Toxic Effects Of The N, N-Dimethylformamide, DMF To Liver Cell Of Human Being And Interference Of Vitamin C

N, N-Dimethylformamide(DMF) is a kind of organic solvent with low noxiousness. DMF is mostly used in extractions, organic synthesises, dyestuffs, fibres, leathers, pharmacies, and so on. With the development of modern industry, DMF is used by more and more areas, but toxic affairs occured on and on because of exposed to it. So it is especially important to prevent such lesions. Vitamin C(VitC) is a water-solubility matter and can resist oxide. It widely exists in people's blood plasma and cells and can directly participate in oxidation-effect or indirectly deoxidize other antioxidant. So people have paid more and more attention to VitC in recent years.In this experiment, liver cells of human being were cultured as the study object. MTT colorimetry was used to observe the effect of different DMF concentrations on multiplication and proliferation of liver cells in different working hours. The next investigation was not only to probe into that DMF did harm to liver cells showed by dose-effect relation, but also to explore different VitC concentrations intervented in such damages. Thus working concentration and hours basis for next experimentation was offered. Comet assay(single-cell gel electrophoresis) was used to observe liver cells' DNA damage by the different DMF concentrations and different VitC concentrations intervented in such damages. This will offer theoretical basis for preventing such occupational harmness from happening and protecting worker's health.Objective1. To investigate the toxic effects of N, N-Dimethylformamide(DMF) of different concentrations on liver cells of human being and the intervention of the Vitamin C of different concentration in such damage.2. To explore DNA damage of liver cells of human being caused by the different doses of dimethylformamide (DMF) exposure, and the effects of Vitamin C (VitC) intervented in such damage.Methods1. The liver cells in logarithmic increasing phase were prepared into suspension of 5xl05cell/ml density, then the suspension was planted in 96-well plates, with each well 200ul suspension. After being cultured 24 hours, the cells were contaminated by DMF, whose eventual concentration is 1.56n 6.25^ 25 and 100 mmol/L respectively. The cell suspension was prepared in the same way and was planted it in culture receptacle, with each well 180ul suspension and 20ul Vitamin C (whose eventual concentration is 0.025 > 0.05 and O.lOmmol/L, respectively). After being cultured 24 hours, the cells were contaminated by DMF, whose concentration is 25 mmol/L and 100 mmol/L. Then the cells were kept on culturing for 6h, 12h>24h respectively. After this, MTT assay was performed to detect the viability and proliferation of the cells, and the intervention of Vitamin C. There were groups of control and blank in this experiment. The viabilityof cell rate = ( the experimentation absorbency - the blank absorbency) / (the negative absorbency-the blank absorbency).2. After being contaminated, the cells were digested and beated upon by trypsin, then were centrifuged and suspended in PBS solution (The cell density was approximately 4x 106cell/ml). The suspension was kept in a 4°C condition. At the same time, trypan blue pigmentation was used to count the cell livability. The Comet Assay (single-cell gel electrophoresis) in vitro was performed to detect the liver cells' DNA damage induced by DMF of 1.56> 6.25> 25 and lOOmmol/L for 6h, 12h> 24h respectively, andthe damage of liver cells (which was pretreated by VitC of 0.025 > 0.05 > 0.10 and 0.25mmol/L) induced by DMF of lOOmmol/L for 61k 24h. In this experiment, there were groups of control and negative, and The data were expressed as the mean tail length (MTL) and the mean tail moment (MTM).Results1. MTT Colorimetry Assay: (Din DMF experiment, the cell proliferation rate of the groups being contaminated was statistically significant lower (PO.01) than the negative group. During each contaminating hour group, the cell proliferation rate decreased as working hours prolonged, DMF did harm to liver cells showed by contaminating time-effect relation. And in the group contaminated by DMF of lOOmmol/L in 24h, 0.56±0.09, the cell proliferation rate was the lowest.(2)In the experiment of VitC intervention, ?The first one is the cell was pretreated by VitC, and then contaminated by DMF of 25mmol/L, the cell proliferation rate in the DMF contaminated groups interfered by lower doses (0.025mmol/L and 0.05mmol/L) VitC were higher than the DMF contaminated control group, but the cell proliferation rate in the DMF contaminated group interfered by hinger (O.Olmmol/L) VitC was much lower than the DMF contaminated control group. ?The second one is the cell was pretreated by VitC, and then contaminated by DMF of lOOmmol/L, the cell proliferation rate in the groups interfered by VitC of 0.025 mmol/L and 0.05 mmol/L in 12h and 24h were higher than the DMF contaminated group, but the cell proliferation rate in the group interfered by VitC of 0.01 mmol/L was much lower than the DMF contaminated control group.2. Comet Assay: (DThe MTL difference of DNA damage in DMF contaminated groups was statistically significant lower (P< 0. 05) in comparation to negative group. That means DMF may induce the DNA damage of liver cells. With the exclusion of 6.25 mmol/LDMF in 6h group, the MTL difference of DNA damage in the 6.25mmol/I^ 25 mmol/L and lOOmmol/L DMF contaminated groups were not statistically significant (P>0.05) respectively conmpared to control group(P>0.05). And the MTL value became larger and larger as contaminated dose increased. There was obvious dose-effect relationship. With the exclusion of lOOmmol/L DMF group, the MTL value of each DMF contaminated group became larger and larger as working hours increased. There was obvious contaminating time-effect relationship. The MTM difference of DNA damage in DMF contaminated groups was statistically significant lower (P< 0. 05) in comparation to negative group.(2) The MTL difference of DNA damage in VitC intervention groups were statistically significant (PO.05) compared to negative group. The MTL difference of DNA damage in 25 mmol/L and lOOmmol/L VitC groups were not statistically significant (P>0.05) respectively compared to control group. The lower doses (0.025mmol/L, 0.05mmol/L and O.lOmmol/L) VitC can reduce the damage of DNA induced by DMF, but the higher (0.25mmol/L) VitC cannot reduce the damage of DNA induced by DMF obviously.Conclusions1. In MTT experiment:(DDMF could inhibit The liver cells proliferation, thereinto DMF concentration and hour, the cell proliferation rate was the lowest in the lOOmmol/L concentration of DMF and the 24 contaminated hours. In each contaminated hour, as the concentration of DMF increased, there appeared that the cell proliferation rate decreased;as working hours extended, there appeared that the cell proliferation rate decreased too. (2)VitC of 0.025 and 0.05 mmol/L concentration could inhibit the toxic effect of DMF contaminating cells for long working time, but VitC of O.lOmmol/L concentration could cooperate the toxic effect of DMF for some degree.2. In comet assay experiment:(DDMF might induce DNA damage of liver cells of human being in spite of its concentration or hour. And as DMF concentration increased, there appeared that the comet tail length prolonged.(2)VitC of 0.025, 0.05 mmol/L and O.lOmmol/L could prevented DNA damage induced by DMF, however, in spite of 24-hour VitC pretreatment.