Okai Yasuji, Sato Eisuke F, Higashi-Okai Kiyoka, Inoue Masayasu
Department of Human Life Science, Osaka Kun-Ei Women's College, Sets City, Osaka 566-8501, Japan.
Environ Health Perspect. 2004 Apr;112(5):553-6. doi: 10.1289/ehp.6584.
Although para-nonylphenol (NP) is known as an endocrine disruptor, the immunologic effect of NP has been poorly analyzed. We found that NP from 5 to 50 microM caused a dose-dependent stimulatory effect on the generation of reactive oxygen species (ROS) in human blood neutrophils, which was measured by using a chemiluminescence reagent, luminol. Furthermore, ROS-scavenging enzymes such as catalase and superoxide dismutase and antioxidative agents alpha-tocopherol and beta-carotene showed strong preventive effects on NP-induced ROS generation. To analyze the biochemical mechanism of NP-induced ROS generation in human neutrophils, we investigated the effects of different types of metabolic inhibitor for the activation pathways of ROS generation in the cells. Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase inhibitor, diphenyl iodonium chloride and the myeloperoxidase inhibitor sodium azide (NaN3) showed remarkable inhibitory effects on ROS generation induced by NP, but an inhibitor against mitochondrial respiratory function, potassium cyanide (KCN), did not exhibit significant effect. Furthermore, the phosphatidylinositol-3 (PI3) kinase inhibitor wortmannin and the tyrosine kinase inhibitor protein phosphorylation inhibitor 1 (PP1) caused strong suppression against NP-induced ROS generation. The selective protein kinase C inhibitor Ro-32-0432, p38 MAP kinase inhibitor SB 203580, and ERK MAP kinase inhibitor PD 98059 also showed significant suppressive effects on NP-induced ROS generation. These results suggest that NP causes an enhancing effect on ROS generation in human blood neutrophils through the activation of signal transduction pathways associated with the respiratory burst function in these cells. Additionally, to examine in vivo effects of NP, we also analyzed the effects of NP itself and the synergistic effects of NP and a typical inflammatory agent, opsonized zymosan, on human whole blood including neutrophils.
虽然对壬基酚（NP）作为一种内分泌干扰物已为人所知，但其免疫效应却鲜有分析。我们发现，5至50微摩尔的NP对人血中性粒细胞中活性氧（ROS）的产生具有剂量依赖性刺激作用，这是通过使用化学发光试剂鲁米诺进行测量的。此外，过氧化氢酶和超氧化物歧化酶等ROS清除酶以及抗氧化剂α-生育酚和β-胡萝卜素对NP诱导的ROS产生具有很强的预防作用。为了分析NP诱导人中性粒细胞产生ROS的生化机制，我们研究了不同类型的代谢抑制剂对细胞内ROS产生激活途径的影响。还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）依赖性氧化酶抑制剂二苯基碘鎓氯化物和髓过氧化物酶抑制剂叠氮化钠（NaN3）对NP诱导的ROS产生具有显著抑制作用，但线粒体呼吸功能抑制剂氰化钾（KCN）则未表现出明显作用。此外，磷脂酰肌醇-3（PI3）激酶抑制剂渥曼青霉素和酪氨酸激酶抑制剂蛋白磷酸化抑制剂1（PP1）对NP诱导的ROS产生有强烈抑制作用。选择性蛋白激酶C抑制剂Ro-32-0432、p38丝裂原活化蛋白激酶抑制剂SB 203580和细胞外信号调节激酶丝裂原活化蛋白激酶抑制剂PD 98059对NP诱导的ROS产生也有显著抑制作用。这些结果表明，NP通过激活与这些细胞呼吸爆发功能相关的信号转导途径，对人血中性粒细胞中ROS的产生具有增强作用。此外，为了研究NP的体内效应，我们还分析了NP本身以及NP与一种典型炎症因子（调理酵母聚糖）对包括中性粒细胞在内的人全血的影响及协同效应。
