Toxic Damage Of C57BL/6J Mice By Gavaged With Emulsifier Polysorbate 80

Backgrounds:Polysorbate 80(PS80)is a fine dispersion of minute droplets of one liquid in another in which it is not soluble or miscible.It not only can be added into various foods such as convenience foods,ice cream,jam,butter and dairy products,but also widely used in drug excipients.Hence,humans are likely to be in a long-term exposure of low concentration PS80 environment.The toxicological safety evaluation of PS80 has been completed in the 1970 s,the traditional toxicology evaluation method identify PS80 as a low-toxicity or nontoxic substances.In recent years,some security incidents caused by PS 80 make the conclusion questioned.Therefore,this article is conducted to evaluate the toxic damage caused by exposure of prolonged low-doses PS80 in mice,and to explore the toxicological mechanism.Methods:1)80(half male and half female)6-weeks-old C57BL-6 mice were divided into 4 groups at random after one week's acclimatization.PS80 was dissolved in water and administered by oral gavage at dose 5,50 and 500mg/kg.Meanwhile,the control group received water equally.Mice were weighed,observed and recorded regularly,and they were passive euthanasia at the end of the 90 th day.The various of organs and blood were excised,weighed,then collected.Some tissues were cut and stained with hematoxylin and eosin(H&E).2)We used commercial kits to analysis the serum total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),and catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),malondialdehyde(MDA)of brain and colon tissue;Examined H&E staining of hippocampus and cortex by microscopic.3)The elisa kits were used to determine the serum tumor necrosis factor(TNF-?),interleukin-6(IL-6)and interleukin-?(IL-1?);the mRNA expression of TNF-?,IL-6,IL-1?,IL-10 and P-glycoprotein were detected by real-time PCR;The expression of P-glycoprotein was detected by immunohistochemical staining and western blot methodResults:1)The influence on physiological indexes in mice by PS80During the experiment,the high dose groups(500mg/kg bw)showed less physical activity and slower brain reaction,and some had symptoms of diarrhea and polyuria.There is no significant difference in weight growth in the same gender of mice.Compared with control group,all the mice display obvious enlargement in brain size,female 5mg/kg?50mg/kg and 500mg/kg dose groups obvious bigger than control group(P<0.05),500mg/kg male dose group has the same results;and partial abnomal in liver and kidney to body weight.Obvious hemorrhage was visually observed in spleen tissue;neurogliocytes was found atrophic in shape and reduced in numbers from pathological slices of hippocampus and cerebral tissue,which is further proved by Nissl staining.2)The inflammatory and oxidative damage in brain and colon by PS80SOD activity of treated groups in both brain and colon tissue decreases with different degrees compared with control group.CAT activity declines in brain tissue,but there is not obvious decrease in colon tissue.GSH activity in brain tissue declines with different degrees compared with control group;in colon tissue,female low dose group decreases significantly(P<0.05)compared with control group,while other groups do not show obvious change.MDA of treated groups in brain tissue rises with different degrees compared with control group,but there is not obvious decrease in colon tissue.According to the results of SOD,CAT,GSH and MDA test,we can infer that chronic low-dose PS80 causes oxidative stress in brain tissue of mice,while it has little effect on the colon tissue.It is detected that the content of infmatory factors is very low in serum,which indicates that PS80 didn't cause serious inflammatory response in mice.However,we found the gene expression of inflammatory factors rised with different degrees.Therefore,we can infer that PS80 increased the gene expression of inflammatory factors in brain tissue.3)The study of toxicological mechanism in brain by PS80By real time PCR detection of P-gp protein gene in the expression of brain tissue,the experimental group compared to control group were very significant(P < 0.01)except male low dose group(5mg/kg bw);P-gp protein immunohistochemical microscopy results indicate the main outside the cells around vascular expression,semi-quantitative results analysis of the experimental group P-gp protein expression have different degree lower than the control group.Western blot results of quantitative analysis,the experimental P-gp protein expression quantity than the control group were significantly(P<0.05)or very significantly(P<0.01)reduced.Illustrate PS80 can restrain brain P-gp gene and protein expression,resulting in the mice brain damage.Conclusion:Prolonged low-doses gavage with emulsifier polysorbate 80 in mice would decrease the number of nerve cells,at the same time cause oxidative stress damage,invoke inflammatory markers genes of brain.It maybe caused by the inhibition of P-gp in blood brain barrier.