| Object: Olaquindox is an antimicrobial growth promoter. As the characteristicsof high efficient, low toxicity and residue, Olaquindox is widely used as feedadditives in China. However, due to improper use of olaquindox, a large number ofanimal poisoning and deaths have occurred, causing widespread concern in social. Inthis study, by observing the effect of Olaquindox on HEK-293(human embryonickidney cells), such as DNA strand breaks, the oxidative stress and lysosomalmembrane and mitochondrial membrane stability, we will explore the possiblegenotoxic mechanism, also to further assess the impact on human health and provideexperimental evidence.Method: In this study, the genotoxic potential of Olaquindox was investigated inhuman embryonic kidney cell line293(HEK-293) using single cell gelelectrophoresis (SCGE) assay. HEK-293cells were pretreated by Desipramine andN-acetylcysteine (NAC), which are intervention agent. The expression level of p53protein, DNA damage-related protein, was detected by Western Blotting. Real-timeRT-PCR detected gene expression levels of ATM and p53(DNA damage-related gene).To explore the possible genotoxic mechanism, Acridine orange (AO) was used tomeasure the intracellular lysosomal membrane stability,2’,7’-dihydro-dichloro-fluorescein (DCFH) and phthalaldehyde (OPT) was used to determine the levels ofintracellular ROS and GSH respectively, and the Rhodamine123was used to detectmitochondrial membrane potential. In order to explore the mechanisms relatedpathways, HEK-293cells were pretreated with Desipramine (50μM) and N-acetylcysteine (N-Acetylcysteine, NAC)(10mM) as lysosomal and oxidative stressprotection agents. The experimental results are all analyzed by using SPSS13.0statistical package.Results: SCGE show that after HEK-293cells treated by200,400and800μg/ml Olaquindox for2h，The result show the tail length is42.88μm,74.10μm,84.16μm respectively, tail DNA percentage is34.06％,36.30％,50.83％respectively, tail moment is15.69μm%,28.72μm%,43.95μm%respectively,compared with the control, the differences is significant. After HEK-293cells weretreated by200,400,800μg/ml Olaquindox for2h, intracellular lysosomal membranestability increase about24.66%,34.93%,48.07%respectively, which is higher thanthat in control group. Compared with the control, the level of ROS in the800μg/mlOlaquindox group statistically increase about67.89%(P＜0.01), and the level of GSHin the400and800μg/ml Olaquindox group obviously decreases about26.92%(P＜0.01) and41.58%(P＜0.01) respectively. After HEK-293cells are treated by400and800μg/ml Olaquindox for12h, compared with the control, mitochondrial membranepotential decreased about30.08%(P＜0.01) and44.95%(P＜0.01) respectively, andalso the expression level of p53protein increase, and the expression levels of p53andATM gene increase in a dose-dependent manner. Compared with HEK-293cellstreated by800μg/ml Olaquindox only, intracellular lysosomal membrane potentialdecrease about42.21%(P＜0.01) in the group of HEK-293cells pretreatmented by50μM desipramine for1h. Compared with HEK-293cells treated by800μg/mlOlaquindox only, ROS levels decreased about36.74%(P＜0.01) and GSH levelsincreased about70.65%(P＜0.01) in the group of HEK-293cells pretreatmented by10mM NAC for1h. Two protective agent can effectively reduce the DNA strandbreaks caused by Olaquindox in comet tail.Conclusion: Olaquindox can induce DNA strand breaks of HEK-293cells,upregulate the expression of the DNA damage-related proteins p53and the genes p53and ATM. Underlying mechanisms of DNA damage induced by Olaquindox may berelated with lysosomal pathway: olaquindox lead to DNA strand breaks by changingthe lysosomal membrane permeability and releasing some lysosomal hydrolases. Desipramine protects lysosomal membrane stability, and inhibits the proteinexpression of p53. DNA damage induced by Olaquindox is related with oxidativestress: As an effective antioxidant, NAC reduce the intracellular levels of oxidativestress, inhibit DNA damage, and downregulate p53protein expression. DNA damageinduced by Olaquindox is related with mitochondria: The low stability ofmitochondrial membrane induces the DNA strand breaks. In this experiment, weproved that the changes of lysosomal membrane stability and generation of oxidativestress are before the changes in mitochondrial membrane potential. |
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