Shafer D A, Xie Y, Falek A
Human and Behavioral Genetics Research Lab, Georgia Mental Health Institute, Atlanta 30306.
Environ Mol Mutagen. 1994;23(1):37-44. doi: 10.1002/em.2850230107.
In previous studies we have shown highly significant increases in chromosome damage and sister chromatid exchanges in heroin addicts, particularly when caffeine and metabolic inhibitors are added to the medium. Using human HUT-78 T-cell cultures, we now find direct in vitro evidence of opiate-induced or opiate-promoted mutagenesis via several assay systems. First, with microgel electrophoresis (MGE), we observed graded, dose-dependent, significant increases (P < .0001) in the frequency of comet tails of fragmented DNA when cells were treated with morphine alone (5 x 10(-9) M up to 10(-7) M) or when co-treated with the more potent mutagen, ethylmethanesulfonate (EMS). There were also dose-dependent increases in the lengths and densities of the comet tails observed. These findings were confirmed by a series of MGE experiments in which several days of morphine exposure preceded a 2-hr pulse of EMS. Second, mutant frequency (MF) assays also indicated significant opiate effects. These studies required separate assessment of cloning efficiencies and the frequencies of TG-resistant, HPRT-deficient mutant clones under four test conditions: no treatment, morphine alone for 4 days, morphine plus EMS, and EMS alone. Prior to the treatment phase, aminopterin was used to eliminate background HPRT mutations. The medium was changed after the treatment phase, the cells were allowed to express mutant phenotypes, and then TG was added and resistant mutant clones counted after 16 days. The background MF level for controls and for cells treated with EMS alone were negligible at 5.12 x 10(-8) and 7.25 x 10(-8), respectively. In the cells treated with morphine alone or morphine plus EMS, MF levels increased very significantly (P < .001) by > 100-fold to 5.1 x 10(-6) and 7.0 x 10(-6), respectively. Cloning efficiency also decreased significantly with both morphine-exposed conditions. Preliminary analysis with the single strand conformational polymorphism (SSCP) procedure following 6-thioguanine (TG) selection, also confirmed the occurrence of Exon 3 mutants of the HPRT gene in cells exposed to morphine plus EMS. It appears that brief EMS exposure can be repaired, whereas, if morphine exposure persists through one or more cell cycles, direct or indirect mutagenesis is initiated.
在先前的研究中，我们已经表明海洛因成瘾者的染色体损伤和姐妹染色单体交换有极显著增加，尤其是当培养基中添加咖啡因和代谢抑制剂时。利用人类HUT - 78 T细胞培养物，我们现在通过几种检测系统发现了阿片类药物诱导或促进诱变的直接体外证据。首先，通过微凝胶电泳（MGE），当细胞单独用吗啡（5×10⁻⁹ M至10⁻⁷ M）处理或与更强效的诱变剂甲磺酸乙酯（EMS）共同处理时，我们观察到碎片化DNA彗星尾频率呈分级、剂量依赖性的显著增加（P <.0001）。观察到的彗星尾长度和密度也有剂量依赖性增加。这些发现通过一系列MGE实验得到证实，在这些实验中，在EMS进行2小时脉冲处理之前先让细胞暴露于吗啡几天。其次，突变频率（MF）检测也表明阿片类药物有显著作用。这些研究需要在四种测试条件下分别评估克隆效率以及对硫代鸟嘌呤（TG）耐药、次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（HPRT）缺陷的突变克隆频率：未处理、单独用吗啡处理4天、吗啡加EMS以及单独用EMS处理。在处理阶段之前，使用氨甲蝶呤消除背景HPRT突变。处理阶段后更换培养基，让细胞表达突变表型，然后添加TG并在16天后计数耐药突变克隆。对照组和单独用EMS处理的细胞的背景MF水平可忽略不计，分别为5.12×10⁻⁸和7.25×10⁻⁸。在单独用吗啡或吗啡加EMS处理的细胞中，MF水平非常显著地增加（P <.001），分别增加超过100倍至5.1×10⁻⁶和7.0×10⁻⁶。在两种吗啡处理条件下克隆效率也显著降低。在6 - 硫代鸟嘌呤（TG）选择后用单链构象多态性（SSCP）程序进行的初步分析也证实，在暴露于吗啡加EMS的细胞中存在HPRT基因的外显子3突变体。似乎短暂的EMS暴露可以被修复，然而，如果吗啡暴露持续经过一个或多个细胞周期，就会引发直接或间接诱变。
