Schild Laura J, Divi Rao L, Beland Frederick A, Churchwell Mona I, Doerge Daniel R, Gamboa da Costa Gonçalo, Marques M Matilde, Poirier Miriam C
Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Cancer Res. 2003 Sep 15;63(18):5999-6003.
The use of the antiestrogen tamoxifen (TAM) is associated with an increase in endometrial cancer. TAM-induced endometrial carcinogenesis may proceed through a genotoxin-mediated pathway, although the detection of endometrial TAM-DNA adducts in exposed women is still controversial. In this study, a monkey model has been used to investigate the question of TAM-DNA adduct formation in primates. Two methods have been used to determine TAM-DNA adducts: a TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), using an antiserum that has specificity for (E)-alpha-(deoxyguanosin-N(2)-yl)-tamoxifen (dG-TAM) and (E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen (dG-desmethyl-TAM) and electrospray ionization tandem mass spectrometry (ES-MS/MS) coupled with on-line sample preparation and high-performance liquid chromatography (HPLC). Mature (19 year old) cynomolgus monkeys were given either vehicle control (n = 1) or TAM (n = 3) twice daily for a total dose of 2 mg of TAM/kg body weight (bw)/day for 30 days by naso-gastric intubation. Tissues were harvested, and DNA was isolated from uterus, ovary, liver, brain cortex, and kidney. By TAM-DNA CIA, values for uterine TAM-DNA adducts in two monkeys were 0.9 and 1.7 adducts/10(8) nucleotides, whereas values for ovarian TAM-DNA adducts in the same animals were 0.4 and 0.5 adducts/10(8) nucleotides. Liver, brain cortex, and kidney DNA samples from the three exposed monkeys had TAM-DNA levels of 2.1-4.2 adducts/10(8) nucleotides, 0.4-5.0 adducts/10(8) nucleotides, and 0.7-2.1 adducts/10(8) nucleotides, respectively. By HPLC-ES-MS/MS, the levels of TAM-DNA adducts detected in all tissues were comparable with those observed by TAM-DNA CIA. Thus, values for uterine TAM-DNA adducts ranged from 0.5 to 1.4 adducts/10(8) nucleotides, whereas values for ovarian TAM-DNA adducts, measurable in two monkeys, were 0.2 and 0.3 adducts/10(8) nucleotides. Liver DNA contained the highest TAM-DNA adduct levels (7.0-11.1 adducts/10(8) nucleotides), whereas brain cortex DNA contained lower adduct levels (0.6-4.8 adducts/10(8) nucleotides) and the lowest levels were measured in the kidney (0.2-0.4 adducts/10(8) nucleotides). This study indicates that cynomolgus monkeys are capable of metabolizing TAM to genotoxic intermediates that form TAM-DNA adducts in multiple tissues.
抗雌激素他莫昔芬（TAM）的使用与子宫内膜癌的增加有关。TAM诱导的子宫内膜致癌作用可能通过基因毒素介导的途径进行，尽管在暴露女性中检测子宫内膜TAM-DNA加合物仍存在争议。在本研究中，使用了一种猴子模型来研究灵长类动物中TAM-DNA加合物形成的问题。采用两种方法测定TAM-DNA加合物：一种是TAM-DNA化学发光免疫分析（TAM-DNA CIA），使用对（E）-α-（脱氧鸟苷-N（2）-基）-他莫昔芬（dG-TAM）和（E）-α-（脱氧鸟苷-N（2）-基）-N-去甲基他莫昔芬（dG-去甲基-TAM）具有特异性的抗血清；另一种是电喷雾电离串联质谱（ES-MS/MS），结合在线样品制备和高效液相色谱（HPLC）。将成年（19岁）食蟹猴通过鼻胃管插管，每天两次给予赋形剂对照（n = 1）或TAM（n = 3），TAM的总剂量为2 mg/kg体重（bw）/天，持续30天。采集组织，从子宫、卵巢、肝脏、大脑皮层和肾脏中分离DNA。通过TAM-DNA CIA，两只猴子子宫中TAM-DNA加合物的值分别为0.9和1.7个加合物/10（8）个核苷酸，而同一动物卵巢中TAM-DNA加合物的值分别为0.4和0.5个加合物/10（8）个核苷酸。三只暴露猴子的肝脏、大脑皮层和肾脏DNA样本中TAM-DNA水平分别为2.1 - 4.2个加合物/10（8）个核苷酸、,0.4 - 5.0个加合物/10（8）个核苷酸和,0.7 - 2.1个加合物/10（8）个核苷酸。通过HPLC-ES-MS/MS，在所有组织中检测到的TAM-DNA加合物水平与TAM-DNA CIA观察到的水平相当。因此，子宫TAM-DNA加合物的值范围为0.5至1.4个加合物/10（8）个核苷酸，而在两只猴子中可测量的卵巢TAM-DNA加合物的值为0.2和0.3个加合物/10（8）个核苷酸。肝脏DNA中TAM-DNA加合物水平最高（7.0 - 11.1个加合物/10（8）个核苷酸），而大脑皮层DNA中加合物水平较低（0.6 - 4.8个加合物/10（8）个核苷酸），肾脏中测量到的水平最低（0.2 - 0.个4加合物/10（8）个核苷酸）。本研究表明，食蟹猴能够将TAM代谢为在多个组织中形成TAM-DNA加合物的基因毒性中间体。
