Dasmahapatra A K, Lee P C
Division of Gastroenterology, Medical College of Wisconsin, MACC Fund Research Center, Milwaukee 53226.
In Vitro Cell Dev Biol Anim. 1993 Aug;29A(8):643-8. doi: 10.1007/BF02634553.
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).
采用虹鳟（Onchorhynchus mykiss）肝细胞短期培养法，在体外研究地塞米松（DEX）对微粒体CYP 1A1蛋白含量和7-乙氧基异吩恶唑酮-O-脱乙基酶（EROD）活性的影响。通过控制胶原酶消化制备肝细胞，并以0.25×10⁶个细胞/cm²的密度接种于预先用鳟鱼皮提取物（7.6微克皮肤蛋白/cm²）包被的塑料培养皿中以促进细胞贴壁，细胞在16℃下培养。细胞在24小时时用DEX（10⁻⁹至10⁻⁷M）或溶剂（二甲基亚砜，DMSO）处理。在72小时时测量微粒体CYP 1A1蛋白含量和EROD活性。与未处理（对照）或DMSO处理的细胞相比，用DEX（10⁻⁸至10⁻⁷M）处理的肝细胞中，使用CYP 1A1特异性抗血清通过蛋白质印迹法测定的CYP 1A1蛋白和EROD活性均显著降低。该效应呈剂量依赖性，即随着DEX剂量增加（10⁻⁸至10⁻⁷M），CYP 1A1蛋白和EROD活性逐渐降低。10⁻⁹M的DEX无效。向用10⁻⁷M DEX处理的肝细胞中同时加入10⁻⁶M RU486（一种II型特异性糖皮质激素受体拮抗剂）可消除DEX的作用。10⁻⁸M的RU486无效。螺内酯（10⁻⁸至10⁻⁶M），一种I型特异性糖皮质激素受体拮抗剂，不能抵消DEX的作用。在相似条件下，单独的RU486或螺内酯（10⁻⁶M）对CYP 1A1无影响。因此，DEX可下调鱼类培养肝细胞中的CYP 1A1，且这种调节是通过II型糖皮质激素受体介导的。
