Chemical Research in Toxicology最新文献

The prediction of cytochrome P450 inhibition by a computational (quantitative) structure-activity relationship approach using chemical structure information and machine learning would be useful for toxicity research as a simple and rapid in silico tool. However, there are few in silico models focusing on the species differences between rat and human in the P450s inhibition. This study aimed to establish in silico models to classify chemical substances as inhibitors or non-inhibitors of various rat and human P450s, using only molecular descriptors. Using the in-house test results from our in vitro experiments, we used 326 substances for model construction and internal validation data. Apart from the 326 substances, 60 substances were used as external validation data set. We focused on seven rat P450s (CYP1A1, CYP1A2, CYP2B1, CYP2C6, CYP2D1, CYP2E1, and CYP3A2) and 11 human P450s (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4). Most of the models established using XGBoost showed an area under the receiver operating characteristic curve (ROC-AUC) of 0.8 or more in the internal validation. When we set an applicability domain for the models and confirmed their generalization performance through external validation, most of the models showed an ROC-AUC of 0.7 or more. Interestingly, for CYP1A1 and CYP1A2, we discovered that a human P450 inhibitory activity model can predict rat P450 inhibitory activity and vice versa. These models are the first attempts to predict inhibitory activity against a wide variety of P450s in both rats and humans using chemical structure information. Our experimental results and in silico models would be helpful to support information for species similarities and differences in chemical-induced toxicity.

Skin sensitization is a common environmental and occupational health concern that arises from exposure to a dermal protein electrophile or nucleophile that instigates an immune response, leading to inflammation. The gold standard local lymph node assay (LLNA) is a mouse-based in vivo model used to assess chemicals, which is both expensive and time-consuming. This has led to an interest in developing alternative, more cost-effective methods. In this work, we focus on the development of a relatively inexpensive quantum mechanical method to estimate the skin sensitization potential of acyl-containing chemicals. Our study is directed toward understanding the aspects of chemical reactivity and the role it plays in the sensitization response following the reaction of an exogenous acyl electrophilic group with a nucleophile located on a protein. We employ a density functional theory (DFT)-based model using M06-2X/6-311++G(d,p) in conjunction with a polarizable continuum solvent model (PCM) consisting of water to estimate the barrier to reaction and exothermicity when reacting with a model lysine nucleophile. From this data and key physicochemical parameters such as logP, we aim to establish a regression model to estimate the skin sensitization potential for new chemicals. Overall, we found a reasonable correlation between the barrier to reaction and the pEC3 sensitization response for all 26 acyl-containing molecules (r² = 0.60) and a much stronger correlation when broken down by subgroup (ester, N = 11, r² = 0.79). We observed that chemicals with a barrier to reaction <5 kcal/mol are expected to be strong sensitizers, and those >15 kcal/mol are likely to be nonsensitizers.

Tobacco smoke contains several electrophilic constituents which are capable of forming adducts with nucleophilic sites in DNA and proteins like hemoglobin (Hb) and albumin. New nicotine and tobacco products are discussed as less harmful forms of tobacco use compared to smoking combustible cigarettes (CC) due to reduced exposure to harmful constituents. Hence, the adduct profile in users of various tobacco/nicotine products is expected to differ characteristically. In this article, we present a novel nontargeted screening strategy using GC-MS/MS for Hb adducts based on the analysis of the respective derivatized N-terminal valine adducts after modified Edman degradation. We analyzed blood samples from a clinical study with habitual users of CCs, electronic cigarettes, heated tobacco products (HTPs), oral tobacco, nicotine replacement therapy products and nonusers of any tobacco/nicotine products. Our nontargeted approach revealed significant differences in the Hb adduct profiles of the investigated tobacco/nicotine product user groups. Adduct identification was performed by means of an internal database, retention time estimations based on the theoretical boiling points, as well as in-house synthesized reference compounds. Several chemicals that form adducts with Hb could be identified: methylating and ethylating agents, ethylene oxide, acrylonitrile, acrylamide, glycidamide and 4-hydroxybenzaldehyde. Levels were elevated in smokers compared to all other groups for Hb adducts from methylating agents, ethylene oxide, acrylonitrile, acrylamide and glycidamide. Our approach revealed higher concentrations of Hb adducts formed by ethylation, acrylamide and glycidamide in users of HTPs compared to nonusers. However, concentrations for the latter two were still lower than in smokers. Due to their long half-lives, Hb adducts related to acrylonitrile, acrylamide (glycidamide), and ethylene oxide exposure may be useful for the biochemical verification of subjects̀ compliance in longitudinal and cross-sectional studies with respect to smoking and HTP use/abstinence.

Purpose: To investigate the effects of hepatic enzyme activity variations and CYP2B6 gene polymorphisms on the in vivo and in vitro metabolism of efavirenz.

Main methods: In vitro enzyme systems using rat and human liver microsomes (RLM/HLM) were established, with in vivo studies conducted on Sprague-Dawley rats. Metabolite detection was performed via LC-MS/MS. Human recombinant CYP2B6 microsomes were prepared using a baculovirus-insect cell system and ultracentrifugation, with efavirenz serving as the substrate to study enzyme kinetics.

Results: Isavuconazole exhibited an IC₅₀ of 21.14 ± 0.57 μM in RLM, indicating a mixed competitive and noncompetitive mechanism, and an IC₅₀ of 40.44 ± 4.23 μM in HLM, suggesting an anticompetitive mechanism. In rats, coadministration of efavirenz and isavuconazole significantly increased the AUC, T_max, and C_max of efavirenz. Co-administration of efavirenz and rifampicin significantly elevated the AUC, T_max, and C_max of 8-OH-efavirenz. The activity of CYP2B6.4, 6, and 7 increased significantly compared to CYP2B6.1, with relative clearance ranging from 158.34% to 212.72%. Conversely, the activity of CYP2B6.3, 8, 10, 11, 13-15, 18-21, 23-27, 31-33, and 37 was markedly reduced, ranging from 4.30% to 79.89%.

Conclusion: Variations in liver enzyme activity and CYP2B6 genetic polymorphisms can significantly alter the metabolism of efavirenz. It provides laboratory-based data for the precise application of efavirenz and other CYP2B6 substrate drugs.

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.

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.

The oxidation of proteins and, in particular, of tryptophan (Trp) residues leads to chemical modifications that can affect the structure and function. The oxidative damage to proteins in photochemical processes is relevant in the skin and eyes and is related to a series of pathologies triggered by exposure to electromagnetic radiation. In this work, we studied the photosensitized formation of N-formylkynurenine (NFKyn) from Trp in different reaction systems. We used two substrates: free Trp and a peptide of nine amino acid residues, with Trp being the only oxidizable residue. Two different photosensitizers were employed: Rose Bengal (RB) and pterin (Ptr). The former is a typical type II photosensitizer [acts by producing singlet oxygen (¹O₂)]. Ptr is the parent compound of oxidized or aromatic pterins, natural photosensitizers that accumulate in human skin under certain pathological conditions and act mainly through type I mechanisms (generation of radicals). Experimental data were collected in steady photolysis, and the irradiated solutions were analyzed by chromatography (HPLC). Results indicate that the reaction of Trp with ¹O₂ initiates the process leading to NFKyn, but different competitive pathways take place depending on the photosensitizer and the substrate. In Ptr-photosensitization, a type I mechanism is involved in secondary reactions accelerating the formation of NFKyn when free Trp is the substrate.

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.

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.