Background:Although tobacco smoking is one of the established risk factors for liver cancer, results from epidemiological studies remain inconclusive on the relationship between second-hand smoke (SHS) and liver cancer, particularly among nonsmokers.Objectives:This study aimed to examine the association between SHS and liver cancer, and to assess its potential interaction with major risk factors such as hepatitis B virus (HBV) infection.Methods:A population-based case-control study was conducted in Jiangsu, China, from 2003 to 2010, including 2011 newly diagnosed primary liver cancer cases and 7933 population-based controls. Data on SHS exposure at home and in the workplace, along with other major liver cancer risk factors such as alcohol consumption, were collected through self-reported questionnaires. SHS exposure was assessed as a dichotomous variable, categorizing participants as either exposed or unexposed to SHS. HBV infection status was determined by testing serum samples for serum hepatitis B virus surface antigen. Multivariable unconditional logistic regression models were used to examine the association between SHS and risk of liver cancer. Besides stratified analyses, interactions on additive and multiplicative scales were further evaluated between SHS and other risk factors for liver cancer.Results:Exposure to SHS was associated with liver cancer, shown as adjusted odds ratios (ORs) of 1.84 (95% confidence interval [CI]: 1.56, 2.18) in the overall population and 2.11 (95% CI: 1.67, 2.65) among never smokers. Stratified analyses showed that the association between SHS and liver cancer was stronger among HBV-positive participants (OR: 2.09, 95% CI: 1.48, 2.93) compared with HBV-negative participants (OR: 1.87, 95% CI: 1.54, 2.27) (P = 0.02). Both super-additive and super-multiplicative interactions were observed between SHS and HBV infection, with relative excess risk due to interaction (RERI) of 12.56 (95% CI: 6.60, 18.53) and ratio of ORs of 1.55 (95% CI: 1.07, 2.23) in the total population, and RERI of 12.87 (95% CI: 4.93, 20.81) and ratio of ORs of 1.78 (95% CI: 1.06, 2.99) among never smokers.Conclusion:SHS is independently associated with liver cancer, and this association is further modified by HBV infection, leading to a substantially elevated risk, particularly among nonsmokers.

Chlorinated polyfluoroalkyl ether sulfonic acids (Cl-PFESAs, F-53B) are widespread environmental pollutants that have been introduced as emerging alternatives to perfluorooctane sulfonate. They are suspected to have thyroid-disrupting effects, however, their impact on human thyroid hormone (TH) synthesis and the underlying mechanisms remain poorly understood. Using Cl-PFESAs at concentrations relevant to children's serum levels, in the present study we found that Cl-PFESA mixture significantly elevated thyroxine (T4) and triiodothyronine (T3) synthesis in human thyroid follicular epithelial cells (Nthy-ori 3-1)-contrasting with the hypothyroid effect reported in animal studies. We further demonstrated that mechanistically Cl-PFESAs activated transcription factor paired box gene 8 (PAX8) at environmentally relevant concentrations, upregulating thyroglobulin, thyroid peroxidase, and catalase while downregulating sodium iodide symporter. PAX8 knockdown completely abolished Cl-PFESA-induced upregulation of thyroid-specific transcription factors and TH hypersecretion, confirming that PAX8 plays a central role in mediating the TH-stimulating effect of Cl-PFESA. Furthermore, co-exposure with iodide exhibited a nonmonotonic effect: low-to-medium iodide levels (100-300 μg/L) enhanced Cl-PFESA-stimulated TH secretion, whereas high levels (≥600 μg/L) suppressed it. Taken together, this study revealed a novel, human thyrocyte-specific hyperthyroid effect of Cl-PFESA at children serum relevant concentrations and a PAX8-mediated molecular mechanism. Our findings provide crucial toxicological evidence for the human health risk that this class of emerging PFAS alternatives may pose and its potential complications by iodine intake status.

The role of multiple elements in esophageal cancer (EC) remains unclear, especially in low-level metal exposure area. To investigate the causal relationship between elements and EC risk, we conducted a case–cohort study nested within the Taizhou Longitudinal Study, in which the baseline fasting serum concentrations of 21 elements in a subcohort of 926 randomly selected subjects and 256 participants with incident EC were measured. Baseline urine levels of four arsenic species were also measured among 826 subcohort subjects and 232 EC cases to further elucidate the potential impact of arsenic on EC risk. Cox proportional hazards regression was used to estimate adjusted hazard ratios for EC associated with elements and false discovery rate (FDR) correction was performed for multiple testing. The results indicated that elevated serum level of arsenic was associated with a reduced risk of EC, with a p-value for trend of 0.002 after FDR correction. When analyzing urinary arsenic species, we found urinary dimethylarsinic acid (DMA) was weakly correlated with serum arsenic. DMA was inversely associated with EC risk after excluding the first two years of follow-up and showed a significant interaction with smoking. Overall, this case–cohort study did not suggest positive associations between any of serum elements examined and EC risk. Nevertheless, the inverse associations of serum arsenic and urinary DMA with EC risk should be interpreted cautiously. Further population-based and experimental studies that incorporate metabolome and consider multiple biomarkers of exposure are required.

OBJECTIVE:To build a multi-center laboratory network in the China National Human Biomonitoring (CNHBM) program and deliver high-quality datasets supporting national human biomonitoring exposure assessment and public health policy-making. METHODS:A systematic Quality Management System (QMS) framework was established, encompassing laboratory network building, standardized policies, resource allocation, and a rigorous Quality Evaluation System (QES). Applicant laboratories underwent interlaboratory comparison investigations (ICI) using identical test samples. Certified network laboratories implemented strict standard operating procedures, analyzed reference materials and program-specific blinded Quality Control (QC) pools, and performed incurred sample reanalysis throughout the analytical process. RESULTS:Through ICI, four laboratories were certified for heavy metals and metalloids (HMMs) analysis. Throughout the measurement of 21,701 blood and 21,704 urine samples, internal quality control (IQC) demonstrated high precision (CV% generally <15%, >80% within the reference range), and the median relative percentage difference (RPD) in sample reanalysis was below 5%. External quality control (EQC) using CNHBM-specific QC pools confirmed excellent inter-laboratory comparability, with overall coefficients of variation for key HMMs maintained below 15%. CONCLUSION:This study presents the first successful implementation of an integrated QMS framework for a national biomonitoring program in China. The framework effectively harmonized multi-laboratory data production, yielding a high-quality, unified national dataset for exposure assessment and public health policy-making, and provides a replicable model for large-scale environmental health surveillance initiatives.

Despite epidemiological links between rare earth elements (REEs) exposure and thyroid dysfunction, health risks of chronic, low-dose mixture exposure in populations remain unknown. We exposed human thyroid follicular epithelial cells Nthy-ori 3-1 to a biomimetic 14-REE mixture, encompassing light (La, Ce, Pr, Nd, Sm, and Eu) and heavy (Gd, Tb, Dy, Ho, Yb, Lu, Sc, and Y) elements, at concentrations reflecting human internal exposure from 1× to 1000× serum background concentration (SBC). REEs significantly inhibited triiodothyronine (T3) secretion without affecting thyroxine (T4). This disruption was associated with dysregulation of the dual oxidase 1 (DUOX1)/catalase (CAT)-mediated H₂O₂ redox system involved in thyroid hormone synthesis, together with impaired deiodinase-mediated T4-to-T3 conversion. Upregulation of DUOX1 and thyroid peroxidase (TPO), alongside NRF2-mediated defense activation, was significantly initiated at the 1× SBC level, indicating high sensitivity to background environmental concentrations. REE-induced oxidative stress triggered mitochondrial damage and ATP depletion, which may be mechanistically linked to the impaired deiodinase-mediated T4-to-T3 conversion. Antioxidant intervention effectively restored mitochondrial integrity, ATP levels, and deiodinase activities, thereby rescuing T3 secretion. This study revealed a thyrotoxic mechanism of REEs under realistic exposure scenarios, providing critical evidence for assessing their environmental health risks.

The global replacement of Bisphenol A (BPA) with structurally similar yet understudied analogues demands precise biomonitoring tools to assess human exposure and health risks. To bridge critical gaps, we developed a novel ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous quantification of 15 emerging BPA analogues including Bisphenol AF (BPAF), Bisphenol AP (BPAP), Bisphenol B (BPB), Bisphenol C (BPC), Bisphenol E (BPE), Bisphenol F (BPF), Bisphenol G (BPG), Bisphenol FL (BPFL), Bisphenol M (BPM), Bisphenol P (BPP), Bisphenol pH (BPPH), Bisphenol S (BPS), Bisphenol TMC (BPTMC), Bisphenol Z (BPZ), and Bisphenol A diglycidyl ether (BFDGE)-in human serum and urine matrices. The method achieved ultrahigh sensitivity with limits of detection (LODs) of 0.008-0.032 μg/L in urine and 0.010-0.030 μg/L in serum, high accuracy (recoveries: 84.58-113.53 %), and reproducibility (relative standard deviation (RSD) ≤14.7 %). Rigorous validation was conducted through uncertainty assessment and green chemistry metrics (GAC) evaluation. The following scores were obtained: Analytical GREEnness metric approach (AGREE):0.65, Analytical GREEnness Metric for Sample Preparation (AGREEprep):0.64, Modified Green analytical procedure index (MoGAPI) :72, Complex modified Green analytical procedure index (ComplexMoGAPI):73, RGB model: 74.2 %, Blue Applicability Grade Index (BAGI):60, Click Analytical Chemistry Index (CACI):69 and Carbon Footprint Reduction Index (CaFRI):71, respectively. These results confirm the method's reliability and environmental sustainability. Applied to 44 paired samples from Chinese children, the study revealed serum biomarkers (BPPH + BPTMC) as superior indicators of internal exposure compared to urinary metabolites, providing the first-reported paired biospecimen data for these analogues. Furthermore, dual-matrix biomonitoring (serum + urine) significantly improved cumulative exposure assessment accuracy. This work establishes a robust analytical framework for tracking emerging BPA substitutes, supporting evidence-based chemical regulation and advancing environmental health research.

The deficiency and overnutrition of both metal and non-metallic elements in human body can pose harmful effects to human health. Therefore, it is necessary to determination of elements in human body for assessing health risk of elementome. In this study, a simple and accurate biomonitoring approach was developed for quantifying 40 elements in biological samples within 6 min using a direct dilution method combined with inductively coupled plasma mass spectrometry (ICP-MS). It had good linearity (R2 ≥ 0.999) and high sensitivity (limit of detection (LOD) as low as 2 ng/L) with spiked recovery rates ranged between 82.53 % and 110.03 % for serum samples and between 81.92 % and 108.66 % for urine samples. The intra-day and inter-day precision are less than 15 %. In addition, uncertainty indicators and green analytical chemistry (GAC) approaches are used to evaluate the sources of uncertainty and greenness of developed analytical procedures. This method was finally used to detect the paired biofluids samples collected from 202 Chinese children. The distribution of element in this population through detection rate and level of body burden was analyzed. Furthermore, the function of paired serum and urine samples for detection of 40 elements is first explored by analyzing pairing samples. These results demonstrated that the proposed method for analysis of paired biological samples provides an alternative quantitative elementomics method to enable the comprehensive assessment of health risk associated with 40 elemental phenotypes of individuals.

Aluminum (Al) is a metal existing in the human body, yet the immunotoxicity of Al remains elusive. To investigate the immunotoxicity of Al, C57BL/6 mice were treated with 200 or 800 ppm Al via drinking water for 3 months, and thereafter the adaptive immune system was evaluated. In addition, mouse splenocytes and human peripheral blood mononuclear cells (PBMC) were treated with Al in vitro to assess the impact of Al in vitro. Treatment with Al reduced the production of IgM and IgG in the serum, and the activation of B cells, CD4 T cells and CD8 T cells in the spleen of mice; treatment with Al in vitro suppressed the production of IgM and IgG, and the activation of B cells, CD4 T cells and CD8 T cells in mouse splenocytes and human PBMC. In vitro co-culture assays suggested that the suppressed adaptive immunity was due to B cells modified by Al. In terms of mechanism, a direct action of Al on B1 cells induced the B1 cells to be IL-10-producing cells and thereby suppressed the adaptive immune system, which was critically dependent on the Jak1/3-STAT signaling. This study reveals that Al suppresses the adaptive immunity via induction of IL-10-producing B1 cells.

Obesity, a significant global health issue, heightens the risk of non-alcoholic fatty liver disease (NAFLD). Its interaction with environmental pollutants might exacerbate NAFLD's severity. Haloacetamides (HAcAms), a group of emerging nitrogenous disinfection byproducts (DBPs) and potent oxidative stressors, are found in chlorinated drinking water. Since oxidative stress is associated with HAcAms-DBP cytotoxicity and a key factor in NAFLD pathogenesis, we hypothesize that HAcAms-DBPs could exacerbate liver injury and NAFLD, particularly with high-fat diets. This study examined HAcAms-DBPs' impact on liver lipid metabolism in mice treated with 1 to 100 times the background drinking water level (13.05 µg/L) for up to 16 weeks of oral administration. Compared to a high-fat-only group, mice co-exposed to a high-fat diet and HAcAms-DBPs for 16 weeks had elevated serum alanine transaminase, aspartate transaminase, triglyceride, hepatic lipid aggregation, and inflammation response. Under high-fat conditions, background drinking water levels of HAcAms significantly upregulated liver Acetyl-CoA carboxylase 1, fatty acid synthase, peroxisome proliferator-activated receptor gamma (PPARγ), PPARγ coactivator-1α, glucose transporter 1 and 4 protein expression in C57BL/6J mice; 10 times background significantly increased expression of inflammatory marker tumor necrosis factor and liver fibrosis marker protein alpha-smooth muscle actin; 100 times further increased both liver damage and markers of early non-alcoholic steatohepatitis phenotypes like steatosis and lobular inflammation. HAcAms-DBPs plus high-fat conditions worsened liver damage. The possible health risks of NAFLD induced by HAcAms in obese individuals deserve further study.

Cadmium (Cd) is a highly toxic metal in human body, and therefore understanding the immunotoxicity of Cd is significant for public health. The aim of this study was to investigate the role of hematopoietic stem cells (HSC) in regulating the immunotoxicity of Cd. After exposure to 10 ppm Cd via drinking water for up to 9 months, C57BL/ 6 mice had a suppressed adaptive immune system at day 135 but had an enhanced adaptive immune system at day 270 during Cd exposure. The biphasic impacts of Cd on the adaptive immune system were correlated to the mitochondrial (MT) activation of HSC. Mechanistically, a direct action of Cd activated the non-canonical Wnt signaling to increase MT activation in HSC in the bone marrow (BM) at day 90, thus resulting in an impaired adaptive immune system in mice at day 135 during Cd exposure; conversely, Cd reduced the production of thrombopoietin (TPO) by osteoblasts in the BM to suppress MT activation in HSC at day 180, which in turn caused an enhanced adaptive immune system in mice at day 270 during Cd exposure. Thus, Cd biphasically impacts the adaptive immune system via regulating MT activation of HSC, providing a novel angle for understanding the immunotoxicology of metals.

Lithium (Li) is a metal that broadly exists in human bodies. To date, the impact of Li on the humoral immune system remains to be defined. In this study, C57BL/6 mice treated with 50 or 200 ppm Li for 3 months and human peripheral blood mononuclear cells (PBMC) treated with Li in vitro were used. Treatment with 200 ppm Li suppressed the production of serum IgG, but not serum IgM in mice; treatment with 200 ppm Li impaired the activation of CD4 T cells and B cells, and the production of germinal center in mice. Mechanistically, treatment with 200 ppm Li impaired the Ras-Raf-MEK-ERK signaling in CD4 T cells and thereby suppressed the humoral immune system in mice. In line with this, in vitro treatment with Li on human PBMC decreased the production of IgG, but not IgM in the supernatants; in vitro treatment with Li on human PBMC suppressed the activation of CD4 T cells and impaired the Ras-Raf-MEK-ERK signaling in CD4 T cells. Collectively, the present study indicated that Li impaired the humoral immune system via repressing the Ras-Raf-MEK-ERK signaling in CD4 T cells, which was a previously unrecognized immunotoxicity of Li.

Unhealthy lifestyles, obesity, and environmental pollutants are strongly correlated with the development of nonalcoholic fatty liver disease (NAFLD). Haloacetaldehyde-associated disinfection byproducts (HAL-DBPs) at various multiples of concentrations found in finished drinking water together with high-fat (HF) were examined to gauge their mixed effects on hepatic lipid metabolism. Using new alternative methods (NAMs), studying effects in human cells in vitro for risk assessment, we investigated the combined effects of HF and HAL-DBPs on hepatic lipid metabolism and lipotoxicity in immortalized LO-2 human hepatocytes. Coexposure of HAL-DBPs at various multiples of environmental exposure levels with HF increased the levels of triglycerides, interfered with de novo lipogenesis, enhanced fatty acid oxidation, and inhibited the secretion of very low-density lipoproteins. Lipid accumulation caused by the coexposure of HAL-DBPs and HF also resulted in more severe lipotoxicity in these cells. Our results using an in vitro NAM-based method provide novel insights into metabolic reprogramming in hepatocytes due to coexposure of HF and HAL-DBPs and strongly suggest that the risk of NAFLD in sensitive populations due to HAL-DBPs and poor lifestyle deserves further investigation both with laboratory and epidemiological tools. We also discuss how results from our studies could be used in health risk assessments for HAL-DBPs.

Iodoacetic acid (IAA) is a halogenated disinfection by-product of growing concern due to its high cytotoxicity, genotoxicity, endocrine disruptor effects, and potential carcinogenicity. However, the data on distribution and excretion of IAA after ingestion by mammals are still scarce. Here, we developed a reliable and validated method for detecting IAA in biological specimens (plasma, urine, feces, liver, kidney, and tissues) based on modified QuEChERS sample preparation combined with gas chromatography-tandem triple quadrupole mass spectrometry (GC–MS/MS). The detection method for IAA exhibited satisfactory recovery rates (62.6–108.0

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder characterized by abnormal lipid deposition, with oxidative stress being a risk factor in its onset and progression. Haloacetamides (HAcAms), as unregulated disinfection by-products in drinking water, may alter the incidence and severity of NAFLD through the production of oxidative stress. We explored whether HAcAms at 1, 10, and 100-fold concentrations in Shanghai drinking water perturbed lipid metabolism in normal human liver LO-2 cells. CRISPR/Cas9 was used to construct a LO-2 line with stable NRF2 knock-down (NRF2-KD) to investigate the mechanism underlying abnormal lipid accumulation and hepatocyte damage caused by mixed exposure to HAcAms. At 100-fold real-world concentration, HAcAms caused lipid deposition and increased triglyceride accumulation in LO-2 cells, consistent with altered de novo lipogenesis. Differences in responses to HAcAms in normal and NRF2-KD LO-2 cells indicated that HAcAms caused hepatocyte lipid deposition and triglyceride accumulation by activation of the NRF2/PPARγ pathway and aggravated liver cell toxicity by inducing ferroptosis. These results indicate that HAcAms are important risk factors for NAFLD. Further observations and verifications of the effect of HAcAms on NAFLD in the population are warranted in the future.

Haloacetaldehyde disinfection by-products (HAL-DBPs) are among the top three unregulated DBPs found in drinking water. The cytotoxicity and genotoxicity of HALs are much higher than that of the regulated trihalomethanes and haloacetic acids. Previous studies have mainly focused on the toxic effects of single HALs, with few examining the toxic effects of mixed exposures to HALs. The study aimed to observe the effects of mixed exposures of 1∼1000X the realistic level of HALs on the hepatotoxicity and lipid metabolism of C57BL/6J mice, based on the component and concentration of HALs detected in the finished water of Shanghai. Exposure to realistic levels of HALs led to a significant increase in phosphorated acetyl CoA carboxylase 1 (p-ACC1) in the hepatic de novo lipogenesis (DNL) pathway. Additionally, exposure to 100X realistic levels of HALs resulted in significant alterations to key enzymes of lipid ab initio synthesis, including ACC1, fatty acid synthase (FAS), and diacylglycerol acyltransferase 2 (DGAT2), as well as key proteins of lipid disposal such as carnitine palmitoyltransferase 1 (CPT-1) and peroxisome proliferator activated receptor α (PPARα). Exposure to 1000-fold realistic levels of HALs significantly increased hepatic and serum triglyceride levels, as well as total cholesterol, low-density lipoprotein, alanine aminotransferase, aspartate transaminase, alkaline phosphatase, and lactate dehydrogenase levels, significantly decreased high-density lipoprotein. Meanwhile, histopathological analysis demonstrated that HALs exacerbated tissue vacuolization and inflammatory cell infiltration in mouse livers, which showed the typical phenotypes of non-alcoholic fatty liver disease (NAFLD). These results suggested that the HALs mixture is a critical risk factor for NAFLD and is significantly highly toxic to C57BL/6J mice.

Human exposures to rare earth elements are increasing with expanded use in aerospace, precision instruments, and new energy batteries, materials, and fertilizers. Individually these elements have low toxicity, although few investigations have examined the health effects of longer-term mixture exposures. We used the LO-2 cell line to examine the effects of graded exposures to lanthanum, cerium, and yttrium (LCY) mixtures at 1-, 100-, and 1000-fold their human background levels (0.31 mu g/L La, 0.25 mu g/L Ce, and 0.12 mu g/L Y) on cell cycle, oxidative stress, and nuclear factor erythroid-2-related factor (NRF2) pathway biomarkers, assessing responses every 10 passages up to 100 passages. Cell migration, concanavalin A, malignant transformation, and tumorigenesis in nude mice were also examined. Mixed LCY exposures activated oxidative stress and the NRF2 pathway by the 30th passage and increased the proportion of cells in the S phase and cell cycle-specific biomarkers by the 40th passage. LCY exposures did not cause malignant transformation of hepatocytes or induced tumorigenesis in nude mice but enhanced cell proliferation, migration, and agglutination. Importantly, LCY mixtures with longer-term exposure activated the NRF2 pathway and altered the hepatocyte cell cycle at doses far below those used in previous toxicological studies. The consequences of LCY mixtures for public health merit further study.

Fipronil is a broad-spectrum insecticide used for plants and poultry. Owing to its widespread use, fipronil and its metabolites (fipronil sulfone, fipronil desulfinyl, and fipronil sulfide), termed FPM, can be frequently detected in drinking water and food. Fipronil can affect the thyroid function of animals, but the effects of FPM on the human thyroid remain unclear. We employed human thyroid follicular epithelial Nthy-ori 3-1 cells to examine combined cytotoxic responses, thyroid-related functional proteins including the sodium-iodide symporter (NIS), thyroid peroxidase (TPO), deiodinases I-III (DIO I-III), and the nuclear factor erythroid-derived factor 2-related factor 2 (NRF2) pathway induced by FPM of 1-1000-fold concentrations detected in school drinking water collected from a heavily contaminated area of the Huai River Basin. Thyroid-disrupting effects of FPM were evaluated by examining biomarkers of oxidative stress and thyroid function and tetraiodothyronine (T4) levels secreted by Nthy-ori 3-1 cells after FPM treatment. FPM activated the expression of NRF2, HO-1 (heme oxygenase 1), TPO, DIO I, and DIO II but inhibited NIS expression and increased the T4 level of thyrocytes, indicating that FPM can disrupt the function of human thyrocytes through oxidative pathways. Given the adverse impact of low FPM concentrations on human thyrocytes, supportive evidence from rodent studies, and the critical importance of thyroid hormones on development, the effects of FPM on the neurodevelopment and growth of children warrant priority attention.

Lithium (Li) has been widely used in clinical therapy and new Li-ion battery industry. To date, the impact of Li on the development of immune cells is largely unknown. The aim of this study was to investigate the impact of Li on hematopoiesis. C57BL/6 (B6) mice were treated with 50 ppm LiCl, 200 ppm LiCl, or the control via drinking water for 3 months, and thereafter the hematopoiesis was evaluated. Treatment with Li increased the number of mature lymphoid cells while suppressing the number of mature myeloid cells in mice. In addition, a direct action of Li on hematopoietic stem cells (HSC) suppressed endoplasmic reticulum (ER) stress to reduce the proliferation of HSC in the bone marrow (BM), thus leading to fewer HSC in mice. On the other hand, the suppression of ER stress by Li exposure increased the expression of Hsp90, which promoted the potential of lymphopoiesis but did not impact that for myelopoiesis in HSC in the BM of mice. Moreover, in vitro treatment with Li also likely disturbed the ER stress-Hsp90 signaling, suppressed the proliferation, and increased the potential for lymphopoiesis in human HSC. Our study reveals a previously unrecognized toxicity of Li on HSC and may advance our understanding for the immunotoxicology of Li.

Haloacetaldehydes(HALs) are the third largest disinfection by-products(DBPs) class by mass in drinking water. Most of them alone in high doses are more cytotoxic and genotoxic than regulated DBPs. However, the toxic effects of mixed exposure to HALs at environmentally relevant levels are still unknown. Given that genotoxicity is critical for risk assessment, we employed multiple genotoxic tests including the Salmonella typhimurium revertant mutation assay(Ames assay), the single cell gel electrophoresis(SCGE) assay, the cytoplasmic blocking micronucleus(CBMN) assay, and the γ -H2AX assay to investigate the genotoxicity of HALs based on the HALs concentrations and components detected in the finished drinking water of Shanghai, China. The results demonstrated the concentrations of HALs were low, ranging from 0.04 µg/L to 4.47 µg/L, and the total concentration was 10.85 µg/L. Although the mutagenicity of HALs was negative even at 1000-fold concentrations in the real world, mixed exposure to 100 and 1000-fold concentrations HALs resulted in DNA and chromosomal damage in human hepotocyte(HepG2) cells. HALs significantly increased the levels of reactive oxygen species(ROS) and γ-H2AX and activated nuclear factor erythroid-derived factor 2-related factor 2(NRF2) pathway-related protein expressions in HepG2 cells. The antioxidant NAC could ameliorate NRF2 pathway-related protein expression and DNA damage caused by HALs, suggesting that the genotoxicity of mixed exposure to HALs involved cellular oxidative stress and NRF2 pathway activation.