Chemical Research in Toxicology最新文献

Stiripentol (SRP) is an antiepileptic agent utilized in managing seizures related to Dravet syndrome. Long-term safety studies have highlighted significant adverse effects in patients including drowsiness, reduced appetite, ataxia, and elevated levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The present study aimed at identifying the reactive metabolite of SRP and defining the potential correlation between its cytotoxicity and metabolic activation. Rat liver microsome incubation of SRP fortified with GSH as a trapping agent produced an α,β-unsaturated ketone metabolite (M1) and a related GSH conjugate (M2). Moreover, both the phase I metabolite and the GSH conjugate were detected in the bile of SRP-treated rats, indicating that both in vivo and in vitro metabolic activation of SRP took place. Notably, SRP exhibited significant cytotoxicity toward rat primary hepatocytes. Pretreatment with ketoconazole, a selective CYP3A enzyme inhibitor, mitigated the susceptibility of hepatocytes to SRP-induced cytotoxicity. These findings suggest that SRP may undergo metabolism to the α,β-unsaturated ketone metabolite, potentially contributing to the cytotoxic effects associated with SRP.

The popularity of electronic cigarettes and vaping products has launched the outbreak of a condition affecting the respiratory system of users, known as electronic-cigarette/vaping-associated lung injury (EVALI). The build-up of vitamin E acetate (VEA), a diluent of some illicit vaping oils, in the bronchoalveolar lavage of patients with EVALI provided circumstantial evidence as a target for investigation. In this work, we provide a fundamental characterization of the interaction of VEA with lung cells and pulmonary surfactant (PS) models to explore the mechanisms by which vaping-related lung injuries may be present. We first confirm the localization and uptake of VEA in pulmonary epithelial cells. Further, as PS is vitally responsible for the biophysical functions of the lungs, we explore the effect of added VEA on three increasingly complex models of PS: dipalmitoylphosphatidylcholine (DPPC), a lipid-only synthetic PS, and the biologically derived extract Curosurf. Using high-resolution techniques of small-angle X-ray scattering, small-angle neutron scattering, neutron spin-echo spectroscopy, and neutron reflectometry, we compare the molecular-scale behaviors of these membranes to the bulk viscoelastic properties of surfactant monolayer films as studied by Langmuir monolayer techniques. While VEA does not obviously alter the structure or organization of PS membranes, a consistent softening of membrane systems─regardless of compositional complexity─provides a biophysical explanation for the respiratory distress associated with EVALI and yields a new perspective on the behavior of the PS system.

Organophosphorus (OP) compounds pose a serious risk to human health by covalently modifying acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Currently approved oxime therapeutics can reactivate OP-inhibited AChE and BChE, despite significant limitations. The OP-inhibited enzymes undergo a secondary O-dealkylation event, known as aging, for which no currently approved therapeutics are effective as treatments. Many decades of research have studied the aging mechanism in AChE and BChE. It has previously been accepted that aging occurs via a spontaneous O-dealkylation event, by loss of a carbocation or by water hydrolysis of the OP-adducted serine residue. Here, we present a novel mechanism of aging in which the catalytic histidine acts as a nucleophile to induce aging and, as a result, becomes alkylated after exposure to methyl paraoxon (MP) and other pesticides. Using bottom-up proteomics, we identify that upon aging of MP-inhibited AChE and BChE, a methyl transfer occurs from the phosphylated serine residue to the catalytic histidine residue. The extent of histidine methylation is pH-dependent as less methylation is observed at lower pH, while increased methylation is observed at higher pH. At near physiological pH (7.5), the ratio of N-MeHis/His is 3:1 for AChE and 1.3:1 for BChE after 24 h. When other OP compounds were also tested for histidine modification, ethyl paraoxon was shown to result in ethylation of the catalytic histidine; however, when the alkoxy group was branched in the case of an isopropoxy group present in diisopropyl fluorophosphate, no alkylation of histidine was observed. Recent advances in the development of quinone methide precursors show promise in the recovery of OP-aged AChE. In this work, we discuss the importance of this novel aging mechanism and its impact on the recoverability of OP-aged AChE or BChE as it appears that the histidine modification limits the overall recovery of active AChE.

Since their discovery as impurities in numerous pharmaceuticals beginning in 2018, there has been a strong push to predict and prevent the formation of mutagenic nitrosamines. Several experimental methods, particularly the Nitrosation Assay Procedure, have been developed to predict a molecule's susceptibility to nitrosation. Here, we have compiled the results of hundreds of these experiments from the literature to construct two structure-activity relationship models: a statistical model and an expert rule-based model. The statistical model has been built with graph neural networks and was trained on a dataset of 207 nitrogen-containing molecules. This model makes a binary call for each nitrogen center, predicting if it is likely to be nitrosated or not. Conversely, the rule-based model labels each possible nitrosamine product as one of four categories, ranging from "unlikely" to "very likely". It makes this determination based on 15 rules, which cover 12 deactivating (inhibit nitrosation) and 3 activating (favor nitrosation) features that have been drawn from the literature. Both models perform remarkably well, with accuracies of ∼80%. The rule-based model is generally biased toward favoring nitrosation while the statistical model is more likely to classify an amine as un-nitrosatable due to the makeup of the dataset. Using the models together can balance these biases and further improve the reliability of both.

Tris(2,4-di-tert-butylphenyl)phosphate (TDTBPP), a novel organophosphate ester (OPE), has been extensively detected in various environmental and biological samples; however, its potential biological effects remain unexplored. In this study, we investigated biotransformation characteristics, alteration of lipid metabolism, and mRNA expression in primary mouse hepatocytes (PMHs) following exposure to TDTBPP. After 36-h exposure in PMHs, TDTBPP exhibited a high stability potential with no statistically significant degradation trend. Subsequently, we analyzed the disruption of lipid homeostasis in PMHs following exposure to 0-4.5 μM TDTBPP. Lipidomic analysis indicated that TDTBPP disrupted lipid homeostasis in PMHs, and several lipid classes were dysregulated, in particular, glycerolipids and glycerophospholipids. Additionally, three lipids were proposed as potential lipid biomarkers of TDTBPP exposure, including triglycerides (TGs) and phosphatidylcholines (PCs). These observations were further supported by transcriptional changes, with significant alteration observed in genes associated with lipid uptake, de novo lipogenesis, β-oxidation of fatty acids, glycerolipid metabolism, and lipid export. Overall, these findings highlight the detrimental effects of TDTBPP on lipid homeostasis, providing important insights for health risk assessments of this abundant OPE in the environment.

It is uncertain whether exposure to environmental concentrations of perfluorobutanesulfonate (PFBS) disrupts the reproductive endocrine system in amphibian tadpoles. In this study, tadpoles (Lithobates catesbeianus) in G26 stage were treated with different levels of PFBS (0, 1, 3, and 10 μg/L) for 60 days to investigate whether and how PFBS affects the reproductive endocrine system and gonadal development in tadpoles. Tadpole testes exhibited structural damage to germ cells and significantly fewer spermatogonia following PFBS exposure, but the sex ratio remained unaffected. Further, PFBS exposure downregulated transcripts of genes associated with ovarian (figla and nobox) and testicular (sox9 and dmrt1) development in tadpoles. Encoding gonadotropin hormone genes were transcriptionally upregulated in the pituitary, and serum gonadotropins (FSH and LH) were elevated. Genes related to testosterone synthesis were transcriptionally upregulated, and serum testosterone concentrations were raised. The transcription of the cyp19a1 gene, which is involved in the synthesis of estradiol (E2), was downregulated, leading to decreased levels of serum E2. Furthermore, the transcript level of the vitellogenin gene was downregulated in the liver. Thus, PFBS exposure appears to disrupt the hypothalamic-pituitary-gonadal-liver axis in tadpoles, subsequently impacting gonadal development. The findings of this study indicate that environmental concentrations of PFBS threaten the reproductive endocrine system in amphibians for the first time. This provides important insights for further investigation into the risk that PFBS poses to the stability of the amphibian population.

Isopropyl glycidyl ether (IPGE) is a member of the large glycidyl ether family frequently used as a reactive diluent during the epoxy resin manufacturing process. Although the toxicity induced by this type of chemical has been investigated in many studies of different aspects (acute, subchronic, genotoxic, reproduction, etc.), there is still little known about their toxicokinetics. To gain information about the attainable systemic concentration, a liquid chromatography─tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of IPGE in rat plasma using its structural analogue tert-butyl glycidyl ether as the internal standard. Two types of atmospheric pressure ionization techniques have been utilized; however, the protonated molecule ion could not be observed in either ionization mode. First, the ammonium adduct form was used for fragmentation, albeit this multiple-reaction monitoring transition proved to be not sensitive enough for real study sample analysis. In order to achieve the desired sensitivity, the Meerwein reaction was applied as an in-source derivatization tool to generate a product by using the ethylnitrilium ion formed from the eluent acetonitrile. This gas-phase reaction enabled us to build up a method with a substantial sensitivity increase (LLOQ of 0.01 μg/mL) compared to that obtained with the ammonium adduct. After method validation, real study samples from a single-dose oral toxicity study were analyzed to evaluate the blood plasma concentration of IPGE at three dose levels. Dose-dependent superproportional systemic exposure was observed in the studied dose range (1000-2000 mg/kg). Additionally, seven metabolites of IPGE were tentatively identified in rat plasma: 3-isopropoxy-2-hydroxy-1-propanol (M1), sulfate-conjugate of IPGE (M3), glucuronide-conjugate of IPGE (M4), 3-isopropoxy-2-hydroxypropionic acid (M5), O-isopropyl-N-acetylserine (M6), O-(2-hydroxy-isopropyl)-N-acetylserine (M7), and glutathione-conjugate of IPGE (M11). Present work may pave the way to other methods that are able to quantify compounds similar to IPGE even in human plasma, which could provide valuable information to assist exposure assessment and biomonitoring in occupational health and safety studies.

Benzo[a]pyrene (BaP) and nicotine exposure have been implicated in lung cancer development. This study aims to elucidate the molecular mechanisms and potential biomarkers associated with this exposure in lung cancer patients. We integrated gene expression data from The Cancer Genome Atlas lung cancer cohort and the Comparative Toxicogenomics Database to identify differentially expressed genes (DEGs) associated with BaP and nicotine exposure. Enrichment analyses, survival analyses, and immune cell infiltration analyses were conducted to interpret the biological significance of these DEGs. A risk score model and a nomogram were constructed for a prognostic evaluation. We identified 163 DEGs related to BaP and nicotine exposure in lung cancer. Enrichment analysis revealed significant biological processes and pathways, including "IL-17 signaling", "cellular senescence", and "p53 signaling". From the DEGs, 34 prognostic genes were identified, with CLDN5, DNASE1L3, and GPR37 being independent prognostic factors. A risk score model based on these genes showed significant prognostic value, with high-risk patients exhibiting poorer survival outcomes. Additionally, a nomogram based on these risk scores demonstrated good predictive accuracy and clinical utility. Kaplan-Meier analyses confirmed that high expression of CLDN5 and GPR37 correlated with poor survival, while high DNASE1L3 expression indicated better survival. Single-gene enrichment analyses linked these genes to immune responses, cell adhesion, and DNA methylation. Immune cell infiltration analysis revealed significant correlations between the expression of these genes and the infiltration of various immune cell types. Our findings highlight the significant role of CLDN5, DNASE1L3, and GPR37 in lung cancer associated with BaP and nicotine exposure. The constructed risk score model and nomogram provide valuable tools for prognostication, and the identified genes offer potential targets for therapeutic intervention. Understanding the influence of toxic exposure on the tumor-immune microenvironment can guide future research and treatment strategies.

Exposure assessment of hazardous volatile organic compounds (VOCs) requires accurate quantification of internal dose when establishing limits or identifying significant differences within and among populations. Even though accurate internal dose can be directly measured in blood, it is not always practical or possible to collect a suitable blood specimen. This work studies the relationship between blood and urine levels for certain smoke biomarkers (e.g., tobacco, marijuana) measured in self-reported cigarette smokers. Urine and blood specimens were collected as matched pairs from individuals at the same time. We used our latest specimen collection and VOC analysis protocols to minimize sample collection, handling, and analysis biases. From these analyses, unmetabolized urine benzene, furan, 2,5-dimethylfuran, isobutyronitrile, and benzonitrile levels were found to trend with blood levels. In addition, we measured urine creatinine levels, which were found to be significantly associated with all blood analyte concentrations (p-value ranging from <0.0063 to <0.0001) except for isobutyronitrile (p = 0.3347). For the analytes that were associated with urine creatinine levels, the ratios of urine-to-blood concentrations were substantially higher than those predicted from the urine/blood partition coefficients (K_urine/blood), which should occur if VOCs can freely equilibrate (i.e., passive diffusion) between the blood and urine. The urine isobutyronitrile concentration, which was the only analyte that was not associated with the urine creatinine level, had a urine-to-blood ratio similar to K_urine/blood. These results suggest either that urine VOC levels for certain VOCs do not equilibrate with blood levels in the urinary tract or that there is a conversion of conjugated to free forms, increasing urine VOC levels. Nevertheless, these deviations from partition theory (e.g., Henry's Law) are analyte-specific and require characterization to establish a relationship between blood and urine levels.

High-throughput cell-based bioassays can fulfill the growing need to assess the hazards and modes of toxic action (MOA) of ionic liquids (ILs). Although nominal concentrations (C_nom) are typically used in an in vitro bioassay, freely dissolved concentrations (C_free) are considered a more accurate dose metric because they account for chemical partitioning processes and are informative about MOA. We determined the C_free of IL cations in AREc32 and AhR-CALUX assays using both mass balance model (MBM) prediction and experimental quantification. Partition coefficients between membrane lipid-water (K_mw), serum albumin-water (K_albumin/w), and cell-water (K_cell/w) as well as potential confounding factors (binding to a test plate and micelle formation) were determined to improve the MBM prediction. IL cations showed a higher affinity for both cell lines than that predicted by the MBM based on K_mw and K_albumin/w. Their affinity for the AhR-CALUX cells was more than 1 order of magnitude higher than for the AREc32, signifying cell line-specific affinity. The MBM with an experimental K_cell/w accurately predicted C_free. Evaluating cytotoxicity based on C_free eliminated the leveling off of toxicity observed for hydrophobic IL cations (side chain cutoff), suggesting that C_nom underestimates the effects of compounds with high affinity for the assay medium. Cell membrane concentrations calculated from C_free using K_mw were compared to the critical membrane burden to identify whether IL cations act as baseline toxicants. The IL cations carrying 16 carbons in the chain in the AREc32 assay and most of the IL cations in the AhR-CALUX assay were classified as excess toxicants. However, since the reasons for the deviation of experimental K_cell/w from MBM prediction remain unexplained, it is uncertain whether the cell membrane concentrations can be well predicted from K_mw used in this study. Therefore, future studies should aim to uncover the underlying causes of differing cell affinities observed across cell lines and model predictions.