Mechanism Study On The Toxic Effect Of Iodoacetic Acid In Drinking Water

Owning to the rapid development of industry and agriculture, wastewater emission is increasing sharply, water pollution is worsening and the quality of raw water is deteriorating. Not only the existing conventional water treatment process can’t completely remove various pollutants in water, but also toxic disinfection by-products are unintentionally generated when the disinfectant reacts with organic matter and inorganic precursors in the source water during the disinfection process of drinking water. Early researches indicated that organic extracts in drinking water had mutagenicity, genotoxicity, developmental and reproductive toxicity, and can induce oxidative stress, apoptosis and cell malignant transformation. In recent years, epidemiological studies also showed that chlorinated drinking water can lead to bladder cancer, colorectal cancer, and various adverse reproductive outcomes (ARO), such as low birth weight children, small-for-gestational-age neonates and premature birth. Since the contaminants in drinking water can be not only from the raw water, but also generated during the process of water disinfection, the components of the contaminants are complex with various species. Previous studies did not know exactly which contaminant caused the effects because of the limitation in analysis method and the complexity of the research. In recent years, accompanied by the improvement in analytical technology and the development of the concept exposomics, great progress has been made in enhanced understanding of pollutant characteristics in drinking water based on the pollutant profiling method GC-MS and LC-MS/MS. The toxicological studies have developed from the traditional animal based high dose exposure to the toxic effect studies based on toxicity pathway and human-derived cell lines, and this concept has significant repercussions in academia community and regulatory agencies. To this end, the US scientific committee published the book "Toxicity testing in the 21 century:a vision and a strategy" in 2007, which highly overviewed the core ideas and vision for chemical toxicity test. Therefore, in view of the analysis results of the contaminant characteristics in drinking water, this study chose one major pollutant iodoacetic acid (IAA). which could potentially affect human health, for mechanism-based toxicological study.The components of the contaminants in drinking water are complex, with the levels ranging from tens of nanograms to milligrams per liter. Therefore, evaluation of the combined effects of pollutants in drinking water is very difficult. However, the toxic effect of contaminants in drinking water still has some rules to follow. For example, our previous studies have shown that oxidative stress plays an important role in the cytotoxicity and genotoxicity effect induced by whole mixture of water pollutants or certain pollutants, indicating that oxidative stress caused by water pollution deserves concern. Since oxidation stress is the basic defense mechanism of body under external or internal environment stimuli, which can directly or indirectly damage DNA, proteins and lipids, thereby inducing gene mutations, protein denaturation and lipid peroxidation. Therefore, oxidative stress plays an important role in the toxic mechanisms. Existing studies have shown that oxidative stress and adaptive response may be a common mechanism for certain pollutants. Therefore, the toxic effect studies based on specific oxidation stress signaling pathways are expected to enhance the understanding of potential impact of low-dose exposure of contaminants in drinking water on human health. Nrf-2 (Nuclear factor E2-related factor 2)/ARE (antioxidant response element) signaling pathway is very critical in the oxidative stress response. As an important nuclear transcription factor, Nrf-2 is involved in the regulation of cellular antioxidant and phase II detoxification enzymes, maintenance of cellular redox homeostasis and inhibiting oxidative damage and inflammation. Nrf2 deficiency or activation barrier will increase the cell sensitivity to stressors, which is related to tumor-promoting processes and cell apoptosis. Studies have shown that oxidative stress plays an important role in the combined toxic effect of animal carcinogenic MX and microcystin, which is associated with human cancer. Because contaminants in drinking water may be stressors in the body, it will provide new ideas for mixed exposure assessment methods by using oxidative stress pathway to study toxicological effects of mixed pollutants in drinking water.In accordance with the above ideas, this study firstly studied the effects of mixed contaminants in drinking water on Nrf2/ARE pathway, based on the characteristics analysis of drinking water. We discovered that when single pollutant did not cause changes of ARE activity, low-level mixed exposure of these pollutants could cause significant changes in Nrf2/ARE pathway. Considering the result of contaminant characteristic analysis of drinking water, we focused on one new disinfection by-product iodoacetic acid (IAA), which has potent toxic effects in preliminary studies although with low levels in drinking water. Our study confirmed that Nrf2 pathway plays an important role in IAA cytotoxicity and genotoxicity. Then, we studied the effects of IAA on gene expression and regulation in HepG2 cells by using gene chip technology, in order to find the molecular mechanisms of IAA toxicity. We expected the results may lay an important foundation for understanding the mechanisms of IAA toxic effect and developing mechanisms based health risk assessments. Meanwhile, this study may provide important basic data and scientific evidence for developing national drinking water standards in the future.1. Effect of mixed exposure of low-level contaminants in drinking water on Nrf2/ARE signaling pathwayLow-level exposure is the real characteristic of human exposure to contaminant in drinking water. Traditional risk assessment face many challenges when extrapolating from high dose to low dose, from animal to human, nor could it characterize the health and addative effect induced by low-level, strong toxic components. Oxidative stress is an important defense mechanism, where Nrf2 plays a critial role in maintaining cellular redox homeostasis, inhibiting oxidative damage and regulating cellular antioxidant enzymes and phase II detoxification enzymes. Upon revealing the whole exposure character by contaminant profile technique besed on GC-MS, GC-ECD and LC-MS/MS, we evaluate the toxicity of organic contaminant (OC) extracts from drinking water by examining activation of Nrf2-mediated antioxidant response, measured as Nrf2 protein accumulation, expression of Nrf2-regulated genes and ARE-luciferase reporter assays. Our study showed that individual contaminant induced Nrf2/ARE response at concentrations as high as 1000 to 100,000 folds of environmental levels, while whole contaminants induced oxidative stress response at only 8 folds of environmental levels. Additionally, a synthetic mixture prepared based on the contamination profiling of drinking water induced ARE activity to the same extent as the real-world mixture, reinforcing our conclusion that Nrf2 pathway measurement is a sensitive method for assessing toxicity of OCs of drinking water, which revealed the characteristics of oxidative stress induced by low-level mixed contaminants after two-factor authentication by simulated and real water samples. This approach should be broadly useful in risk assessment of mixed environmental exposure.According to the pollution profiling of drinking water contaminants, we clearly identified the major pollutants in drinking water. Upon inquiring the toxicological data, we chose iodoacetic acid, a new disinfection by-product with high detection rate in drinking water of Shanghai and strong toxicity, for further in-depth study.2. Comparative study of the cytotoxic and genotoxic effects of IAA in multiple human-derived cell linesIAA is a new emerging DBPs formed when iodide ions oxidized by disinfectant chlorine or chloramines. Although the concentration of IAA in drinking water is hundreds of nanograms to micrograms per liter, which is relatively lower than the main disinfection by-products THMs and HAAs. However, IAA has strong mutagenicity on Salmonella typhimurium, potent cytotoxicity and genotoxicity on mammalian cells, could induce malignant transformation of NIH/3T3 cells, which can in turn cause tumors in nude mice, and people exposure to it throughout life by drinking water. Therefore, the potential impact of IAA on human health deserves concern. Gordon research conference on disinfection by-products in 2012 also considered IAA and NDMAs as noteworthy disinfection by-products. Owning to the lack of epidemiological and toxicological data, IRCA has not classified IAA into any carcinogenicity categories, nor is there any limit requirement for IAA in either World Health Organization (WHO) guidelines for drinking water quality or any countries, including China. However, the U.S. Environmental Protection Agency (EPA) has started to pay attention to IAA’s potential hazards.Importantly, Shanghai is located in the Yangtze River estuary, which makes it easily affected by the salt tide, sea water intrusion increases halide in raw water, which in turn generate IAA during disinfection. Therefore, the toxic mechanism study of IAA is of great value for further health risk assessment of IAA and the establishment of standards for IAA drinking water quality.Evidence of IAA’s cytotoxicity and genotoxicity is mainly from CHO, TK6 and HepG2 cells, but the genotoxicity results of IAA are not consistent. Considering IAA can be transfered with the blood to other organs and tissues once enter human body, cytotoxicity and genotoxicity studies of IAA on different human-derived cell line is important for understanding the sensitive target organ of IAA. Therefore, our study focuses on 10 different human cell lines to compare their sensitivities to IAA induced cytotoxicity and genotoxicity. The result showed that different human cell lines exhibit cell type-specific sensitivity to IAA-induced cytotoxicity and genotoxicity. Human normal cell line HEK-a was most susceptible to the cytotoxic effects of IAA, and the cytotoxicity of IAA on HEK-a was 14×more potent than Eca-109 cells; the rank of cytotoxicity of cell treated with the same concentration of IAA was HEK-a> SW579> 5637> MGC 80-3> HepG2> HaCAT> CaCo-2≈PBL> 293> Eca-109 cells. Micronuclei assay showed that the rank order of relative genotoxic potency was PBL> SW579> HepG2> MGC 80-3> 293> CaCo2> HaCAT> HEK-a cells with Eca-109 and 5637 cells not genotoxic. Moreover, immunofluorescence staining of centromere protein A (CENP-A) revealed a high proportion of micronuclei contained centromere. This suggests that IAA may disrupt the process of sister chromatid separation and chromosome segregation during mitosis and induce genotoxicity by exhibiting an aneugenic mode of action.3. Study on the role of Nrf2/ARE signaling pathway in the mechanism of toxic effect of IAAOxidative stress and adaptive response is the basic mechanism of poison toxicity and body defense. Previous studies found that IAA can lead to increased ROS, indicating oxidative stress plays an important role in the toxic effect of IAA, while the exact mechanism is unclear. Since xenobiotics primarily metabolized by the liver, thus liver cells may well reflect the essential toxic characteristics of the test substance, and HepG2 cells have DNA repair systems and phase II detoxification enzymes, therefore, we chose human-derived HepG2 cell line for research on Nrf2-mediated antioxidant s response, hoping to understand the role of Nrf2/ARE pathway in the mechanism of toxic effect of IAA. Here, we evaluated the toxicity of IAA by examining the activation of Nrf2-mediated antioxidant response, ARE luciferase reporter activity, intracellular glutathione in HepG2 cells. IAA showed a significant activation effect on ARE-luciferase reporter, both mRNA/protein expression of Nrf2 and its downstream genes (GCLC, NQO1 and HO-1) and glutathione levels increased in a concentration-dependent manner. Subsequently, we confirmed the specific role of Nrf2 in IAA induced toxicity using NRF2-knockdown cells. Deficiency of NRF2 significantly enhanced sensitivity to IAA toxicity, and Knockdown of NRF2 led to an increase of the rate of micronucleus formation. To determine whether IAA-induced oxidatant reaction can be blocked by a natural antioxidant curcumin, we pretreated HepG2 cells with 10μM curcumin for 1 h before exposure to IAA, the result showed that curcumin pre-treatment can effectively reduce IAA-induced cytocoxicity and genotoxicity, suggesting that activation of Nrf2 pathway may reduce the toxicity of IAA. To verify the reliability of the results, SD rats were exposed to IAA for 24 h. The result showed that acute exposure of IAA at 100 times of environmental concentration significantly increased Nrf2 and Gclc gene expression in rat liver, and 1000 times of environmental concentration of IAA could augment the expression of Nrf2, Keapl Ho-1, Nqol and Gclc. All the results above indicating that Nrf2-mediated antioxidant response plays an important role in the toxic effects of IAA, and Nrf2 could be a biomarker of IAA induced toxicity.4. Molecular mechanism study on the toxic effect of iodoacetic acid based on gene chip data analysisClarifying the phenomena of life from the genome or system level is the basic imperative of human post-genome era. Although IAA has proved to have a strong cytotoxicity, genotoxicity and tumorigenicity, the mechanism of its toxicity remains unclear, the only high-throughput test of IAA is PCR array with respect to DNA damage and oxidative stress signaling pathways, there hasn’t been any toxicogenomic study to analyze the molecular mechanisms of IAA toxicity as a whole. Since the occurrence of any toxitic effects is the result of integral disorder of cell regulatory pathways and networks, investigating the cell responses to environmental stimuli from a single signaling pathway or a functional "modules" is rather limited. In this study, we applied the toxicogenomics methods to invertigate the influence of IAA on HepG2 cell gene expression and regulation. Through "cDNA chip-miRNA chip" joint analysis, we screened negatively related and differentially expressed genes and miRNAs. Then we builded relationships through interaction between genes, sorted out the regulation of genes in the form of signal transduction networks, and discovered the context of signal transduction genes, especially those core regulatory factors that closely related to IAA exposure, have high connectivity in the network and play an important role in the stability of the network. The results of the bioinformatics analysis showed that the majority of the altered transcriptome profiles were associated with genes responding to cell proliferation and those regulating apoptosis. The major pathways involved negative correlation gene were colorectal cancer, prostate cancer, apoptosis, TGF-beta signaling pathway, Wnt signaling pathway, etc.