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A cytotoxicity study was conducted with a primary culture of the nervous system cells, including brain microvascular endothelial cells (BMECs) and astrocytes, which are important components of the blood-brain barrier. The real-time cell analysis (RTCA) was used to determine the cytotoxicity of copper-oxide nanoparticles (CuO NPs). The IC50 values of CuO NPs in astrocytes and BMECs were determined by the RTCA at different exposure times and were used as base values for further research. DNA damage after exposure to CuO NPs for 3 and 24 h was assessed using comet assay at the IC50 obtained from RTCA. The onset time of cytotoxicity induced by CuO NPs was 2 and 2-4 h post-exposure in BMECs and astrocytes, respectively. Furthermore, the degree of cytotoxicity induced by exposure to CuO NPs for 24-48 h in the BMECs and astrocytes was similar. Treatment with CuO NPs at 1/2*IC50 and 1/5*IC50 for 3 h induced genotoxicity in both cells as assessed by a measurement of DNA damage, although no cytotoxicity was observed. However, significant DNA damage was observed at all concentrations of CuO NPs used in this study, when the treatment time was 24 h. [ABSTRACT FROM AUTHOR]

DNA lesion bypass facilitates DNA synthesis across bulky DNA lesions, playing a critical role in DNA damage tolerance and cell survival after DNA damage. Assessing lesion bypass efficiency in the cell is important to better understanding of the mechanism of carcinogenesis and chemoresistance. Here we developed a chromatin immunoprecipitation (ChIP)-based method to measure lesion bypass activity across cisplatin-induced intrastrand crosslinks in cancer cells. DNA lesion bypass enables the replication to continue in the presence of replication blocks. Thus, the successful lesion bypass should result in the coexistence of DNA lesions and the newly synthesized DNA fragment opposite to this lesion. Using ChIP, we precipitated the cisplatin-induced intrastrand crosslinks, and quantitated the precipitated newly synthesized DNA that was labeled with BrdU. We validated this method on ovarian cancer cells with inhibited TLS activity. We then applied this method to show that ovarian cancer stem cells exhibit high lesion bypass activity relative to bulk cancer cells from the same cell line. In conclusion, this novel ChIP-based lesion bypass assay can detect the extent to which cisplatin-induced DNA lesions are bypassed in live cells. Our study may be applied more broadly to the study of other DNA lesions, as specific antibodies to these specific lesions are available. • A ChIP-based assay was developed to determine the DNA lesion bypass. • Cancer cells with inhibited TLS show reduced lesion bypass after cisplatin treatment. • Ovarian CSCs exhibit high lesion bypass activity compared to bulk cancer cells. [ABSTRACT FROM AUTHOR]