Chlorpyrifos alters functional integrity and structure of an in vitro BBB model: co-cultures of bovine endothelial cells and neonatal rat astrocytes.
The blood-brain barrier (BBB) is a structural and functional interface between the circulatory system and the brain. Organophosphorous compounds such as chlorpyrifos (CPF) may cross the BBB and disrupt BBB integrity and function. To determine events that may contribute to CPF toxicity, we used an in vitro BBB model in which bovine microvascular endothelial cells (BMEC) and neonatal rat astrocytes were co-cultured. We hypothesized that CPF is metabolized by the BBB leading to an inhibition of esterase activity and a disruption of the BBB. The co-culturing of BMECs and astrocytes resulted in tight junction formation as determined by electron microscopy, electrical resistance and western blot analysis of two tight junction-associated proteins (ZO-1 and e-cadherin). We observed time dependent increases in ZO-1 and e-cadherin expression and electrical resistance during BBB formation, which were maximal after 9-13 days of co-culturing. The CPF concentration and production of its metabolites were monitored by HPLC following 24 h exposure to CPF on the luminal side of the BBB. We found that the BBB metabolized CPF, with the metabolite 2,3,6-trichloro-2-pyridinol being the major product. CPF and its metabolites were detected on the abluminal side of the BBB suggesting that CPF crossed this barrier. CPF was also detected intracellularly and on the membrane inserts. At tested concentrations (0.1-10 microM), CPF inhibited both carboxylesterase (CaE) and cholinesterase (ChE) activities in BMECs by 43-100%, while CPF-oxon totally inhibited CaE and ChE activity in concentrations as low as 0.1 microM. CPF also caused a concentration-dependent decrease in electrical resistance, with significant inhibition observed at 1 nM and complete loss at 1 microM. These data show that low concentrations of CPF and its metabolites are present within the BBB. CPF and its metabolites, especially CPF-oxon, contribute to the inhibition of CaE and ChE activity, as well as the alteration of BBB integrity and structure.
Parran
Damani K
DK
Laboratory for Neurotoxicity Studies, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Tech, 1 Duckpond Drive, Blacksburg, VA 24061-0442, USA.
Magnin
Geraldine
G
Li
Wen
W
Jortner
Bernard S
BS
Ehrich
Marion
M
D000818:Animals; D001253:Astrocytes / Q000187:drug effects*; D001711:Biotransformation; D001812:Blood-Brain Barrier / Q000187:drug effects*; D015153:Blotting, Western; D043182:Carboxylesterase / Q000378:metabolism; D002417:Cattle; D002469:Cell Separation; D002470:Cell Survival / Q000187:drug effects; D004390:Chlorpyrifos / Q000493:pharmacokinetics / Q000633:toxicity*; D002800:Cholinesterase Inhibitors / Q000633:toxicity*; D002851:Chromatography, High Pressure Liquid; D018920:Coculture Techniques; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects*; D007306:Insecticides / Q000493:pharmacokinetics / Q000633:toxicity*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D051381:Rats
D002800:Cholinesterase Inhibitors; D007306:Insecticides; D007770:L-Lactate Dehydrogenase; D043182:Carboxylesterase; D004390:Chlorpyrifos