Down regulation of CYP 1A1 by glucocorticoids in trout hepatocytes in vitro.
Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of 0.25 x 10(6) cells/cm2 in plastic culture dishes precoated with trout skin extract (7.6 micrograms skin protein/cm2) to facilitate cell attachment were maintained at 16 degrees C. Cells were treated with DEX (10(-9) to 10(-7) M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72 h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly lower in DEX (10(-8) to 10(-7) M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10(-8) to 10(-7) M). DEX at 10(-9) M was ineffective. Concomitant addition of 10(-6) M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10(-7) M DEX abolished the DEX effect. RU486 at 10(-8) M was ineffective. Spironolactone (10(-8) to 10(-6) M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10(-6) M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and this regulation is mediated through the type II glucocorticoid receptor(s).
Division of Gastroenterology, Medical College of Wisconsin, MACC Fund Research Center, Milwaukee 53226.
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Dasmahapatra
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Lee
D000818:Animals; D015153:Blotting, Western; D002467:Cell Nucleus / Q000201:enzymology; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D003576:Electron Transport Complex IV / Q000096:biosynthesis*; D004790:Enzyme Induction; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D015735:Mifepristone / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology; D010088:Oxidoreductases / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000037:antagonists & inhibitors / Q000502:physiology; D014337:Trout
D011965:Receptors, Glucocorticoid; D015735:Mifepristone; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019363:Cytochrome P-450 CYP1A1; D003576:Electron Transport Complex IV