Morris S M, Chen J J, Domon O E, McGarrity L J, Bishop M E, Manjanatha M G, Casciano D A
Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, AR 72079, USA.
Mutat Res. 1998 Aug 31;405(1):41-56. doi: 10.1016/s0027-5107(98)00126-2.
The phytoestrogen, genistein, is a naturally occurring isoflavone found in soy products. On a biochemical basis, genistein is a competitive inhibitor of tyrosine kinases and the DNA synthesis-related enzyme, topoisomerase-II (topo-II). Exposure of mammalian cells to genistein results in DNA damage that is similar to that induced by the topo-II inhibitor and chromosomal mutagen, m-amsa. In order to determine the potential genotoxicity of genistein, human lymphoblastoid cells which differ in the functional status of the tumor suppressor gene, p53, were exposed to genistein and the induction of micronuclei quantified by microscopic analysis. In addition, the mutant fraction at the thymidine kinase (tk) locus (both the normal-growth and slow-growth phenotypes) was determined by resistance to trifluorothymidine (TFT) and at the hypoxanthine phosphoribosyl transferase (hprt) locus by resistance to 6-thioguanine (6-TG). Flow cytometric analysis of the percentage of viable, apoptotic and degenerating cells was utilized to determine the rate and kinetics of cell death after genistein exposure. The detection of micronuclei in both cell lines indicated that genistein-induced damage had occurred in both AHH-1 tk+/- and L3. Linear regression analysis detected a significant increase in the number of 6-TG-resistant clones in both AHH-1 tk+/- (p53+/-) and L3 (p53+/+). A comparison of slopes revealed no difference between the lines. In contrast, a significant, concentration-dependent increase in the number of TFT-resistant clones with the slow-growth phenotype was detected in AHH-1 tk+/- (mutant p53), but not in L3 (wild-type p53). Cell death occurred primarily by apoptosis in both cell lines; however, a concentration-dependent decrease in the percentage of viable cells was detected immediately after exposure in L3, but not until 32 h after exposure in AHH-1 tk+/-. A comparison of the slopes of the concentration-response curves for the percentage of viable cells revealed no difference between the cell lines in the effect of genistein on cell viability. Our results may be interpreted that genistein is a chromosomal mutagen and that p53 functional status affects the recovery of chromosomal mutants, possibly by signalling cells into the apoptosis pathways.
植物雌激素染料木黄酮是一种存在于豆制品中的天然异黄酮。从生化角度来看，染料木黄酮是酪氨酸激酶和与DNA合成相关的酶拓扑异构酶II（topo-II）的竞争性抑制剂。将哺乳动物细胞暴露于染料木黄酮会导致DNA损伤，这种损伤与拓扑异构酶II抑制剂及染色体诱变剂m-amsa所诱导的损伤相似。为了确定染料木黄酮的潜在遗传毒性，将具有不同肿瘤抑制基因p53功能状态的人淋巴母细胞暴露于染料木黄酮，并通过显微镜分析对微核的诱导情况进行定量。此外，通过对三氟胸苷（TFT）的抗性来确定胸苷激酶（tk）基因座（正常生长和缓慢生长表型）的突变率，通过对6-硫鸟嘌呤（6-TG）的抗性来确定次黄嘌呤磷酸核糖转移酶（hprt）基因座的突变率。利用流式细胞术分析活细胞、凋亡细胞和退化细胞的百分比，以确定染料木黄酮暴露后细胞死亡的速率和动力学。在两种细胞系中微核的检测表明，染料木黄酮诱导的损伤在AHH-1 tk+/-和L3细胞中均有发生。线性回归分析检测到AHH-1 tk+/-（p53+/-）和L3（p53+/+）中6-TG抗性克隆的数量均显著增加。斜率比较显示两细胞系之间无差异。相反，在AHH-1 tk+/-（突变型p53）中检测到具有缓慢生长表型的TFT抗性克隆数量显著增加且呈浓度依赖性，而在L3（野生型p53）中未检测到。两种细胞系中细胞死亡主要通过凋亡发生；然而，在L3细胞中暴露后立即检测到活细胞百分比呈浓度依赖性下降，而在AHH-1 tk+/-细胞中直到暴露后32小时才检测到。对活细胞百分比的浓度-反应曲线斜率进行比较，结果显示染料木黄酮对细胞活力的影响在两细胞系之间无差异。我们的结果可以解释为染料木黄酮是一种染色体诱变剂，并且p53的功能状态可能通过将细胞信号传导至凋亡途径来影响染色体突变体的恢复。
