AMS: BINDING OF ANTIOESTROGEN, TAMOXIFEN TO DNA AS CHEMOPREVENTIVE AGENT IN WOME

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 他莫昔芬[(Z)-trans-1，2-diphenyl-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-butene]可以有效地抑制雌激素受体阳性乳腺癌的生长，并具有可接受的风险：益处概况。在美国进行的化学预防试验表明，他莫昔芬可将罹患乳腺癌的高危健康女性的乳腺癌发病率降低49%。然而，流行病学证据表明，服用他莫昔芬的主要不良反应是乳腺癌患者和健康女性子宫内膜癌的发生率增加。一些报告还表明，他莫昔芬治疗与胃肠道肿瘤发病率的增加有关。在大鼠中，他莫昔芬会导致DNA损伤，导致肝脏肿瘤的发展。他莫昔芬如何导致子宫内膜癌仍然存在争议，需要确定该药物是否会导致女性子宫和胃肠道的DNA损伤。托瑞米芬是他莫昔芬的结构类似物，他莫昔芬也用于乳腺癌的治疗，目前正在接受作为化学预防疗法的评估。这种药物可能被证明是一种比他莫昔芬更安全的替代品，如果它对女性没有或更低水平的DNA损伤。这项研究项目的最终目的是确定他莫昔芬或托瑞米芬是否在人体组织中形成DNA加合物。这两种药物造成的损害程度(如果有的话)将与在大鼠肝脏中检测到的损害程度进行比较，这可能表明人类患肿瘤的潜在风险。我们选择使用AMS，因为常规的DNA加合物形成方法被发现缺乏在人体组织中检测他莫昔芬加合物所需的灵敏度。AMS的高灵敏度允许使用伦理上可接受的低剂量14C放射性标记给人类志愿者来调查目标器官中潜在的DNA损伤。目前的资源是唯一一种具有检测人类受试者低水平DNA损伤所需的灵敏度的资源。 在进行人体研究之前，我们进行了实验，以验证AMS在我们的研究中的使用，并证明AMS在我们的应用中将是有用的。为了确定AMS是否可以在治疗剂量和亚治疗剂量下检测与DNA结合的他莫昔芬，我们研究了大鼠激活他莫昔芬与DNA结合物种的能力，并将结果与32P-后标记试验结果进行了比较。[14C]用AMS检测单次给药后大鼠肝脏中的三苯氧胺DNA加合物。此外，在处理的大鼠的肝外组织中也可以检测到加合物的形成，这是32P-后标记试验所不可能的。对于人体研究，到目前为止，我们已经招募了两种方案的患者，在手术前给他们一次治疗剂量的[14C]他莫昔芬(20 mg胶囊)。在第一项研究中，一组接受子宫切除术的患者(n=6)和一组接受乳腺癌手术的患者(n=5)服用他莫昔芬0.37MBq。然而，这个放射性同位素剂量太低，无法检测到共价结合，因此进行了第二项研究，子宫切除术患者(n=10)接受了1.85MBq的他莫昔芬。手术切除组织，分离DNA和蛋白质，用AMS法测定14C-三苯氧胺结合浓度。在接受治疗的患者的子宫肌层和子宫内膜中，他莫昔芬被发现与蛋白质和DNA不可逆转地结合，尽管水平很低。子宫内膜和子宫肌层组织的DNA损伤程度分别为0.024±0.008和0.049±0.011加合物/108个核苷酸(平均扫描电子显微镜)。因此，这些结果表明，他莫昔芬可以与人子宫DNA结合，但DNA损伤水平比在继续发展为肝脏肿瘤的大鼠身上检测到的水平低3至4个数量级。最近完成了一项类似的研究，其中10名子宫切除患者接受了[14C]托瑞米芬的治疗，结果正在准备发表。另一项旨在调查他莫昔芬是否与人结肠结合的研究也已经进行。10名患者每人接受20毫克[14C]他莫昔芬(1.85MBq)，样本的处理方式与子宫研究中的相同。初步结果表明，与子宫相比，他莫昔芬和代谢物在结肠的组织浓度更高，DNA加合物水平高出数倍。 作为这项工作的延伸，我们还进行了体外研究，以确定哪些人细胞色素P450能够将他莫昔芬转化为与DNA结合的反应性代谢物。这包括进行含有特定的人类重组P450、[14C]他莫昔芬和DNA的培养。提取DNA并用AMS分析其与三苯氧胺的结合程度。通过这种方式，我们已经证明了人的CYP3A4能够产生已知的三苯氧胺的反应性代谢物，并诱导高水平的DNA加合物形成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Tamoxifen [(Z)-trans-1,2-diphenyl-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-butene] is effective in inhibiting the growth of oestrogen receptor positive breast cancers in women and has an acceptable risk:benefit profile. Chemoprevention trials based in the U.S. have demonstrated tamoxifen reduces by 49%, the incidence of breast cancer in healthy women who are at high-risk of developing the disease. However, epidemiological evidence has shown that the main adverse effect of tamoxifen administration is an increased occurrence of endometrial cancer in both breast cancer patients and healthy women. Some reports also indicate tamoxifen therapy is associated with an increased incidence of tumours in the GI tract. In rats, tamoxifen causes DNA damage leading to the development of liver tumours. It remains controversial how tamoxifen causes endometrial cancer and there is a need to establish whether this drug causes DNA damage in the uterus and GI tract of women. Toremifene is a structural analogue of tamoxifen, which is also used in the treatment of breast cancer and is undergoing evaluation as a chemopreventive therapy. This drug may prove to be a safer alternative to tamoxifen if it causes no, or lower levels of DNA damage in women. The ultimate aims of this research project are to determine whether tamoxifen or toremifene form DNA adducts in human tissues. The extent of damage, if any, induced by the two drugs will be compared to that detected in rat liver and this may give an indication of the potential risk of developing tumours in humans. We have chosen to use AMS because routine methods for the measurement of DNA adduct formation have been found to lack the sensitivity required to detect tamoxifen adducts in human tissues. The high sensitivity of AMS permits the use of ethically acceptable low doses of 14C radiolabel to be administered to human volunteers to investigate potential DNA damage in target organs. The present resource is the only one with the degree of sensitivity needed to detect low levels of DNA damage in human subjects. Prior to performing studies in humans, we undertook experiments to validate the use of AMS in our research and to prove that AMS would be useful in our applications. To determine if AMS could detect tamoxifen bound to DNA at therapeutic and sub-therapeutic doses we investigated the ability of rats to activate tamoxifen to DNA binding species and compared the results with those obtained using the 32P-postlabelling assay. [14C]Tamoxifen DNA adducts were detected in rat liver after a single dose, by AMS. In addition, adduct formation was also detectable in extrahepatic tissues of treated rats, which had not been possible using the 32P-postlabelling assay. For the human studies, to date, we have recruited patients on two protocols in which they were given a single therapeutic dose of [14C]tamoxifen (20mg capsule) prior to surgery. In the first study, a group of patients (n=6) undergoing hysterectomy and a group undergoing breast cancer surgery (n=5) received 0.37MBq tamoxifen. However, this radioisotope dose was too low to detect covalent binding therefore a second study was undertaken in which hysterectomy patients (n=10) received 1.85MBq tamoxifen. Tissue was removed at surgery, the DNA and protein isolated, and then the concentration of bound 14C-tamoxifen was determined by AMS. Tamoxifen was found to bind irreversibly to protein and DNA in both the myometrium and endometrium of treated patients although at very low levels. The extent of DNA damage detected was 0.024 ¿ 0.008 and 0.049 ¿ 0.011 adducts/10^8 nucleotides (mean ¿ SEM) in endometrial and myometrial tissues respectively. These results therefore demonstrate that tamoxifen can bind to human uterine DNA but the level of DNA damage is 3 to 4 orders of magnitude lower than that detected in rats that go on to develop liver tumours. An analogous study in which 10 hysterectomy patients were administered [14C]toremifene has recently been completed and results are being prepared for publication. An additional study designed to investigate whether tamoxifen binds to human colon has also been carried out. Ten patients each received 20mg of [14C]tamoxifen (1.85MBq) and samples were processed in the same manner as in the uterus studies. Initial results indicate higher tissue concentrations of tamoxifen and metabolites in the colon compared to the uterus and DNA adduct levels several fold higher. As an extension to this work we have also conducted in vitro studies to identify which human cytochrome P450 is capable of converting tamoxifen to reactive metabolites, which bind to DNA. This involved performing incubations containing specific human recombinant P450s, [14C]tamoxifen and DNA. The DNA was extracted and analyzed by AMS to measure the extent of covalent tamoxifen binding. In this way we have shown that human CYP3A4 is able to generate a known reactive metabolite of tamoxifen and induce high levels of DNA adduct formation.