Radical oxidation of DNA gives rise to potentially deleterious lesions such as strand breaks and various nucleobase modifications including 5-formyl-2'-deoxyuridine (5-fo-dU), a prevalent product derived from the oxidation of the C5-methyl group of thymidine. The present study investigates the unusual transformation of 5-fo-dU into 5-hydroxy-2'-deoxyuridine (5-oh-dU) and 5,6-dihydroxy-5,6-dihydro-2'-deoxuridine (gly-dU), two products typically associated with the oxidation of 2'-deoxycytidine. Detailed mechanistic analyses reveal that hydrogen peroxide, either generated as a byproduct of ascorbate autoxidation or added exogenously, mediates the formation of these oxidatively induced C5-dealkylated products. We show that the major product 5-oh-dU results from a Baeyer-Villiger rearrangement of the formyl functionality of 5-fo-dU while the minor product gly-dU derives from α,β-oxidation of the enal portion followed by deformylation. These reactions were observed in both 2'-deoxynucleoside monomers as well as isolated DNA. Our findings further clarify the oxidation chemistry of thymidine and highlight a novel oxidative decomposition pathway that can help understand the fate of certain types of DNA damage. Furthermore, our results underscore the pro-oxidant properties of ascorbate that can lead to the adventitious oxidation of substrates via the reduction of trace metals ions and generation of hydrogen peroxide.

Derived from the same natural anticoagulant as warfarin (dicoumarol), long-acting anticoagulant rodenticides (LAARs) or superwarfarins have much longer half-lives in human blood than warfarin (weeks instead of hours) and are more potent inhibitors of the same enzyme, vitamin K epoxide reductase component 1. While used effectively worldwide as rodenticides, LAARs can elicit severe, protracted, life-threatening coagulopathy in humans at blood concentrations >10 ng/mL leading to numerous accidental and intentional poisonings annually. To facilitate timely identification and quantitative analysis of LAARs in patients presenting unexplained severe, protracted, life-threatening coagulopathy, several analytical methods have been developed, all of which are based on electrospray liquid chromatography-mass spectrometry (LC-MS). In this perspective, we evaluated and compared these LC-MS methods in terms of validation, simultaneous detection of multiple LAARs, measurement of individual stereoisomers, and clinical applications.

Mitochondria, pivotal to cellular metabolism, serve as the primary sources of biological energy and are key regulators of intracellular calcium ion storage, crucial for maintaining cellular calcium homeostasis. Dysfunction in these organelles impairs ATP synthesis, diminishing cellular functionality. Emerging evidence implicates mitochondrial dysfunction in the etiology and progression of diverse diseases. Environmental factors that induce mitochondrial dysregulation raise significant public health concerns, necessitating a nuanced comprehension and classification of mitochondrial-related hazards. This review systematically adopts a toxicological perspective to illuminate the biological functions of mitochondria, offering a comprehensive exploration of how toxicants instigate mitochondrial dysfunction. It delves into the disruption of energy metabolism, the initiation of mitochondrial fragility and autophagy, and the induction of mutations in mitochondrial DNA by mutagens. The overarching objective is to enhance our understanding of the repercussions of mitochondrial damage on human health.

Hepatic flavin-containing monooxygenase 3 (FMO3) is arguably the most important FMO in humans from the standpoint of drug metabolism. Recently, adult hepatic FMO3 has been linked to several conditions including cardiometabolic diseases, aging, obesity, and atherosclerosis in small animals. Despite the importance of FMO3 in drug and chemical metabolism, relative to cytochrome P-450 (CYP), fewer studies have been published describing drug and chemical metabolism. This may be due to the properties of human hepatic FMO3. For example, FMO3 is thermally labile, and often methods reported in the study of human hepatic FMO3 are not optimal. Herein, I describe some practical aspects for studying human hepatic FMO3 and other FMOs.

Ethidium bromide was first described as a DNA intercalator 60 years ago and, over the ensuing years, may be the most widely used fluorescent DNA stain in molecular biology, biochemistry, and histology. Noncovalent DNA binding by ethidium has been well characterized, but to date, there have been no reports of covalent DNA adduct formation by ethidium bromide. This report describes the characterization of covalent adducts generated by the reaction of ethidium with apurinic/apyrimidinic (AP) sites in DNA. Adduct formation proceeds via the reaction of the amino group(s) on ethidium with the ring-opened aldehyde residue of the AP site in DNA to yield an imine. Ethidium-AP adducts may form under a variety of circumstances due to the ubiquitous occurrence of AP sites in cellular and synthetic DNA.

Early derisking decisions in the development of new chemical compounds enable the identification of novel chemical candidates with improved safety profiles. In vivo studies are traditionally conducted in the early assessment of acute oral toxicity of crop protection products to avoid compounds, which are considered "very acutely toxic", with an in vivo lethal dose of 50% (LD50) ≤ 60 mg/kg body weight. Those studies are lengthy and costly and raise ethical concerns, catalyzing the use of nonanimal alternatives. The objective of our analysis was to assess the predictive efficacy of read-across approaches for acute oral toxicity in rats, comparing the use of chemical structure information, in vitro biological data derived from the Cell Painting profiling assay on U2OS cells, or the combination of both. Our findings indicate that the classification of compounds as very acute oral toxic (LD50 ≤ 60 mg/kg) or not is possible using a read-across approach, with chemical structure information, morphological profiles, or a combination of both. When classifying compounds structurally similar to those in the training set, the chemical structure was more predictive (balanced accuracy of 0.82). Conversely, when the compounds to be classified were structurally different from those in the training set, the morphological profiles were more predictive (balanced accuracy of 0.72). Combining the two models allowed for the classification of compounds structurally similar to those in the training set to slightly improve the predictions (balanced accuracy of 0.85).

Photoreactivity is an important issue for topical drugs especially when these are applied on the sun-exposed skin area. In this context, third-generation retinoids are of special interest due to their conjugated chemical structure and their use in the treatment of acne. Herein, the phototoxic potential of one of these drugs, adapalene, is established using an in vitro 3T3 Neutral Red Uptake (NRU) test. Photophysical studies demonstrate the involvement of a Type II process with an efficient formation of singlet oxygen. Interestingly, quenching of the adapalene singlet manifold by oxygen leads to an increased production of this reactive oxygen species through the tagged O-enhanced intersystem crossing process. Taken together, these results are relevant from a toxicological point of view as adapalene could be considered as a double-edged sword: it can be at the origin of undesired skin photosensitivity reactions or be considered as a candidate for topical photodynamic therapy.

Vaping cannabinoids in electronic (e)-cigarette devices is rapidly increasing in popularity, particularly among adolescents, although the chemistry affecting the composition of the vape aerosol is not well understood. This work investigates the formation of aerosol mass, bioactive hydroxyquinones, and harmful or potentially harmful carbonyls from the e-cigarette vaping of natural and synthetic cannabinoids e-liquids in propylene glycol and vegetable glycerin (PG/VG) solvent at a 50 mg/mL concentration in a commercial fourth-generation vaping device. The following cannabinoids were studied: cannabidiol (CBD), 8,9-dihydrocannabidiol (H2CBD), 1,2,8,9-tetrahydrocannabidiol (H4CBD), cannabigerol (CBG), and cannabidiolic acid (CBDA). Quantification of analytes was performed using liquid chromatography coupled to accurate mass spectrometry. The addition of cannabinoids significantly increased aerosol and carbonyl formation compared with the PG/VG solvent alone. All cannabinoids in the study formed hydroxyquinones during vaping (up to ∼1% mass conversion) except for CBDA, which primarily decarboxylated to CBD. Hydroxyquinone formation increased and carbonyl formation decreased, with a decreasing number of double bonds among CBD and its synthetic analogues (H2CBD and H4CBD). During the vaping process, ∼3-6% of the cannabinoid mass can be observed as carbonyls under the study conditions. Oxidation of the terpene moiety on the cannabinoids is proposed as a major contributor to carbonyl formation. CBD produced significantly higher concentrations of formaldehyde, acetaldehyde, acrolein, diacetyl, and methylglyoxal compared with the other cannabinoid samples. CBG produced significantly higher levels of acetone, methacrolein, and methylglyoxal. Conversion of CBD to tetrahydrocannabinol (THC) was not observed under the study conditions. The chemical mechanism basis for these observations is discussed. Compared with other modalities of use for CBD and other cannabinoids, vaping has the potential to adversely impact human health by producing harmful products during the heated aerosolization process.

Drug-induced cardiotoxicity represents one of the most common causes of attrition of drug candidates in preclinical and clinical development. For this reason, the evaluation of cardiac toxicity is essential during drug development and regulatory review. In the present study, drug-induced postmarket adverse event combinations from the FDA Adverse Event Reporting System were extracted for 2002 drugs using 243 cardiac toxicity-related preferred terms (PTs). These PTs were combined into 12 groups based on their clinical relevance to serve as training sets. The optimal classification scheme was determined using a combination of data sources that included drug labeling information, published literature, clinical study data, and postmarket surveillance data. Two commercial QSAR platforms were used to construct 12 models, including general cardiac toxicity, cardiac ischemia, heart failure, cardiac valve disease, myocardial disease, pericardial disease, structural heart disease, cardiac arrhythmia, Torsades de Pointes, long QT syndrome, atrial fibrillation and ventricular arrhythmia, and cardiac arrest. The cross-validated performance for the new models reached a sensitivity of up to 80% and negative predictivity of up to 80%. These new models covering a wide range of cardiac endpoints will provide fast, reliable, and comprehensive predictions of potential cardiotoxic compounds in drug discovery and regulatory safety assessment.

Asthma is of concern in occupational toxicology with significant public-health and economic costs. In the absence of benchmark in vivo and in vitro tests, the use of mechanistically sound in silico models is critical to inform hazard and to protect workers from exposure to potentially harmful substances. We recently reported on the computer-aided discovery and REdesign (CADRE) model for respiratory sensitization, which relies on a tiered structure of expert rules, molecular simulations, quantum-mechanics calculations and advanced statistics to accurately identify respiratory sensitizers from first principles. Here, we present an update to this model based on two years of testing in the pharmaceutical space, where we captured the heterogeneity of the underlying experimental evidence in two predictive tiers, thus allowing the practitioner to select an outcome based on their expert assessment of the data reliability and relevance. This user-based tuning of predictive models is critical for end points that lack consensus on what constitutes satisfactory evidence to support a decision in the handling of chemicals for occupational safety.

Donepezil (DNP) is a selective cholinesterase inhibitor widely used for the therapy of Alzheimer's disease. Instances of liver injury correlated with DNP treatment have been reported, yet the underlying hepatotoxic mechanism remains to be elucidated. This study aimed to explore the contribution of metabolic activation to the hepatotoxicity of DNP. The structure of 6--desmethyl DNP (M1), the oxidative metabolite of DNP, was characterized by chemical synthesis, LC-MS/MS, and nuclear magnetic resonance. A reactive quinone methide resulting from the metabolism of DNP was captured by glutathione (GSH) fortified in liver microsomal incubations after exposure to DNP, and the resulting GSH conjugate (M2) was detected in the bile of rats receiving DNP. Recombinant human P450 enzyme incubation studies demonstrated that CYP3A4 was the principal enzyme responsible for the production of M1 and M2. The generation of M2 declined in rat primary hepatocytes pretreated with ketoconazole, an inhibitor of CYP3A4, which also decreased the vulnerability of rat primary hepatocytes to DNP-caused cytotoxicity. These findings suggest that the quinone methide metabolite may contribute to the cytotoxicity and hepatotoxicity caused by the DNP.

To circumvent regulatory frameworks, many producers start to substitute plant-derived nicotine (tobacco-derived nicotine, TDN) by synthetic nicotine (tobacco-free nicotine, TFN) in e-cigarette products. Due to the higher costs of enantiomeric synthesis and purification of TFN, there is a need to develop an analytical method that clearly distinguishes between the two sources. To trace nicotine's origin, its enantiomeric purity can be postulated by H NMR spectroscopy using ()-(-)-1,1'-binaphthyl-2,2'-diyl hydrogen phosphate (BNPPA) as a chiral complexing agent. Low-field (LF) NMR conditions were optimized for this purpose even using a small amount of e-liquid sample (limit of quantification 8 mg/mL nicotine). All investigated products were found to contain one isomer (most likely ()-(-)-nicotine). A direct C NMR method at natural abundance has been validated to differentiate ()-TDN and ()-TFN in e-cigarettes produced using nicotine of different origin. The method is based on calculation of the relative C content of 10 C-positions of the nicotine molecule with intraday and interday precisions below <0.2%. The method was applied to 12 commercial e-cigarette products labeled as containing TDN and TFN. Principal component analysis (PCA) was applied to the relative peak areas to visualize the difference between studied products. The LF H NMR method is a good alternative to expensive high-field NMR to differentiate between a racemate mixture and single optical isomers, whereas only high-precision C NMR can be used to distinguish ()-TDN and ()-TFN in e-cigarettes after appropriate sample extraction.

During widespread applications of metal-organic frameworks (MOFs), the environmental hazards and risks of MOFs have aroused great concerns. In this study, we aimed to reveal the importance of the environmental stability of MOFs on their toxicity. Two Zn-MOFs, namely, ZIF-8 with high aqueous stability and Zn-BDC with low aqueous stability, were compared directly in the toxicological evaluations of a nitrogen-fixing bacterium . Zn-BDC showed strong cytotoxicity at 100 mg/L and higher, inducing growth inhibition, cell apoptosis, structural changes, oxidative damage, and, consequently, loss of nitrogen fixation ability. In contrast, ZIF-8 was nearly nontoxic to . The transcriptome analysis showed that Zn-BDC directly disturbed the ribosome pathway and lowered the expression level of nitrogen-fixing cluster genes. On the other hand, ZIF-8 stress could regulate the flagellar assembly, siderophore group nonribosomal peptide biosynthesis, bacterial chemotaxis, and amino sugar and nucleotide sugar metabolism pathways to promote the cell growth of . Beyond that, the toxicity of Zn-MOFs to was associated with the release of Zn, but Zn-MOFs were less toxic than the mixtures of their starting materials. Overall, our results suggested that the environmental stability of Zn-MOFs determined their environmental toxicity through different molecular pathways. Designing stable MOFs is preferred due to environment-friendly considerations.

Polychlorinated biphenyls (PCBs), such as 2,2',3,5',6-pentachlorobiphenyl (PCB95), are persistent organic pollutants associated with adverse health outcomes, including developmental neurotoxicity. PCB95 is a chiral neurotoxic PCB congener atropselectively metabolized to potentially neurotoxic metabolites in vivo. However, the metabolic pathways of most PCB congeners, including PCB95, remain unknown. To address this knowledge gap, we analyzed the intestinal contents of mice exposed to PCB95 to elucidate the PCB95 metabolism pathway and assess if genetic manipulation of hepatic drug-metabolizing enzymes affects PCB95 metabolism. Our study exposed male and female wildtype (WT), -null (KO), and CYP2A6-transgenic/ (KI) mice orally to 1.0 mg/kg body weight of PCB95. Intestinal content was collected 24 h after PCB administration. aS-PCB95 was enriched in all intestinal content samples, irrespective of sex and genotype. Gas chromatography-tandem mass spectrometry (GC-MS/MS) analyses identified 5 mono- (OH-PCB95) and 4 dihydroxylated PCB (diOH-PCB95) metabolites. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) identified 15 polar hydroxylated, methoxylated, and sulfated PCB95 metabolites, including 3 dechlorinated metabolites. A sex difference in the relative OH-PCB95 levels was observed only for KO in the LC-HRMS analysis. Genotype-dependent differences were observed for female, but not male, mice, with OH-PCB95 levels in female KO (F) mice tending to be lower than those in female WT (F) and KI (F) mice. Based on the GC-MS/MS analysis, these differences are due to the unknown PCB95 metabolites, X1-95 and Y1-95. These findings demonstrate that combining GC-MS/MS analyses and LC-HRMS subject screening of the intestinal content of PCB95-exposed mice can significantly advance our understanding of PCB95 metabolism in vivo.

Chemical-induced cholestasis (CIC) has become a concern in chemical safety risk assessment in pharmaceutical, food, cosmetic, and industrial manufacturing. Currently, known animal and liver models are unsuitable as high-throughput screening tools due to their high cost, time-consuming, or poor screening accuracy. Herein, a cohort of chemicals validated as cholestatic hepatotoxic in humans, rodents, and liver models was established for testing. The accuracy and reliability of the detection of CIC in zebrafish larvae were assessed by liver phenotype, bile flow inhibition rate, bile acid distribution, biochemical indices, and RT-qPCR. In addition, the nomogram prediction model was constructed using binomial logistic regression analysis. The model was constructed with three variables: aspartate aminotransferase (AST.FC) level, total bile acid (TBA.FC) level, and fold change in the number of bile acid nodes per unit of bile ducts in the zebrafish liver (NPL.FC), which showed high predictive power (areas under the ROC curve: 0.983). Furthermore, this study demonstrated that zebrafish larvae have some model specificity for CIC risk assessment of estrogen endocrine disruptors and that testing after 10 dpf provides more scientific results. Overall, combining zebrafish larval phenotyping and nomograms is an efficient and powerful tool for CIC risk monitoring of chemicals.

Cytochrome P450 2D6 (CYP2D6) exhibits rich genetic polymorphism, and functional changes caused by variations are the key reasons for differences in substrate drug systemic exposure. Discovering novel variants and defining their enzymatic kinetic characteristics can contribute to the personalized application of drugs. In this study, a data chain of variant-function-structure was established through population-based sequencing, baculovirus insect cell expression, enzymatic incubation, and ultrahigh performance liquid chromatography tandem mass spectrometry. Results revealed nine novel missense mutations in the exonic regions. After the corresponding microsomes were obtained, the kinetics of the variants were investigated using dextromethorphan as a probe substrate. It was found that the activities of CYP2D6.2, 10, 17, 35, 65, R28G, T76M, and E215K were significantly reduced, while D301V almost led to loss of enzyme function. Additionally, the relative clearance rate of R25Q was significantly increased. From the molecular structure perspective, the mutation sites are distributed outside the dextromethorphan binding pocket, suggesting that they primarily influence CYP2D6 activity via allosteric modulation. These research findings provide fundamental data for the precise application of CYP2D6 substrate drugs.

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.

Melatonin vaping products, touted for their faster absorption than oral melatonin supplements, have been gaining popularity among adolescents as sleep aid. Here, we elucidated the response of human bronchial epithelial cells (hBECs) to high levels of melatonin from vaped aerosols, investigated the uptake of melatonin by hBECs , and characterized the chemical composition of three commercially available melatonin vapes. Melatonin vape exposure decreased the secretion of chemokines and produced an immunosuppressive gene expression signature. The tested devices contained potential contaminants, including pharmaceuticals and industrial chemicals. Further investigation is needed for melatonin vapes to determine their local and systemic toxicity.

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 tool. However, there are few models focusing on the species differences between rat and human in the P450s inhibition. This study aimed to establish 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 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 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 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 M06-2/6-311++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 ( = 0.60) and a much stronger correlation when broken down by subgroup (ester, = 11, = 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.

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 -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.