The Preliminary Study Of Anticoagulation Induced By Sodium Dehydroacetate On Rats And Rabbits

Objective:Sodium Dehydroacetate, as a safe fungicide, is widely used in many fields such as food, feed and cosmetic products because of its broad spectrum antibiotic and good antifungal effects. However, high dose of sodium dehydroacetate could cause animal bleeding tendency. Our previous studies had also found that sodium dehydroacetate could cause multiple organs bleeding of rats. In this paper, we studied the bleeding tendency of rats and rabbits treated with sodium dehydroacetate by measuring PT and APTT of clotting time, detecting VK1, concentration of sodium dehydroacetate and VKORC1 protein expression in blood, and analyzing HE stain histopathology of main organs after rats and rabbits were administrated with different dose of sodium dehydroacetate.Methods:Wistar rats, half males and half females, were given a gavage of sodium dehydroacetate once a day for 13d at doses of 50 mg/kg,100 mg/kg,150 mg/kg,200 mg/kg, saline as a control group,14 rats in each group. Like the rats, rabbits were given a gavage of sodium dehydroacetate at doses of 100 mg/kg and 200 mg/kg, and saline as a control. Blood and main organs were collected after administrated for 3d,5d,7d,9d, 11d, and 13d,4 rats or rabbits in each group. Then we measured PT and APTT, analyzed histopathology of tissues of the heart,liver, spleen, lungs and kidneys, detected concentration of sodium dehydroacetate in blood by HPLC method, VK1 levels of serum by ELISA technology and VKORC1 protein expression with immunohistochemical (IHC) staining. When HPLC method was used to detect concentration of sodium dehydroacetate in serum, blood samples were extracted with acetonitrile, concentrated, then detected in 293nm, which the chromatographic condition was X Bridge C18 column and methanol -0.02 mol/L ammonium acetate (35/65, v/v) as the mobile phase.Results:After Rats were oral administrated different doses of sodium dehydroacetate, the PT and APTT were prolonged significantly (P<0.001) compared to control group, and PT and APTT values of male rats were significantly higher than that of female rats (P<0.05). By analysis of HE-staining rat tissue section, obvious bleeding in 200 mg/kg group were saw in organs of heart, liver, spleen, lungs and kidneys, and the bleeding extent in liver may be related to doses of sodium dehydroacetate used. The VK1 levels in blood of 100-200 mg/kg sodium dehydroacetate groups were significantly lower than that of control group (P<0.01). VKORC1 protein level in 200 mg/kg group was lower than that in control group in organs of heart, liver, spleen, lungs and kidneys based on the IHC analysis (P<0.001). Concentration of sodium dehydroacetate detected by HPLC method was 20-50 mg/L in rat blood. The correlation coefficients between VK1 levels in blood and PT or APTT were 0.719 and 0.599 respectively; the correlation coefficients between serum concentration of sodium dehydroacetate and PT or APTT were 0.900-0.988 and 0.806-0.852. respectivelyAfter rabbits were administrated with sodium dehydroacetate with 150-200 mg/kg for 7d, the PT and APTT values were prolonged remarkably (P< 0.001) compared to control group. And histopathology analysis of 200 mg/kg group showed that obvious bleeding in 200 mg/kg group were saw in organs of liver, spleen, lungs and kidneys. Concentration of sodium dehydroacetate detected by HPLC method was 5-20 mg/L in rabbit blood. Correlation analysis showed that correlation coefficients of serum concentration of sodium dehydroacetate and PT or APTT were 0.837 and 0.351 respectively.Conclusion:High dose of sodium dehydroacetate (150-200 mg/kg) could prolong the coagulation parameters of rats and rabbits significantly, and caused bleeding in main organs; the VK] levels in bloods and VKORC1 expression in organs of rats and rabbits decreased obviously. The prolonged PT or APTT caused by sodium dehydroacetate in rats and rabbits appeared high relationship with the serum concentration of sodium dehydroacetate and VK1 levels in serum. In conclusion, sodium dehydroacetate could cause bleeding of rats and rabbits with probable mechanism that sodium dehydroacetate inhibited VKORC1 expression, then influenced the circulation of VK and reduced the VK1 level.