Chemical Research in Toxicology最新文献

2-Methylfuran (2-MF) is a process-related contaminant found primarily in heat-treated foods, such as coffee or canned food. The oxidative metabolic activation of 2-MF is supposed to follow the pathway established for furan, which is known to generate the highly reactive metabolite butenedial (BDA). In the case of 2-MF, generation of the BDA homologue 3-acetylacrolein (AcA) is to be expected. 2-MF metabolism to AcA was investigated in two model systems: commercial microsomal preparations and primary rat hepatocytes (pRH). To scavenge the generated 2-MF, two model nucleophils, N-acetyl-l-cysteine (AcCys) and N-α-acetyl-l-lysine (AcLys), were used, and the formation of the corresponding adducts was measured in the supernatants. The metabolic activation of 2-MF to AcA was studied using human liver microsomes as well as rat liver microsomes. Incubation of 2-MF in Supersomes allowed to identify the cytochrome P450 isoenzyme primarily responsible for 2-MF. In addition, primary rat hepatocytes were incubated with 2-MF or AcA and AcLys adduct of AcA (N-α-acetyl-l-lysine-acetylacrolein, AcLys-AcA) determined in the cell supernatants by UHPLC-MS/MS. In model experiments, AcA formed adducts with AcCys and AcLys. The structures of both adducts were characterized. For incubations in biological activating systems, CYP 2E1 was found to be a key enzyme for the conversion of 2-MF to AcA in Supersomes. When pRH were incubated with 2-MF and AcA, AcLys-AcA was detected in the cell supernatants in a time- and dose-dependent manner. The results showed that AcA was indeed formed at the cellular level. In contrast to the AcLys-AcA adduct, no N-acetyl-l-cysteine-acetylacrolein (AcCys-AcA) adduct could be detected in pRH. AcA was determined as a reactive metabolite of 2-MF in vitro, and its adduct formation with nucleophilic cellular components was evaluated. The metabolites were characterized, and AcLys-AcA was identified as potential biomarker.

ZLY06 is a dual agonist of peroxisome proliferator-activated receptor (PPAR) δ/γ, showing potential therapeutic effects on metabolic syndrome. However, our research has revealed that ZLY06 exhibits hepatotoxicity in normal C57BL/6J mice, though the precise mechanism remains unclear. This study aims to investigate the manifestations and mechanisms of ZLY06-induced hepatotoxicity. We administered ZLY06 via oral gavage to C57BL/6J mice (once daily for six weeks) and monitored various indicators to preliminarily explore its hepatotoxicity. Additionally, we further investigate the specific mechanisms of ZLY06-induced hepatotoxicity using PPAR inhibitors (GW9662 and GSK0660) and the Protein kinase B (AKT) activator (SC79). Results showed that ZLY06 led to increased serum ALP, ALT and AST, as well as elevated liver index and hepatic lipid levels. There was upregulation in the gene and protein expression of lipid metabolism-related molecules Acc, Scd1, Cd36, Fabp1 and Fabp2 in hepatocytes, with Cd36 showing the most significant change. Furthermore, cotreatment with SC79 significantly reduced ZLY06-induced hepatotoxicity in AML12 cells, evidenced by decreased intracellular TG levels and downregulation of CD36 expression. Specific knockdown of CD36 also mitigated ZLY06-induced hepatotoxicity. The study found that ZLY06 may bind to AKT1, inhibiting its phosphorylation activation, with the downregulation of p-AKT1 preceding the upregulation of CD36. In summary, ZLY06 mediates the upregulation of CD36 by potentially binding to and inhibiting the phosphorylation of AKT1, leading to hepatic lipid metabolism disorder and inducing liver toxicity.

Histones and their posttranslational modifications (PTMs) are critical regulators of gene expression. Differentiation, environmental stressors, xenobiotics, and major human diseases cause significant changes in histone variants and PTMs. Western blotting is the mainstay methodology for detection of histones and their PTMs in the majority of studies. Surprisingly, despite their high abundance in cells, immunoblotting of histones typically involves loading of large protein amounts that are normally used for detection of sparse cellular proteins. We systematically examined technical factors in the Western-blotting-based detection of human histones with >30 antibodies. We found that under multiple protein transfer conditions, many histone epitopes on polyvinylidene fluoride (PVDF) membranes had a very low antibody accessibility, which was dramatically increased by the addition of a simple denaturation step. Denaturation of membrane-bound proteins also enhanced the specificity of some histone antibodies. In comparison to standard PVDF membranes, the sensitivity of histone detection on standard nitrocellulose membranes was typically much higher, which was further increased by the inclusion of the same denaturation step. Optimized protocols increased by >100-times detection sensitivity for the genotoxic marker γ-H2AX with two monoclonal antibodies. The impact of denaturation and nitrocellulose use varied for different histones, but for each histone, it was generally similar for antibodies targeting N-terminal and C-terminal regions. In summary, denaturation of membrane-bound histones strongly improves their detection by Westerns, resulting in more accurate measurements and permitting analyses with small biological samples.

Proton pump inhibitors (PPIs) are a commonly used class of drugs with a good safety profile. However, their use is associated with rare cases of severe skin reaction. Herein, we present details of a patient who developed two episodes of omeprazole-induced delayed-onset hypersensitivity (atypical drug reaction with eosinophilia and systemic symptoms [DRESS]). Lymphocytes from the patient were stimulated to proliferate and secrete cytokines and cytolytic molecules when treated with the drug. T-cell cross-reactivity was observed with structurally related PPIs. Hence, other PPIs have the potential to cause further serious immune-related adverse events in patients who present with hypersensitivity to a primary PPI.

Short-chain fatty acids (SCFA) are an important energy source for colonocytes and crucial messenger molecules both locally in the intestine and systemically. Butyrate, one of the most prominent and best-studied SCFA, was demonstrated to exert anti-inflammatory effects, improve barrier integrity, enhance mucus synthesis in the intestine, and promote cell differentiation of intestinal epithelial cells in vitro. While the physiological relevance is undisputed, it remains unclear if and to what extent butyrate can influence the effects of xenobiotics, such as food-grade titanium dioxide (E171, _fgTiO₂), in the intestine. TiO₂ has been controversially discussed for its DNA-damaging potential and banned as a food additive within the European Union (EU) since 2022. First, we used enterocyte Caco-2 monocultures to test if butyrate affects the cytotoxicity and inflammatory potential of _fgTiO₂ in a pristine state or following pretreatment under simulated gastric and intestinal pH conditions. We then investigated pretreated _fgTiO₂ in intestinal triple cultures of Caco-2, HT29-MTX-E12, and THP-1 cells in homeostatic and inflamed-like state for cytotoxicity, barrier integrity, cytokine release as well as gene expression of mucins, oxidative stress markers, and DNA repair. In Caco-2 monocultures, butyrate had an ambivalent role: pretreated but not pristine _fgTiO₂ induced cytotoxicity in Caco-2 cells, which was not observed in the presence of butyrate. Conversely, _fgTiO₂ induced the release of interleukin 8 in the presence but not in the absence of butyrate. In the advanced in vitro models, butyrate did not affect the characteristics of the healthy or inflamed states and caused negligible effects in the investigated end points following _fgTiO₂ exposure. Taken together, the effects of _fgTiO₂ strongly depend on the applied testing approach. Our findings underline the importance of the experimental setup, including the choice of in vitro model and the physiological relevance of the exposure scenario, for the hazard testing of food-grade pigments like TiO₂.

Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.

Cytochromes P450 (P450s or CYPs) are the most important phase I metabolic enzymes in the human body and are responsible for metabolizing ∼75% of the clinically used drugs. P450-mediated metabolism is also closely associated with the formation of toxic metabolites and drug–drug interactions. Therefore, it is of high importance to predict if a compound is the substrate of a given P450 in the early stage of drug development. In this study, we built the multitask learning models to simultaneously predict the substrates of five major drug-metabolizing P450 enzymes, namely, CYP3A4, 2C9, 2C19, 2D6, and 1A2, based on the collected substrate data sets. Compared to the single-task model and conventional machine learning models, the multitask fingerprints and graph neural networks model achieved superior performance with the average AUC values of 90.8% on the test set. Notably, the multitask model demonstrated its good performance on the small amount of substrate data sets such as CYP1A2, 2C9, and 2C19. In addition, the Shapley additive explanation and the attention mechanism were used to reveal specific substructures associated with P450 substrates, which were further confirmed and complemented by the substructure mining tool and the literature.

Drug-induced liver injury (DILI) stands as a significant concern in drug safety, representing the primary cause of acute liver failure. Identifying the scientific literature related to DILI is crucial for monitoring, investigating, and conducting meta-analyses of drug safety issues. Given the intricate and often obscure nature of drug interactions, simple keyword searching can be insufficient for the exhaustive retrieval of the DILI-relevant literature. Manual curation of DILI-related publications demands pharmaceutical expertise and is susceptible to errors, severely limiting throughput. Despite numerous efforts utilizing cutting-edge natural language processing and deep learning techniques to automatically identify the DILI-related literature, their performance remains suboptimal for real-world applications in clinical research and regulatory contexts. In the past year, large language models (LLMs) such as ChatGPT and its open-source counterpart LLaMA have achieved groundbreaking progress in natural language understanding and question answering, paving the way for the automated, high-throughput identification of the DILI-related literature and subsequent analysis. Leveraging a large-scale public dataset comprising 14 203 training publications from the CAMDA 2022 literature AI challenge, we have developed what we believe to be the first LLM specialized in DILI analysis based on LLaMA-2. In comparison with other smaller language models such as BERT, GPT, and their variants, LLaMA-2 exhibits an enhanced out-of-fold accuracy of 97.19% and area under the ROC curve of 0.9947 using 3-fold cross-validation on the training set. Despite LLMs’ initial design for dialogue systems, our study illustrates their successful adaptation into accurate classifiers for automated identification of the DILI-related literature from vast collections of documents. This work is a step toward unleashing the potential of LLMs in the context of regulatory science and facilitating the regulatory review process.