Study On Synergistic Oxidative Stress And Genotoxicity Caused By Trace DDT And Nanoparticle Titanium Dioxide In Human Derived Fetal Hepatocytes

DDT is an organochlorine insecticide which could cause chromosomal damage, leukemia and lung cancer in humans. International Agency for Research on Cancer (IARC) has placed DDT on the list of possibly human carcinogen. It has been widely used in agriculture in the past and now still exists at high concentrations along with its metabolites throughout the world for the reason of its long half-lives in soil, water and adipose tissue of animals. Conventional water treatment plants are typically not effective in remediating organochlorine contaminants. Many technologies have been developed to deal with DDT-polluted earth and surface waters in recent years. TiO2-assisted photocatalysis stands out from various environmental technologies. However, little was known about toxicity changes of intermediates with time during the process of degradation. In the experiment, the toxic effects of DDT degradation intermediates on human derived fetal hepatocytes were investigated. The results showed that the survival rate and DNA damage of cells gradually decreased in the time period 0– 12 h after the start of illumination using a simulated sun. But coexistence of DDT and nano-TiO2 in the absence of light was not considered yet. When organisms exposed to a mixture of different toxicants, their simultaneous presence that biologically modify cellular conformation and metabolic activity might induce non-overlapping toxic effects although the toxicity of the single compounds might be well known. The interaction may happen during the process of absorption, distribution, metabolic transformation and excretion or depend among other things on the affinity to target sites at cellular level. Therefore, studies of interactions among nanoparticles and other toxicants are of fundamental interest and practical importance to evaluate the exact health impact.In general, TiO2 was thought as a relatively low toxic compound. But recently numerous studies showed adverse effect of TiO2 on health. Several studies found that nano-TiO2 were more toxic than equivalent larger fine particles of the same chemical composition. Nano-TiO2 could stimulate immune response and cause inflammation, DNA damage and lipid peroxidation. The mechanism of nanoparticles induced DNA damage is not understood, but appears to be related to inflammation and oxidative stress. Nanomaterials have massive interface and strong adsorption ability. Thus nanomaterials can adsorb mental ions or organic compounds efficiently. Because of nanomaterials'special surface effect, they will combine with existed pollutants after they enter the environment. DDT in environment can be easily absorbed on the surface of TiO2 and interact with DDT. In addition to facilitating the absorption of pollutants, the physicochemical characteristics of nanoparticles enable them to cross various biological barriers without a specific transporter. But to the best of our knowledge, no study focused on health effect resulted from DDT and nano-TiO2 was reported yet.Environmental pollutants normally exist in trace concentration, therefore the co-effects of trace nano-TiO2 and DDT in human fetal hepatocytes (L-02) cells were studied in this study. This paper mainly discuss the effect of nano-TiO2 and DDT on ROS generation, DNA strand breaks, chromosome damage and lipid peroxidation in L-02 cells by a series of in vitro experiments, which were used to explore the oxidative stress level. Based on departed researches on toxicity of DDT and nano-TiO2, the interaction of both compounds was studied and potential toxicological mechanisms were also evaluated in vitro studies, further consummated the toxicology evaluation of photocatalyzed reactions, then provided the theory basis for photodegradation mediated by nano-TiO2 and provide experiment evidences to the health impact of nanoparticles.The whole study was composed of the following three parts: PartⅠ: Degradation of DDT in water using nano-TiO2 photocatalyst and DNA damage effect caused by intermediate productsObjective: To observe the toxic effects of DDT degradation intermediates on human derived fetal hepatocytes.Methods: An initial DDT concentration of 50μg/mL in water was degraded by nano-TiO2 photocatalyzed reactions under simulated sun for 12 h. Human derived fetal hepatocytes were exposed to DDT degradation intermediates. Survival rate and DNA damage were measured.Results: 50μg/mL DDT could decrease the cell viability (P < 0.05) and induce obvious DNA damage effect (P < 0.05). After being exposed to DDT degradation intermediates in the time period 0-2 h after the start of illumination, the survival rate of L-02 cells significantly decreased in comparison with the control (P < 0.05). Cell viability increased with time and the intermediates was similar to the control after 3 h. The comet assay indicated that significant DNA damage was observed exposed intermediates before 40 min (P < 0.05) and the damage effect decreased with time.Conclusion: DDT could cause cytotoxic effect and DNA damage to L-02 cells. With the development of photocatalyzed reaction, the toxicity of DDT degradation intermediates decreased.PartⅡ: Oxidative stress induced by DDT and nano-TiO2 in human derived fetal hepatocytesObjective: To investigate the levels of oxidative stress induced by DDT, nano-TiO2, DDT+nano-TiO2 in human derived fetal hepatocytes.Methods: L-02 cells were exposed to single DDT (0.001, 0.01, 0.1μmol/L), single nano-TiO2 (0.01, 0.1, 1μg/mL), combined different concentration of DDT with nano-TiO2 respectively. DMSO (1 mL/L) was used as solvent control. Reactive oxygen species (ROS) level was determined by flow cytometry. SOD, MDA, which were the product of the representative of antioxidative enzyme, lipid peroxidation respectively, were detected in L-02 cells after exposure.Results: It can be observed that cells treated with 0.01μmol/L or above concentration of DDT or 1μg/mL nano-TiO2 alone respectively showed a significantly increased level of ROS production compared with control cells (P < 0.05). ROS level generated by a mixture of nano-TiO2 and DDT was markedly elevated (P < 0.05). 0.1μg/mL or above concentration of nano-TiO2 alone and 0.1μmol/L DDT alone significantly decreased SOD activity (P < 0.001) in comparison with solvent control. The content of MDA was significantly increased in L-02 cells exposed to nano-TiO2 alone at the concentration of 0.01μg/mL or above concentration (P<0.05, P<0.001, P<0.001). Cells treated with greater than 0.01μmol/L DDT showed a significantly increased level of MDA (P < 0.001,P < 0.001). Mixture of trace amount of DDT and nano-TiO2 was synergistic to decrease SOD level (P < 0.05), increase MDA generation (P < 0.05) and ROS formation (P < 0.05). The results of factorial analysis showed an interaction between both compounds (P < 0.05). Response surface model indicated that the interaction was synergism (P < 0.05). During the concentration tested, there was a negative association between SOD level and ROS generation (r = -0.86901,P < 0.01) and a striking positive association between MDA content and 8-OHdG level (r = 0.91175,P < 0.01).Conclusion: Our study indicated that coexistence of trace amount of DDT and nano-TiO2 could induce oxidative stress in L-02 cells including increase of lipid peroxidation and decrease of antioxidative enzyme. Oxidative damage in L-02 cells might be ascribed to DDT and nano-TiO2 induced lipid peroxidation and the weakening of antioxidation ability.PartⅢ: Synergistic genotoxicity caused by DDT and nano-TiO2 in human derived fetal hepatocytesObjective: To investigate the effects of DDT, nano-TiO2, DDT+nano-TiO2 on DNA and chromosome damage level in human derived fetal hepatocytes.Methods: L-02 cells were exposed to single DDT (0.001, 0.01, 0.1μmol/L), single nano-TiO2 (0.01, 0.1, 1μg/mL), combined different concentration of DDT with nano-TiO2 respectively. DMSO (1 mL/L) was used as solvent control. DNA damage was measured by comet assay and 8-OHdG which is the marker of DNA oxidative damage. And chromosome change was investigated using micronucleus test.Results: No significant effects on DNA damage were observed in single nano-TiO2 groups (P > 0.05) and 0.01μmol/L or above concentration of DDT could induce DNA damage (P < 0.05). There was no statistically significant increase of OTM in all groups compared with control group in neutral comet assay (P > 0.05). 1μg/mL nano-TiO2 could increase 8-OHdG formation significantly (P < 0.05). And 0.001μmol/L or above concentration of DDT alone significantly increase 8-OHdG level (P<0.05, P<0.001, P<0.001). Nano-TiO2 could not induce MN frequency in concentrations ranging from 0 to 1μg/mL (P > 0.05). MN frequency significantly increased in the cells treated with greater than 0.01μmol/L DDT (P<0.001). Coexistence of both compounds could enhance the MN frequency. Factorial design showed that there was an interaction between both tested substances (P < 0.05) and response surface model indicated that the interaction was synergism (P < 0.05). There was a striking positive association between DNA single strand breaks and ROS generation (r = 0.85838, P < 0.01), between 8-OHdG level and ROS generation (r = 0.86691, P < 0.01) and between MN frequency and ROS generation (r = 0.80569, P < 0.01) in L-02 cells exposed to DDT and nano-TiO2 with concentrations tested.Conclusion: Exposure with trace amount of DDT and nano-TiO2 synergistically enhanced their capability in DNA damage and chromosome change on L-02 cells. The primary mechanism of toxicity may be that DDT and nano-TiO2 can induce the free radical generation, followed by DNA damage and result in chromosome damage via aneugenic activity.