| Summary: | 碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 97 === Cigarette consists of oxidants, free radicals, and metal ions that cause cellular oxidative damage. Carbonylation is one kind of cellular oxidative damage that makes proteins lose their correct conformation and functions. Therefore, the detection of carbonylation can be an indicator of the oxidative damage of proteins. In this study, we treated human monocyte (THP-1) with cigarette smoke extract (CSE) and then analyzed the carbonylation of the cells using dot blot with 2, 4-dinitrophenyl hydrazine. Cigarette smoking extract (20%) induced protein carbonylation over a wide range of time course in THP-1 cells was analyzed by immuno-dot blot assay. Maximum induction of protein carbonylation was observed at 0.5 h after cigarette smoke extract (CSE) exposure. The induction fold was 9.0 ± 5.1. The protein carbonylation levels showed a transitory decrease over 0.5 hour of treatment with CSE. The induction folds of protein carbonylation induced by 20% CSE were 5.7 ± 2.7, 4.3 ± 1.0, 3.3 ± 0.8, and 1.9 ± 0.5 at 1h, 3h, 6h, and 24h, respectively. We further characterized the metal-ion-mediated oxidative stress by testing whether metal ion chelators (10-phenanthroline (PHE), neocuproline (NC), N-acetyl-L-cysteine (NAC), D-penicillamine (D-Pen), and EDTA) or antioxidants (vitamin C (vit C), trolox, S-allyl-L-cysteine (SAC), and α-tocopherol) could block CSE-induced protein carbonylation in THP-1 cells. The results showed that D-Pen and NAC could significantly inhibit CSE-induced protein carbonylation in THP-1 cells, and the inhibition percentages were 82.7% and 78.6%, respectively. To determine the distribution and protein profiles in CSE-induced protein carbonylation in THP-1 cells, carbonylated proteins were analyzed by immuno-dot blot assay and Western blot combined with a cell fractionation system. The results showed that the CSE-induced protein carbonylation was much higher in nuclear subfraction than that in cytosolic one. The proteins with molecular weight of 100 kDa, 110-121, and 121-211 in nuclear subfraction exhibited high protein carbonylation signals.In conclusion, our findings suggest that copper-ion-mediated oxidant stress and the nuclear subfraction play an important role in CSE-induced protein carbonylation. To identify specific carbonylated proteins induced by CSE and its relationship with smoking-related diseases need further investigations.
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