Research Article: Toxicological assessment of multi-walled carbon nanotubes combined with nonylphenol in male mice

Date Published: July 20, 2018

Publisher: Public Library of Science

Author(s): Hao Fang, Yibin Cui, Zhuang Wang, Se Wang, Tobias Stoeger.


Carbon nanotubes have attracted increasing attention attributable to their widespread application. To evaluate the joint toxicity of multi-walled carbon nanotubes (MWCNTs) and nonylphenol (NP), we investigated the toxicological effects of NP, pristine MWCNTs, and MWCNTs combined with NP in male mice. After exposing male mice by gavage for 5 days, intracellular superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity, as well as malondialdehyde (MDA) and glutathione (GSH) levels in tissues were determined to evaluate in vivo oxidative stress. In addition, genotoxicity was assessed by examining DNA damage in mouse liver and sperm via the comet assay, and transmission electron microscopy (TEM) was used for direct visual observations of mitochondrial damage in the liver. Results from the oxidative damage and DNA damage experiments indicate that after adsorbing NP, MWCNTs at a high dose induce oxidative lesions in the liver and cause DNA damage in mouse sperm; these data offer new insights regarding the toxicological assessment of MWCNTs.

Partial Text

There has been a rapid growth in the application of nanoscale materials, attributable to the development of nanotechnologies [1]. Carbon nanotubes (CNTs), which were discovered in 1991 [2], have attracted a great deal of attention owing to their unique structural, electrical, and mechanical properties, which make them potentially useful in extremely small-scale biological, electrical, and mechanical applications. Evidence suggests that CNTs adsorb organic pollutants effectively. For instance, CNTs adsorb environmental endocrine disruptors (EEDs), neutral dissolved organic matter, and trihalomethanes more extensively than activated carbon materials do [3–5]. Therefore, CNTs are potential adsorbents of these organic pollutants.

In this study, we compared the anti-oxidative damages of NP, pristine MWCNTs, and MWCNTs+NP in mice. In addition, we evaluated genotoxic effects using the comet assay during acute toxicity tests. Although no differences in body weight and organ coefficients were observed in all five groups (data not shown), the enzyme and genotoxic results indicate that MWCNTs+NP exposure for 5 days in male mice results in greater toxicity than exposure to NP or MWCNTs alone. As a common mechanism for intracellular damage, oxidative stress has been clearly implicated in the induction of inflammation in many studies examining CNTs both in vivo and in vitro [1, 27, 42]. CNTs stimulate the generation of reactive oxygen species (ROS), which can damage lipids, carbohydrates, proteins, and DNA [43, 44]. ROS-mediated toxicity has also been observed in vitro for single-walled CNTs with a diameter of 8 nm and length of < 5 μm [30]. Normally, antioxidant enzymes such as SOD and GSH-Px reduce H2O2 and superoxide radicals, protecting polyunsaturated fatty acids from lipid peroxidation and further preserving the structure of the cell membrane. However, excess ROS production destroys the natural antioxidant defense system and leads to several sub-cellular injuries, including protein denaturation, membrane damage, and DNA damage [1]. In the present study, we investigated changes in SOD and GSH-Px activity, as well as GSH levels to compare the toxicity of NP, MWCNTs, and MWCNTs+NP. Our results suggest that MWCNTs+NP induce significant changes in GSH-Px activity and deplete GSH in the liver, although evidence suggests that pristine CNTs are not toxic or little toxic to animals when administered by gavage (50 mg/kg) or by the intraperitoneal route (250 mg/kg) [45, 46]. In this study, decreased SOD levels in the liver were observed in both the MWCNTs and MWCNTs+NP groups. This is likely attributable to the high concentration of MWCNTs (100 mg/kg) used in this study. It has been reported that the oral no-observed-adverse-effect-level value of NP seems to range from approximately 50 to 100 mg/kg [47]. In this study, we set a high concentration of MWCNTs (100 mg/kg) and a relatively low dose of NP (5 mg/kg). This is mainly due to the limitation of adsorbing capacity of NP on MWCNTs in our pilot study, which indicated that the largest extent of NP adsorption on MWCNTs was approximately 58 mg/g. In the meanwhile, although previous study showed animals were successfully administered via the intraperitoneal route by using an extremely high dose (250 mg/kg) [46], in this study we still chose the oral route for the sake of safety. The oxidative damage appeared to be higher in the 100 mg/kg MWCNTs+ 5 mg/kg NP group than in the other treatment groups. The adsorptive properties of MWCNTs may explain this phenomenon. The addition of NP to the MWCNTs may have exacerbated the induction of intracellular ROS generation by simultaneously exerting adverse effects on the antioxidant defense system. Although studies have suggested that NP is an environmental contaminant that results in adverse environmental health effects attributable to oxidative stress both in vivo and in vitro [14, 48–50], in our study, there were no significant differences in toxicity between the NP and CK groups. We inferred from these data that the NP exposure dose and time used in this study were not sufficient to stimulate ROS generation and induce oxidative damage; however, when NP adsorbs to MWCNTs, it remains in the tissues for a longer duration. To investigate the toxicological effects induced by NP, MWCNTs, and MWCNTs+NP in mice, several anti-oxidative defense system parameters were examined, with the comet assay used specifically to study genotoxicity. No obvious acute toxicity was observed 5 days after exposure to NP at a dose of 5 mg/kg in mice. In addition, high doses of MWCNTs+NP induced more oxidative lesions in the liver and caused more DNA damage in the sperm than pristine MWCNTs, as shown by measuring changes in markers of oxidative damage and via the comet assay.   Source: