Aly Hamdy A A, Domènech Oscar
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt.
Toxicol Lett. 2009 Dec 1;191(1):79-87. doi: 10.1016/j.toxlet.2009.08.008. Epub 2009 Aug 15.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant that induces hepatic and extrahepatic oxidative stress and the mechanisms of TCDD-induced reactive oxygen species are not fully investigated. Moreover, the potential toxicity of TCDD in isolated rat hepatocytes is not fully explored. The aim of the current study was to explore the possible cytotoxic effect of TCDD on primary rat hepatocytes and to explore the impact of mitochondria in TCDD-induced toxicity. Hepatocytes were isolated from adult rat liver and incubated with 0, 5, 10 or 15 nM of TCDD for 24, 48 and 72 h. Cell viability, lactate dehydrogenase (LDH) leakage into media along with reactive oxygen species (ROS) generation and hydrogen peroxide (H(2)O(2)) production, mitochondrial membrane potential (Deltapsi(m)), superoxide dismutase (SOD), catalse (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), total thiol contents, hepatic aryl hydrocarbon hydroxylase (AHH), and ethoxyresorufin O-deethylase (EROD) were performed in hepatocytes. In addition, superoxide anion generation, lipid peroxidation (LPO), mitochondrial protein carbonyl content and respiratory chain complexes II and IV were assayed in hepatocyte mitochondria. Cell viability was significantly decreased while LDH leakage into media was significantly increased in a dose and time related manner. ROS generation and H(2)O(2) production along with EROD and AHH activities were significantly increased in hepatocytes in the same pattern. The antioxidant enzymes SOD, CAT, GPx and GR and the non-enzymatic protein thiols, in addition to Deltapsi(m) were significantly decreased in hepatocytes in a concentration and time dependent pattern. On the other side, mitochondrial superoxide anion along with LPO and mitochondrial protein carbonyl content were significantly increased while the respiratory chain complexes II and IV activities were significantly decreased in hepatocyte mitochondria. This effect may lead to disruption in the functional integrity of hepatocytes and hepatocyte mitochondria. In conclusion, our data clearly show that TCDD induces hepatocyte toxicity and mitochondrial dysfunction by a mechanism involving generation of ROS. Mitochondria might be the primary source of (or at least contribute to) the oxidative stress response and resulting toxicological outcomes elicited by TCDD.
2,3,7,8-四氯二苯并-对-二恶英（TCDD）是一种环境污染物，可诱导肝脏和肝外氧化应激，而TCDD诱导活性氧生成的机制尚未得到充分研究。此外，TCDD对分离的大鼠肝细胞的潜在毒性也未得到充分探索。本研究的目的是探讨TCDD对原代大鼠肝细胞可能的细胞毒性作用，并探讨线粒体在TCDD诱导的毒性中的影响。从成年大鼠肝脏中分离肝细胞，并与0、5、10或15 nM的TCDD孵育24、48和72小时。检测肝细胞的细胞活力、乳酸脱氢酶（LDH）泄漏到培养基中的情况以及活性氧（ROS）生成和过氧化氢（H₂O₂）产生、线粒体膜电位（ΔΨm）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GPx）、谷胱甘肽还原酶（GR）、总硫醇含量、肝芳烃羟化酶（AHH）和乙氧基异吩恶唑酮O-脱乙基酶（EROD）。此外，还检测了肝细胞线粒体中的超氧阴离子生成、脂质过氧化（LPO）、线粒体蛋白羰基含量以及呼吸链复合物II和IV。细胞活力显著降低，而LDH泄漏到培养基中的情况以剂量和时间相关的方式显著增加。肝细胞中的ROS生成和H₂O₂产生以及EROD和AHH活性以相同模式显著增加。抗氧化酶SOD、CAT、GPx和GR以及非酶蛋白硫醇，除了ΔΨm外，在肝细胞中以浓度和时间依赖性模式显著降低。另一方面，肝细胞线粒体中的线粒体超氧阴离子以及LPO和线粒体蛋白羰基含量显著增加，而呼吸链复合物II和IV的活性显著降低。这种效应可能导致肝细胞和肝细胞线粒体功能完整性的破坏。总之，我们的数据清楚地表明，TCDD通过涉及ROS生成的机制诱导肝细胞毒性和线粒体功能障碍。线粒体可能是TCDD引发的氧化应激反应和由此产生的毒理学结果的主要来源（或至少有贡献）。
