Multiwall carbon nanotubes exhibit a cytotoxic but not directly DNA-damaging potential in human LP9/TERT-1 peritoneal mesothelial cells

Multiwall carbon nanotubes (MWCNT) are discussed to exhibit an asbestos-like toxic potential depending on their length and fiber-like shape. For this reason, potential adverse biological effects of MWCNT were investigated in vitro in a German BMBF funded project (contract No. 03X0109A). In this project tailor made MWCNT with different length and diameter were produced to be able to correlate effects with MWCNT characteristics. The present in vitro experiments were performed with the human peritoneal mesothelial cell line LP9/TERT-1 as a model system. Cells were exposed for 24 h to 5 <my>g/cm2 of 6 different MWCNT and data were compared to long amosite asbestos as positive control and more particle-like MWCNT (tangled MWCNT) and particles (milled MWCNT, Printex 90) as negative controls. Pre-experiments demonstrated that incubation of cells at 5 <my>g/cm2 for 24 h differentiated best between the various treatments. For in vitro experiments MWCNT were directly suspended in cell culture medium by using a sonotrode (HD2070; VS70T) twice for 5 minutes at 90 % duty cycle and 100 % amplitude. Suspension, size, and distribution of MWCNT were monitored by scanning electron microscope (SEM). The cytotoxic potential of MWCNT was estimated by means of "relative increase in cell counts" (RICC) and lactate dehydrogenase (LDH)-release. An enzyme (hOGG1)-modified comet assay was used to determine the direct genotoxic potential. In the present study, only CNT3 (length: 8.57 <my>m; diameter: 0,085 <my>m) mediated a significant increase in LDH-release and thus disturbances of membrane integrity, whereas all other MWCNT and reference materials demonstrated no marked effect, compared to the medium control. In the hOGG1-modified comet assay, which detects DNA strand breaks and oxidative DNA-base lesion, no significant direct DNA-damaging effect of the investigated MWCNT was obvious, independent of length and diameter. In contrast, using RICC as an endpoint strong reduction in cell number/proliferation could be demonstrated for CNT1 (length: 7.91 <my>m; diameter: 0.037 <my>m), CNT2 (length: 10.24 <my>m; diameter: 0.04), CNT3, CNT3a (length: 9.3 <my>m; diameter: 0.061 <my>m), and long amosite asbestos (length: 13.95 <my>m; diameter: 0.39 <my>m), as compared to the particulate reference items and the medium control. In conclusion, certain MWCNT exhibit a marked cytotoxic/anti-proliferative activity in human mesothelial cells (LP9/TERT-1) in vitro, without indications for a direct DNA-damaging potential.