Sharma Minoti, Shubert David E, Sharma Moheswar, Lewis Jennifer, McGarrigle Barbara P, Bofinger Diane P, Olson James R
Department of Molecular and Cellular Biophysics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA.
Chem Biol Interact. 2003 Dec 15;146(3):237-49. doi: 10.1016/j.cbi.2003.06.002.
Although long-term tamoxifen therapy is associated with increased risk of endometrial cancer, little is known about the ability of endometrial tissue to biotransform tamoxifen to potentially reactive intermediates, capable of forming DNA adducts. The present study examined whether explant cultures of human endometrium provide a suitable in vitro model to investigate the tissue-specific biotransformation of tamoxifen. Fresh human endometrial tissue, microscopically uninvolved in disease, was cut into 1 x 2-mm uniform explants and incubated with media containing either 25 or 100 microM tamoxifen in a 24-well plate. Metabolites were analyzed by reversed-phase HPLC using postcolumn, online, photochemical activation and fluorescence detection. Three metabolites, namely, alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen were identified in culture medium and tissue lysates. N-desmethyltamoxifen was found to be the major metabolite in both tissue and media extracts of tamoxifen-exposed explants. Incubations of tamoxifen with recombinant human cytochrome P-450s (CYPs) found that CYP2C9 and CYP2D6 produced all three of the above tamoxifen metabolites, while CYP1A1 and CYP3A4 catalyzed the formation of alpha-hydroxytamoxifen and N-desmethyltamoxifen, and CYP1A2 and CYP1B1 only formed the alpha-hydroxy metabolite. CYP2D6 exhibited the greatest activity for the formation of all three tamoxifen metabolites. Western immunoblots of microsomes from human endometrium detected the presence of CYPs 2C9, 3A, 1A1 and 1B1 in fresh endometrium, while CYPs 2D6 and 1A2 were not detected. Immunohistochemical (IHC) analysis also confirmed the presence of CYPs 2C9, 3A and 1B1 in fresh human endometrium and in viable tissue cultured for 24 h with or without tamoxifen. Together, the results support the use of explant cultures of human endometrium as a suitable in vitro model to investigate the biotransformation of tamoxifen in this target tissue. In addition, the results support the role of CYPs 2C9, 3A, 1A1 and 1B1 in the biotransformation of tamoxifen, including the formation of the DNA reactive alpha-hydroxytamoxifen metabolite, in human endometrium.
尽管长期他莫昔芬治疗与子宫内膜癌风险增加相关，但对于子宫内膜组织将他莫昔芬生物转化为潜在反应性中间体（能够形成DNA加合物）的能力却知之甚少。本研究检测了人子宫内膜外植体培养物是否能提供一个合适的体外模型，以研究他莫昔芬的组织特异性生物转化。将肉眼未见疾病的新鲜人子宫内膜组织切成1×2毫米的均匀外植体，在24孔板中与含有25或100微摩尔他莫昔芬的培养基一起孵育。代谢产物通过反相高效液相色谱法进行分析，采用柱后在线光化学活化和荧光检测。在培养基和组织裂解物中鉴定出三种代谢产物，即α-羟基他莫昔芬、4-羟基他莫昔芬和N-去甲基他莫昔芬。在暴露于他莫昔芬的外植体的组织和培养基提取物中，N-去甲基他莫昔芬均为主要代谢产物。他莫昔芬与重组人细胞色素P-450（CYP）的孵育实验发现，CYP2C9和CYP2D6产生上述所有三种他莫昔芬代谢产物，而CYP1A1和CYP3A4催化α-羟基他莫昔芬和N-去甲基他莫昔芬的形成，CYP1A2和CYP1B1仅形成α-羟基代谢产物。CYP2D6在所有三种他莫昔芬代谢产物的形成中表现出最大活性。人子宫内膜微粒体的蛋白质免疫印迹检测发现，新鲜子宫内膜中存在CYP 2C9、3A、1A1和1B1，而未检测到CYP 2D6和1A2。免疫组织化学（IHC）分析也证实，新鲜人子宫内膜以及在有或无他莫昔芬的情况下培养24小时的存活组织中存在CYP 2C9、3A和1B1。总之，这些结果支持将人子宫内膜外植体培养物用作合适的体外模型，以研究他莫昔芬在该靶组织中的生物转化。此外，这些结果支持CYP 2C9、3A、1A1和1B1在人子宫内膜中他莫昔芬生物转化中的作用，包括DNA反应性α-羟基他莫昔芬代谢产物的形成。
