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.
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