Daré Elisabetta, Tofighi Roshan, Vettori Maria Vittoria, Momoi Takashi, Poli Diana, Saido Takaomi C, Mutti Antonio, Ceccatelli Sandra
The National Institute of Environmental Medicine, Division of Toxicology and Neurotoxicology, Karolinska Institutet, Box 210, S-171 77, Stockholm, Sweden.
Brain Res. 2002 Apr 12;933(1):12-22. doi: 10.1016/s0006-8993(02)02274-6.
Neurobehavioral changes have been described in workers occupationally exposed to styrene vapors. Alterations of neurotransmitters and loss of neurons have been observed in brains of styrene-exposed rats. However, the mechanisms of neuronal damage are not yet clearly understood. We have characterized the cellular alterations induced by the main reactive intermediate of styrene metabolism, styrene 7,8-oxide (SO) in the human neuroblastoma SK-N-MC cell line and primary culture of rat cerebellar granule cells (CGC). SK-N-MC cells exposed to SO (0.3-1 mM) displayed apoptotic morphology, together with chromatin condensation and DNA cleavage into high molecular weight fragments of regular size. These features were accompanied by the activation of class II caspases, as detected with the DEVD assay, by following the cleavage of the caspase-substrate poly (ADP-ribose) polymerase (PARP) and by detection of the active fragment of caspase-3. Pre-incubation of the cells with the caspase inhibitor z-VAD-fmk reduced the cellular damage induced by SO, suggesting that caspases play an important role in SO toxicity. Increased proteolysis by class II caspases was detected also in primary culture of CGC exposed to SO. In addition, the presence of the 150-kDa cleavage product of alpha-fodrin suggests a possible activation of calpains in SK-N-MC cells. Moreover, SO did not affect the level of expression of the p53 protein, even though it is known to cause DNA damage. The identified intracellular pathways affected by SO exposure provides end-points that can be used in future studies for the evaluation of the neurotoxic effect of styrene in vivo.
职业接触苯乙烯蒸气的工人已出现神经行为变化。在接触苯乙烯的大鼠大脑中观察到神经递质改变和神经元丧失。然而，神经元损伤的机制尚未完全清楚。我们已对苯乙烯代谢的主要反应中间体苯乙烯7,8 - 氧化物（SO）在人神经母细胞瘤SK - N - MC细胞系和大鼠小脑颗粒细胞（CGC）原代培养物中诱导的细胞变化进行了表征。暴露于SO（0.3 - 1 mM）的SK - N - MC细胞呈现凋亡形态，伴有染色质凝聚和DNA裂解为规则大小的高分子量片段。这些特征伴随着II类半胱天冬酶的激活，通过DEVD分析检测到，通过追踪半胱天冬酶底物聚（ADP - 核糖）聚合酶（PARP）的裂解以及检测半胱天冬酶 - 3的活性片段。用半胱天冬酶抑制剂z - VAD - fmk对细胞进行预孵育可减少SO诱导的细胞损伤，表明半胱天冬酶在SO毒性中起重要作用。在暴露于SO的CGC原代培养物中也检测到II类半胱天冬酶引起的蛋白水解增加。此外，α - 血影蛋白150 kDa裂解产物的存在表明SK - N - MC细胞中钙蛋白酶可能被激活。此外，尽管已知SO会导致DNA损伤，但它并未影响p53蛋白的表达水平。所确定的受SO暴露影响的细胞内途径提供了可用于未来体内评估苯乙烯神经毒性研究的终点。
