Combined Toxicity Research Articles

Molecular insights into acetyl triethyl citrate (ATEC) induced toxic effect in HepG2 cells based on multi‐omics integrative analysis

AbstractCitrate esters have become the main alternatives to traditional plasticizers in food packaging materials. However, there is a lack of understanding of their toxic effects, particularly the combined effects and inner mechanisms has not been well studied. Our group pioneered the study on combined toxicity of tributyl citrate (TBC) and acetyl triethyl citrate (ATEC), two commonly co‐used citrate esters in food packaging materials. The results showed that exposure to TBC and ATEC can decrease the viability of HepG2 cells in a dose dependent manner. When the mixtures of ATEC and TBC exposed to HepG2 cells, they exhibited antagonism effect. Therefore, ATEC was selected to investigate the molecular mechanisms with multi‐omics techniques at its 1/4 EC50 concentration. A total of 31 metabolites with significant changes were found as potential biomarkers. The LIPG (Lipase G, endothelial type) and GCLM (glutamate‐cysteine ligase modifier subunit) were identified as differentially expressed genes based on transcriptomic analysis. Moreover, downregulated l‐glutamate and l‐glutamine which participate in TCA cycle, resulting in the collapse of energy production and cytotoxicity. These findings on major metabolic pathways will provide insight into the mechanism of cytotoxicity of HepG2 cells after ATEC exposure.

Microplastics from face masks: Unraveling combined toxicity with environmental hazards and their impacts on food safety.

Microplastics (MPs) refer to tiny plastic particles, typically smaller than 5mm in size. Due to increased mask usage during COVID-19, improper disposal has led to masks entering the environment and releasing MPs into the surroundings. MPs can absorb environmental hazards and transfer them to humans and animals via the food chain, yet their impacts on food safety and human health are largely neglected. This review summarizes the release process of MPs from face masks, influencing factors, and impacts on food safety. Highlights are given to the prevalence of MPs and their combined toxicities with other environmental hazards. Control strategies are also explored. The release of MPs from face masks is affected by environmental factors like pH, UV light, temperature, ionic strength, and weathering. Due to the chemical active surface and large surface area, MPs can act as vectors for heavy metals, toxins, pesticides, antibiotics and antibiotic resistance genes, and foodborne pathogens through different mechanisms, such as electrostatic interaction, precipitation, and bioaccumulation. After being adsorbed by MPs, the toxicity of these environmental hazards, such as oxidative stress, cell apoptosis, and disruption of metabolic energy levels, can be magnified. However, there is a lack of comprehensive research on both the combined toxicities of MPs and environmental hazards, as well as their corresponding control strategies. Future research should prioritize understanding the interaction of MPs with other hazards in the food chain, their combined toxicity, and integrating MPs detection and degradation methods with other hazards.

Prevalence and relevance of H558R in the efficacy and toxicity of flecainide in patients with atrial fibrillation: a cohort study.

The SCN5A gene polymorphism histidine-558-to-arginine (H558R) has been associated with atrial fibrillation (AF) and may affect the therapeutic effects of flecainide. This study aimed to assess the prevalence of the H558R polymorphism in a European cohort of patients with AF and examine its association with flecainide's effects on AF recurrence and toxicity. This cohort study included patients diagnosed with AF and prescribed flecainide between 2017 and 2021 in a regional health area. Patients without the polymorphism (H558R-/-) were compared with heterozygous patients (H558R+/-) for a primary outcome of combined 6-month AF recurrence or toxicity. Secondary analyses evaluated the long-term outcomes and compared the prevalence of H558R in the AF cohort to a general population sample (n=3401). A total of 104 patients were enrolled, with 57% H558R-/-, 37% H558R+/- and 6% H558R+/+. The prevalence of the H558R polymorphism was significantly higher in the AF cohort than in the general population (43.27% vs 24.37%, prevalence ratio 1.78, 95% CI 1.41 to 2.23, p<0.01). H558R+/- patients had a significantly lower risk of 6-month AF recurrence or toxicity (p=0.023, risk ratio 0.423, 95% CI 0.189 to 0.947), corresponding to an absolute risk difference of 21.5%. These findings were similar in the multivariable analysis. In long-term follow-up, H558R+/- patients continued to demonstrate a lower risk of AF recurrence or toxicity (p=0.039, HR 0.53, 95% CI 0.276 to 0.999). The H558R polymorphism is more prevalent in patients with AF compared with the general population and its presence is associated with a more favourable response to flecainide treatment.

Mitochondrial damage and associated combined toxicity induced by deoxynivalenol and Alternaria toxins co-exposure

Mitochondrial damage and associated combined toxicity induced by deoxynivalenol and Alternaria toxins co-exposure

Evidence that cadmium aggravate the toxicity of triphenyl phosphate in aquatic sediments to Corbicula fluminea

The ubiquitous co-existence of triphenyl phosphate (TPhP) and heavy metals in sediments raises significant biotoxicity concerns. However, uncertainty still exists regarding their combined toxicity to benthic organisms. Therefore, this research was conducted to elucidate the influences of cadmium (Cd) on TPhP toxicity to Corbicula fluminea (C. fluminea) in sediments. As a result, Cd promoted the accumulation of TPhP in C. fluminea and enhanced TPhP toxicity, manifested by damaged cell membranes and pronounced histological alterations. Molecular docking revealed that TPhP-Cd complexes exhibit greater binding affinity to cytochrome P4501A1 (CYP1A1) compared to TPhP alone. With the activity of CYP1A1 increasing, the biotransformation of TPhP was promoted in low-TPhP+Cd treatments (T5C0/T5C5/T5C35). Additionally, metabolites related to antioxidant defence and repair processes were reinforced to alleviate the toxicity of TPhP and Cd. However, excessive oxidative stress impaired the CYP1A1 activity in high-TPhP+Cd treatments (T35C0/T35C5/T35C35). Furthermore, metabolic pathway analysis revealed significant perturbations in the citrate cycle, alanine, aspartate and glutamate metabolism, purine metabolism, and pyrimidine metabolism. These disruptions weakened the repair capacity and aggravated apoptosis in digestive glands, potentially contributing to the synergistic toxicity of TPhP and Cd. The results highlight the ecological risks posed by TPhP in combination with heavy metals to benthic organisms.

Importance of tailored non-clinical safety testing of novel antimalarial drugs: Industry best-practice

Malaria is an acute, debilitating parasitic illness. There were 249 million cases of malaria in 2022, resulting in 608,000 deaths globally, 76% of which were children ≤5 years. The unique nature of this disease (recurrences leading to re-treatments and numerous organ systems affected), specific clinical treatment regimens, poor compliance, and diversity of affected populations (predominantly pediatrics, women of childbearing potential, pregnant and lactating women), often makes standard testing approaches inadequate, and tailor-made safety assessments are more appropriate.We provide best practice recommendations based on company experience for the non-clinical safety assessment of antimalarial drugs, with a focus on small molecules since they represent the majority of drug candidates for this illness. We focus on specific testing considerations for repeat dose toxicity studies, including combination toxicity assessments, since new drug treatment regimens typically foresee short treatment durations to improve compliance (i.e., 1 day) with combinations of compounds to improve efficacy and limit potential resistance. Due to the target population, the timing of reproductive, developmental, and juvenile toxicity studies may be earlier than general testing roadmaps for other small molecule drugs.In conclusion, key recommendations presented should enable a more effective and efficient development path whilst protecting clinical trial participants and patients.

Effects of single or combined exposure to tralopyril and ocean acidification on energy metabolism response and sex development in Pacific oysters (Crassostrea gigas)

The combined effects of the novel antifouling biocide tralopyril (TP) nitrile and ocean acidification (OA) on marine organisms are still not well understood, despite the increasing attention given to the toxic effects of emerging pollutants and OA on marine organisms in recent years. In this study, Crassostrea gigas (C. gigas) was exposed to TP, OA, and a combination of TP and OA for 21 days with a 14-day depuration. This study investigated the inter-tissue variability in energy metabolism responses and the impacts on gonadal development in C. gigas under both single and combined exposures to TP and OA. The results indicate that TP exposure and OA resulted in up-regulation of energy metabolism genes in the C. gigas, with tissues exhibiting enhanced aerobic metabolism. Furthermore, OA influences the sex determination of C. gigas, promoting the development of female individuals. Moreover, following depuration, C. gigas is able to restore normal energy metabolism and sexual development through the accumulation of suitable energy reserves. This study provides a valuable reference for the environmental and ecological risk assessment of TP, addressing the research gap in understanding the combined toxicity of TP and OA on aquatic organisms.

Evaluating the impact of the combined acute toxicity of iron (Fe) and microplastics on Namalycastis jaya

The rising concern over heavy metals (HMs) and microplastics (MPs) pollution in marine ecosystems, primarily driven by anthropogenic activities, poses significant threats to ecological health. Understanding the combined exposure of HMs and MPs aids in toxicity assessment. In this study, we examined the combined effects of polystyrene microplastics (MPs) and iron (Fe) on oxidative stress, bioaccumulation, histopathology, and genotoxicity in Namalycastis jaya. Oxidative stress was assessed by analyzing the levels of Superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD), Malondialdehyde (MDA), and Bicinchoninic acid (BCA), while genotoxicity was evaluated using the comet assay. Bioaccumulation analysis, conducted via Inductively coupled plasma-optical emission spectrometry (ICP-OES), indicated that the highest values (4.790 µg/ml) were observed in combined exposure, emphasizing the significant increase in iron (Fe) accumulation in polychaetes facilitated by MPs. Biochemical analysis revealed that oxidative damage in polychaetes became evident within 48 h of exposure to individual contaminants. However, in the case of combined exposures, elevated stress levels were observed within just 24 h. The genotoxic assay further demonstrated a higher degree of DNA damage in the combined exposure compared to individual exposures. Similarly, histopathology revealed mild alterations in the gut epithelium in combined exposures. It is evident that MPs intensify both oxidative and DNA damage induced by Fe in polychaetes. The insights gained from this study provide valuable information for the risk assessment of Fe and MPs in environmental safety, contributing to our understanding of the complex interactions between these pollutants in marine ecosystems.

Combined toxicity of microplastics and antibiotics towards aquatic ecosystems: A case study in Beiluo River, China

Microplastics (MPs) can adsorb antibiotics to form complex pollutants that seriously threaten the health of freshwater ecosystems. However, few studies have examined the combined ecotoxicity of MPs and multiple antibiotics in natural aquatic environments. In this study, we examined the effects of combined exposure to antibiotics and MPs on aquatic community structure in natural settings in the Beiluo River using eDNA analysis. The results revealed that antibiotic pollution in the Beiluo River mainly originates from animal husbandry and agriculture, whereas MPs mainly originate from agriculture and urban sewage. The community structure of aquatic organisms was significantly correlated with the concentrations of antibiotics and MPs and that the combined effects of antibiotics and MPs were highly interpretable for cyanobacteria and phytoplankton. The effect of MPs on antibiotic toxicity is highly dependent on the antibiotic type and MP particle size. These insights highlight that co-exposure to MPs and antibiotics in the natural environment is a common phenomenon and that co-exposure to MPs can alter the toxicological profile of antibiotics. The results enhance our comprehensive understanding of the complex combined effects of MPs and antibiotics on aquatic organisms, emphasising the necessity of informed scientific management of these emerging contaminants.

Negative effects of polyvinyl chloride microplastics and the plasticizer DnOP on earthworms: Co-exposure enhances oxidative stress and immune system damage in earthworms

Polyvinyl chloride microplastics (PVC-MPs) are the most used plastics in agriculture. Di-n-octyl phthalate (DnOP), a commonly used plasticizer in PVC-MPs, may be released from plastic and coexist with PVC-MPs. The effects of DnOP alone and coexisting with PVC-MPs are not known. We evaluated the effects of DnOP or/and PVC-MPs on earthworms, and used integrated biomarker response (IBR) to assess the combined toxicity. Molecular docking and transcriptomics were employed for further interpretation of possible toxicity mechanisms. The results showed that exposure to DnOP or/and PVC-MPs caused oxidative damage and interfered with reproduction, adversely affecting the growth and reproduction of earthworms. IBR results showed that toxicity of DnOP+PVC-MPs exposure was greater than that of DnOP and PVC-MPs exposure alone. DnOP has the ability to directly bind to proteins that are associated with antioxidant enzymes and alter their structure. The transcriptomics results indicated that DnOP and PVC-MPs exposure alone mainly affected growth and development-related pathways, while co-exposure affected apoptosis and immune system-related pathways more. To the best of our knowledge, this is the first comprehensive investigation of the combined toxicity of DnOP or/and PVC-MPs to earthworms from different perspectives, which gives scientifically sound evidence for the rational use of plasticizers DnOP and PVC-MPs.

Dichlorvos evokes systemic lipid dysmetabolism in Wistar rats: rescindment influence of curcumin

Organophosphorus pesticides such as dichlorvos (DDVP) are often employed to eradicate pests, especially in low and medium-income countries. However, they have several negative impacts on the visceral organs. Astonishingly, curcumin protects organs from the detrimental effects of xenobiotics via the maintenance of redox homeostasis; unfortunately, its role in dichlorvos-provoked multi-organ impairment vis-à-vis lipid homeostasis has not been examined. Therefore, this undertaking probed the remedial efficacy of curcumin on DDVP-prompted combined systemic toxicity and major lipid distribution. Randomization was engaged to dedicate rats (40) into seven groups (6 rats/group): control, DDVP only (20 mg kg-1day-1), DDVP administered with either curcumin (50 and 100 mg kg-1day-1) or reference medication atropine (0.2 mg kg-1day-1), and curcumin only (50 and 100 mg kg-1day-1). DDVP was dispensed orally for one week, followed by two weeks of curcumin treatment. Twenty-four hours after the last administration, we sacrificed the rats and collected their blood and viscera (liver, kidney, heart, lung, and brain) for bioassays. Curcumin remarkably (p&lt;0.05) rescinded DDVP-mediated increases in plasma, LDL, and systemic cholesterol; markedly (p&lt;0.05) attenuated DDVP-elicited decreases in HDL-cholesterol and TAG contents in all the compartments except erythrocyte and liver; and significantly (p&lt;0.05) abated DDVP-induced plasma phospholipidaemia, multi-organ phospholipidosis, and up-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA-R) activity. This finding demonstrated that curcumin reverses DDVP-triggered anomalous lipid dynamics by abating cholesterogenesis and phospholipidosis and restoring HMG-CoA-R activity.

Combined toxicity of perfluoroalkyl substances and microplastics on the sentinel species Daphnia magna: Implications for freshwater ecosystems

Persistent chemicals from industrial processes, particularly perfluoroalkyl substances (PFAS), have become pervasive in the environment due to their persistence, long half-lives, and bioaccumulative properties. Used globally for their thermal resistance and repellence to water and oil, PFAS have led to widespread environmental contamination. These compounds pose significant health risks with exposure through food, water, and dermal contact. Aquatic wildlife is particularly vulnerable as water bodies act as major transport and transformation mediums for PFAS. Their co-occurrence with microplastics may intensify the impact on aquatic species by influencing PFAS sorption and transport. Despite progress in understanding the occurrence and fate of PFAS and microplastics in aquatic ecosystems, the toxicity of PFAS mixtures and their co-occurrence with other high-concern compounds remains poorly understood, especially over organisms’ life cycles.Our study investigates the chronic toxicity of PFAS and microplastics on the sentinel species Daphnia, a species central to aquatic foodwebs and an ecotoxicology model. We examined the effects of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and polyethylene terephthalate microplastics (PET) both individually and in mixtures on Daphnia ecological endpoints. Unlike conventional studies, we used two Daphnia genotypes with distinct histories of chemical exposure. This approach revealed that PFAS and microplastics cause developmental failures, delayed sexual maturity and reduced somatic growth, with historical exposure to environmental pollution reducing tolerance to these persistent chemicals due to cumulative fitness costs. We also observed that the combined effect of the persistent chemicals analysed was 59% additive and 41% synergistic, whereas no antagonistic interactions were observed. The genotype-specific responses observed highlight the complex interplay between genetic background and pollutant exposure, emphasizing the importance of incorporating multiple genotypes in environmental risk assessments to more accurately predict the ecological impact of chemical pollutants.

Pharmacokinetic predictions of ROS-mediated targets and genotoxin combinations via multiple ligand simultaneous docking and ROS evaluation in vitro using HepG2 cell lines.

Although combination therapy is known for its high efficacy, reduced side effects and drug resistance, toxicity remains a major drawback. Some of the genes are likely to induce hepatotoxicity through ROS-mediated mechanisms when a drug is metabolized alone or in combination in the liver. To address this, we have developed a scientific approach to predict the toxicity of different genotoxin combinations and validate their interactions with various targets. The current study is an extensive study of our previous set of in vivo rat liver microarray data processed using R studio for their functional analysis. About five combinations of genotoxins such as CPT/ETP, CPT/CPL, ETP/CPL, CP/CPT and EES/CP along with their differential gene expression targeting Chemical carcinogenesis-ROS are chosen for this study. We aim to examine the binding affinity of different genotoxin combinations using in silico multiple ligand simultaneous docking (MLSD) and are then bio-evaluated for cytotoxicity in vitro using human hepatocellular carcinoma cell lines (HepG2) with the MTT assay. As a result, dose-response cytotoxicity with its strength of interactions and a significant variance in ROS levels in the treated cells is observed compared to their IC50 values. Out of 5 combinations such as CPT/CPL, ETP/CPL and EES/CP are found not only to be significantly cytotoxic but also induce oxidative stress specifically above their IC50 values with good and moderate binding interactions ensuring their toxicity. On the contrary, the safe combinations are found to be CTP/ETP and CP/CPT possibly with no and tolerable adverse effects standing as preliminary information for researchers in drug design and development.

A comparative study on the toxic effects of lead pollution and nanoplastic-lead mixed pollution on red drum and their detoxification strategies

Nanoplastics and heavy metals pose various adverse effects on marine organisms. However, the combined toxicity of nanoplastics and lead pollution to marine fish is not fully understood. This study investigates the toxic effects and detoxification strategies of lead pollution (p07) compared to nanoplastic-lead mixed pollution (m07) in red drum during exposure and recovery phases. Under m07 pollution, the maximum lead content in muscle was 22.61 mg/kg, which was significantly higher than the 15.82 mg/kg observed under p07 pollution. This finding demonstrated that nanoplastics significantly enhance lead accumulation, leading to more severe toxic effects on red drum. Histological analyses revealed that lipid droplets in the liver and epithelial lifting in the gills were the primary lesion types. During the exposure periods, red drum primarily detoxified lead through cellular renewal and the removal of damaged proteins under p07 pollution. Conversely, under m07 pollution, detoxification relied on cellular senescence, apoptosis, endocytosis, and the removal of damaged proteins. In the recovery phases, red drum predominantly recovered through cell proliferation and antioxidant responses under p07 pollution. Under m07 pollution, the focus shifted to functional protein synthesis, apoptosis, endocytosis, and lipid metabolism. This study offers valuable insights into the monitoring and management of combined environmental pollution.

Adsorption behavior of ZIF-67 to bisphenol compounds affects combined toxicity on Photobacterium phosphoreum

ZIF-67, as a typical MOF material, is considered a new type of high-potential adsorbent due to its ample surface area and tunable surface chemistry, which has the potential to interact with other contaminants in unforeseen ways, resulting in combined toxicity. To further elucidate this possibility, we chose typical bisphenol compound (BP) which is widely used in commercial manufacturing, to explore the combined toxicity with MOF. MOF showed a high adsorption capacity for BPAF (> 80%) and the weakest adsorption capacity for BPA (< 10%), and DFT confirmed the different interaction strengths of MOF for BPs. The difference in adsorption capacity for BPs resulted in different amounts of free BPs, contributing to combined toxicity. Based on flow cytometry and TEM, the results showed that membrane damage was reduced and the ability of ZIF-67 to enter the cell was decreased in the low-concentration ZIF-67 mixing group, and the ability of ZIF-67 to enter the cell was increased in the high-concentration ZIF-67 mixing group, and the membranes were severely damaged. RT-PCR and biochemical indicators measurements helped to explain the underlying toxicity mechanism. This study is of practical significance for the development of environmental guidelines for mixed contaminant effects and accurate risk assessments.

Effects of coexisting nanomaterials on the photodegradation behavior and ecotoxicity of antibiotics in the aqueous

Nanomaterials (NPs) and antibiotics, as two emergent pollutants, forms a complex contamination through their interaction, potentially causing adverse effects on the organism. This study systematically examined the influence of two NPs (CuO NPs and carbon nanotubes, CNTs) on the photodegradation behavior of tetracycline (TC) and their combined toxic effects on Chlorella vulgaris. The results showed that CuO NPs significantly accelerated TC photodegradation compared to CNTs, increasing the TC photodegradation rate constant by187.6%. Electron spin resonance (ESR) indicated that under the coexistence of CuO NPs or CNTs, 1O2、O2•- and •OH were the main active species promoting TC photodegradation. Probe and quenching experiments confirmed the predominant role of O2•- and 1O2 in the presence of CuO NPs and CNTs. Additionally, three possible TC photodegradation pathways were proposed for the coexistence of CuO NPs and CNTs. In the Chlorella vulgaris growth inhibition experiment, the combined toxicity of CuO NPs or CNTs and TC was higher than that of individual substance, indicating significant synergistic effects, especially with the combination of CNTs and TC. This study provides a new perspective on accurately assessing the environmental behaviors and risks when NPs and antibiotics coexist.

Multigenerational effects of combined exposure of triphenyltin and micro/nanoplastics on marine medaka (Oryzias melastigma): From molecular levels to behavioral response

In natural environments, micro/nanoplastics (MNP) inevitably coexist with various pollutants, making it essential to examine their combined toxicity and intergenerational effects on marine organisms. This study investigated the combined toxicity and intergenerational effects of exposure to triphenyltin (T), microplastics (M), nanoplastics (N), a combination of microplastics and triphenyltin (MT), and a combination of nanoplastics and triphenyltin (NT) on marine medaka. The results showed that all treatments had adverse and intergenerational effects on marine medaka. Regarding oxidative stress and energy metabolism, smaller sized plastic particles caused more significant damage to the organisms. However, MT inflicted greater gonadal system damage than NT, leading to imbalanced sex hormone levels. Additionally, T induced hyperactivity in fish, whereas MNP tended to induce behavioral depression. Notably, large plastic particles in the F0 generation had a more pronounced impact on depressive behaviors compared to smaller particles. These findings suggest that both individual and combined exposures to TPT and MNP can detrimentally affect marine medaka from the molecular to behavioral levels, posing risks to population sustainability. This study provided a robust theoretical foundation and deeper insights into the ecotoxicological impacts and risk assessments of coexisting pollutants.

Biotransformation and oxidative stress markers in yellowfin seabream (Acanthopagrus latus): Interactive impacts of microplastics and florfenicol

This study investigates the combined toxicity of microplastics (MPs) and florfenicol (FLO) on biotransformation enzymes and oxidative stress biomarkers in the liver and kidney of yellowfin seabream (Acanthopagrus latus). Fish were fed 15 mg kg−1 of FLO and 100 or 500 mg kg−1 of MPs for 10 days. Biomarkers, including ethoxyresorufin-O-deethylase, glutathione-S-transferase, superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde (MDA), and protein carbonylation (PC), were measured in both organs at 1, 7, and 14 days post-exposure. FLO levels peaked on day 1 and declined after that. Liver biomarkers were more responsive to pollutants, with the combined exposure of FLO and MPs leading to more pronounced toxicity. By day 14, only the FLO group showed a return to baseline biomarker levels, while MDA and PC levels remained elevated in MPs and co-exposed groups. These findings highlight the importance of considering the interactive effects of multiple pollutants in addressing marine environmental stressors.

A mechanistic study on the interaction effects between legacy and pollutants of emerging concern: A case study with B[a]P and diclofenac

To study the intricate toxicological mechanisms triggered by exposure to mixed pollutants, we exposed zebrafish embryos to legacy and emerging pollutants through binary mixtures of benzo[a]pyrene (B[a]P) and diclofenac (DFC). The combination of next-generation transcriptomics and toxicopathology disclosed instances where exposure to mixtures did not attain the expected sum of acute effects of individual toxicants, indicating potential antagonism. Despite overall higher mortality in DFC treatments, the same antagonistic trend was noted in genotoxicity and molecular pathways related to RNA turnover, cell proliferation, apoptosis and cell-cycle control. The formation of oedemas in the heart cavity and yolk sac can be an adverse outcome (AO) resulting from exposure to DFC isolated or combined, whose potential key events (KEs) may involve cell cycle arrest and apoptosis via p53 and MAPK pathways. From the findings it can be hypothesised that, rather than genotoxicity, the molecular initiating event (MIE) maybe inflammation triggered by oxidative stress. Nonetheless, the exact role of ROS in the process needs further clarification. Impaired eye function by action of DFC and B[a]P combined may be another AO, in the case caused by ocular degeneration following the suppression of biologic processes and molecular functions involved in eye development and its functionalities, possibly linked to hindered regulation of the expression of hsf4 and cryaa. Altogether, toxicopathology suggests predominance of antagonistic effects, but its integration with mechanism suggests that interactions between DFC and B[a]P in environmentally-relevant concentrations that may lead to hindrance of key functions such as the control of inflammation and cell cycle. These outcomes suggest potentially severe implications for health and survival, in case of prolonged chronic exposure to combined toxicants.

Combined environmental relevant exposure to perfluorooctanoic acid and zinc sulfate enhances apoptosis through binding with endogenous antioxidants in Daphnia magna

Perfluorooctanoic acid (PFOA) is a long-chain legacy congener of the per- and polyfluoroalkyl substances (PFAS) family, notorious as a "forever chemical" owing to its environmental persistence and toxic nature. Essential elements such as zinc (Zn) can cause toxic effects when they change their metal speciation and become bioavailable, such as zinc sulphate (ZnSO4). Combined toxicity assessment is a realistic approach and a challenging task to evaluate chemical interactions and associated risks. Therefore, the present study aims to elucidate the acute mixture toxicity (12–48 hours) of PFOA and ZnSO4 in Daphnia magna at environment-relevant concentrations (ERCs, low dose: PFOA 10 μg/L ZnSO4 20 μg/L; high dose: PFOA 20 μg/L ZnSO4 50 μg/L) in terms of developmental impact, apoptosis induction, and interaction with major endogenous antioxidants. Our results showed that deformity rates significantly increased (p < 0.05) with increasing exposure duration and exposure concentrations, compared to the control group. Further, lack of antenna, tale degeneration, and carapace alterations were the most commonly observed deformities following combined exposure to PFOA and ZnSO4, and these malformations were particularly pronounced after 48 h of exposure. Acridine orange (AO) staining was employed to examine apoptosis in D. magna, and apoptotic cells in terms of bright green fluorescence were detected in the abdominal claw carapace, heart, and post-abdominal area following exposure to a high dose of PFOA and ZnSO4. The molecular docking results revealed that both PFOA and ZnSO4 showed strong binding affinities with endogenous antioxidants CAT and GST, where PFOA was more strongly bound with CAT and GST with higher docking scores of -9.59 Kcal/mol and -7.49 Kcal/mol than those with ZnSO4 (-6.70 Kcal/mol and -6.55 Kcal/mol, respectively). In conclusion, the mixture exposure to PFOA and ZnSO4 at the environmental level induce developmental impacts and apoptosis through binding with major endogenous antioxidants in D. magna.