Research Article: Evaluation of Complex Toxicity of Canbon Nanotubes and Sodium Pentachlorophenol Based on Earthworm Coelomocytes Test

Date Published: January 26, 2017

Publisher: Public Library of Science

Author(s): Yang Yang, Yao Xiao, Mei Li, Funian Ji, Changwei Hu, Yibin Cui, Zhen-guang Yan.


As a standard testing organism in soil ecosystems, the earthworm Eisenia fetida has been used widely in toxicity studies. However, tests at the individual level are time- and animal-consuming, with limited sensitivity. Earthworm coelomocytes are important for the assimilation and elimination of exogenous compounds and play a key role in the processes of phagocytosis and inflammation. In this study, we explored an optimal condition to culture coelomocytes of E. fetida in vitro and investigated the cytotoxicity of multiwalled carbon nanotubes (MWCNTs) and sodium pentachlorophenol (PCP-Na) using coelomocytes via evaluating lethal toxicity, oxidative stress, membrane damage, and DNA damage. The results showed that coelomocytes can be successfully cultured in vitro in primary under the RPMI-1640 medium with 2–4×104 cells/well (1–2×105 cells/mL) in 96-well plates at 25°C without CO2. Both MWCNTs and PCP-Na could cause oxidative damage and produce ROS, an evidence for lipid peroxidation with MDA generation and SOD and CAT activity inhibition at high stress. The two chemicals could separately damage the cell membrane structure, increasing permeability and inhibiting mitochondrial membrane potential (MMP). In addition, our results indicate that PCP-Na may be adsorbed onto MWCNTs and its toxicity on earthworm was accordingly alleviated, while a synergetic effect was revealed when PCP-Na and MWCNTs were added separately. In summary, coelomocyte toxicity in in vitro analysis is a sensitive method for detecting the adverse effects of carbon nanotubes combined with various pollutants.

Partial Text

Earthworms play a key role in nutrient mineralization, decomposition, and soil structure improvement [1]. They are considered as bioindicators of soil quality and health due to their sensitivity to various chemicals, such as nanomaterials, pesticides, and heavy metals [2, 3]. Therefore, standard methods have been established to measure the responses of individual earthworm species by determining mortality, behavior, pathological symptoms, body weight change, and reproductive activity [4–6]. However, large quantities earthworms were demanded for the observation of the end point of their death and it was necessary to consume much more chemicals in the experiment. Moreover, it takes a long time to prepare the artificial soil, e.g., the stable time and the exposure time of soil. Consequently, there has been attempted to develop more efficient methods to evaluate earthworm responses at a cellular level.

Cell survival rate of coelomocytes was lower than 30% after cultivation of 24 h at 37°C with 5% CO2. Cultivation at 25°C without CO2 was a more suitable method. A previous study has shown that the optimum temperature for earthworm growth was 20–30°C, with a pH range between 7 and 8 [27]. The presence of 5% CO2 might decrease the pH of the medium, resulting in unsuitable conditions for cell growth. Cell survival rate decreased when inoculation density exceeded 1 × 105/ml, possibly due to increased metabolic requirements [14]. Cell survival rate was almost 100% in 24 h for high-glucose DMEM and RPMI-1640 medium, but after 72h exposure, cell survival rate of RPMI-1640 medium remained over 80%, suggesting that RPMI-1640 was more suitable for coelomocyte cultivation. In addition, our results indicate that 24 h incubation rendered the highest and the less variable signal in overtime culture. Incubation time is critical and differs substantially between earthworm species [28]. Accordingly, the cells were thus incubated for 24 h at room temperature (21°C) in darkness after in vivo exposure to nanoparticles [13].

Cell survival rate, oxidative stress, membrane damage, and DNA damage of coelomocytes exposed to PCP-Na, MWCNTs, and their complex in vitro were associated with exposure concentration and methods. Overall, both CNTs and PCP-Na can cause oxidative damage and produce ROS, resulting in lipid peroxidation with MDA generation and SOD and CAT activity inhibition at high stress, which is consistent with the individual results. In comparison, higher susceptibility was obtained in in vivo cytotoxicity tests in this study, compared to traditional endpoints. Moreover, CNTs and PCP-Na could damage the membrane structure of the cells, increasing permeability and inhibiting MMP. In addition, our results indicate that the toxicity of PCP-Na may be alleviated by the appearance of MWCNTs after adsorption, while PCP-Na and MWCNTs added separately had a synergetic effect based on the cytotoxicity toxicity study. In summary, coelomocyte toxicity in vitro analysis is a new sensitive method for detecting the adverse effects of various pollutants on earthworms, and the cytotoxicity indexes are positively correlated with the indexes for individual levels. Earthworm cytotoxicity tests are expected to replace individual level toxicity tests and can become a fast, efficient, and sensitive method to evaluate toxicity of pollutants on soil organisms.




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