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

AMS：抗雌激素、他莫昔芬与 DNA 的结合作为女性的化学预防剂

• 批准号: 7724079
• 负责人: Karen A Brown
• 金额: $ 2.8万
• 依托单位: UNIVERSITY OF CALIF-LAWRNC LVRMR NAT LAB
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724079
• 关键词: Adverse effects; Benefits and Risks; Binding; Biological Assay; Breast Cancer Treatment; CYP3A4 gene; Cancer Patient; Chemoprevention; Chemopreventive Agent; Colon; Computer Retrieval of Information on Scientific Projects Database; Cytochrome P450; DNA; DNA Adduction; DNA Adducts; DNA Binding; DNA Damage; Development; Disease; Dose; Endometrial; Endometrial Carcinoma; Endometrium; Estrogen receptor positive; Evaluation; Extrahepatic; Funding; Gastrointestinal tract structure; Genus Cola; Grant; Growth; Human; Human Volunteers; Hysterectomy; In Vitro; Incidence; Institution; Liver; Liver neoplasms; Measurement; Measures; Methods; Myometrial; Nucleotides; Operative Surgical Procedures; Organ; Patients; Pharmaceutical Preparations; Process; Proteins; Protocols documentation; Publications; Radioisotopes; Radiolabeled; Rattus; Recombinants; Recruitment Activity; Reporting; Research; Research Design; Research Personnel; Research Project Grants; Resources; Risk; Sampling; Source; Tamoxifen; Therapeutic; Tissues; Toremifene; United States National Institutes of Health; Uterus; Woman; Work; adduct; analog; base; cancer surgery; capsule; diphenyl; human study; human subject; human tissue; malignant breast neoplasm; myometrium; radiotracer; research study; tumor

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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 他莫昔芬[（Z）-反式-1，2-二苯基-1-[4-[2-（二甲氨基）乙氧基]苯基]-1-丁烯]可有效抑制女性雌激素受体阳性乳腺癌的生长，并且具有可接受的风险：获益特征。在美国进行的化学预防试验表明，他莫昔芬可以降低49%的健康女性乳腺癌发病率，这些女性有患乳腺癌的高风险。然而，流行病学证据表明，他莫昔芬给药的主要不良反应是增加乳腺癌患者和健康女性子宫内膜癌的发生率。一些报告还指出，他莫昔芬治疗与胃肠道肿瘤发病率增加有关。在大鼠中，他莫昔芬导致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，需要确定这种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一种结构类似物，也用于治疗乳腺癌，目前正在评估作为一种化学预防疗法。这种药物可能被证明是一个更安全的替代他莫昔芬，如果它不会导致，或较低水平的DNA损伤的妇女。 该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。将比较这两种药物引起的损害程度（如果有的话）与在大鼠肝脏中检测到的损害程度，这可能表明人类发生肿瘤的潜在风险。我们选择使用AMS，因为常规的DNA加合物形成的测量方法被发现缺乏检测人体组织中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14 C放射性标记给予人类志愿者，以研究靶器官中潜在的DNA损伤。目前的资源是唯一一个具有检测人类受试者中低水平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在我们的应用中是有用的。为了确定AMS是否可以检测到他莫昔芬结合到DNA在治疗和亚治疗剂量，我们研究了大鼠激活他莫昔芬的DNA结合物种的能力，并将结果与使用32 P-后标记试验获得的结果进行比较。单次给药后，通过AMS在大鼠肝脏中检测到[14 C]他莫昔芬DNA加合物。此外，加合物的形成也可检测到治疗大鼠的肝外组织，这是不可能使用32 P-后标记试验。对于人体研究，迄今为止，我们已经招募了两种方案的患者，其中他们在手术前给予单一治疗剂量的[14 C]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）接受了0.37 MBq他莫昔芬。 然而，该放射性同位素剂量太低而不能检测共价结合，因此进行了第二项研究，其中子宫切除术患者（n=10）接受1.85 MBq他莫昔芬。在手术中取出组织，分离DNA和蛋白质，然后通过AMS测定结合的14 C-他莫昔芬的浓度。他莫昔芬被发现不可逆地结合蛋白质和DNA在治疗患者的子宫肌层和子宫内膜，虽然在非常低的水平。 在子宫内膜和子宫肌层组织中检测到的DNA损伤程度分别为0.024 <$0.008和0.049 <$0.011加合物/10^8核苷酸（平均<$SEM）。因此，这些结果表明，他莫昔芬可以与人类子宫DNA结合，但DNA损伤的水平比在继续发展肝肿瘤的大鼠中检测到的水平低3至4个数量级。最近完成了一项类似的研究，其中10名子宫切除术患者服用[14 C]托瑞米芬，结果正在准备发表。另一项旨在研究他莫昔芬是否与人类结肠结合的研究也已进行。10名患者各接受20 mg [14 C]他莫昔芬（1.85 MBq），并以与子宫研究相同的方式处理样本。初步结果表明，与子宫相比，结肠中他莫昔芬和代谢物的组织浓度更高，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结合的活性代谢物。这涉及进行含有特定人重组P450、[14 C]他莫昔芬和DNA的孵育。提取DNA并通过AMS分析以测量共价他莫昔芬结合的程度。以这种方式，我们已经表明，人CYP 3A 4能够产生他莫昔芬的已知反应性代谢产物，并诱导高水平的DNA加合物形成。