Roy D, Pathak D N
Department of Environmental Health Sciences, University of Alabama, Birmingham 35294-0008, USA.
J Toxicol Environ Health. 1995 Apr;44(4):449-59. doi: 10.1080/15287399509531973.
We demonstrate for the first time that diethylstilbestrol (DES), a synthetic estrogen, is converted by nuclei to histone-binding metabolite(s). Reaction of [3H]DES with nuclei in the presence of cumene hydroperoxide or NADPH revealed binding of [3H]DES to histone nuclear proteins. Gel electrophoresis experiments revealed that all five histones, 1, 2A, 2B, 3, and 4, were irreversibly bound to [3H]DES. Histones 1 and 3 were more susceptible to the attack by [3H]DES quinone, a metabolite of DES, than histones 2A, 2B, or 4. The kinetic constants, Km and Vmax, of this binding reaction in the presence of cumene hydroperoxide were 10 microM and 750 pmol/mg protein/30 min, respectively. This binding was significantly inhibited by cytochromes P-450 inhibitors. Low-molecular-weight thiols, such as glutathione and cysteine, or thiol modifiers, such as n-ethylmaleimide, dithionitrobenzoic acid, and hydroxymercuric benzoate, drastically inhibited binding of [3H]DES quinone to histone 3. The binding of [3H]DES metabolites to both transcriptionally active and inactive chromatin histone proteins was observed. We conclude that DES is metabolized to histone-binding metabolites, presumably by nuclear cytochrome P-450. DES quinone may be one of the histone-binding DES metabolites. These data suggest that an analogous in vivo modification in the transcriptionally active chromatin histones by DES metabolites may influence gene function.
我们首次证明，合成雌激素己烯雌酚（DES）可被细胞核转化为与组蛋白结合的代谢产物。在异丙苯过氧化氢或烟酰胺腺嘌呤二核苷酸磷酸（NADPH）存在的情况下，[³H]DES与细胞核反应显示[³H]DES与组蛋白核蛋白结合。凝胶电泳实验表明，所有五种组蛋白，即组蛋白1、2A、2B、3和4，都与[³H]DES不可逆地结合。与组蛋白2A、2B或4相比，组蛋白1和3更容易受到DES的代谢产物[³H]DES醌的攻击。在异丙苯过氧化氢存在的情况下，这种结合反应的动力学常数Km和Vmax分别为10微摩尔和750皮摩尔/毫克蛋白质/30分钟。这种结合受到细胞色素P-450抑制剂的显著抑制。低分子量硫醇，如谷胱甘肽和半胱氨酸，或硫醇修饰剂，如N-乙基马来酰亚胺、二硫代硝基苯甲酸和苯甲酸羟汞，能极大地抑制[³H]DES醌与组蛋白3的结合。观察到[³H]DES代谢产物与转录活性和非活性染色质组蛋白都有结合。我们得出结论，DES可能通过细胞核细胞色素P-450代谢为与组蛋白结合的代谢产物。DES醌可能是与组蛋白结合的DES代谢产物之一。这些数据表明，DES代谢产物在转录活性染色质组蛋白中进行的类似体内修饰可能会影响基因功能。
