductive toxicity, or for a major part of toxicokinetics. In response to these challenges, the Methodology Working Group of the Scientific Committee on Consumer Safety organised a dedicated workshop in December 2024 to discuss advances in the application of Next Generation Risk Assessment (NGRA) as a strategic animal-free approach for the safety assessment of cosmetic ingredients. The workshop focused on a number of important key issues for the practical application of NAMs and NGRA, their regulatory acceptance and identification of possible (partial) solutions to overcome existing limitations.

T-2 toxin can induce the development of cartilage diseases. N6-methyladenosine (m6A) modification mediated by methyltransferase-like 3 (METTL3) is involved in the occurrence and progression of bone-related diseases. However, the mechanism of action of METTL3 in T-2 toxin-caused cartilage injury has not been fully revealed. It is unclear whether METTL3 regulates cartilage injury by modulating its downstream target macrophage colony-stimulating factor (Csf1). Here, we aimed to investigate the mechanism by which the T-2 toxin induces cartilage damage. We performed histological staining, western blotting, immunohistochemistry, immunofluorescence, RNA-seq, MeRIP-seq, and qRT-PCR. T-2 toxin was found to down- and upregulate METTL3 and Csf1 expression levels in the articular cartilage tissue of mice, respectively. According to the in vitro experiments, the knockdown of METTL3 exacerbates the extracellular matrix (ECM) degradation associated with the HT-2 toxin (the deacetylated form of the T-2 toxin). Based on bioinformatics analysis and related experiments, we found that Csf1 interacts with METTL3 through m6A modification, serving as a METTL3 target to promote the degradation of the cartilage extracellular matrix of chondrocytes. Summarily, gene regulation of the METTL3/m6A/Csf1 axis in the extracellular matrix of chondrocytes can promote the development of T-2 toxin-induced cartilage damage, providing a reference for the effective treatment of cartilage-related diseases. These findings reveal the regulatory mechanism of METTL3/m6A/Csf1 axis as a potential therapeutic target for the treatment of cartilage-associated diseases.

Polychlorinated biphenyl-153 (PCB-153) is a representative organic pollutant that can accumulate in the human body, particularly in adipose tissue, for long periods due to its environmental persistence and high lipid solubility. Existing risk assessments primarily rely on simple extrapolations based on blood concentrations, and a quantitative approach that considers internal exposure at the tissue level is lacking. The main objective of this study was to establish a human physiologically based pharmacokinetic (PBPK) model for PCB-153 and to quantitatively predict tissue accumulation in the human body based on this model, thereby suggesting the possibility of expanding its application as a precise and scientific human risk assessment tool in the future. Based on existing mouse experimental data, a PBPK model was constructed and validated, and a whole-body human PBPK model was established through interspecies extrapolation. Monte Carlo simulations were then performed, considering physiological variations in the adult population, to predict concentration-time curves for major human tissues (fat, liver, brain, skin, and plasma) following a single dose (20 mg/kg) and repeated exposures at the reference dose (RfD) level (5.86 ng/kg/day). The simulation results based on the human PBPK model established in this study showed that adipose tissue showed the highest accumulation pattern with an average partition coefficient (K_p) of 795,725 after a single dose (20 mg/kg), and even under repeated exposure conditions (RfD), the average K_p in adipose tissue was found to be 4961, the highest among all tissues. In addition to adipose tissue, PCB-153 was widely distributed in the brain, skin, liver, etc., and the same pattern was observed in terms of steady-state concentrations upon repeated administration. These results strongly suggest the possibility of PCB-153 accumulation in major organ tissues even at relatively low external exposure doses (RfD), and support the need for a conservative and quantitative risk assessment, especially for the possibility of chronic toxicity due to long-term exposure. The human PBPK model established in this study establishes a foundation for numerically quantifying the tissue-specific accumulation characteristics of PCB-153, suggesting an alternative approach that overcomes the limitations of existing, limited risk assessments. This model can serve as a scientific basis for future human exposure assessments and standardization of highly soluble environmental pollutants.