Journal of Xenobiotics

This study aimed to evaluate the effects of the water-soluble fraction of crude oil (WSFO) on Indian carp (Labeo rohita) with and without treatment with zinc oxide nanoparticles (Nano-ZnO). A total of 225 fish were randomly assigned to five groups in triplicate for 21 days. Group I served as the control group. Groups II and III were exposed to 0.5% and 1% untreated WSFO, respectively. Groups IV and V received 5% and 10% WSFO treated with Nano-ZnO, while Groups VI and VII received 5% and 10% WSFO treated without Nano-ZnO. No blood samples were obtained from fish exposed to untreated WSFO, due to increased hemolysis. Exposure to treated WSFO increased creatine phosphokinase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, and gamma-glutamyl transferase activities, while alanine aminotransferase activity decreased. Although a significant decrease was observed in total protein, globulin, and triglyceride levels, albumin and cholesterol increased. Thiol groups and glutathione peroxidase activity significantly decreased, while superoxide dismutase, catalase, total antioxidant capacity, and malondialdehyde levels increased. The findings showed that exposure to WSFO, whether treated or untreated, induces significant biochemical and oxidative stress responses in Labeo rohita. Although WSFO treated with Nano-ZnO mitigated hemolysis, it was unable to prevent enzyme and antioxidant imbalances, indicating persistent physiological stress. Full article

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals used in various products, such as firefighting foams and non-stick cookware, due to their resistance to heat and degradation. However, these same properties make them persistent in the environment and human body, raising public health concerns. This study selected eleven PFAS commonly found in drinking water and exposed Caenorhabditis elegans to concentrations ranging from 0.1 to 200 µM to assess neurodevelopmental toxicity using a high-throughput, high-content screening (HTS) platform coupled with artificial intelligence for image analysis. Our findings showed that PFAS such as 6:2 FTS, HFPO-DA, PFBA, PFBS, PFHxA, and PFOS inhibited dopaminergic neuron activity, with fluorescence intensity reductions observed across concentrations from 0.1 to 100 µM. PFOS and PFBS also disrupted synaptic transmission, causing reduced motility and increased paralysis in aldicarb-induced assays, with the most pronounced effects at higher concentrations. These impairments in both neuron activity and synaptic function led to behavioral deficits. Notably, PFOS was one of the most toxic PFAS, affecting multiple neurodevelopmental endpoints. These results emphasize the developmental risks of PFAS exposure, highlighting the impact of both individual compounds and mixtures on neurodevelopment. This knowledge is essential for assessing PFAS-related health risks and informing mitigation strategies. Full article

Background: Glyphosate has been extensively used as herbicide since the early 1970s. The daily exposure limit is set at 0.3 mg/kg bw/d in Europe and 1.75 mg/kg bw/d in the USA. Among its derivatives, aminomethylphosphonic acid is the most stable and abundant. Understanding their biological effects then requires reliable methods for quantification in biological samples. Methods: We developed and validated a fast, low-cost, and reliable chromatographic method for determining glyphosate and aminomethylphosphonic acid concentrations. The validation included following parameters: specificity, selectivity, matrix effect, accuracy, precision, calibration performance, limit of quantification, recovery, and stability. Sample extraction employed an anion exchange resin with elution using hydrochloric acid 50.0 mmol/L. For HPLC analysis, analytes were derivatized, separated on a C18 column with a mobile phase of phosphate buffer (0.20 mol/L, pH 3.0) and acetonitrile (85:15), and detected at 240 nm. Results: The method demonstrated high reliability and reproducibility across various matrices. Its performance met all validation criteria, confirming its suitability for quantifying glyphosate and aminomethylphosphonic acid in different biological and experimental setups. Conclusions: This method can offer a practical resource for applications in experimental research, medical diagnostics, quality control, and food safety. Full article

The study of ecotoxicity induced by vanadium (V) represents an area of increasing interest due to the growing use of V in both the industrial and pharmaceutical areas. This leads to its introduction into water environments, marking a developing problem, especially since rising global temperatures appear to intensify its toxic properties. Cytotoxicological approaches carried out on whole marine embryos represent a valid research tool since they grow directly in contact with the pollutants and are equipped with highly responsive cells to stressors. Here, we discuss the detrimental impact on Paracentrotus lividus sea urchin embryos resulting from the combination of V and higher temperatures, reflecting the effects of climate variation. The results demonstrate the remodeling of embryonic architecture at the morphometric level, revealing developmental delays and anomalies. These malformations involve variations in the total skeletal mass due to the almost total absence of the skeleton, with the exception of small calcareous aggregates. Furthermore, both a modulation in total tissue remodeling enzymatic activities and a variation in the amount of three MMP-like gelatinases (MMP-2, -9, and -14) were observed. This research demonstrates that climate change significantly increases the harmful effects of V, emphasizing the necessity for comprehensive toxicity assessments in environmental evaluations. Full article

The soil environment has been considered capable of storing toxic substances without serious consequences for the inhabitants since plants are able to bioaccumulate pollutants without compromising their survival. The application of chemicals to increase soil productivity and the dumping of waste have worsened soil quality. Recently, following a greater awareness of the importance of monitoring the damage deriving from the consumption of contaminated crops for humans and of the protection of biodiversity, studies aimed at identifying the effects of soil contamination on terrestrial animals have increased considerably. Studies using field lizards as model organisms fit into this scenario; this research has shed light on the uptake, accumulation, and toxicity of soil pollutants on reptiles. This review summarizes data collected on lizards of the Podarcis genus, a group of resilient wild species capable of living in both pristine and anthropized areas; the data reveal that many of the effects recorded in lizard tissues at the molecular, biochemical, and histological levels are independent of the chemical composition of the contaminants and are mostly linked to the type of cellular response. Overall, these studies confirm Podarcis lizards as a good model system in ecotoxicological and cytotoxicological research, providing an accurate description of the effects of pollutants, clarifying the defense mechanisms activated in relation to different exposure routes and, finally, providing predictive information on the risks faced by other animals. Since the effects recorded in lizards have often also been observed in mammals, it can be concluded that the results obtained from studies on these animals can be translated to other terrestrial vertebrates, including mammals. Full article

In this study, to better understand the mechanisms of the profound impact of alcohol consumption on drug pharmacokinetics, efficacy, and toxicity, we characterized the alcohol-induced changes in the ensemble of drug-metabolizing enzymes and transporters (DMETs) in the human liver by performing global proteomic analysis of human liver microsomes from 94 donors. DMET protein levels were analyzed concerning alcohol consumption, smoking history, and sex using non-parametric tests, which were further strengthened by correlational analysis. To this end, we used a provisional index of alcohol exposure formulated based on the relative abundances of four marker proteins best correlating with the level of alcohol consumption. Alcohol-induced changes in the cytochrome P450 pool include significant increases in CYP2E1, CYP2B6, CYP2J2, and NADPH-cytochrome P450 reductase levels and the lowering of CYP1A2, CYP2C8, CYP2C9, CYP4A11, and cytochrome b₅. Changes in UDP-glucuronosyltransferase (UGT) abundances comprise elevated UGT1A6, UGT1A9, and UGT2A1, and reduced UGT1A3, UGT1A4, UGT2B7, UGT2B10, and UGT2B15 levels. Tobacco smokers showed elevated CYP1A2, UGT1A6, and UGT2B4 and reduced FMO3, FMO4, and FMO5 levels, while in females, CYP1A2, UGT2B17, and UGT2B15 levels were lower, and UGT2A3 and STS were higher compared to males. The alcohol-induced changes in the DMET ensemble at the protein level reported herein provide deep insights into how alcohol impacts drug and xenobiotic metabolism. Full article

The lack of new antibacterial medicines and the rapid rise in bacterial resistance to antibiotics pose a major threat to individuals and healthcare systems. Despite the availability of various antibiotics, bacterial resistance has emerged for almost every antibiotic discovered to date. The increasing prevalence of multidrug-resistant bacterial strains has rendered some infections nearly untreatable, posing severe challenges to health care. Thus, the development of alternatives to conventional antibiotics is critical for the treatment of both humans and food-producing animals. Endolysins, which are peptidoglycan hydrolases encoded by bacteriophages, represent a promising new class of antimicrobials. Preliminary research suggests that endolysins are more effective against Gram-positive bacteria than Gram-negative bacteria when administered exogenously, although they can still damage the cell wall of Gram-negative bacteria. Numerous endolysins have a modular domain structure that divides their binding and catalytic activity into distinct subunits, which helps maximize their bioengineering and potential drug development. Endolysins and endolysin-derived antimicrobials offer several advantages as antibiotic substitutes. They have a unique mechanism of action and efficacy against bacterial persisters (without requiring an active host metabolism); subsequently, they target both Gram-positive and Gram-negative bacteria (including antibiotic-resistant strains), and mycobacteria. Furthermore, there has been limited evidence of endolysin being resistant. Because these enzymes target highly conserved links, resistance may develop more slowly compared to traditional antibiotics. This review provides an overview and insight of the potential applications of endolysins as novel antimicrobials. Full article

Agroecosystems provide habitats, food, and water for many pollinators and insects, but they are also heavily exposed to threats from the widespread use of pesticides and fertilizers. Managed honeybees and wild bees encounter pesticides in vineyards by collecting morning dew from vine leaves and accessing sugars from grapes, particularly during dry periods. This study assessed the toxicological effects of the commercial fungicide formulations Fantic FNCWG^® and Ramedit combi^®, both individually and in combination, on honeybees. Using a multi-biomarker approach, we evaluated neurotoxicity, metabolic disturbances, phase II detoxification processes, and immune system function. Our findings revealed that commercial fungicide mixtures with multiple active ingredients affect bees differently than single active compounds. Biomarker responses highlighted how these complex mixtures disrupt various enzymatic pathways; including immune function; altering critical enzyme kinetics involved in detoxification and potentially impairing essential bee functions. This study emphasizes the need for more comprehensive research into the sublethal effects of commercial pesticides, particularly those used in vineyards, which are understudied compared to pesticides used in orchards. Full article

Infertility and environmental pollution are two globally prevalent and related issues. To explore women’s reproductive health, the composition of follicular fluid (FF) has been studied and it was found that changes to its composition, including the presence of exogenous chemicals, can adversely affect the fertilization process. Two groups of women (idiopathic infertility and controls) who were patients at a fertility clinic were recruited for this study. Samples of urine and FF were gathered from each participant to determine the concentration of 14 common phenols (four parabens, six bisphenols, two benzophenones, and two naphthols). Associations between phenol concentrations (free and total) in both matrices were described using Spearman’s correlation coefficient and were compared between two groups by the Mann–Whitney U test. Eight phenols were quantified in more than 50% of the urine samples, while only three parabens were quantified in hydrolyzed FF samples, and only methylparaben was quantified in non-hydrolyzed FF samples. Conjugates were the predominant form in FF samples. However, a significant correlation of 0.533 (p < 0.0001) was observed between free and total methylparaben concentrations in FF. Differences in concentrations between cases and controls in both matrices were not statistically significant, except for benzophenone-3 in urine, with a higher median observed in the control group (p = 0.04). The total paraben concentrations in urine and FF samples were rather weakly correlated (r = 0.232–0.473), implying that urine concentrations may not be appropriate for predicting their concentration in FF. Full article

Type 2 diabetes mellitus (T2DM) is an intricate disease correlated with many metabolic deregulations, including disordered glucose metabolism, oxidative stress, inflammation, and cellular apoptosis due to hepatic gluconeogenesis aberrations. However, there is no radical therapy to inhibit hepatic gluconeogenesis disturbances yet. We thus sought to probe the effectiveness and uncover the potential mechanism of quercetin (QCT) and silk sericin (SS) in mitigating hyperglycemia-induced hepatic gluconeogenesis disorder, which remains obscure. Administration of QCT and SS to diabetic male albino rats markedly restored the levels of glucose, insulin, advanced glycation end-products (AGEs), liver function enzymes, alpha-fetoprotein (AFP), globulin, and glycogen, in addition to hepatic carbohydrate metabolizing enzymes and gluconeogenesis in comparison with diabetic rats. Furthermore, treatment with QCT and SS modulated hepatic malondialdehyde (MD), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), nitric oxide, tumor necrosis factor-alpha (TNF-α), and interleukin-1β (IL-1β), in addition to serum interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), implying their effectiveness in safeguarding cells against oxidative impairment and inflammation. Remarkably, QCT and SS treatments led to the upregulation of expression of phosphatidylinositol 3-kinases (PI3K), phospho-Akt (p-Akt), and forkhead box-O1 (FOXO1) genes in hepatic tissues compared to diabetic rats, orchestrating these singling pathways for curtailing hyperglycemia and pernicious consequences in hepatic tissues. Importantly, immunohistochemical investigations exhibited downregulation of caspase-3 expression in rats treated with QCT and SS compared to diabetic animals. Beyond that, the histopathological results of hepatic tissues demonstrated notable correlations with biochemical findings. Interestingly, the in silico results supported the in vivo findings, showing notable binding affinities of QCT and SS to PI3K, GPx, and TNF-α proteins. These results imply that QCT and SS could mitigate oxidative stress and inflammation and regulate hepatic gluconeogenesis in diabetic rats. However, QCT revealed greater molecular interactions with the studied proteins than SS. Overall, our results emphasize that QCT and SS have significant therapeutic effects on attenuating hyperglycemia-induced hepatic gluconeogenesis, with QCT showing superior effectiveness. Full article

Cognitive enhancement through stimulants such as modafinil is becoming increasingly popular, with many individuals using prescription stimulants for non-medical purposes to improve alertness, attention, and mood. The misuse of such substances has raised concerns, particularly in forensic toxicology. The UHPLC-QqQ-MS/MS method was developed to quantify modafinil in evidentiary samples and biological materials. Additionally, the authors noted the presence of sodium adducts during the analysis of samples with high concentrations of modafinil. The method was validated for accuracy, precision, and linearity, with a concentration range of 0.1–10.0 µg/mL for the evidences and 1.0–100.0 ng/mL for blood. The method successfully detected modafinil as the sole substance in all evidences, with concentrations ranging from 90.7 to 120.8 mg, corresponding to 45.5% to 80.5% of the labeled dose. The method was applied to real post-mortem human cases, where, among others, the concentration of modafinil in blood was 110 ng/mL, whereas, in another case, the concentration of modafinil in the putrefaction fluid exceeded 1000 ng/mL. The developed UHPLC-QqQ-MS/MS method is effective for the quantification of modafinil in evidentiary samples and biological materials, offering a reliable tool for forensic toxicology applications. This method can be used to evaluate modafinil use in both legal and illicit contexts, including cases of overdose or misuse. Full article

Dietary exposure to the plasticiser bisphenol A (BPA), an obesogenic and endocrine disruptor from plastic and epoxy resin industries, remains prevalent despite regulatory restriction and food safety efforts. BPA can be accumulated in humans and animals, potentially exerting differential health effects based on individual metabolic capacity. This pilot study examines the impact of direct ex vivo BPA exposure on the gut microbiota of obese and normal-weight children, using 16S rRNA amplicon sequencing and anaerobic culturing combined methods. Results showed that direct xenobiotic exposure induced modifications in microbial taxa relative abundance, community structure, and diversity. Specifically, BPA reduced the abundance of bacteria belonging to the phylum Bacteroidota, while taxa from the phylum Actinomycetota were promoted. Consistently, Bacteroides species were classified as sensitive to BPA, whereas bacteria belonging to the class Clostridia were identified as resistant to BPA in our culturomics analysis. Some of the altered bacterial abundance patterns were common for both the BPA-exposed groups and the obese non-exposed group in our pilot study. These findings were also corroborated in a larger cohort of children. Future research will be essential to evaluate these microbial taxa as potential biomarkers for biomonitoring the effect of BPA and its role as an obesogenic substance in children. Full article

Endocrine-disrupting chemicals (EDCs) are natural or synthetic substances that are able to interfere with hormonal systems and alter their physiological signaling. EDCs have been recognized as a public health issue due to their widespread use, environmental persistence and the potential levels of long-term exposure with implications in multiple pathological conditions. Their reported adverse effects pose critical concerns about their use, warranting their strict regulation. This is the case of bisphenol A (BPA), a well-known EDC whose tolerable daily intake (TDI) was re-evaluated in 2023 by the European Food Safety Authority (EFSA), and the immune system has been identified as the most sensitive to BPA exposure. Increasing scientific evidence indicates that EDCs can interfere with several hormone receptors, pathways and interacting proteins, resulting in a complex, cell context-dependent response that may differ among tissues. In this regard, the neuronal and immune systems are important targets of hormonal signaling and are now emerging as critical players in endocrine disruption. Here, we use BPA and its analogs as proof-of-concept EDCs to address their detrimental effects on the immune and nervous systems and to highlight complex interrelationships within the immune–neuroendocrine network (INEN). Finally, we propose that Receptor for Activated C Kinase 1 (RACK1), an important target for EDCs and a valuable screening tool, could serve as a central hub in our toxicology model to explain bisphenol-mediated adverse effects on the INEN. Full article

Chlorpyrifos (CPF) is a broad-spectrum organophosphate insecticide. Long-term exposure to low levels of CPF is associated with neurodevelopmental and neurodegenerative disorders. The mechanisms leading to these effects are still not fully understood. Normal NMDA receptor (NMDAR) function is essential for neuronal development and higher brain functionality, while its inappropriate stimulation results in neurological deficits. Thus, the current study aimed to investigate the role of NMDARs in CPF-induced neurotoxicity. We show that NMDARs mediate CPF-induced excitotoxicity in differentiated human fetal cortical neuronal ReNcell CX stem cells. In addition, by using two-electrode voltage clamp electrophysiology of Xenopus oocytes expressing NMDARs, we show CPF potentiation of both GluN1-1a/GluN2A (EC₅₀ ≈ 40 nM) and GluN1-1a/GluN2B (EC₅₀ ≈ 55 nM) receptors, as well as reductions (approximately halved) in the NMDA EC₅₀s and direct activation by 10 μM CPF of both receptor types. In silico molecular docking validated CPF’s association with NMDARs through relatively high affinity binding (−8.82 kcal/mol) to a modulator site at the GluN1–GluN2A interface of the ligand-binding domains. Full article

Food is essential for human survival; however, food can be an important route of exposure to contaminants. This study investigated the presence and distribution of anthropogenic contaminants in food consumed by families with small children in South Florida, United States, evaluating seasonal and socio-economic variabilities in chemical composition. QuEChERS protocols, followed by non-targeted analysis (NTA) using an LC-Orbitrap HRMS system, were used for the comprehensive screening of organic contaminants. The compounds were annotated and identified with the Compound Discoverer (CD) software, and contaminant distributions were analyzed using boxplots and Principal Component Analysis (PCA). The results showed significant seasonal and socio-economic differences in contaminant distributions (p < 0.05). In the wet season, a predominance of polymers and surfactants, such as dodecanedioic acid and N-dodecylacrylamide, were found in food, which might be due to increased transport of industrial pollutants during increased precipitation, while plasticizers (e.g., bis(2-ethylhexyl) phthalate) and drugs (e.g., warfarin) were more prevalent during the dry season, which could be related to less dilution effects in this period. A higher abundance of 1-nitrosopiperidine, present in cured meats, was noted in food from upper socio-economic classes, while the lower class showed higher abundance of benzocaine, a common topical anesthetic. Full article

Nanoplastics pose a growing threat to marine ecosystems, particularly affecting the early developmental stages of marine organisms. This study investigates the effects of amino-modified polystyrene nanoparticles (PS-NH₂, 50 nm) on the embryonic development of Phallusia mammillata, a model ascidian species. Both chorionated and dechorionated embryos were exposed to increasing concentrations of PS-NH₂ so morphological alterations could be assessed with a high-content analysis of the phenotypes and genotoxicity. PS-NH₂ induced the same morphological alterations in both chorionated and dechorionated embryos, with dechorionated embryos being more sensitive (EC₅₀ = 3.0 μg mL⁻¹) than chorionated ones (EC₅₀ = 6.26 μg mL⁻¹). Interestingly, results from the morphological analysis showed two concentration-dependent mechanisms of action: (i) at concentrations near the EC₅₀, neurodevelopmental abnormalities resembling the ones induced by exposure to known endocrine disruptors (EDs) were observed, and (ii) at higher concentrations (15 μg mL⁻¹ and 7.5 μg mL⁻¹ for chorionated and dechorionated embryos, respectively), a nonspecific toxicity was evident, likely due to general oxidative stress. The phenotypes resulting from the PS-NH₂ treatment were not related to DNA damage, as revealed by a genotoxicity assay performed on neurula embryos. Our data suggest that PS-NH₂-induced toxicity is primarily mediated through oxidative stress, probably triggered by interactions between the positive charges of the PS NPs and the negative charges on the cell membranes. The lack of a protective chorion further exacerbated these effects, highlighting its role in mitigating/protecting against NP-induced damage. Full article

Chemotherapy-induced cardiotoxicity is a critical issue in cardio-oncology, as cancer treatments often lead to severe cardiovascular complications. Approximately 10% of cancer patients succumb to cardiovascular problems, with lung cancer patients frequently experiencing arrhythmias, cardiac failure, tamponade, and cardiac metastasis. The cardiotoxic effects of anti-cancer treatments manifest at both cellular and tissue levels, causing deformation of cardiomyocytes, leading to contractility issues and fibrosis. Repeated irradiation and chemotherapy increase the risk of valvular, pericardial, or myocardial diseases. Multi-OMICs analyses reveal that targeting specific pathways as well as specific protein modifications, such as ubiquitination and phosphorylation, could offer potential therapeutic alternatives to current treatments, including Angiotensin converting enzymes (ACE) inhibitors and beta-blockers that mitigate symptoms but do not prevent cardiomyocyte death, highlighting the need for more effective therapies to manage cardiovascular defects in cancer survivors. This review explores the xenobiotic nature of chemotherapy agents and their impact on cardiovascular health, aiming to identify novel biomarkers and therapeutic targets to mitigate cardiotoxicity. Full article

Engineered light-sensitive molecules offer a sophisticated toolkit for the manipulation of biological systems with both spatial and temporal precision. Notably, artificial “caged” compounds can activate specific receptors solely in response to light exposure. However, the uncaging process can lead to the formation of potentially harmful byproducts. For example, the photochemical release of adrenaline (epinephrine) is accompanied by the formation of adrenochrome, which has neuro- and cardiotoxic effects. To investigate this effect in detail, we synthesized and compared two “caged” epinephrine analogs. The first was a classical compound featuring an ortho-nitrobenzyl protecting group attached to the amino group of epinephrine. The second analog retained the ortho-nitrobenzyl group but included an additional carbamate linker. The photolysis of both compounds was conducted under identical conditions, and the resulting products were analyzed using UV–Vis spectroscopy, chromatography, and NMR techniques. Surprisingly, while the classical compound led to the formation of adrenochrome, the carbamate-type caged epinephrine did not produce this byproduct, resulting in the clean release of the active substance. Subsequently, we assessed the novel compound in an in vitro platelet activation assay. The results demonstrated that the uncaging of epinephrine significantly enhances platelet activation, making it a valuable tool for advanced signaling studies. Full article

Background: Chronic stress, characterized by sustained activation of physiological stress response systems, is a key risk factor for numerous health conditions. Allostatic load (AL), a biomarker of cumulative physiological stress, offers a quantitative measure of this burden. Lifestyle habits such as alcohol consumption and smoking, alongside environmental exposures to toxic metals like lead, cadmium, and mercury, were individually implicated in increasing AL. However, the combined impact of these lifestyle habits and environmental factors remains underexplored, particularly in populations facing co-occurring exposures. This study aims to investigate the joint effects of lifestyle habits and environmental factors on AL, using data from the NHANES 2017–2018 cycle. By employing linear regression and Bayesian Kernel Machine Regression (BKMR), we identify key predictors and explore interaction effects, providing new insights into how cumulative exposures contribute to chronic stress. Results from BKMR analysis underscore the importance of addressing combined exposures, particularly the synergistic effects of cadmium and alcohol consumption, in managing physiological stress. Methods: Descriptive statistics were calculated to summarize the dataset, and multivariate linear regression was performed to assess associations between exposures and AL. BKMR was employed to estimate exposure–response functions and posterior inclusion probabilities (PIPs), focusing on identifying key predictors of AL. Results: Descriptive analysis indicated that the mean levels of lead, cadmium, and mercury were 1.23 µg/dL, 0.49 µg/dL, and 1.37 µg/L, respectively. The mean allostatic load was 3.57. Linear regression indicated that alcohol consumption was significantly associated with increased AL (β = 0.0933; 95% CI [0.0369, 0.1497]; p = 0.001). Other exposures, including lead (β = −0.1056; 95% CI [−0.2518 to 0.0408]; p = 0.157), cadmium (β = −0.0001, 95% CI [−0.2037 to 0.2036], p = 0.999), mercury (β = −0.0149; 95% CI [−0.1175 to 0.0877]; p = 0.773), and smoking (β = 0.0129; 95% CI [−0.0086 to 0.0345]; p = 0.508), were not significant. BKMR analysis confirmed alcohol’s strong importance for AL, with a PIP of 0.9996, and highlighted a non-linear effect of cadmium (PIP = 0.7526). The interaction between alcohol and cadmium showed a stronger effect on AL at higher exposure levels. In contrast, lead, mercury, and smoking demonstrated minimal effects on AL. Conclusions: Alcohol consumption and cadmium exposure were identified as key contributors to increased allostatic load, while other exposures showed no significant associations. These findings emphasize the importance of addressing lifestyle habits and environmental factors in managing physiological stress. Full article