Kodavanti P R, Shin D S, Tilson H A, Harry G J
Cellular and Molecular Toxicology Branch, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711.
Toxicol Appl Pharmacol. 1993 Nov;123(1):97-106. doi: 10.1006/taap.1993.1226.
Some polychlorinated biphenyls (PCBs) have been reported to alter locomotor activity and decrease brain dopamine function in laboratory animals. PCBs with ortho- and/or parachlorine substitutions and varying number of chlorinations are known to decrease cell dopamine content in vitro and have been detected in brains of animals exposed to PCBs, suggesting that the neurotoxicity could be mediated by ortho-substituted congeners. Dopamine or other neurotransmitter uptake and release phenomena are dependent on the maintenance of intracellular Ca2+ homeostasis, and perturbations in Ca2+ homeostasis could lead to altered cell function and/or death. We compared the effects of two PCB congeners on Ca2+ homeostasis in cerebellar granule cells: 2,2'-dichlorobiphenyl (DCBP), a putative neurotoxic congener, and 3,3',4,4',5-pentachlorobiphenyl (PCBP), a presumed nonneurotoxic congener. In cerebellar granule cells (6-8 days in vitro), DCBP was cytotoxic as indicated by a significant increase in LDH leakage at 200 microM after 2 hr of exposure and at 100 microM after 4 hr exposure. PCBP, on the other hand, did not affect LDH leakage even at 200 microM for up to 4 hr. Although both congeners increased cerebellar granule cell [Ca2+]i, DCPB was more effective in increasing [Ca2+]i to a greater extent than PCBP. The increase in [Ca2+]i produced by both congeners was not transient, but a steady rise was observed with time. To understand cellular Ca(2+)-buffering capacity, Ca2+ sequestration and Ca2+ extrusion were studied in mitochondria, microsomes, and synaptosomes, isolated from adult rat cerebellum. DCBP was a potent inhibitor of 45Ca2+ uptake by mitochondria (IC50 = 6.17 +/- 0.53 microM) and microsomes (IC50 = 7.61 +/- 0.35 microM). PCBP inhibited Ca2+ sequestration by mitochondria (68% of control) and microsomes (72% of control), but the effects were much less than those produced by equivalent concentrations of DCBP. Synaptosomal Ca(2+)-ATPase was inhibited by DCBP, but not by PCBP. These results indicate that at concentrations where cytotoxicity in cerebellar granule cells was not observed, DCBP increased intracellular [Ca2+]i, and at the same concentrations, Ca2+ sequestration by intracellular organelles and Ca(2+)-ATPase in synaptic plasma membrane were inhibited. Although PCBP increased [Ca2+]i in cerebellar granule cells to some extent, it was not potent in affecting Ca2+ sequestration or Ca2+ extrusion in adult cerebellar components. Hence, PCBP-induced slight increase of [Ca2+]i levels in the cells might have been associated with effective Ca2+ sequestration by intracellular organelles, as seen in cerebellar preparations. The results of this study support the hypothesis that the position of chlorine substitution on the biphenyl ring and/or number of chlorine substitutions may have significant implications for predicting potential effects of PCB congeners in the nervous system, and perturbations in Ca2+ homeostasis might play a significant role in the neuroactivity of PCBs.
据报道，一些多氯联苯（PCBs）会改变实验动物的运动活性并降低脑内多巴胺功能。已知具有邻位和/或对位氯取代以及不同氯原子数的多氯联苯会在体外降低细胞多巴胺含量，并且在接触多氯联苯的动物大脑中也已检测到，这表明神经毒性可能由邻位取代的同系物介导。多巴胺或其他神经递质的摄取和释放现象依赖于细胞内Ca2+稳态的维持，而Ca2+稳态的扰动可能导致细胞功能改变和/或死亡。我们比较了两种多氯联苯同系物对小脑颗粒细胞Ca2+稳态的影响：2,2'-二氯联苯（DCBP），一种假定的神经毒性同系物，以及3,3',4,4',5-五氯联苯（PCBP），一种假定的非神经毒性同系物。在体外培养6 - 8天的小脑颗粒细胞中，DCBP具有细胞毒性，暴露2小时后200 microM时乳酸脱氢酶（LDH）泄漏显著增加，暴露4小时后100 microM时也显著增加。另一方面，即使在200 microM下长达4小时，PCBP也不影响LDH泄漏。尽管两种同系物都增加了小脑颗粒细胞的[Ca2+]i，但DCBP在增加[Ca2+]i方面比PCBP更有效，且增加幅度更大。两种同系物引起的[Ca2+]i增加并非短暂的，而是随时间持续上升。为了解细胞的Ca(2+)缓冲能力，对从成年大鼠小脑中分离的线粒体、微粒体和突触体中的Ca2+螯合和Ca2+外排进行了研究。DCBP是线粒体（IC50 = 6.17 +/- 0.53 microM）和微粒体（IC50 = 7.61 +/- 0.35 microM）摄取45Ca2+的强效抑制剂。PCBP抑制线粒体（对照的68%）和微粒体（对照的72%）的Ca2+螯合，但作用远小于同等浓度的DCBP。突触体Ca(2+)-ATP酶受到DCBP抑制，但不受PCBP抑制。这些结果表明，在未观察到小脑颗粒细胞细胞毒性的浓度下，DCBP增加了细胞内[Ca2+]i，且在相同浓度下，细胞内细胞器的Ca2+螯合和突触质膜中的Ca(2+)-ATP酶受到抑制。尽管PCBP在一定程度上增加了小脑颗粒细胞的[Ca2+]i，但在影响成年小脑成分中的Ca2+螯合或Ca2+外排方面并不显著。因此，如在小脑制剂中所见，PCBP诱导的细胞内[Ca2+]i水平轻微升高可能与细胞内细胞器有效的Ca2+螯合有关。本研究结果支持以下假设：联苯环上氯取代的位置和/或氯取代的数量可能对预测多氯联苯同系物在神经系统中的潜在影响具有重要意义，并且Ca2+稳态的扰动可能在多氯联苯的神经活性中起重要作用。
