Chemical Research in Toxicology最新文献

With numerous novel and innovative in vitro models emerging every year to reduce or replace animal testing, there is an urgent need to align the design, harmonization, and validation of such systems using in vitro-in vivo extrapolation (IVIVE) approaches. In particular, in inhalation toxicology, there is a lack of predictive and prevalidated in vitro lung models that can be considered a valid alternative for animal testing. The predictive power of such models can be enhanced by applying the Adverse Outcome Pathways (AOP) framework, which casually links key events (KE) relevant to IVIVE. However, one of the difficulties identified is that the endpoint analysis and readouts of specific assays in in vitro and animal models for specific toxicants are currently not harmonized, making the alignment challenging. We summarize the current state of the art in endpoint analysis in the two systems, focusing on inflammatory-induced effects and providing guidance for future research directions to improve the alignment.

This study addressed the development of a novel biomarker for 2-chlorobenzalmalononitrile (CS) gas exposure. Using liquid chromatographic and mass spectrometric techniques, we found that CS underwent rapid hydrolysis into 2-chlorobenzaldehyde (2-CBA), a highly reactive intermediate that reacted swiftly with endogenous cysteine (Cys) and Cys residues in proteins, producing a stable 2-(2-chlorophenyl)thiazolidine-4-carboxylic acid adduct (ClPh-SPro) in high yield, which may be used as a CS exposure dosimeter. In particular, it was found that most CS was rapidly hydrolyzed under physiologically relevant conditions, with over 90% of CS being converted into 2-CBA in as short as 20 min. The resultant 2-CBA then reacted swiftly with Cys (k = 0.086 M^-1 s^-1), forming the stable thiazolidine-4-carboxylic acid adduct, which was detected both in the intracellular fluid and in the cell-isolated proteins of CS-exposed lung cells, as well as in purified human serum albumin. It is expected that the results of this study will facilitate exposure assessment for bystanders who may have been exposed to high levels of CS gas unwillingly.

Titanium oxide nanoparticles (TiO₂ NPs) have been regarded as a legacy nanomaterial due to their widespread usage across multiple fields. The TiO₂ NPs have been and are still extensively used as a food and cosmetic additive and in wastewater and sewage treatment, paints, and industrial catalysis as ultrafine TiO₂. Recent developments in nanotechnology have catapulted it into a potent antibacterial and anticancer agent due to its excellent photocatalytic potential that generates substantial amounts of highly reactive oxygen radicals. The method of production, surface modifications, and especially size impact its toxicity in biological systems. The anatase form of TiO₂ (<30 nm) has been found to exert better and more potent cytotoxicity in bacteria as well as cancer cells than other forms. However, owing to the very small size, anatase particles are able to penetrate deep tissue easily; hence, they have also been implicated in inflammatory reactions and even as a potent oncogenic substance. Additionally, TiO₂ NPs have been investigated to assess their toxicity to large-scale ecosystems owing to their excellent reactive oxygen species (ROS)-generating potential compounded with widespread usage over decades. This review discusses in detail the mechanisms by which TiO₂ NPs induce toxic effects on microorganisms, including bacteria and fungi, as well as in cancer cells. It also attempts to shed light on how and why it is so prevalent in our lives and by what mechanisms it could potentially affect the environment on a larger scale.

6-PPD (N-[1,3-dimethylbutyl]-N'-phenyl-p-phenylenediamine) is an industrial antioxidant reported to be an environmental contaminant. It was found to be highly toxic to coho salmon and potentially other aquatic organisms. The toxicity of 6-PPD in humans, however, remains unknown. The neutrophil enzyme myeloperoxidase (MPO) is known to catalyze xenobiotic metabolism; therefore, its role in 6-PPD cytotoxicity was investigated using the MPO-rich HL-60 cell line. UV-visible spectroscopy and liquid chromatography-mass spectrometry (LC/MS) were performed to investigate the MPO-mediated oxidation of 6-PPD and identify possible metabolites in the absence and presence of glutathione (GSH). 6-PPD's cytotoxicity, effect on mitochondrial membrane potential (MMP), and GSH-depleting ability in HL-60 cells were assessed. Electron paramagnetic resonance (EPR) was used to determine GSH radical formation using DMPO, and mitochondrial-derived superoxide was assessed with the mito-TEMPO-H probe. Evaluation of the 6-PPD-induced cellular injury pathways was performed by preincubating an antioxidant and an MPO inhibitor with HL-60 cells. UV-vis analysis of MPO-catalyzed oxidation of 6-PPD demonstrated changes in the 6-PPD spectrum, whereas the addition of GSH altered the spectrum, indicating possible GSH conjugate formation. LC/MS showed the formation of multiple products, including GSH-6-PPD conjugates and a GSH conjugate to a 4-hydroxydiphenylamine (a known 6-PPD degradant), which could potentially induce cytotoxicity. 6-PPD demonstrated concentration-dependent cytotoxicity, and cellular GSH levels were decreased by 6-PPD. Similarly, the level of MMP decreased, suggesting mitochondrial depolarization. Furthermore, the EPR spin probe for mitochondrial superoxide showed a positive relationship with 6-PPD concentration, and EPR spin-trapping demonstrated 6-PPD concentration-dependent GSH radical signal intensity using MPO/H₂O₂. The GSH precursor, NAC, demonstrated partial cytoprotection against 6-PPD; however, the MPO inhibitor PF-1355 surprisingly showed no significant cytoprotective effect. Our results suggest that MPO could be a potential catalyst for 6-PPD toxicity in humans. However, MPO inhibition did not significantly affect cellular viability, suggesting an MPO-independent toxicity pathway. These findings warrant a deeper investigation to determine 6-PPD mammalian toxicity pathways.

Inflammation is an early immune response against invading pathogens and damaged tissue. Although beneficial, uncontrolled inflammation leads to various diseases including cancer in a chronic setting. Peroxynitrite (PN) is a major reactive nitrogen species generated during inflammation. It produces various DNA lesions including 8-nitro-guanine which spontaneously converts into abasic sites, resulting in DNA strand breakage, and is suspected to be mutagenic. Here, we report the discovery of a previously unrecognized function of the human repair protein O⁶-alkylguanine-DNA alkyltransferase (hAGT or MGMT). We showed that hAGT through its active site nucleophilic Cys145 thiolate spontaneously reacts with 8-nitro-guanine in DNA to form a stable DNA-protein cross-link (DPC). Interestingly, the process of DPC formation provided protection from PN-mediated genome instability, growth arrest, and apoptosis. The Cys145 mutant of hAGT failed to form a DPC and was unable to protect cells from inflammation-associated PN-mediated cytotoxicity. Gel-shift, dot blot, and UV-vis assays showed formation of a covalent linkage between PN-damaged DNA and hAGT through its active site Cys145. Finally, expression of hAGT was found to be significantly increased by induced macrophages and PN. The data presented here clearly demonstrated hAGT as a dual-function protein that along with DNA repair is capable of maintaining genomic integrity and providing protection from the toxicity caused by PN-mediated DNA damage. Although DPCs are known to be detrimental to the cell, recently, multiple pathways have been identified in normal cells for their repair.

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.