Characterization of the Interaction between Cadmium and Chlorpyrifos with Integrative Techniques in Incurring Synergistic Hepatoxicity

et al. (2013) Characterization of the Interaction between Cadmium and Chlorpyrifos with Integrative Techniques in Incurring Synergistic Hepatoxicity. PLoS ONE 8(3): e59553. doi:10.1371/journal.pone.0059553 Characterization of the Interaction between Cadmium and Chlorpyrifos with Integrative Techniques in Incurring Synergistic Hepatoxicity Sijin Liu 0 Liqun Chen 0 Guangbo Qu 0 Xue Sun 0 Shuping Zhang 0 Lei Wang 0 Nan Sang 0 Yuguo Du 0 Jun Liu 0 Joseph J. Barchi, National Cancer Institute at Frederick, United States of America 0 1 State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences , Beijing , China , 2 College of Environment and Resource, Shanxi University , Taiyuan, Shanxi , China Mixture toxicity is an important issue for the risk assessment of environmental pollutants, for which an extensive amount of data are necessary in evaluating their potential adverse health effects. However, it is very hard to decipher the interaction between compounds due to limited techniques. Contamination of heavy metals and organophosphoric insecticides under the environmental and biological settings poses substantial health risk to humans. Although previous studies demonstrated the co-occurrence of cadmium (Cd) and chlorpyrifos (CPF) in environmental medium and food chains, their interaction and potentially synergistic toxicity remain elusive thus far. Here we integrated the approaches of thin-layer chromatography and 1H NMR to study the interaction between Cd2+ and CPF in inducing hepatoxicity. A novel interaction was identified between Cd2+ and CPF, which might be the bonding between Cd2+ and nitrogen atom in the pyridine ring of CPF, or the chelation formation between one Cd2+ and two CPF molecules. The Cd-CPF complex was conferred with distinct biological fate and toxicological performances from its parental components. We further demonstrated that the joint hepatoxicity of Cd ion and CPF was chiefly due to the Cd-CPF complex-facilitated intracellular transport associated with oxidative stress. - Funding: This work was supported by grants from the Chinese Academy of Sciences (KZCX2-EW-404), National Natural Science Foundation of China (grant numbers: 21077128, 20921063, 21177151), and from the program of Hundreds Talents of from the Chinese Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The co-existence of various pollutants in environment and food chains is considerably concerned due to the fused impact on environment and public health [1,2], especially if the joint toxicity of pollutants poses adverse health effects on humans [2]. Mounting evidence suggests that simultaneous exposure of pollutants on organisms can potentiate the toxicity of individual components [3,4]. Thus far, it has been difficult to study the joint toxicity of pollutants, in particular to determine the interaction between compounds, due to limited technical approaches [5]. Only limited interactions have been fully characterized, such as the interaction of chlorpyrifos (CPF) with methyl mercury [6]. The heavy metal cadmium (Cd) has broad industrial applications, such as battery production and electroplating, and it is substantially dispersed in the environment [7,8]. Excretion of Cd ion in human body is about 12 mg/day, and the half-life is 2030 years. Exposure to Cd could cause serious diseases, such as itai-itai disease or even cancers [9]. As a typical environmental hazard, Cd is ranked eighth within the top 20 in the priority;list of hazardous substances by the ATSDR (Agency for Toxic Substances and Disease Registry) [10]. CPF is one of the most widely used organophosphoric insecticides worldwide under agricultural and residential settings in the last few decades [11,12]. Although CPF has been banned for very long time, it still largely remains in water, air and soil, as well as in many dwellings. CPF was found in 100% of indoor air samples and 6470% of blood samples from mothers and newborns [13]. Large amount of CPF can cause acute toxicity, and even a trace amount of CPF can induce neurological toxicity in fetuses and children [13]. Cd ion and CPF are often jointly present in the same environmental media and food chains, and are simultaneously exposed on organisms [14,15], leading to pronounced environmental and health problems [16]. They incur common sensitive targets of toxicity, such as carcinogenicity and hepatoxicity [17,18,19,20], and oxidative stress is assumed to be the principal molecular basis underlying cytotoxicity caused by Cd and CPF [21]. Despite the cooccurrence of these two chemicals in environmental medium and food chains, their toxicity and human risk assessment were predominantly based on the toxicological performances of single chemical. The interaction of Cd ion with other organophosphorus pesticide (such as fenitrothion) has been suggested by other studies [22]; whereas the toxic effects of CPF have also been demonstrated to be modulated by metals, such as zinc [23]. However, the synergistic interaction between Cd ion and CPF has not been established thus far, and the corresponding molecular mechanism is largely unknown as well. Figure 1. The MTT-based cell survival assay of Hep G2 cells exposed to various concentrations of Cd2+ or CPF. The cell viability was assessed by the MTT assay in Hep G2 cells treated with up to 128 mM Cd2+ (a) and up to 5,120 mM CPF (b) after 24 h exposure (n = 6). doi:10.1371/journal.pone.0059553.g001 In the current study, to elucidate the reciprocal impact between CPF and Cd ions, we here addressed their joint hepatoxicity using a few in vitro assessments in a representative human hepatocyte cell line Hep G2. We embarked on their synergistic molecular interaction by integrating techniques, such as thin-layer chromatography (TLC) and 1H NMR. Overall, we demonstrated the formation of the Cd-CPF complex, which was conferred with distinct biological fate and toxicological performances from its parental chemicals. Results and Discussion To evaluate the potential synergistic effect, we first evaluated the joint cytotoxic effect of Cd2+ and CPF on Hep G2 cells by assessing the cell viability with the MTT assay. After 24 h, no toxicity was observed to Hep G2 cells treated with up to 32 mM Cd2+ and up to 1,280 mM CPF, respectively (Fig. 1a&b). To intensively study the synergistic effect, we chose the concentration of 10 mM for both Cd2+ and CPF, at which neither of them caused damage to cell viability (Fig. 1 and Fig. 2a). It should be noted that CPF was dissolved in DMSO and the concentration of 10 mM CPF in culture medium contained only 0.001% DMSO which caused no toxicity to cells compared to the blank control (data not shown). The concomitant exposure of the Cd2+ and CPF mix at 10 (...truncated)