Magnetic bead-based approach to monitoring of cigarette smoke-induced DNA oxidation damage and screening of natural protective compounds.
Cigarette smoking can damage DNA and induce spontaneous mutagenesis or carcinogenesis. Here, we describe a novel strategy for in situ monitoring of cigarette smoke-induced DNA oxidation damage and offer a method for screening natural compounds that protect DNA against tobacco smoke. The present protocol takes advantage of a fast and simple magnetic separation/mixing method and a highly sensitive chemiluminescence (CL) ELISA. The DNA immobilized on the magnetic beads was oxidized by the smoke in the absence or presence of natural compounds, and then oxidative DNA was conveniently held by magnetic force, whereas the complex tobacco smoke matrix and any remaining compounds were completely eliminated by extensive washing, and possible interferences were thus removed and oxidative damage was then sensitively monitored by CL ELISA. A library of 32 natural products was then screened and three were found to protect DNA from oxidative damage and thus may be promising compounds for the development of new drugs. Moreover, the protection effect of these three natural compounds against DNA oxidation damage was successfully classified by directly spiking them in the reference cigarettes. In addition, the potential to screen a mixture in a complex sample matrix, such as crude extracts, was also demonstrated, and hence the proposed technique can screen compounds within a complex matrix and enhance the screening throughput.
Chen
Hongjun
H
School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China.
Chu
Guohai
G
Cao
Zhijuan
Z
Liang
Jianying
J
Lu
Jianzhong
J
Zhou
Guojun
G
D000818:Animals; D001483:Base Sequence; D001688:Biological Products / Q000494:pharmacology*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004353:Drug Evaluation, Preclinical; D005240:Feasibility Studies; D016014:Linear Models; D008163:Luminescent Measurements; D008280:Magnetics; D010084:Oxidation-Reduction; D012907:Smoking / Q000009:adverse effects*; D014026:Nicotiana
D001688:Biological Products; D004247:DNA