The Role Of NOX And GSH In Cadmium-induced DNA Double-strand Breaks

The heavy metal cadmium (Cd) is a classified carcinogen and can cause DNA double-strand breaks by oxidative stress. Since intracellular reduced glutathione (GSH) is an antioxidant and has a thiol group with the ability to combine Cd, GSH is likely to have a protective effect on Cd-induced DNA double-strand breaks, by decreasing the content of reactive oxygen species (ROS) in the cell. Meanwhile, nicotinamide adenine dinucleotide phosphate oxidase (NOX) can also produce ROS in the cell. Cd may have effect on the enzyme activity to regulate the level of ROS, which may influence Cd-induced DNA double-strand breaks. To test these hypothesis, in this paper we use immunofluorescence technique and flow cytometry to detect the role of NOX and GSH on cadmium-induced DNA double-strand breaks in human fibroblasts. The results were as follows:1. Cd inhibited the cell viability of human fibroblasts. MTT test was used for detecting the cell viability of human fibroblasts. Exposure to Cd (10,20,40,80,160, 320 μM) for 8 h significantly reduced the cell viability of human fibroblasts (P< 0.05).2. Different concentration and time of Cd exposure caused a significant increase in DNA double-strand breaks level. Human fibroblasts were exposed to different concentrations of Cd (0,5,10,20 and 40 μM) for 8 h or to 20 μM Cd for 0,4,8,16 and 24 h, both the result showed that Cd could significantly increase the level of DNA double-strand breaks (P< 0.01).3. Cadmium caused ROS production in human fibroblasts. We detected intracellular ROS production in human fibroblasts by flow cytometry. The results showed that intracellular ROS level was much higher than that in control group when cells were treated with 20 μM cadmium for 1 and 8 h (P< 0.05). However, there were no significant differences (P> 0.05) between control and cadmium treatment groups (2 and 4 h) after exposure with Cd.4. Cadmium exposure could significantly increase the activity of NOX, influencing the genetic toxicity of cadmium via regulating the level of ROS. Different concentration of cadmium (0,10,20 μM) treatment lead to significantly increase of NOX activity. Diphenyleneiodonium chloride (DPI, an inhibitor of NOX activity,10 μM) combined treatment with cadmium could significantly relieve Cd-induced DNA double-strand breaks, and they influence Cd-induced DNA double-strand breaks via regulating the level of intracellular ROS.5. Cadmium exposure could significantly decrease the level of intracellular GSH, which further weaken the genetic toxicity of cadmium via regulating the content of ROS. Cadmium treatment at different concentration (0,5,10,20,40 μM) lead to the change of GSH level in human fibroblasts. N-acetyl-cysteine (NAC, as a precursor of GSH,1 mM) pretreatment significantly increased intracellular GSH and reduced the Cd-induced DNA double-strand breaks, while L-buthionine-sulfoximine (BSO, a depletion agent for GSH,50 μM) pretreatment significantly decreased intracellular GSH and increased DNA double-strand breaks level, proving that GSH has a protective role in Cd-induced DNA damage. And it influenced Cd-induced DNA double-strand breaks via regulating the level of ROS in the cell.In conclusion, the present study demonstrated that cadmium exposure could significantly increase the activity of NOX, subsequently adjust the level of ROS and influence Cd-induced DNA double-strand breaks. And GSH played a protective role in Cd-induced DNA double-strand breaks via regulating the level of ROS in human fibroblasts.