Effect Of β-carotene In The A549 Cell DNA Damage Induced By Cigarette Smoke

[Objective] p-carotene is one kind of phytochemicals and a precursor of vitamin A, being rich in vegetables and fruits. As being an antioxidant, P-carotene has been regarded as the potential chemoprevention activity, it can act as an immune modulator, quench singlet oxygen, and reduce peroxyl radicals at a low partial oxygen pressure, p-carotene also enhances cell gap-junction communication and induces hepatic enzymes which detoxify carcinogens. A large body of observational epidemiologic studies has consistently demonstrated that individuals who eat more fruits and vegetables, which are rich in carotenoids, and people who have higher serum p-carotene levels have a lower risk of cancer, particularly lung cancer. In contrast to these observations, two large human intervention studies that used high-dose synthetic P-carotene supplements reported an increased incidence of lung cancer among smokers and asbestos-exposed people. The possible mechanism is that P-carotene itself may act as an antioxidant, but in the rich radical lung it can be converted into an oxidant and induce DNA damage directly or indirectly. So its oxidized products may facilitate carcinogenesis.Recently the phosphorylation of histone H2AX denoted γH2AX has gained attention for its relationship with DNA damage. The phosphorylated form of histone variant H2AX plays an important role in the recruitment of DNA repair and(DSBs). Many relation proteins of maintaining, protecting, repairing of chromosome structure can form a foci organically, when they combine with the site of DNA damage, to check and repair the DNA damage. In this process, it can induce cell cycle to stop and cell to apoptosis according to the degree of DNA damage.With the characteristic of yH2AX foci formation as an indicator for DNA damage, particularly the DSBs, yH2AX can be a new identity of DNA damage. This study combines the ATBC, CARET human intervention studies, then use the technology of yH2AX antibody recognition, to explore the different effect of B-carotene in the A549 cell DNA damage which induced by cigarette smoke solution. So that we can make an overall evaluation of the safety of P-carotene, then offer scientific evidence for reasonable supplement p-carotene.[Methods] In vitro experiment, the A549 cell were cultured in complete Dulbecco's modified minimum essential medium(DMEM). First we should to choose an appropriate cigarette smoke solution dosage, and ensure the dosage can induce the right cell DNA damage. The culture medium was replaced with fresh varied concentrations of p-carotene after 48h of culture, and the negative cells received with 0.5% DMSO, and continue to culture 24 hours. Finish the cell culture, and give the cigarette smoke solution. The positive cells received with the 0.5mmol/L H2O2, all cells incubated for lhour to induce cell DNA damage. Then use the yH2AX immunofluorescencer to measure fluorescence intensity. Statistical evaluation of the data was analyzed by Cochran-Mantel-Haenszel. Each experiment was run in duplicate or triplicate. All experiments were repeated at least three times. [Results] The result from immunofluorescence microscope shown that there is no difference between the blank group, the varied P-carotene group and DMSO treated group, indicating that the PC alone group can not induce the A549 cell to produce DNA damage. But the exposed cigarette smoke solution group has the significant difference between the DMSO group, showing that the cigarette smoke solution groupcan induce the A549 cell to produce DNA damage. There is no difference between the 0.1(jmol/L PC together exposed cigarette smoke solution and the cigarette smoke solution alone, so its shown O.lumol/L PC has no function to the cigarette smoke solution induce the A549 cell DNA damage. There is significant difference between the group of 0.5nmol/L PC exposed cigarette smoke solution simultaneously to group of lO.Oumol/L pC exposed cigarette smoke solution simultaneously and the cigarette smoke solution alone, showing that the PC concentration from 0.5 umol/L to lO.Oumol/L can accelerate the A549 DNA damage by the cigarette smoke solution, particularly, the bad function is increasing with the PC concentration enhanced.There is a same found by the flow cytometer examined. All experiments can be repeated.[Conclussion] Exposure of A549 cells to cigarette smoke solution can induce H2AX phosphorylation. 0.5umol/L and 10.0|imol/L of PC have no protective effective against cigarette smoke solution induced DNA damage, but they can accelerate the A549 cell DNA damage, especially the degree of damage is enchanced with the PC concentration increasing.