Study On The Immunotoxicity Induced By NP In Female Sprague-dawley Rats

Objective: Nonylphenol （NP） is the final biodegradation product of nonylphenolpolyethoxylates （NPEs）, which are nonionic surfactants and widely used in themanufacture of detergents, herbicides, insecticides, plastic additives and emulsifiers. Asa representative environmental endocrine disruptor, its adverse effects on organismsincludes those affecting the endocrine system, reproductive system, immune system,growth and tumor promotion. Due to its estrogenic activity, low biodegradability andbio-accumulation, a very low concentration of nonylphenol can pose harmful effects onbiological systems. However, most previous studies have focused on the toxicity of NPto developmental process and reproductive system, especially in aquatic ecosystems.Little is known about the toxicological effects of NP on immune system, especially onreproductive immunology. To address this issue, the effects of nonylphenol on spleenand thymus tissues and splenocytes of female SD rats were studied and its effects onimmunological function of female SD rats were discussed, which provided a scientificbasis to further explore the mechanisms of NP-induced immune injury.Methods: Thirty-two female SD rats were randomly divided into four groups,8ineach, treated with NP through gavage at the doses of0,20,80and200mg/kgrespectively, once two days for60consecutive days. In the end of the experiment, thespleen and thymus were aseptically excised and weighed in order to calculate the radiobetween the organ and body weight for each animal; blood samples were taken from theabdominal aorta and processed for haematological investigations; the pathologicalexamination of the spleen and thymus was done; the level of natural killer （NK） cells,natural killer T （NKT） cells in the spleen, thymus and peripheral blood were detected byflow cytometry （FCM）, and the level of interleukin-4（IL-4）, interferon-gamma （IFN-γ）in serum was measured with enzyme linked immunosorbent assay. Meanwhile, the levelof reactive oxygen species （ROS）, intracellular free calcium concentration ([Ca²⁺]i),mitochondrial membrane potential （MMP） and apoptosis in spleen and thymiclymphocytes were detected by flow cytometry. Results:（1） Compared with the control, the spleen and thymus weight of highdose group were decreased （P<0.05）, and both of them were in a dose-dependentmanner（spleen: r=-0.480, P<0.05; thymus: r=-0.317, P<0.05）, but no any changewas observed in spleen and thymus index;（2） The predominant haematological findingwas a highly significant increase of the number of white blood cell, lymphocyte andneutrophil granulocyte and the percentage of neutrophil granulocyte of female ratsexposed to200mg/kg NP compared to control group. Hemoglobin content of rats in200mg/kg NP treated groups were slightly elevated when compared to control levels （P <0.05）;（3） Histopathological changes were found among the spleen of the experimentalgroups that characterized with white pulp reduced, splenic corpuscle shrunk and spleensinus vascular dilatation and congestion. However, no abnormalities or treatment relatedchanges were observed in any of the thymus sections of the treatment groups whencompared with the control and significant alternation in the ratio of cortex: medulla wasnot observed in the treatment groups;（4）NP induced reduction of the level of IL-4andIFN-γ in serum and the differences were statistically significant, whereas, the IFN-γ/IL-4ratio did not show a statistically significant difference in the treatment groupswhen compared with the control;（5） NP had no effect on the level of NK and NKT cellsin the thymus and spleen, but the percentage of NK and NKT cells in peripheral bloodwas slightly higher with NP increasing;（6） Compared with the control, the levels ofROS and calcium in spleen lymphocytes were increased dramatically in the200mg/kgNP treated group. Both of them were in a dose-dependent manner(ROS: r=0.488,P<0.05;[Ca²⁺]i: r=0.497, P<0.05); NP induced reduction of the level of MMP andnegative relation between the level and dose was found（r=-0.565, P<0.05）; NP onlyinduced increase of the level of ROS in thymic lymphocytes and positive relationbetween the level and exposure dose was found （r=0.486, P<0.05）;（7）Concentration-dependent relationship between NP and apoptosis was shown.Conclusion:（1） NP can induce the spleen and thymus weights decreased greatly inhigh dose group, and both of them were in a dose-dependent manner, which indicatedthat NP can affect the development of immune system in rats;（2） The predominanthaematological finding indicated that NP can seriously affect the animal hematologicalparameters and cause adverse effects on blood system;（3） Histopathological changeswere found among the spleen of the experimental groups that characterized with white pulp reduced and spleen sinus vascular dilatation and congestion, which may affect theimmune function of female rats;（4） In our study, we found that the percentage of NKand NKT cells in peripheral blood were slightly higher with NP increasing, which maycause certain effects on body. Thus, we need more researches in future. In our presentstudy, Th1/Th2cytokine imbalance was not observed, but NP induced reduction of thelevel of IL-4and IFN-γ in serum, which may affect immune responses and causeadverse effects on immune system function in female rats;（5） By flow cytometry,results showed that, NP induced increase of the levels of ROS and calcium in spleenlymphocytes and enhanced ROS production in thymic lymphocytes; NP aslo inducedreduction of the level of MMP in spleen lymphocytes. Both of them were in adose-dependent manner. NP aslo induced apoptosis in rat spleen and thymic lymphocyteto a certain extent. The results indicated NP may induce adverse effects on the spleenand thymic lymphocyte in rats, which may affect the immune function of female rats.