Drug metabolizing enzyme activities and superoxide formation in primary and immortalized rat brain endothelial cells.
The activities of several enzymes involved in drug metabolism, NADPH-cytochrome P450 reductase, cytochrome P450 isoforms CYP1A and CYP2B, and uridine diphosphate glucuronosyltransferase (UGT) have been measured in primary cultures of rat cerebrovascular endothelial cells and in the immortalized rat brain endothelial cell line RBE4. These drug metabolizing activities were similar in the microsomes prepared from both cell types, even after 20 passages for RBE4 cells. These results were confirmed by Western immunoblotting analysis, using polyclonal antibodies raised against rat liver enzymes. The superoxide production observed during NADPH-cytochrome P450 reductase-dependent monoelectronic reduction of four xenobiotics, menadione, anthraquinone, nitrofurazone and diquat, was also investigated in these cultured cells at confluence. The rates of radical production were concentration-dependent. The superoxide formation induced by quinone metabolism was comparable in both cell cultures, and high amounts of superoxide radicals were produced even after 20 passages of RBE4 cells. On the other hand, nitrofurazone and diquat metabolism produced weak amounts of superoxide radicals in both cell types. Taken together, these results suggest that RBE4 cell line seems to constitute a valuable in vitro model for studies on the activity of some enzymatic systems involved in drug metabolism at the blood-brain barrier and the functional consequences of their activity.
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CNRS URA 1288, Laboratoire de Pharmacologie, Faculté de Médecine, Vandoeuvre-lès-Nancy, France.
Bayol-Denizot
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Suleman
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Roux
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Minn
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D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D008297:Male; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species; D013481:Superoxides / Q000378:metabolism*; D015262:Xenobiotics / Q000633:toxicity
D017382:Reactive Oxygen Species; D015262:Xenobiotics; D013481:Superoxides; D006899:Mixed Function Oxygenases