Cytotoxicity Induced By MWCNTs With Different Length And Functional Groups

Multi-walled carbon nanotubes (MWCNTs) have been widely used in biology and medicine fields as a kind of nano-materials with large specific surface area, small size and low biological toxicity. Many experimental studies indicated that MWCNTs have the potential to induce cytotoxicity. However, the results are often inconsistent and even contradictory among different researchers. ATP-binding casette (ABC) transporter plays an important role in multi-drug resistance and protecting cell from xenobiotic pollutants.In this study, human hepatoma cell line HepG2 and human colonic cell line Caco2 were used as testing objects and six MWCNTs with different length (0.5-2 μm and 10-30μm) and functional groups (pristine, carboxyl modified and hydroxyl modified) were selected to study their cytotoxicity. These MWCNTs were abbreviated to M-L-COOH, M-S-COOH, M-L-OH, M-S-OH, M-Land M-S hereafter. Cell viability, reactive oxygen species (ROS) generation and mitochondrial membrane potential (JC1) et.al were used to systemically compare adverse effects of six MWCNTs and study their potential toxicity mechanism. The inhibition of ABC transporters and chemosensitization to As was studied in low concentrations (< 2 μg/mL) which couldn’t cause cytotoxicity. Potential toxicity mechanism also be uncovered by analysing protein and membrane damage. The results are summarized as follows:1) Cytotoxicity of six MWCNTs with different length and functional groups all showed a concentration-dependent response. M-L-COOH and M-L-OH at 2 μg/mL and higher concentrations caused the significant decreases in cell viability. The lowest effective concentrations for M-L, M-S-COOH, and M-S were 5 μg/mL, while that for M-S-OH was 10μg/mL. M-S induced higher cytotoxicity than M-L, mainly resulting from that M-S could easily enter cells. Functionalization of long MWCNTs increased the cytotoxicity, but the functionalization of short MWCNTs decreased their cytotoxicity. It might be due to that functionalization could increase the solubility of long MWCNTs and reduce their aggregation, resulting in an increase in cytotoxicity. However, functionalization allows short MWCNTs to easily enter cells and thus improves their biocompatibility, resulting in a decrease in cytotoxicity.2) The exposure of MWCNTs with low concentrations (0.01,0.05,0.2,0.5,2 and 5 μg/mL) when they didn’t cause cytotoxicity on HepG2 indicated that the six MWCNTs, at non-toxic concentration, might not be environmentally safe as they inhibited ABC transporters’ efflux capabilities. This inhibition was observed even at very low concentrations 0.01 μg/mL. The concentrations of CAM accumulation were 40-1000 times lower than their effective concentrations on cytotoxicity. The lowest effective concentrations in membrane fluidity were very similar with the lowest effective concentrations of CAM accumulation, indicating that changes in membrane fluidity might be the potential reason for inhibition of ABC transporter activity.3) Two cell lines HepG2 and Caco2 were used to analyze the chemosensitization on As and its bioavailability influenced by MWCNTs at low concentrations. The co-exposure of As and MWCNTs on HepG2 found that MWCNTs could increase the cytotoxicity of As. The results indicated that the inhibition of ABC transporters caused a chemosensitizing effect of MWCNTs. The co-exposure of As and MWCNTs on Caco2 found ABC transporter can increase the transmittance of As.This study found that MWCNTs with different length and functional groups had different cytotoxicity and their toxicity increased with higher concentrations. This study also found that MWCNTs had toxicity in low concentrations which they couldn’t cause cytotoxicity and could inhibit the ABC transporter’ efflux capabilities to cause chemosensitization. It provides insight to risk assessments of low levels of MWCNTs in the environment.