9,10-Phenanthrenequinone provokes dysfunction of brain endothelial barrier through down-regulating expression of claudin-5.

Chronic exposure to diesel exhaust particle (DEP) is considered to provoke dysfunction of the blood-brain barrier, but the detailed molecular mechanism remains unclear. In this study, we investigated the toxic effects of five DEP components against human vascular cells and found that, among them, 9,10-phenanthrenequinone (9,10-PQ), a major tricyclic quinone in DEP, most potently elicits the cellular toxicities. Additionally, treatment with 9,10-PQ at its cytolethal concentrations (more than 2 µM) facilitated the production of reactive oxygen species (ROS), caspase activation, and DNA fragmentation in human brain microvascular endothelial (HBME) cells, inferring that high concentrations of 9,10-PQ elicit the cell apoptosis through the ROS-dependent mechanism. Measurement of trans-endothelial electrical resistance and paracellular permeability showed that treatment with sublethal concentrations (less than 1 µM) of 9,10-PQ elevates permeability across HBME cell monolayer. Immunofluorescence observation and Western blotting analysis also revealed that the 9,10-PQ treatment remarkably down-regulated the intercellular localization and expression of claudin-5 (CLDN5), a tight junctional protein that plays a key role in function of the blood-brain barrier, and the down-regulation was markedly recovered by pretreatment with a proteasome inhibitor Z-Leu-Leu-Leu-CHO. This result may indicate that sublethal concentrations of 9,10-PQ facilitate the dysfunction of the endothelial cell barrier through lowering in the expression and proteasomal proteolysis of CLDN5. The treatment with 9,10-PQ promoted nitric oxide (NO) production presumably through the induction of inducible NO synthase. In addition, the 9,10-PQ-mediated down-regulation of CLDN5 was ameliorated and deteriorated by pretreating with a scavenger and donor, respectively, of NO. Similarly to the 9,10-PQ treatment, treatment with a donor of peroxynitrite, a highly reactive oxidant formed by the reaction of NO and superoxide anion, resulted in the marked reduction of CLDN5 expression and elevation of 26S proteasome-based proteolytic activities. Thus, it is suggested that the formation of NO and peroxynitrite participates in the mechanism of brain endothelial cell barrier dysfunction elicited by 9,10-PQ.

Protective Effect of Aldo-keto Reductase 1B1 Against Neuronal Cell Damage Elicited by 4'-Fluoro-α-pyrrolidinononanophenone.

Chronic exposure to cathinone derivatives increases the risk of severe health hazards, whereas little is known about the detailed pathogenic mechanisms triggered by the derivatives. We have recently shown that treatment with α-pyrrolidinononanophenone (α-PNP, a highly lipophilic cathinone derivative possessing a long hydrocarbon main chain) provokes neuronal cell apoptosis and its 4'-fluorinated analog (F-α-PNP) potently augments the apoptotic effect. In this study, we found that neuronal SK-N-SH cell damage elicited by F-α-PNP treatment is augmented most potently by pre-incubation with an AKR1B1 inhibitor tolrestat, among specific inhibitors of four aldo-keto reductase (AKR) family members (1B1, 1C1, 1C2, and 1C3) expressed in the neuronal cells. In addition, forced overexpression of AKR1B1 remarkably lowered the cell sensitivity to F-α-PNP toxicity, clearly indicating that AKR1B1 protects from neurotoxicity of the derivative. Treatment of SK-N-SH cells with F-α-PNP resulted in a dose-dependent up-regulation of AKR1B1 expression and activation of its transcription factor NF-E2-related factor 2. Metabolic analyses using liquid chromatography/mass spectrometry/mass spectrometry revealed that AKR1B1 is hardly involved in the F-α-PNP metabolism. The F-α-PNP treatment resulted in production of reactive oxygen species and lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) in the cells. The enhanced HNE level was reduced by overexpression of AKR1B1, which also lessened the cell damage elicited by HNE. These results suggest that the AKR1B1-mediated neuronal cell protection is due to detoxification of HNE formed by F-α-PNP treatment, but not to metabolism of the derivative.

Facilitation of 9,10-phenanthrenequinone-elicited neuroblastoma cell apoptosis by NAD(P)H:quinone oxidoreductase 1.

9,10-Phenanthrenequinone (PQ), a major quinone component in diesel exhaust particles, is considered to provoke damage of respiratory and vascular cells through highly producing reactive oxygen species (ROS), but little is known about its pathophysiological role in neuronal cell damage. In this study, we found that incubation with 1,2-naphthoquinone, 1,4-naphthoquinone and PQ, major quinone components in diesel exhausts, provokes apoptosis of human neuroblastoma cell lines. SK-N-SH cell treatment with a lethal concentration of PQ facilitated ROS production within 6 h. The treatment also promoted formation of 8-hydroxy-deoxyguanosine, p53 activation, elevation of Bax/Bcl-2 ratio, lowering of mitochondrial membrane potential, and resultant activation of caspase-9 and caspase-3, inferring that ROS production, DNA damage and mitochondrial dysfunction are crucial processes of the PQ-triggered SK-N-SH cell apoptosis. The PQ treatment of SK-N-SH cells elevated the level of 4-hydroxynonenal (HNE), a cytotoxic reactive aldehyde generated from lipid peroxidation. The treatment with PQ and HNE also decreased cellular levels of total and reduced glutathiones, and the damage elicited by HNE was ameliorated and deteriorated by pretreating with cell-permeable glutathione analog and the depletor, respectively. Moreover, the treatment with PQ and HNE decreased the proteasomal proteolytic activities, suggesting a contribution of decrease in the antioxidant abilities to the ROS-mediated neuroblastoma cell apoptosis. Our comparative analyses of 17 cells showed a positive correlation between the PQ reductase and NAD(P)H:quinone oxidoreductase 1 (NQO1) activities. In addition, overexpression and knockdown of NQO1 augmented and lowered, respectively, the ROS production through PQ redox-cycling and the quinone toxicity. Furthermore, the treatment with PQ and HNE up-regulated the NQO1 expression. Taken together, PQ exposure produces large amounts of ROS in neuroblastoma cells via NQO1 up-regulation and resultant acceleration of its redox-cycling, followed by activation of the ROS-dependent apoptotic mechanism.

Enhancement of Endothelial Barrier Permeability by Mitragynine.

Persistent inhalation of mitragynine (MG), a major alkaloid in the leaves of Mitragyna speciosa, causes various systemic adverse effects such as seizure, diarrhea and arthralgias, but its toxicity to endothelial cells and effects on barrier function of the cells are poorly understood. In this study, we compared toxicities of MG and mitraphylline, another constituent of the leaves, against human aortic endothelial (HAE), bronchial BEAS-2B, neuronal SK-N-SH, hepatic HepG2, kidney HEK293, gastric MKN45, colon DLD1, lung A549, breast MCF7 and prostate LNCaP cells, and found that MG, but not mitraphylline, shows higher toxicity to HAE cells compared to the other cells. Forty-eight-hours incubation of HAE cells with a high concentration of MG (60 µM) provoked apoptotic cell death, which was probably due to signaling through enhanced reactive oxygen species (ROS) generation and resultant caspase activation. Treatment of the cells with MG at sublethal concentrations less than 20 µM significantly lowered transendothelial electrical resistance and elevated paracellular permeability, without affecting the cell viability. In addition, the MG-elicited lowering of the resistance was abolished by a ROS inhibitor N-acetyl-L-cysteine and augmented by H2O2 and 9,10-phenanthrenequinone, which generates ROS through its redox cycle. These results suggest the contribution of ROS generation to the increase in endothelial barrier permeability.