Multi-model Analysis of the Combined Toxic Effects of Ultrafine Carbon Black and Amisulbrom on A549 Cells

Multi-model Analysis of the Combined Toxic Effects of Ultrafine Carbon Black and Amisulbrom on A549 Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multi-model Analysis of the Combined Toxic Effects of Ultrafine Carbon Black and Amisulbrom on A549 Cells Wenyuan Xu, Xingyu Yue, Zengxue Liu, Zhuang Ye, Zhenghan Wang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9386037/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Ultrafine carbon black (UFCB), a typical nanoscale component of PM₂.₅, is environmentally persistent and prone to forming composite pollutants. Amisulbrom, a widely used pesticide, can migrate synergistically with atmospheric contaminants, yet their combined health risks remain unclear. This study evaluated the individual and combined toxicities of UFCB and amisulbrom in A549 cells. Both agents induced dose-dependent cytotoxicity, with 24 h IC₅₀ values of 19.79 and 54.45 µg/mL, respectively. Multiple models including Bliss independence, weighted quantile sum, and Bayesian kernel machine regression consistently showed an additive effect rather than synergism or antagonism, with UFCB as the main contributor. Combined exposure significantly increased intracellular ROS and apoptosis, but glutathione and malondialdehyde showed no synergistic changes. Measured indicators were comparable to theoretical values, further verifying the additive joint effect. Earth and environmental sciences/Environmental sciences Health sciences/Risk factors UFCB Amisulbrom Combined exposure Multi-model analysis Oxidative stress Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights • UFCB and amisulbrom induce dose-dependent toxicity in A549 cells. • Multi-model analysis verifies additive toxicity, with UFCB as the main contributor. • Oxidative stress and apoptosis mediate the additive toxicity of mixed pollutants. 1. Introduction With the acceleration of industrialization and the widespread application of modern pesticides, the coexistence of atmospheric nanoparticles and pesticides has become a global environmental issue 1 – 4 . Ultrafine carbon black (UFCB), a primary emission product generated by human activities such as industrial production and transportation, features particle sizes ≤ 100 nm and an enormous specific surface area. UFCB readily adsorbs persistent organic pollutants such as polycyclic aromatic hydrocarbons and can directly penetrate the alveolar barrier via the respiratory tract, inducing lung tissue inflammation, oxidative stress, and even cell apoptosis. Consequently, UFCB represents a significant risk factor for respiratory diseases 5 – 9 . Amisulbrom is a novel sulfonamide fungicide that is widely employed to control oomycete diseases in crops such as cucumbers and tomatoes 10 , 11 . Reports indicate that amisulbrom exhibits a half-life (DT50) of 143 days in soil and 100 days in aquatic sediments 10 , 12 . Over the long time, its environmental residues can persist in environmental media via different pathways, including atmospheric deposition and soil volatilization 13 . UFCB nanoparticles occur widely in the atmosphere. The coexistence of both substances in the atmosphere may lead to the generation of mixed pollutants 14 , with potential combined respiratory exposure and health risks. However, the associated joint health risks of amisulbrom and UFCB remain unclear. Current studies on the individual toxicity of UFCB or amisulbrom have made some progress, confirming that both can induce cellular damage through inducing oxidative stress. For example, Chu, et al. 5 demonstrated that UFCB induced cytotoxicity and apoptosis in mouse lung fibroblasts by activating the mitochondrial pathway via oxidative stress. Similarly, Kim, et al. 11 confirmed that amisulbrom caused apoptosis in human trophoblast and endometrial cells by inducing mitochondrial dysfunction through oxidative stress. However, organisms in real-world environments often face combined exposure to multiple pollutants. The combined toxic effects of multiple pollutants are not simply the sum of individual effects but may exhibit complex characteristics, spanning synergistic, additive, or antagonistic interactions 15 , 16 . Existing studies have not yet clarified the toxic impact of combined exposure to UFCB and amisulbrom. The accurate evaluation of combined toxic effects requires scientifically grounded modeling approaches. For example, Liu, et al. 17 utilized the Bliss independence model to assess the dose–response relationship of drug interactions. Gennings, et al. 18 introduced improved lag-weighted quantile and regression models to analyze the combined exposure effects of multi-component environmental chemicals over time. Liang, et al. 19 employed the Bayesian kernel machine regression (BKMR) to explore the combined impact of multiple metal elements on population hearing loss. Accordingly, this study employed multi-model analysis to explore the toxicological effects of amisulbrom and UFCB. A549 cells, a representative human lung adenocarcinoma cell line, exhibit functional characteristics highly similar to those of alveolar epithelial cells, making them a classical in-vitro model for evaluating the toxicity of respiratory pollutants 20 – 22 . This cell line is widely used in respiratory toxicity mechanism studies. In previous work, Diao et al. utilized A549 cells to investigate avermectin-induced DNA damage and the resulting apoptosis and autophagy processes. Ramushu et al. investigated lithium exposure-induced oxidative stress, apoptosis, and G2/M phase arrest. Kong et al. employed transcriptomic analysis to reveal the biological effects of carbon black exposure on A549 cells. Among these toxicity mechanisms, oxidative stress is recognized as a core pathway for pollutant-induced cellular damage. Changes in related markers such as reactive oxygen species (ROS), glutathione (GSH), and malondialdehyde (MDA) can characterize the extent of oxidative injury. Furthermore, the close association between oxidative stress and apoptosis provides crucial clues for elucidating the mechanisms and patterns underlying the toxic effects 5 , 11 , 16 . In this study, A549 cells were used as the research subject to determine the physicochemical properties of UFCB based on physicochemical characterization. CCK-8 assays were conducted to evaluate the impact of UFCB and amisulbrom exposure, both individually and in combination, on cell viability through determining the half-maximal inhibitory concentration (IC 50 ) values. Three models (Bliss 23 , WQS 24 , and BKMR 25 ) were used to analyze the interaction patterns, dominant factors, and independent effect profiles. To further validate the synergistic toxicity effects, oxidative stress and apoptosis-related indicators were calculated using the Bliss model. This study aimed to reveal the toxic characteristics and synergistic effects of combined exposure to UFCB and amisulbrom, providing experimental data and theoretical support for health risk assessments of combined exposure to environmental pollutants. 2. Materials and Methods 2.1 Chemicals Amisulbrom (purity 95%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., and the stock solution was prepared using DMSO, stored at − 20°C after packaging, and diluted to the required concentration using the medium during the experiment. UFCB granules were purchased from Anhui Black Cat New Material Co., Ltd., 5 mg of UFCB was irradiated by ultraviolet, dissolved in 5mL complete medium, prepared into 1000 µg/mL mother liquor, sonicated before use and diluted to working concentration 26 . 2.2 Characterization of UFCB UFCB was dispersed in ultrapure water, filtered by ultrasound and 0.22 µm filter membrane, and the content of endotoxin was detected by limulus lysate kinetic turbidimetry. Morphology characterization was performed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The hydrodynamic particle size and Zeta potential were measured by dynamic light scattering (DLS) in 20 µg/mL UFCB complete medium solution. In addition, 16 polycyclic aromatic hydrocarbons (PAHs) were quantitatively analyzed by high performance liquid chromatography-fluorescence method (HPLC-FLD) to evaluate their organic pollutant loading. 2.3 Cell viability measurement A549 lung epithelial cells (NTCC No.: CVCL-6926) were purchased from Wuhan Hua’erna Company and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded at 1×10 4 cells/well in a 96-well plate. After 24 hours, cells were exposed to different concentrations of UFCB, amisulbrom, and DMSO, respectively, and cultured for an additional 24 hours. Subsequently, 10 µL of CCK-8 reagent was added to each well, incubated for 45–60 minutes, and absorbance was measured at 450 nm. All experimental groups were independently replicated at least three times, with 3–6 parallel replicates per well. 2.4 Determination of ROS Levels in A549 Cells DCFH-DA staining was used to detect intracellular ROS levels. A549 cells were seeded in 6-well plates at a rate of 4×105 per well, cultured for 24 h and adhered to the wall, and then subjected to exposure treatment for 24 h, respectively. Three parallel wells were set for each dose. After treatment, 10 µM DCFH-DA was incubated in 1 mL serum-free medium at 37 ℃ in the dark for 30 minutes, and the fluorescence intensity was detected in the FITC channel of flow cytometry. 2.5 Determination of Intracellular GSH and MDA Contents Collect cells treated with the toxin for 24 hours (3 replicates per dose), resuspend in pre-chilled PBS, and disrupt cells. Centrifuge and collect the supernatant. GSH content was quantified using the Nanjing Jiancheng kit via the thiol colorimetric method; MDA content was determined using the Biyuntian lipid peroxidation assay kit based on the TBA condensation reaction, measured colorimetrically at 532 nm. 2.6 Cell Apoptosis Apoptosis was detected using the Annexin V-FITC/PI dual staining method. Cells treated with the toxic agent for 24 hours were collected, resuspended in Annexin V binding buffer, and then sequentially incubated with 5 µL Annexin V-FITC and 5 µL PI at room temperature in the dark for 10 minutes. The samples were analyzed using a CytoFlex flow cytometer, and the proportion of apoptotic cells was determined via FlowJo software. 2.7 Statistical analysis All data analysis and graphing were performed in GraphPad Prism software. Experimental data were derived from three independent replicate experiments, with the results expressed as the mean ± standard deviation (SD). Differences among multiple groups were compared using one-way analysis of variance (ANOVA), with p < 0.05 indicating statistically significant differences. Quantitative analysis of the interaction between UFCB and amisulbrom was performed using the Bliss independence model, weighted quantile sum (WQS) regression model, and BKMR model. The Bliss independence model calculates the theoretical combined inhibition rate (PE = E1 + E2 − E1 × E2) based on the cell inhibition rates of individual exposures (E1 and E2). The interaction type is then determined using the combined interaction index (CI = PE/OE), where the predicted effect (PE) represents the predicted value for the combined exposure group; and the observed effect (OE) is the measured value for the combined exposure group. CI 1.1 indicates antagonism. WQS regression and BKMR analyses were implemented using the "gWQS" and "bkmr" packages in R software, respectively. The synergistic effect of mixture toxicity was assessed by comparing the PE with the OE at each endpoint. The OE, defined as the sum of the toxicity effect values produced by individual chemical exposures, was calculated using the method reported by Li, et al. 27 . One-way ANOVA was conducted to test the significance of differences between the observed and theoretical values, thereby identifying interactions. The interactive effects of amisulbrom and UFCB were classified according to the following criteria: (1) no significant difference between the observed values and theoretical values indicated additive interaction; (2) observed values significantly higher than theoretical values indicated synergistic interaction; and (3) significantly lower observed values compared to theoretical values indicated antagonistic interaction. 3. Results and Discussion 3.1 Characterization of UFCB Defining the physicochemical properties of UFCB is crucial for investigating its toxic effects on biological organisms. Therefore, this study systematically characterized the key properties of UFCB (Table 1 ). The measured particle size of UFCB was 20 ± 2 nm, with a Brunauer–Emmett–Teller (BET) surface area of 273 ± 5 m 2 /g. A larger BET surface area enhances the pollutant adsorption capacity of UFCB particles as well as their interactions with cells, thereby influencing the toxic effects of UFCB. Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) observations (Fig. 1 A) revealed that nanoscale spherical particles of UFCB were prone to chain-like agglomeration in media such as ethanol. This morphology aligns with the findings of Chu, et al. 5 regarding carbon black nanoparticles and reflects common agglomeration patterns of nanoparticles in media. In complete medium (20 µg/mL), UFCB exhibited a peak hydrodynamic diameter of 251.27 ± 12.63 nm (Fig. 1 B), a volume-average diameter of 261.5 ± 13.3 nm, and a polydispersity index of 0.4 ± 0.07, indicating moderate dispersion without significant agglomeration. The endotoxin detection result was 123 EU/g, lower than the level reported by Bourdon JA et al. in previous research on the toxicity of carbon black 28 , thus ruling out its systemic impact on the experiment. Additionally, the total content of 16 polycyclic aromatic hydrocarbons was 231.52 ng/g, including 10.25 ng/g benzo(a)pyrene. This fell below the relevant safety limits set by the German Commission on Product Safety 29 , indicating that contaminant residues within the UFCB would exert minimal interference during toxicity testing, making them suitable for subsequent toxicological evaluation. 3.2 Effects of Single Exposure on Cell Viability We assessed the viability of A549 cells treated with different concentrations of amisulbrom, UFCB, and the solvent dimethyl sulfoxide (DMSO) after 24 h. DMSO showed no significant effect on A549 cell viability at concentrations ≤ 0.5% ( p > 0.05), but it significantly inhibited cell survival at concentrations ≥ 2% ( p < 0.05). Therefore, the final DMSO concentration in the amisulbrom exposure group was strictly controlled below 0.05% in subsequent experiments (Fig. 2 A). UFCB exhibited typical dose-dependent cytotoxic effects within the 5–150 µg/mL concentration range, with an IC 50 of 19.79 µg/mL (Fig. 2 B). This concentration aligns with the cellular survival rate reported by Chu, et al. 5 in previous research on the toxicity of 20-nm UFCB to lung cells. Amisulbrom also exhibited significant dose effects within the 5–150 µg/mL range, with an IC 50 of 54.45 µg/mL (R 2 = 0.966) (Fig. 2 C). Sensitivity to amisulbrom varies among cell lines. Kim, et al. 11 reported IC 50 values of 20.37 µg/mL for HTR-8/SVneo cells and 16.18 µg/mL for ThESCs after 48 hours of exposure to amisulbrom. The current study is the first to demonstrate that amisulbrom exhibits distinct pulmonary cytotoxicity in the A549 cell line, suggesting that respiratory exposure may pose potential human health risks. Overall, UFCB exhibited significantly greater toxicity than amisulbrom, with individual exposure to both compounds producing clear dose-dependent cytotoxic effects in A549 cells. These findings provide a scientific basis for designing dose levels in subsequent combined exposure experiments. 3.3 Effects of Combined Exposure on Cell Viability and Multi-Model Analysis The effects of combined pollutant exposure vary in nature. Taenzler, et al. 30 found that the acute toxicity of mixed pesticide formulations to honeybees predominantly exhibited additive effects. In contrast, Wu, et al. 16 observed that co-exposure to lead and copper exacerbated oxidative stress and apoptosis in neuronal cells via synergistic interactions, while Zhang, et al. 31 found that synergistic hepatotoxicity was induced by the combination of avermectin and cyantraniliprole. Therefore, based on the toxicity results from individual exposure tests, this study employed combined exposures at concentrations of 0.125 ×, 0.25 ×, 0.5 ×, and 1 × the IC 50 . The results showed that the cell survival rates in the combined exposure groups at all concentration combinations were significantly lower than those in the corresponding single-exposure groups ( p < 0.01). Under combined exposure to both agents at a concentration of 1 × IC 50 , the survival rate in the combined exposure group dropped to 26.47%, significantly lower than both the UFCB alone group (55.36%) and the amisulbrom alone group (54.37%), demonstrating that combined exposure exerted a stronger toxic effect. To accurately identify the patterns of synergistic interaction between the two components and overcome the limitations of single-model analysis, this study employed a multi-model integration strategy involving cross-validation with three statistical models: the Bliss independence model, WQS regression, and BKMR. This approach enabled the analysis of combined toxicity characteristics from multiple dimensions, thus significantly enhancing the reliability of mixture toxicity assessment. The Bliss independent action model results yielded a CI value of 0.95 (95% CI: 0.82–1.08). This fell within the additive range, suggesting that combined toxicity exerted an additive effect. The WQS model analysis revealed that the weight of UFCB in the mixed exposure was 0.90, significantly higher than that of amisulbrom (0.10). Furthermore, the interaction term between the WQS index and UFCB dose showed no statistical significance ( p > 0.05), supporting the additive nature of their combined effect. The results of BKMR modeling further validated these conclusions. The interaction coefficient was 0.27 ( p > 0.05), confirming no significant interaction between the two compounds. Variable importance analysis showed that the posterior inclusion probability of UFCB was 1.00, identifying it as a key toxicity factor. In contrast, the posterior inclusion probability of amisulbrom was only 0.12, indicating its weaker and less stable influence on combined toxicity. Univariate analysis also revealed that UFCB significantly inhibited cell viability ( p 0.05). The three models reached consistent conclusions across the three dimensions of overall toxicity assessment, component contribution analysis, and interaction identification. The results confirmed that combined exposure to UFCB and amisulbrom exerted a UFCB-dominant additive effect on A549 cells, with no synergistic or antagonistic enhancement observed. 3.4 Effects of Combined Exposure on Oxidative Stress and Apoptosis in A549 Cells Oxidative stress serves as a common key indicator of nanoparticle- and pesticide-induced cytotoxicity 5 , 11 , which typically manifests as elevated intracellular ROS levels, depletion of the antioxidant GSH, and accumulation of the lipid peroxidation product MDA. Oxidative damage that exceeds the cellular repair capacity can further trigger apoptosis. To validate the toxic effects and additive characteristics of combined exposure to UFCB and amisulbrom at the molecular and cellular levels, this study selected ROS, GSH, MDA, and apoptosis rate as detection indicators to systematically evaluate the oxidative damage and cellular fate responses induced by combined exposure. Regarding ROS levels, all exposure groups exhibited significantly higher ROS levels than the control group ( p < 0.05). Amisulbrom monotherapy induced a greater increase in ROS than UFCB (Fig. 3 A). The combined exposure group (20 µg/mL UFCB + amisulbrom) exhibited a further significant elevation in ROS levels, surpassing both single exposure groups and indicating an additive effect in ROS induction. Similarly, Chu, et al. 5 reported that UFCB synergistically increased ROS levels in mouse lung fibroblasts when combined with lead exposure, suggesting potential interactions between UFCB and other environmental pollutants at the oxidative stress level. In terms of the antioxidant capacity, GSH levels were significantly reduced in all exposure groups ( p < 0.05). UFCB exposure resulted in a dose-dependent decrease, while the depletion effect of amisulbrom was more pronounced. The combined exposure group exhibited a decrease in GSH intermediate between the two single-exposure groups, without demonstrating synergistic enhancement (Fig. 4 B). Regarding lipid peroxidation, MDA levels were significantly enhanced in all exposure groups ( p UFCB). More intriguingly, the MDA levels in the combined exposure group were significantly lower than those in the high-dose UFCB-alone group, despite exhibiting significantly higher ROS levels than the UFCB-alone group. The examination of apoptosis revealed that after 24 hours of single exposure to UFCB or amisulbrom, the total apoptosis rate showed no significant difference compared to the control group. This suggested that at the doses used in this experiment, single exposure primarily induced reversible cellular stress without triggering large-scale apoptosis (Fig. 4 D). However, the combined exposure group (20 µg/mL UFCB + amisulbrom) exhibited a significant increase in the total apoptosis rate, which reached 11.86% ( p < 0.05), substantially higher than that of either single-exposure group. This result strongly correlated with cell viability assays, which showed that cell survival dropped to 26.47% under combined exposure at the IC 50 concentration, further confirming the combined toxic effect of combined exposure. Two seemingly contradictory phenomena emerged in the above results. First, although UFCB had a stronger inhibitory effect on cell viability than amisulbrom, its ROS induction capacity was weaker. Second, while UFCB alone induced greater MDA accumulation than amisulbrom, combined exposure resulted in lower MDA levels than exposure to UFCB alone. These findings suggest that the toxic mechanisms of UFCB and amisulbrom are fundamentally different. As a type of particulate matter, the cytotoxicity of UFCB stems from the interaction between its unique physical properties and potential chemical perturbations. The TEM observations of Li, et al. 32 indicate that UFCB can be internalized by cells via endocytosis, forming vesicular structures within the cytoplasm that directly interfere with organelle function and induce mechanical damage. This physical disruption may be the primary driver of UFCB-induced cell viability decline, while oxidative stress plays only a secondary or auxiliary role in its toxic effects. Although UFCB exhibits a relatively weak ROS-inducing capacity, the physical damage it causes may compromise cell membrane integrity and enhance lysosomal membrane permeability, leading to the release of pro-oxidants (such as free iron ions). This explains why exposure to UFCB alone induces strong lipid peroxidation—a process that can result from both direct ROS attack and secondary oxidative events following membrane structural damage 33 . The significantly lower MDA levels in the combined exposure group compared to the UFCB group can be explained by a shift in the injury pattern. When cells are exposed to UFCB alone, physical damage may lead to the extensive exposure of lipid peroxidation substrates (polyunsaturated fatty acids), resulting in increased MDA production. Under combined exposure with amisulbrom, cells may initiate alternative death pathways (such as apoptosis). Apoptosis is relatively "clean," preserving membrane integrity early in the process and consequently reducing the availability of lipid peroxidation substrates 34 . In contrast, as a pesticide molecule, amisulbrom tends to exert its toxic effects via biochemical pathways such as inducing oxidative stress and interfering with metabolic enzyme activit 35 , consistent with its characteristics of strongly inducing ROS and significantly depleting GSH. To quantify synergistic toxicity, this study calculated the theoretical summation value using the Bliss model and compared it with the measured values. The results showed no significant differences between the measured and theoretical values for all detected indicators, further confirming the additive effect of combined amisulbrom and UFCB exposure on the induction of oxidative stress and apoptosis (Fig. 5 ). Collectively, these findings suggest that under the experimental conditions, amisulbrom and UFCB jointly mediate cellular damage by exacerbating oxidative stress, disrupting redox homeostasis, and initiating apoptotic programs through an additive effect model. 4. Conclusions This study systematically evaluated the toxic effects and mechanisms of UFCB and the novel fungicide amisulbrom, both individually and in combination, on human lung adenocarcinoma A549 cells. The results showed that both UFCB and amisulbrom exposure inhibited A549 cell proliferation in a dose-dependent manner. Cross-validation using multiple models (Bliss, WQS, and BKMR) confirmed an additive effect due to combined exposure, with UFCB being the primary contributor to combined toxicity. The underlying mechanism was closely associated with the induction of excessive intracellular ROS production and apoptosis under combined exposure. Furthermore, no significant differences were observed between the measured and theoretical values for all assessed indicators, further supporting the additive nature of combined UFCB and amisulbrom toxicity. This study provides critical toxicological data for the health risk assessment of combined exposure to atmospheric nanoparticles and pesticides while offering methodological references for multi-model combined toxicity analysis of complex pollutants. In future work, subsequent in-vivo experiments and deeper molecular mechanism studies are required to refine the health risk assessment system and validate its relevance to human health. Declarations Data availability All data generated or analysed during this study are included in this published article. Funding This work was supported by the National Natural Science Foundation of China (32402409). Author contributions Wenyuan Xu (First Author): Writing - Original Draft, Methodology, Conceptualization, Data Curation; Xingyu Yue (Co-first Author): Writing - Review & Editing, Supervision, Validation; Zengxue Liu: Investigation; Formal analysis; Zhuang Ye: Software, Visualization; Zhenghan Wang: Resources; Xiaofan Li: Supervision; Yanhong Shi (Corresponding Author): Conceptualization, Funding Acquisition, Supervision. Declaration of Competing Interest s The authors declare no competing financial interests. References Lin, Z. et al. PM10 and PM2.5 chemical source profiles of road dust over China: Composition, spatio-temporal distribution, and source apportionment. Urban Clim. 51 https://doi.org/10.1016/j.uclim.2023.101672 (2023). Ni, J., Cai, M., Lin, Y., Li, T. & Ma, J. Occurrence, seasonal variations, and spatial distributions of current-use organoamine pesticides in the atmosphere of Shanghai, China. Atmospheric Pollution Res. 15 https://doi.org/10.1016/j.apr.2024.102187 (2024). 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Cell Death Dis. 15 https://doi.org/10.1038/s41419-024-07244-x (2024). Sule, R. O., Condon, L., Gomes, A. V. & Zhou, X. A. Common Feature of Pesticides: Oxidative Stress—The Role of Oxidative Stress in Pesticide-Induced Toxicity. Oxidative Medicine and Cellular Longevity (2022). (2022) https://doi.org/10.1155/2022/5563759 Tables Table 1 Physicochemical properties of UFCB and parameters of its dispersion system in complete medium Dimensions declared by the manufacturer(nm) UFCB 20 ± 2 BET surface area(m 2 /g) 273 ± 5 Endotoxin content(EU/g) 123 Dispersion in complete medium: 20 µg/mL (DLS-verified). Zeta Potential (mV) -4.63 ± 0.71 Peak hydrodynamic diameter(nm) 251.27 ± 12.63 volume-based peak diameter(nm) 261.5 ± 13.3 PDI 0.4 ± 0.07 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 May, 2026 Reviews received at journal 06 May, 2026 Reviews received at journal 27 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers invited by journal 16 Apr, 2026 Editor assigned by journal 15 Apr, 2026 Editor invited by journal 15 Apr, 2026 Submission checks completed at journal 14 Apr, 2026 First submitted to journal 14 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9386037","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":626414573,"identity":"cadf128a-951c-4f60-91d8-ba06e4c41faf","order_by":0,"name":"Wenyuan Xu","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Wenyuan","middleName":"","lastName":"Xu","suffix":""},{"id":626414574,"identity":"5072e0b9-f14e-457d-bdcc-35d86057033f","order_by":1,"name":"Xingyu Yue","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xingyu","middleName":"","lastName":"Yue","suffix":""},{"id":626414576,"identity":"74489537-8990-4be6-b63f-92bd15ec9990","order_by":2,"name":"Zengxue Liu","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zengxue","middleName":"","lastName":"Liu","suffix":""},{"id":626414577,"identity":"5c61f9f2-cae5-43d7-8b67-b00713451109","order_by":3,"name":"Zhuang Ye","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhuang","middleName":"","lastName":"Ye","suffix":""},{"id":626414578,"identity":"dec425ce-df8b-4ee1-93f2-a20d8b4a113f","order_by":4,"name":"Zhenghan Wang","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhenghan","middleName":"","lastName":"Wang","suffix":""},{"id":626414579,"identity":"5ee7f72a-433a-4a97-8605-568cb10910e9","order_by":5,"name":"Xiaofan Li","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaofan","middleName":"","lastName":"Li","suffix":""},{"id":626414580,"identity":"1ba8dd59-c797-4361-a9ba-6a570c290dfd","order_by":6,"name":"Yanhong Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtElEQVRIiWNgGAWjYFACHsYHjA1glgHRWpgNSNbCJkGaFvkZuccqfu6wS2xgb94mwVBzh7AWgxt5aTd7zyQnNvAcK5NgOPaMCC0SOWY3eNuYExuADKALDxPjsByzwr9t9YkN8m+I1MJwI8eMmbftMNAWHiK1GJx5Yywt23bcuI0nrdgi4RgxDmvPMfz4tq1atp/98MYbH2qIcZhAAoRmAxEJRGhgYOA/QJSyUTAKRsEoGMkAAGFcOAHpvJAuAAAAAElFTkSuQmCC","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Yanhong","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2026-04-11 08:38:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9386037/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9386037/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107741337,"identity":"d47f8e62-dcdb-41e7-a4d6-9624a430eb18","added_by":"auto","created_at":"2026-04-24 15:01:23","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":467581,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of ultrafine carbon black (UFCB). (A) Morphological characterization of UFCB. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and scanning transmission electron microscopy (STEM) images are shown from left to right. (B) Particle size distribution curve of UFCB at a concentration of 20 μg/mL in complete medium. (C) Content distribution of polycyclic aromatic hydrocarbons (PAHs) in UFCB, with data points being the average values of three replicate detections.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9386037/v1/fb34c5bc661a219f0d146037.jpeg"},{"id":107869115,"identity":"ace34b96-4c2c-428c-9107-e80c2019529f","added_by":"auto","created_at":"2026-04-27 07:36:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":281314,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of solvent, ultrafine carbon black (UFCB), and amisulbrom alone and their combined exposure on the viability of A549 cells. (A) Effect of dimethyl sulfoxide (DMSO) on A549 cell viability. (B) Effect of UFCB on A549 cell viability. (C) Effect of amisulbrom on A549 cell viability (24 hours of exposure). The statistical significance of all quantitative data is indicated by asterisks as follows: * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9386037/v1/9a58d20fb82dd55f7544b177.jpeg"},{"id":107741339,"identity":"18592e52-80b9-42d9-b5fc-4db47ee26cb1","added_by":"auto","created_at":"2026-04-24 15:01:23","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":521668,"visible":true,"origin":"","legend":"\u003cp\u003eMulti-model analysis of A549 cell viability under single and combined exposure to ultrafine carbon black (UFCB) and amisulbrom (Am). (A) Effects of UFCB, Am, and their combination (UFCB + Am) on A549 cell viability at different multiples of the half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) (n ≥ 3). (B) Heatmap of cell viability under combined exposure to different concentrations of UFCB and Am. (C) Weight distribution of UFCB and Am in the weighted quantile sum (WQS) model. (D–E) Interactive effects between the WQS mixture index and the dosage of UFCB (D) or Am (E) on cell viability. (F–J) Bayesian kernel machine regression (BKMR) model analysis of the overall effect of combined dose quantiles on cell viability (F), dose–response curves for single exposures (G), and interactive effects between the two agents (J).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9386037/v1/0cd7148ca43fd3d55adab092.jpeg"},{"id":107741340,"identity":"6e15bd9d-e4c9-4885-8756-a3557240c8f6","added_by":"auto","created_at":"2026-04-24 15:01:23","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":392592,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ultrafine carbon black (UFCB) and amisulbrom (Am) alone and their combined exposure on oxidative stress-related indicators in A549 cells. (A) Flow cytometry results for intracellular reactive oxygen species (ROS) levels. (B) Intracellular reduced glutathione (GSH) content. (C) Intracellular malondialdehyde (MDA) content. The statistical significance of all quantitative data is indicated by asterisks as follows: * \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; **** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9386037/v1/15a418aa727650d6e76e0968.jpeg"},{"id":107869498,"identity":"dff80969-4fae-4f46-944a-7da6e2ef9636","added_by":"auto","created_at":"2026-04-27 07:37:11","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":159813,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of observed effect (OE) and predicted effect (PE) values of oxidative stress and apoptosis indicators under combined exposure to amisulbrom and ultrafine carbon black (UFCB). (A) Reactive oxygen species (ROS). (B) Malondialdehyde (MDA) content. (C) Glutathione (GSH) levels. (D) Apoptosis rate. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 indicates a significant difference between OE and PE (synergism); ns, no significant difference (additive effect).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9386037/v1/b36fa8e061a120896d73f9a4.jpeg"},{"id":108181044,"identity":"23e922db-45a2-463d-aad4-950e3e8f598a","added_by":"auto","created_at":"2026-04-30 08:56:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2113327,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9386037/v1/c18e92ef-aedb-4732-afea-52151d4d8905.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multi-model Analysis of the Combined Toxic Effects of Ultrafine Carbon Black and Amisulbrom on A549 Cells","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; UFCB and amisulbrom induce dose-dependent toxicity in A549 cells.\u003c/p\u003e\u003cp\u003e\u0026bull; Multi-model analysis verifies additive toxicity, with UFCB as the main contributor.\u003c/p\u003e\u003cp\u003e\u0026bull; Oxidative stress and apoptosis mediate the additive toxicity of mixed pollutants.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eWith the acceleration of industrialization and the widespread application of modern pesticides, the coexistence of atmospheric nanoparticles and pesticides has become a global environmental issue\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Ultrafine carbon black (UFCB), a primary emission product generated by human activities such as industrial production and transportation, features particle sizes\u0026thinsp;\u0026le;\u0026thinsp;100 nm and an enormous specific surface area. UFCB readily adsorbs persistent organic pollutants such as polycyclic aromatic hydrocarbons and can directly penetrate the alveolar barrier via the respiratory tract, inducing lung tissue inflammation, oxidative stress, and even cell apoptosis. Consequently, UFCB represents a significant risk factor for respiratory diseases\u003csup\u003e\u003cspan additionalcitationids=\"CR6 CR7 CR8\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Amisulbrom is a novel sulfonamide fungicide that is widely employed to control oomycete diseases in crops such as cucumbers and tomatoes\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Reports indicate that amisulbrom exhibits a half-life (DT50) of 143 days in soil and 100 days in aquatic sediments\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Over the long time, its environmental residues can persist in environmental media via different pathways, including atmospheric deposition and soil volatilization\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. UFCB nanoparticles occur widely in the atmosphere. The coexistence of both substances in the atmosphere may lead to the generation of mixed pollutants\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, with potential combined respiratory exposure and health risks. However, the associated joint health risks of amisulbrom and UFCB remain unclear.\u003c/p\u003e \u003cp\u003eCurrent studies on the individual toxicity of UFCB or amisulbrom have made some progress, confirming that both can induce cellular damage through inducing oxidative stress. For example, Chu, et al. \u003csup\u003e5\u003c/sup\u003e demonstrated that UFCB induced cytotoxicity and apoptosis in mouse lung fibroblasts by activating the mitochondrial pathway via oxidative stress. Similarly, Kim, et al. \u003csup\u003e11\u003c/sup\u003e confirmed that amisulbrom caused apoptosis in human trophoblast and endometrial cells by inducing mitochondrial dysfunction through oxidative stress. However, organisms in real-world environments often face combined exposure to multiple pollutants. The combined toxic effects of multiple pollutants are not simply the sum of individual effects but may exhibit complex characteristics, spanning synergistic, additive, or antagonistic interactions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Existing studies have not yet clarified the toxic impact of combined exposure to UFCB and amisulbrom. The accurate evaluation of combined toxic effects requires scientifically grounded modeling approaches. For example, Liu, et al. \u003csup\u003e17\u003c/sup\u003e utilized the Bliss independence model to assess the dose\u0026ndash;response relationship of drug interactions. Gennings, et al. \u003csup\u003e18\u003c/sup\u003e introduced improved lag-weighted quantile and regression models to analyze the combined exposure effects of multi-component environmental chemicals over time. Liang, et al. \u003csup\u003e19\u003c/sup\u003e employed the Bayesian kernel machine regression (BKMR) to explore the combined impact of multiple metal elements on population hearing loss. Accordingly, this study employed multi-model analysis to explore the toxicological effects of amisulbrom and UFCB.\u003c/p\u003e \u003cp\u003eA549 cells, a representative human lung adenocarcinoma cell line, exhibit functional characteristics highly similar to those of alveolar epithelial cells, making them a classical in-vitro model for evaluating the toxicity of respiratory pollutants \u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. This cell line is widely used in respiratory toxicity mechanism studies. In previous work, Diao et al. utilized A549 cells to investigate avermectin-induced DNA damage and the resulting apoptosis and autophagy processes. Ramushu et al. investigated lithium exposure-induced oxidative stress, apoptosis, and G2/M phase arrest. Kong et al. employed transcriptomic analysis to reveal the biological effects of carbon black exposure on A549 cells. Among these toxicity mechanisms, oxidative stress is recognized as a core pathway for pollutant-induced cellular damage. Changes in related markers such as reactive oxygen species (ROS), glutathione (GSH), and malondialdehyde (MDA) can characterize the extent of oxidative injury. Furthermore, the close association between oxidative stress and apoptosis provides crucial clues for elucidating the mechanisms and patterns underlying the toxic effects \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, A549 cells were used as the research subject to determine the physicochemical properties of UFCB based on physicochemical characterization. CCK-8 assays were conducted to evaluate the impact of UFCB and amisulbrom exposure, both individually and in combination, on cell viability through determining the half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values. Three models (Bliss\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, WQS\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and BKMR\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e) were used to analyze the interaction patterns, dominant factors, and independent effect profiles. To further validate the synergistic toxicity effects, oxidative stress and apoptosis-related indicators were calculated using the Bliss model. This study aimed to reveal the toxic characteristics and synergistic effects of combined exposure to UFCB and amisulbrom, providing experimental data and theoretical support for health risk assessments of combined exposure to environmental pollutants.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals\u003c/h2\u003e \u003cp\u003eAmisulbrom (purity 95%) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., and the stock solution was prepared using DMSO, stored at \u0026minus;\u0026thinsp;20\u0026deg;C after packaging, and diluted to the required concentration using the medium during the experiment. UFCB granules were purchased from Anhui Black Cat New Material Co., Ltd., 5 mg of UFCB was irradiated by ultraviolet, dissolved in 5mL complete medium, prepared into 1000 \u0026micro;g/mL mother liquor, sonicated before use and diluted to working concentration\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Characterization of UFCB\u003c/h2\u003e \u003cp\u003eUFCB was dispersed in ultrapure water, filtered by ultrasound and 0.22 \u0026micro;m filter membrane, and the content of endotoxin was detected by limulus lysate kinetic turbidimetry. Morphology characterization was performed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The hydrodynamic particle size and Zeta potential were measured by dynamic light scattering (DLS) in 20 \u0026micro;g/mL UFCB complete medium solution. In addition, 16 polycyclic aromatic hydrocarbons (PAHs) were quantitatively analyzed by high performance liquid chromatography-fluorescence method (HPLC-FLD) to evaluate their organic pollutant loading.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell viability measurement\u003c/h2\u003e \u003cp\u003eA549 lung epithelial cells (NTCC No.: CVCL-6926) were purchased from Wuhan Hua\u0026rsquo;erna Company and cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded at 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well in a 96-well plate. After 24 hours, cells were exposed to different concentrations of UFCB, amisulbrom, and DMSO, respectively, and cultured for an additional 24 hours. Subsequently, 10 \u0026micro;L of CCK-8 reagent was added to each well, incubated for 45\u0026ndash;60 minutes, and absorbance was measured at 450 nm. All experimental groups were independently replicated at least three times, with 3\u0026ndash;6 parallel replicates per well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Determination of ROS Levels in A549 Cells\u003c/h2\u003e \u003cp\u003eDCFH-DA staining was used to detect intracellular ROS levels. A549 cells were seeded in 6-well plates at a rate of 4\u0026times;105 per well, cultured for 24 h and adhered to the wall, and then subjected to exposure treatment for 24 h, respectively. Three parallel wells were set for each dose. After treatment, 10 \u0026micro;M DCFH-DA was incubated in 1 mL serum-free medium at 37 ℃ in the dark for 30 minutes, and the fluorescence intensity was detected in the FITC channel of flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Determination of Intracellular GSH and MDA Contents\u003c/h2\u003e \u003cp\u003eCollect cells treated with the toxin for 24 hours (3 replicates per dose), resuspend in pre-chilled PBS, and disrupt cells. Centrifuge and collect the supernatant. GSH content was quantified using the Nanjing Jiancheng kit via the thiol colorimetric method; MDA content was determined using the Biyuntian lipid peroxidation assay kit based on the TBA condensation reaction, measured colorimetrically at 532 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Cell Apoptosis\u003c/h2\u003e \u003cp\u003eApoptosis was detected using the Annexin V-FITC/PI dual staining method. Cells treated with the toxic agent for 24 hours were collected, resuspended in Annexin V binding buffer, and then sequentially incubated with 5 \u0026micro;L Annexin V-FITC and 5 \u0026micro;L PI at room temperature in the dark for 10 minutes. The samples were analyzed using a CytoFlex flow cytometer, and the proportion of apoptotic cells was determined via FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data analysis and graphing were performed in GraphPad Prism software. Experimental data were derived from three independent replicate experiments, with the results expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences among multiple groups were compared using one-way analysis of variance (ANOVA), with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating statistically significant differences.\u003c/p\u003e \u003cp\u003eQuantitative analysis of the interaction between UFCB and amisulbrom was performed using the Bliss independence model, weighted quantile sum (WQS) regression model, and BKMR model. The Bliss independence model calculates the theoretical combined inhibition rate (PE\u0026thinsp;=\u0026thinsp;E1\u0026thinsp;+\u0026thinsp;E2\u0026thinsp;\u0026minus;\u0026thinsp;E1 \u0026times; E2) based on the cell inhibition rates of individual exposures (E1 and E2). The interaction type is then determined using the combined interaction index (CI\u0026thinsp;=\u0026thinsp;PE/OE), where the predicted effect (PE) represents the predicted value for the combined exposure group; and the observed effect (OE) is the measured value for the combined exposure group. CI\u0026thinsp;\u0026lt;\u0026thinsp;0.9 indicates synergism, CI \u0026isin; [0.9, 1.1] indicates additive effects, and CI\u0026thinsp;\u0026gt;\u0026thinsp;1.1 indicates antagonism. WQS regression and BKMR analyses were implemented using the \"gWQS\" and \"bkmr\" packages in R software, respectively.\u003c/p\u003e \u003cp\u003eThe synergistic effect of mixture toxicity was assessed by comparing the PE with the OE at each endpoint. The OE, defined as the sum of the toxicity effect values produced by individual chemical exposures, was calculated using the method reported by Li, et al. \u003csup\u003e27\u003c/sup\u003e. One-way ANOVA was conducted to test the significance of differences between the observed and theoretical values, thereby identifying interactions. The interactive effects of amisulbrom and UFCB were classified according to the following criteria: (1) no significant difference between the observed values and theoretical values indicated additive interaction; (2) observed values significantly higher than theoretical values indicated synergistic interaction; and (3) significantly lower observed values compared to theoretical values indicated antagonistic interaction.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of UFCB\u003c/h2\u003e \u003cp\u003eDefining the physicochemical properties of UFCB is crucial for investigating its toxic effects on biological organisms. Therefore, this study systematically characterized the key properties of UFCB (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The measured particle size of UFCB was 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm, with a Brunauer\u0026ndash;Emmett\u0026ndash;Teller (BET) surface area of 273\u0026thinsp;\u0026plusmn;\u0026thinsp;5 m\u003csup\u003e2\u003c/sup\u003e/g. A larger BET surface area enhances the pollutant adsorption capacity of UFCB particles as well as their interactions with cells, thereby influencing the toxic effects of UFCB. Transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) revealed that nanoscale spherical particles of UFCB were prone to chain-like agglomeration in media such as ethanol. This morphology aligns with the findings of Chu, et al. \u003csup\u003e5\u003c/sup\u003e regarding carbon black nanoparticles and reflects common agglomeration patterns of nanoparticles in media. In complete medium (20 \u0026micro;g/mL), UFCB exhibited a peak hydrodynamic diameter of 251.27\u0026thinsp;\u0026plusmn;\u0026thinsp;12.63 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), a volume-average diameter of 261.5\u0026thinsp;\u0026plusmn;\u0026thinsp;13.3 nm, and a polydispersity index of 0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, indicating moderate dispersion without significant agglomeration. The endotoxin detection result was 123 EU/g, lower than the level reported by Bourdon JA et al. in previous research on the toxicity of carbon black\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, thus ruling out its systemic impact on the experiment. Additionally, the total content of 16 polycyclic aromatic hydrocarbons was 231.52 ng/g, including 10.25 ng/g benzo(a)pyrene. This fell below the relevant safety limits set by the German Commission on Product Safety \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, indicating that contaminant residues within the UFCB would exert minimal interference during toxicity testing, making them suitable for subsequent toxicological evaluation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effects of Single Exposure on Cell Viability\u003c/h2\u003e \u003cp\u003eWe assessed the viability of A549 cells treated with different concentrations of amisulbrom, UFCB, and the solvent dimethyl sulfoxide (DMSO) after 24 h. DMSO showed no significant effect on A549 cell viability at concentrations\u0026thinsp;\u0026le;\u0026thinsp;0.5% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), but it significantly inhibited cell survival at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;2% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Therefore, the final DMSO concentration in the amisulbrom exposure group was strictly controlled below 0.05% in subsequent experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). UFCB exhibited typical dose-dependent cytotoxic effects within the 5\u0026ndash;150 \u0026micro;g/mL concentration range, with an IC\u003csub\u003e50\u003c/sub\u003e of 19.79 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This concentration aligns with the cellular survival rate reported by Chu, et al. \u003csup\u003e5\u003c/sup\u003e in previous research on the toxicity of 20-nm UFCB to lung cells. Amisulbrom also exhibited significant dose effects within the 5\u0026ndash;150 \u0026micro;g/mL range, with an IC\u003csub\u003e50\u003c/sub\u003e of 54.45 \u0026micro;g/mL (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.966) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Sensitivity to amisulbrom varies among cell lines. Kim, et al. \u003csup\u003e11\u003c/sup\u003e reported IC\u003csub\u003e50\u003c/sub\u003e values of 20.37 \u0026micro;g/mL for HTR-8/SVneo cells and 16.18 \u0026micro;g/mL for ThESCs after 48 hours of exposure to amisulbrom. The current study is the first to demonstrate that amisulbrom exhibits distinct pulmonary cytotoxicity in the A549 cell line, suggesting that respiratory exposure may pose potential human health risks. Overall, UFCB exhibited significantly greater toxicity than amisulbrom, with individual exposure to both compounds producing clear dose-dependent cytotoxic effects in A549 cells. These findings provide a scientific basis for designing dose levels in subsequent combined exposure experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of Combined Exposure on Cell Viability and Multi-Model Analysis\u003c/h2\u003e \u003cp\u003eThe effects of combined pollutant exposure vary in nature. Taenzler, et al. \u003csup\u003e30\u003c/sup\u003e found that the acute toxicity of mixed pesticide formulations to honeybees predominantly exhibited additive effects. In contrast, Wu, et al. \u003csup\u003e16\u003c/sup\u003e observed that co-exposure to lead and copper exacerbated oxidative stress and apoptosis in neuronal cells via synergistic interactions, while Zhang, et al. \u003csup\u003e31\u003c/sup\u003e found that synergistic hepatotoxicity was induced by the combination of avermectin and cyantraniliprole.\u003c/p\u003e \u003cp\u003eTherefore, based on the toxicity results from individual exposure tests, this study employed combined exposures at concentrations of 0.125 \u0026times;, 0.25 \u0026times;, 0.5 \u0026times;, and 1 \u0026times; the IC\u003csub\u003e50\u003c/sub\u003e. The results showed that the cell survival rates in the combined exposure groups at all concentration combinations were significantly lower than those in the corresponding single-exposure groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Under combined exposure to both agents at a concentration of 1 \u0026times; IC\u003csub\u003e50\u003c/sub\u003e, the survival rate in the combined exposure group dropped to 26.47%, significantly lower than both the UFCB alone group (55.36%) and the amisulbrom alone group (54.37%), demonstrating that combined exposure exerted a stronger toxic effect.\u003c/p\u003e \u003cp\u003eTo accurately identify the patterns of synergistic interaction between the two components and overcome the limitations of single-model analysis, this study employed a multi-model integration strategy involving cross-validation with three statistical models: the Bliss independence model, WQS regression, and BKMR. This approach enabled the analysis of combined toxicity characteristics from multiple dimensions, thus significantly enhancing the reliability of mixture toxicity assessment.\u003c/p\u003e \u003cp\u003eThe Bliss independent action model results yielded a CI value of 0.95 (95% CI: 0.82\u0026ndash;1.08). This fell within the additive range, suggesting that combined toxicity exerted an additive effect. The WQS model analysis revealed that the weight of UFCB in the mixed exposure was 0.90, significantly higher than that of amisulbrom (0.10). Furthermore, the interaction term between the WQS index and UFCB dose showed no statistical significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), supporting the additive nature of their combined effect. The results of BKMR modeling further validated these conclusions. The interaction coefficient was 0.27 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), confirming no significant interaction between the two compounds. Variable importance analysis showed that the posterior inclusion probability of UFCB was 1.00, identifying it as a key toxicity factor. In contrast, the posterior inclusion probability of amisulbrom was only 0.12, indicating its weaker and less stable influence on combined toxicity. Univariate analysis also revealed that UFCB significantly inhibited cell viability (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while amisulbrom had no significant effect (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eThe three models reached consistent conclusions across the three dimensions of overall toxicity assessment, component contribution analysis, and interaction identification. The results confirmed that combined exposure to UFCB and amisulbrom exerted a UFCB-dominant additive effect on A549 cells, with no synergistic or antagonistic enhancement observed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effects of Combined Exposure on Oxidative Stress and Apoptosis in A549 Cells\u003c/h2\u003e \u003cp\u003eOxidative stress serves as a common key indicator of nanoparticle- and pesticide-induced cytotoxicity \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, which typically manifests as elevated intracellular ROS levels, depletion of the antioxidant GSH, and accumulation of the lipid peroxidation product MDA. Oxidative damage that exceeds the cellular repair capacity can further trigger apoptosis. To validate the toxic effects and additive characteristics of combined exposure to UFCB and amisulbrom at the molecular and cellular levels, this study selected ROS, GSH, MDA, and apoptosis rate as detection indicators to systematically evaluate the oxidative damage and cellular fate responses induced by combined exposure.\u003c/p\u003e \u003cp\u003eRegarding ROS levels, all exposure groups exhibited significantly higher ROS levels than the control group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Amisulbrom monotherapy induced a greater increase in ROS than UFCB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The combined exposure group (20 \u0026micro;g/mL UFCB\u0026thinsp;+\u0026thinsp;amisulbrom) exhibited a further significant elevation in ROS levels, surpassing both single exposure groups and indicating an additive effect in ROS induction. Similarly, Chu, et al. \u003csup\u003e5\u003c/sup\u003e reported that UFCB synergistically increased ROS levels in mouse lung fibroblasts when combined with lead exposure, suggesting potential interactions between UFCB and other environmental pollutants at the oxidative stress level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn terms of the antioxidant capacity, GSH levels were significantly reduced in all exposure groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). UFCB exposure resulted in a dose-dependent decrease, while the depletion effect of amisulbrom was more pronounced. The combined exposure group exhibited a decrease in GSH intermediate between the two single-exposure groups, without demonstrating synergistic enhancement (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Regarding lipid peroxidation, MDA levels were significantly enhanced in all exposure groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, MDA accumulation induced by UFCB alone was significantly higher than that induced by amisulbrom exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), in sharp contrast with their ROS-inducing capacities (amisulbrom\u0026thinsp;\u0026gt;\u0026thinsp;UFCB). More intriguingly, the MDA levels in the combined exposure group were significantly lower than those in the high-dose UFCB-alone group, despite exhibiting significantly higher ROS levels than the UFCB-alone group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe examination of apoptosis revealed that after 24 hours of single exposure to UFCB or amisulbrom, the total apoptosis rate showed no significant difference compared to the control group. This suggested that at the doses used in this experiment, single exposure primarily induced reversible cellular stress without triggering large-scale apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). However, the combined exposure group (20 \u0026micro;g/mL UFCB\u0026thinsp;+\u0026thinsp;amisulbrom) exhibited a significant increase in the total apoptosis rate, which reached 11.86% (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), substantially higher than that of either single-exposure group. This result strongly correlated with cell viability assays, which showed that cell survival dropped to 26.47% under combined exposure at the IC\u003csub\u003e50\u003c/sub\u003e concentration, further confirming the combined toxic effect of combined exposure.\u003c/p\u003e \u003cp\u003eTwo seemingly contradictory phenomena emerged in the above results. First, although UFCB had a stronger inhibitory effect on cell viability than amisulbrom, its ROS induction capacity was weaker. Second, while UFCB alone induced greater MDA accumulation than amisulbrom, combined exposure resulted in lower MDA levels than exposure to UFCB alone. These findings suggest that the toxic mechanisms of UFCB and amisulbrom are fundamentally different.\u003c/p\u003e \u003cp\u003eAs a type of particulate matter, the cytotoxicity of UFCB stems from the interaction between its unique physical properties and potential chemical perturbations. The TEM observations of Li, et al. \u003csup\u003e32\u003c/sup\u003e indicate that UFCB can be internalized by cells via endocytosis, forming vesicular structures within the cytoplasm that directly interfere with organelle function and induce mechanical damage. This physical disruption may be the primary driver of UFCB-induced cell viability decline, while oxidative stress plays only a secondary or auxiliary role in its toxic effects. Although UFCB exhibits a relatively weak ROS-inducing capacity, the physical damage it causes may compromise cell membrane integrity and enhance lysosomal membrane permeability, leading to the release of pro-oxidants (such as free iron ions). This explains why exposure to UFCB alone induces strong lipid peroxidation\u0026mdash;a process that can result from both direct ROS attack and secondary oxidative events following membrane structural damage \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe significantly lower MDA levels in the combined exposure group compared to the UFCB group can be explained by a shift in the injury pattern. When cells are exposed to UFCB alone, physical damage may lead to the extensive exposure of lipid peroxidation substrates (polyunsaturated fatty acids), resulting in increased MDA production. Under combined exposure with amisulbrom, cells may initiate alternative death pathways (such as apoptosis). Apoptosis is relatively \"clean,\" preserving membrane integrity early in the process and consequently reducing the availability of lipid peroxidation substrates \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In contrast, as a pesticide molecule, amisulbrom tends to exert its toxic effects via biochemical pathways such as inducing oxidative stress and interfering with metabolic enzyme activit \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, consistent with its characteristics of strongly inducing ROS and significantly depleting GSH.\u003c/p\u003e \u003cp\u003eTo quantify synergistic toxicity, this study calculated the theoretical summation value using the Bliss model and compared it with the measured values. The results showed no significant differences between the measured and theoretical values for all detected indicators, further confirming the additive effect of combined amisulbrom and UFCB exposure on the induction of oxidative stress and apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Collectively, these findings suggest that under the experimental conditions, amisulbrom and UFCB jointly mediate cellular damage by exacerbating oxidative stress, disrupting redox homeostasis, and initiating apoptotic programs through an additive effect model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThis study systematically evaluated the toxic effects and mechanisms of UFCB and the novel fungicide amisulbrom, both individually and in combination, on human lung adenocarcinoma A549 cells. The results showed that both UFCB and amisulbrom exposure inhibited A549 cell proliferation in a dose-dependent manner. Cross-validation using multiple models (Bliss, WQS, and BKMR) confirmed an additive effect due to combined exposure, with UFCB being the primary contributor to combined toxicity. The underlying mechanism was closely associated with the induction of excessive intracellular ROS production and apoptosis under combined exposure. Furthermore, no significant differences were observed between the measured and theoretical values for all assessed indicators, further supporting the additive nature of combined UFCB and amisulbrom toxicity. This study provides critical toxicological data for the health risk assessment of combined exposure to atmospheric nanoparticles and pesticides while offering methodological references for multi-model combined toxicity analysis of complex pollutants. In future work, subsequent in-vivo experiments and deeper molecular mechanism studies are required to refine the health risk assessment system and validate its relevance to human health.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32402409).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWenyuan Xu (First Author): Writing - Original Draft, Methodology, Conceptualization, Data Curation; Xingyu Yue (Co-first Author): Writing - Review \u0026amp; Editing, Supervision, Validation; Zengxue Liu: Investigation; Formal analysis; Zhuang Ye: Software, Visualization; Zhenghan Wang: Resources; Xiaofan Li: Supervision; Yanhong Shi (Corresponding Author): Conceptualization, Funding Acquisition, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLin, Z. et al. 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(2022) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2022/5563759\u003c/span\u003e\u003cspan address=\"10.1155/2022/5563759\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of UFCB and parameters of its dispersion system in complete medium\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eDimensions declared by the manufacturer(nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUFCB\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBET surface area(m\u003csup\u003e2\u003c/sup\u003e/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e273\u0026thinsp;\u0026plusmn;\u0026thinsp;5\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEndotoxin content(EU/g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e123\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDispersion in complete medium: 20 \u0026micro;g/mL (DLS-verified).\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eZeta\u0026nbsp;Potential\u0026nbsp;(mV)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-4.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePeak hydrodynamic diameter(nm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e251.27\u0026thinsp;\u0026plusmn;\u0026thinsp;12.63\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003evolume-based peak diameter(nm)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e261.5\u0026thinsp;\u0026plusmn;\u0026thinsp;13.3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePDI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"UFCB, Amisulbrom, Combined exposure, Multi-model analysis, Oxidative stress, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-9386037/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9386037/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUltrafine carbon black (UFCB), a typical nanoscale component of PM₂.₅, is environmentally persistent and prone to forming composite pollutants. Amisulbrom, a widely used pesticide, can migrate synergistically with atmospheric contaminants, yet their combined health risks remain unclear. This study evaluated the individual and combined toxicities of UFCB and amisulbrom in A549 cells. Both agents induced dose-dependent cytotoxicity, with 24 h IC₅₀ values of 19.79 and 54.45 \u0026micro;g/mL, respectively. Multiple models including Bliss independence, weighted quantile sum, and Bayesian kernel machine regression consistently showed an additive effect rather than synergism or antagonism, with UFCB as the main contributor. Combined exposure significantly increased intracellular ROS and apoptosis, but glutathione and malondialdehyde showed no synergistic changes. Measured indicators were comparable to theoretical values, further verifying the additive joint effect.\u003c/p\u003e","manuscriptTitle":"Multi-model Analysis of the Combined Toxic Effects of Ultrafine Carbon Black and Amisulbrom on A549 Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-24 15:01:17","doi":"10.21203/rs.3.rs-9386037/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T18:45:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T09:42:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T15:18:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192761974692084837993940369050287173103","date":"2026-04-20T07:32:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13367073345009994732238058457864146211","date":"2026-04-17T10:31:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T03:05:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T21:25:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-15T16:45:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-14T08:03:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-14T07:43:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9152159-281d-49d4-a673-b0dfe96a1188","owner":[],"postedDate":"April 24th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T18:45:01+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-06T09:42:07+00:00","index":35,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":66668849,"name":"Earth and environmental sciences/Environmental sciences"},{"id":66668850,"name":"Health sciences/Risk factors"}],"tags":[],"updatedAt":"2026-05-16T06:53:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-24 15:01:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9386037","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9386037","identity":"rs-9386037","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}