Reactive Metabolites Research Articles

Metabolic Activation and Cytotoxicity of Gramine Mediated by CYP3A in Rats.

Gramine (GRM), which occurs in Gramineae plants, has been developed to be a biological insecticide. Exposure to GRM was reported to induce elevations of serum ALT and AST in rats, but the mechanisms of the observed hepatotoxicity have not been elucidated. The present study aimed to identify reactive metabolites that potentially participate in the toxicity. In rat liver microsomal incubations fortified with glutathione or N-acetylcysteine, one oxidative metabolite (M1), one glutathione conjugate (M2), and one N-acetylcysteine conjugate (M3) were detected after exposure to GRM. The corresponding conjugates were detected in the bile and urine of rats after GRM administration. CYP3A was the main enzyme mediating the metabolic activation of GRM. The detected GSH and NAC conjugates suggest that GRM was metabolized to a quinone imine intermediate. Both GRM and M1 showed significant toxicity to rat primary hepatocytes.

Development of a Novel In Silico Classification Model to Assess Reactive Metabolite Formation in the Cysteine Trapping Assay and Investigation of Important Substructures.

Predicting whether a compound can cause drug-induced liver injury (DILI) is difficult due to the complexity of drug mechanism. The cysteine trapping assay is a method for detecting reactive metabolites that bind to microsomes covalently. However, it is cumbersome to use 35S isotope-labeled cysteine for this assay. Therefore, we constructed an in silico classification model for predicting a positive/negative outcome in the cysteine trapping assay. We collected 475 compounds (436 in-house compounds and 39 publicly available drugs) based on experimental data performed in this study, and the composition of the results showed 248 positives and 227 negatives. Using a Message Passing Neural Network (MPNN) and Random Forest (RF) with extended connectivity fingerprint (ECFP) 4, we built machine learning models to predict the covalent binding risk of compounds. In the time-split dataset, AUC-ROC of MPNN and RF were 0.625 and 0.559 in the hold-out test, restrictively. This result suggests that the MPNN model has a higher predictivity than RF in the time-split dataset. Hence, we conclude that the in silico MPNN classification model for the cysteine trapping assay has a better predictive power. Furthermore, most of the substructures that contributed positively to the cysteine trapping assay were consistent with previous results.

THE BIOCHEMICAL RESPONSES OF BIVALVE MOLLUSCS TO NEUROLEPTIC CHLORPROMAZINE ARE COMPARABLE WITH THE RESPONSES OF HIGHER VERTEBRATES

Pharmaceuticals such as chlorpromazine (Cpz) are emerging aquatic pollutants with potential effects on non-target organisms. However, its effects on aquatic organisms remain limited and inconclusive. The aim of this study was to compare the responses to Cpz in marine and freshwater bivalve molluscs. Methods. Mytilus galloprovincialis and Unio tumidus were exposed to pM and nM concentrations of Cpz for 14 days and analysed 16 parameters, including cytotoxicity, oxidative/reductive stress responses, metallothionein concentration and biotransformation enzymes in the digestive gland. Results. In both species, Cpz increased EROD activity, possibly leading to the formation of reactive metabolites. Marine mussels exposed to Cpz I and II showed increased GST activity, suggesting detoxification, whereas freshwater mussels showed decreased GST activity, suggesting higher Cpz toxicity. Activation of the GTPase dynamin in U. tumidus exposed to Cpz I confirmed the effect of Cpz on endocytosis. Oxidative stress responses were observed in both species, with changes in oxidative/reductive stress responses, suggesting toxic effects of Cpz. GSH and metallothionein concentrations increased in all exposures, while the NADH/NAD+ ratio increased significantly in U. tumidus. Loss of lysosomal membrane stability was observed in all exposed groups, with CtD efflux detected only in mussels. Differences in caspase-3 activity were also observed between species. Conclusion. Cpz showed adverse effects on vertebrates at μM concentrations, while mussels were affected at pM to nM concentrations, indicating concentration-dependent interspecies effects. Stress responses were similar between species, suggesting that marine molluscs are a potential model for assessing the adverse effects of Cpz on higher vertebrates.

Changes in biomarkers of the redox status in whole blood and red blood cell lysates in canine hypothyroidism

Hypothyroidism is the most commonly diagnosed endocrine disease in dogs. The objective of this study was to evaluate the changes in the redox status in canine hypothyroidism using whole blood (WB) and red blood cell (RBCs) lysates. For this purpose, a panel of five antioxidants and five oxidants biomarkers was measured in WB and RBCs lysates of 30 dogs with hypothyroidism, 26 dogs with non-thyroidal illnesses and 15 healthy dogs. The antioxidants measured were cupric reducing antioxidant capacity (CUPRAC), ferric reducing ability of plasma (FRAP), Trolox equivalent antioxidant capacity (TEAC), thiol and paraoxonase type-1 (PON-1). Oxidants measured include the total oxidant status (TOS), peroxide-activity (POX-Act), reactive oxygen-derived metabolites (d-ROMs), advanced oxidation protein products (AOPP) and thiobarbituric acid reactive substances (TBARS). WB showed a significant decrease of the antioxidants CUPRAC, TEAC and thiol, and also an increase in TBARS and a decrease in AOPP in dogs with hypothyroidism compared to healthy dogs. Meanwhile, RBCs lysates showed a significant increase in FRAP and PON-1 in dogs with hypothyroidism. The changes in the redox biomarkers in this study show that WB in canine hypothyroidism had a higher number of changes in biomarkers of the redox status than RBCs lysates, making it a promising sample type for the evaluation of the redox status in this disease. In addition, WB is easier and simpler to process than RBCs lysates and unlike serum, it does not have any hemolysis interference.

AKR1C2 genetic variants mediate tobacco carcinogens metabolism involving bladder cancer susceptibility.

Tobacco carcinogens metabolism-related genes (TCMGs) could generate reactive metabolites of tobacco carcinogens, which subsequently contributed to multiple diseases. However, the association between genetic variants in TCMGs and bladder cancer susceptibility remains unclear. In this study, we derived TCMGs from metabolic pathways of polycyclic aromatic hydrocarbons and tobacco-specific nitrosamines, and then explored genetic associations between TCMGs and bladder cancer risk in two populations: a Chinese population of 580 cases and 1101 controls, and a European population of 5930 cases and 5468 controls, along with interaction and joint analyses. Expression patterns of TCMGs were sourced from Nanjing Bladder Cancer (NJBC) study and publicly available datasets. Among 43 TCMGs, we observed that rs7087341 T > A in AKR1C2 was associated with a reduced risk of bladder cancer in the Chinese population [odds ratio (OR) = 0.84, 95% confidence interval (CI) = 0.72-0.97, P = 1.86 × 10-2]. Notably, AKR1C2 rs7087341 showed an interaction effect with cigarette smoking on bladder cancer risk (Pinteraction = 5.04 × 10-3), with smokers carrying the T allele increasing the risk up to an OR of 3.96 (Ptrend < 0.001). Genetically, rs7087341 showed an allele-specific transcriptional regulation as located at DNA-sensitive regions of AKR1C2 highlighted by histone markers. Mechanistically, rs7087341 A allele decreased AKR1C2 expression, which was highly expressed in bladder tumors that enhanced metabolism of tobacco carcinogens, and thereby increased DNA adducts and reactive oxygen species formation during bladder tumorigenesis. These findings provided new insights into the genetic mechanisms underlying bladder cancer.

The role of corticosterone in nevirapine-induced idiosyncratic drug-induced liver injury.

Nevirapine, an antiretroviral used in the treatment of HIV, is associated with idiosyncratic drug-induced liver injury (IDILI), a potentially life-threatening adverse drug reaction. Its usage has decreased due to this concern, but it is still widely used in lower-resource settings. In general, the mechanisms underlying idiosyncratic drug reactions (IDRs) are poorly understood, but evidence indicates that most are immune-mediated. There is very limited understanding of the early immune response following administration of drugs associated with IDRs, which likely occurs due to reactive metabolite formation. In this work, we aimed to characterize the links between covalent binding of nevirapine, the development of an early immune response, and the subsequent liver injury using a mouse model. We describe initial attempts to characterize an early immune response to nevirapine followed by the discovery that nevirapine induced the release of corticosterone. Corticosterone release was partially associated with the degree of drug covalent binding in the liver but was also likely mediated by additional mechanisms at higher drug doses. Transcriptomic analysis confirmed metabolic activation, glucocorticoid signaling, and decreased immune activation; GDF-15 also warrants further investigation as part of the immune response to nevirapine. Finally, glucocorticoid blockade preceding the first dose of nevirapine attenuated nevirapine-induced liver injury at 3 weeks, suggesting that acute glucocorticoid signaling is harmful in the context of nevirapine-induced liver injury. This work demonstrates that nevirapine induces acute corticosterone release, which contributes to delayed-onset liver injury. It also has implications for screening drug candidates for IDILI risk and preventing nevirapine-induced IDILI.

Effects of psychogenic stress on oxidative stress and antioxidant capacity at different growth stages of rats: Experimental study.

This study examined the psychogenic stress (PS) effects on changes in oxidative stress and the antioxidant capacity of an organism at different growth stages. The experimental animals were male Wistar rats of five different ages from growth periods (GPs) to old age. The growth stages were randomly classified into control (C) and experimental (PS) groups. The PS was performed using restraint and water immersion once daily for 3 h for 4 weeks. Reactive oxygen metabolites (d-ROMs) and the biological antioxidant potential (BAP) were measured before and after the experiment. In addition, the liver and adrenal glands were removed, and the wet weight was measured. The d-ROM and BAP of all growth stages given PS increased significantly. The d-ROM in the C group without PS increased significantly in GPs while decreased significantly in old-aged rats. In addition, the BAP of the C group in GP and early adulthood were all significantly elevated. There were significant differences in organ weights between the C and PS groups at all growth stages. Oxidative stress and antioxidant capacity differed depending on the organism's developmental status and growth stage, and PS also showed different effects. In particular, the variability in oxidative stress was remarkable, suggesting that the effect of PS was more significant in the organism's immature organs.

Cyanide Trapping of Iminium Ion Reactive Metabolites: Implications for Clinical Hepatotoxicity.

Reactive metabolite formation is a major mechanism of hepatotoxicity. Although reactive electrophiles can be soft or hard in nature, screening strategies have generally focused on the use of glutathione trapping assays to screen for soft electrophiles, with many data sets available to support their use. The use of a similar assay for hard electrophiles using cyanide as the trapping agent is far less common, and there is a lack of studies with sufficient supporting data. Using a set of 260 compounds with a defined hepatotoxicity status by the FDA, a comprehensive literature search yielded cyanide trapping data on an unbalanced set of 20 compounds that were all clinically hepatotoxic. Thus, a further set of 19 compounds was selected to generate cyanide trapping data, resulting in a more balanced data set of 39 compounds. Analysis of the data demonstrated that the cyanide trapping assay had high specificity (92%) and a positive predictive value (83%) such that hepatotoxic compounds would be confidently flagged. Structural analysis of the adducts formed revealed artifactual methylated cyanide adducts to also occur, highlighting the importance of full structural identification to confirm the nature of the adduct formed. The assay was demonstrated to add the most value for compounds containing typical structural alerts for hard electrophile formation: half of the severe hepatotoxins with these structural alerts formed cyanide adducts, while none of the severe hepatotoxins with no relevant structural alerts formed adducts. The assay conditions used included cytosolic enzymes (e.g., aldehyde oxidase) and an optimized cyanide concentration to minimize the inhibition of cytochrome P450 enzymes by cyanide. Based on the demonstrated added value of this assay, it is to be initiated for use at GSK as part of the integrated hepatotoxicity strategy, with its performance being reviewed periodically as more data is generated.

Effect of different doses of camelina cake inclusion as a substitute of dietary soyabean meal on growth performance and gut health of weaned pigs.

Camelina cake (CAM) is a co-product proposed as an alternative protein source; however, piglet data are still limited. This study aimed to evaluate the effect of different doses of CAM in substitution of soyabean meal on the growth, health and gut health of weaned pigs. At 14 d post-weaning (d0), sixty-four piglets were assigned either to a standard diet or to a diet with 4 %, 8 % or 12 % of CAM. Piglets were weighed weekly. At d7 and d28, faeces were collected for microbiota and polyamine and blood for reactive oxygen metabolites (ROM) and thyroxine analysis. At d28, pigs were slaughtered, organs were weighed, pH was recorded on gut, colon was analysed for volatile fatty acids (VFA) and jejunum was used for morphological and gene expression analysis. Data analysis was carried out using a mixed model including diet, pen and litter as factors; linear and quadratic contrasts were tested. CAM linearly reduced the average daily gain from d0-d7, d0-d14, d0-d21 and d0-d28 (P ≤ 0·01). From d0-d7 increasing CAM linearly decreased feed intake (P = 0·04) and increased linearly the feed to gain (P = 0·004). CAM increased linearly the liver weight (P < 0·0001) and affected the cadaverine (P < 0·001). The diet did not affect the ROM, thyroxine, intestinal pH, VFA and morphology. All doses of CAM increased the α diversity indices at d28 (P < 0·05). CAM at 4 % promoted the abundance of Butyricicoccaceae_UCG-008. Feeding with CAM enhanced resilience in the gut microbiome and can be evaluated as a potential alternative protein source with dose-dependent limitations on piglet growth performance.

Effect of Ginger Root Extract on Intestinal Oxidative Status and Mucosal Morphometrics in Broiler Chickens.

This study was designed to assess the effect of ginger root extract (GRE) supplementation on the oxidative status and intestinal mucosal development in broiler chickens for 6 weeks. Day-old chicks (Ross 708 strain, n = 432) were distributed into six treatments with six replicate of twelve birds each: Negative CON (basal), MX (basal diet + bacitracin methylene disalicylate (BMD) 0.055 g/kg diet), GRE-1 (basal diet + 0.375% GRE), GRE-2 (basal diet + 0.75% GRE), GRE-3 (basal diet + 1.5% GRE), GRE-4 (basal diet + 3% GRE). Growth indices, goblets cell count, mucin (MUC2) in ileum tissue, antioxidant (SOD, CAT, and GPX) in ileum and liver, biological antioxidant potential (BAP), and reactive oxygen metabolite level in blood and intestinal villi measurement were determined. Body weight (BW) was highest (p < 0.05) in all groups except GRE-4, body weight gain (BWG) was best in GRE-1, while FCR was least in all groups except GRE-4. Optimum MUC2 gene expression, SOD, CAT, blood antioxidants, and intestinal morphometric values were observed in GRE-3. The inclusion of ginger root extract up to 1.5% improved growth and reduced oxidative stress while enhancing mucosal development in broiler chicks.

Anthocyanins attenuate vascular and inflammatory responses to a high fat high energy meal challenge in overweight older adults: A cross-over, randomised, double-blind clinical trial

Postprandial metabolic imbalances are important indicators of later developing cardiovascular disease (CVD)(1). This study investigated the effects of food anthocyanins on vascular and microvascular function, and CVD associated biomarkers following a high fat high energy (HFHE) meal challenge in overweight older adults. Sixteen subjects (13 female, 3 male, mean age 65.9 SD 6.0 and body mass index 30.6 kg/m 2 SD 3.9) participated in a crossover, randomised, controlled, double-blind clinical trial (Australian New Zealand Clinical Trials Registry # ACTRN12620000437965). Participants consumed a HFHE breakfast meal (65g total fat; 33g saturated fat) together with a 250 mL dose of either intervention (Queen Garnet Plum providing 201 mg anthocyanins) or control (apricot) juice. A wash-out period of 14 days occurred between meal challenges, with a 4-day run-in period for juice consumption before each challenge. Blood samples and blood pressure measures were collected at baseline, 2 h and 4 h following the HFHE meal. Vascular function, assessed using flow mediated dilatation (FMD), and microvascular cutaneous vascular reactivity, measured using Laser Speckle Contrast Imaging (LSCI), were evaluated at baseline and 2 h after the HFHE meal. Participants had a higher 2 h postprandial FMD (+1.14%) and a higher microvascular post-occlusive reactive hyperaemia (+0.10 perfusion units per mmHg) when allocated to the anthocyanin compared to the control arm (P = 0.019 and P = 0.049, respectively). C-reactive protein was lower 4 h postprandially in the anthocyanins (1.80 mg/L, IQR 0.90) vs control arm (2.30 mg/L, IQR 1.95) (P = 0.026), accompanied by a trend for lower concentrations of interleukin-6 (P = 0.075). No significant postprandial differences were observed between treatments for blood pressure, triacylglycerol, total cholesterol, serum derivatives of reactive oxidative metabolites, tumor necrosis factor α, interleukin-1 β, or maximum microvascular perfusion following iontophoresis of acetylcholine. Fruit-based anthocyanins attenuated the potential postprandial detrimental effects of a HFHE challenge on parameters of vascular and microvascular function, and inflammatory biomarkers in overweight older adults. Anthocyanins may reduce cardiovascular risk associated with endothelial dysfunction and inflammatory responses to a typical high fat ‘Western’ meal.

De-risking idiosyncratic adverse drug reactions and toxicities in drug discovery utilizing a qualitative in-vitro high-throughput reactive metabolite trapping method

De-risking idiosyncratic adverse drug reactions and toxicities in drug discovery utilizing a qualitative in-vitro high-throughput reactive metabolite trapping method

A new trapping method to quantitatively assess the formation of reactive metabolites from drugs which cause agranulocytosis

A new trapping method to quantitatively assess the formation of reactive metabolites from drugs which cause agranulocytosis

Development of a novel in silico classification model to assess reactive metabolite formation in cysteine trapping assay and investigation of important substructures

Development of a novel in silico classification model to assess reactive metabolite formation in cysteine trapping assay and investigation of important substructures

Insight into chemically reactive metabolites of aliphatic amine pollutants: A de novo prediction strategy and case study of sertraline

The uncommon metabolic pathways of organic pollutants are easily overlooked, potentially leading to idiosyncratic toxicity. Prediction of their biotransformation associated with the toxic effects is the very purpose that this work focuses, to develop a de novo method to mechanistically predict the reactive toxicity pathways of uncommon metabolites from start aliphatic amine molecules, which employed sertraline triggered by CYP450 enzymes as a model system, as there are growing concerns about the effects on human health posed by antidepressants in the aquatic environment. This de novo prediction strategy combines computational and experimental methods, involving DFT calculations upon sequential growth, in vitro and in vivo assays, dissecting chemically reactive mechanism relevant to toxicity, and rationalizing the fundamental factors. Significantly, desaturation and debenzylation-aromatization as the emerging metabolic pathways of sertraline have been elucidated, with the detection of DNA adducts of oxaziridine metabolite in mice, highlighting the potential reactive toxicity. Molecular orbital analysis supports the reactivity preference for toxicological-relevant C-N desaturation over N-hydroxylation of sertraline, possibly extended to several other aliphatic amines based on the Bell-Evans-Polanyi principle. It was further validated toward some other wide-concerned aliphatic amine pollutants involving atrazine, ε-caprolactam, 6PPD via in silico and in vitro assays, thereby constituting a complete path for de novo prediction from case study to general applications.

Effect of substrate concentration on reactive metabolite assessment in drug discovery research

Effect of substrate concentration on reactive metabolite assessment in drug discovery research

Methylglyoxal induces cardiac dysfunction through mechanisms involving altered intracellular calcium handling in the rat heart

Methylglyoxal (MGO) is an endogenous, highly reactive dicarbonyl metabolite generated under hyperglycaemic conditions. MGO plays a role in developing pathophysiological conditions, including diabetic cardiomyopathy. However, the mechanisms involved and the molecular targets of MGO in the heart have not been elucidated. In this work, we studied the exposure-related effects of MGO on cardiac function in an isolated perfused rat heart ex vivo model. The effect of MGO on calcium homeostasis in cardiomyocytes was studied in vitro by the fluorescence indicator of intracellular calcium Fluo-4. We demonstrated that MGO induced cardiac dysfunction, both in contractility and diastolic function. In rat heart, the effects of MGO treatment were significantly limited by aminoguanidine, a scavenger of MGO, ruthenium red, a general cation channel blocker, and verapamil, an L-type voltage-dependent calcium channel blocker, demonstrating that this dysfunction involved alteration of calcium regulation. MGO induced a significant concentration-dependent increase of intracellular calcium in neonatal rat cardiomyocytes, which was limited by aminoguanidine and verapamil. These results suggest that the functionality of various calcium channels is altered by MGO, particularly the L-type calcium channel, thus explaining its cardiac toxicity. Therefore, MGO could participate in the development of diabetic cardiomyopathy through its impact on calcium homeostasis in cardiac cells.

Application of Covalent Binding Body Burden in the HμREL Human Hepatocyte Coculture Model for Reactivity Risk Assessment of Metabolically Low Turnover Drugs.

The human hepatocyte suspension model has been a valuable tool to study covalent binding (CVB) for compounds that form reactive metabolites. However, accurately measuring CVB values with the suspension model becomes challenging for metabolically low turnover compounds. In this study, we evaluated the HμREL human hepatocyte coculture model relative to existing literature using human hepatocyte suspension for drugs of known drug-induced liver injury category. Our results indicate that this coculture model provides ample metabolic turnover to reproducibly measure CVB. It is sufficiently robust to apply a predefined 1 mg/day CVB body burden threshold for risk assessment to guide our discovery programs, allowing for expanded coverage to include metabolically low turnover compounds.

Systemic Oxidative Balance Reflects the Liver Disease Progression Status for Primary Biliary Cholangitis (Pbc): The Narcissus Fountain.

Biological antioxidant potential (BAP) and Reactive Oxygen Metabolites (dROMs) are two tests complementarily assessing systemic oxidative statuses (SOSs) that are never applied in chronic liver disorders (CLDs). We enrolled 41 ursodeoxycholic acid (UDCA)-naïve Primary Biliary Cholangitis (PBC) patients [age: 58.61 ± 11.26 years; females (F): 39], 40 patients with metabolic-dysfunction-associated steatotic livers (age: 54.30 ± 11.21; F: 20), 52 patients with HBV (age: 52.40 ± 8.22; F: 34), 50 patients with (age: 56.44 ± 7.79, F: 29), and 10 controls (age: 52.50 ± 9.64; F: 7). Liver fibrosis and the steatosis severity were determined using transient elastography, and the SOS was balanced using d-ROMs and the BAP test. The gene expressions of superoxide dismutase (SOD1; SOD2) and glutathione peroxidase (GPx1) were evaluated using real-time PCR in advanced fibrosis (AF: F3F4) in patients with PBC. In contrast to other CLDs, in PBC the dROMs and BAP levels were, respectively, directly and inversely correlated with hepatic fibrosis (dROMs, R: 0.883; BAP, R: -0.882) and steatosis (dROMs, R: 0.954; BAP, R: -0931) severity (p < 0.0001 all). Patients with PBC also revealed a progressively increasing trend of d-ROMs (F0-F2 vs. F3: p = 0.0008; F3 vs. F4: p = 0.04) and reduction in BAP levels (F0-F2 vs. F3: p = 0.0007; F3 vs. F4 p = 0.04) according to the worsening of liver fibrosis. In AF-PBC, the SOD1, SOD2, and GPx1 expressions were significantly downregulated in patients presenting SOS imbalance (SOD1, p = 0.02; SOD2, p = 0.03; GPx1, p = 0.02). SOS disequilibrium represents a leitmotiv in patients with PBC, perfectly reflecting their liver disease progression status.

Deciphering the Molecular Mechanisms of Reactive Metabolite Formation in the Mechanism-Based Inactivation of Cytochrome p450 1B1 by 8-Methoxypsoralen and Assessing the Driving Effect of phe268.

This study provides a comprehensive computational exploration of the inhibitory activity and metabolic pathways of 8-methoxypsoralen (8-MP), a furocoumarin derivative used for treating various skin disorders, on cytochrome P450 (P450). Employing quantum chemical DFT calculations, molecular docking, and molecular dynamics (MD) simulations analyses, the biotransformation mechanisms and the active site binding profile of 8-MP in CYP1B1 were investigated. Three plausible inactivation mechanisms were minutely scrutinized. Further analysis explored the formation of reactive metabolites in subsequent P450 metabolic processes, including covalent adduct formation through nucleophilic addition to the epoxide, 8-MP epoxide hydrolysis, and non-CYP-catalyzed epoxide ring opening. Special attention was paid to the catalytic effect of residue Phe268 on the mechanism-based inactivation (MBI) of P450 by 8-MP. Energetic profiles and facilitating conditions revealed a slight preference for the C4'=C5' epoxidation pathway, while recognizing a potential kinetic competition with the 8-OMe demethylation pathway due to comparable energy demands. The formation of covalent adducts via nucleophilic addition, particularly by phenylalanine, and the generation of potentially harmful reactive metabolites through autocatalyzed ring cleavage are likely to contribute significantly to P450 metabolism of 8-MP. Our findings highlight the key role of Phe268 in retaining 8-MP within the active site of CYP1B1, thereby facilitating initial oxygen addition transition states. This research offers crucial molecular-level insights that may guide the early stages of drug discovery and risk assessment related to the use of 8-MP.