Wolf Federica I, Torsello Angela, Covacci Valeria, Fasanella Silvia, Montanari Micaela, Boninsegna Alma, Cittadini Achille
School of Medicine, Institute of General Pathology and Giovanni XXIII Cancer Research Center, Catholic University of Sacred Heart, L. go F. Vito 1, 00168 Rome, Italy.
Exp Gerontol. 2002 May;37(5):647-56. doi: 10.1016/s0531-5565(02)00005-0.
Cause-effect relationships between oxidative stress, DNA damage and aging were investigated in WI-38 human diploid fibroblasts at 21, 41 or 58 population doublings (PDs), corresponding to young, middle age or old fibroblasts, respectively. Oxidative DNA damage was evaluated by immunohistochemical detection of 8-hydroxy-2'deoxyguanosine (8-OHdG) adducts or by single cell microgel electrophoresis (COMET assay). Aging was evaluated by growth rate, senescence-associated-beta-galactosidase (SA-beta galactosidase) activity, cell cycle distribution, and expression of p21. Our results demonstrate that (i) oxidative DNA damage is proportional to the age of cells (ii) DNA damage in old/58 PDs cells reflects both an increased susceptibility to oxidative stress, induced by acute exposure to sub-lethal concentrations of hydrogen peroxide (H(2)O(2)), and a reduced efficiency of repair mechanisms. We also show that mild chronic oxidative stress, induced by prolonged exposure to 5 microM H(2)O(2), accelerates aging in fibroblasts. In fact, this treatment increased 8-OHdG levels, SA-beta-galactosidase activity, and G0/G1 cell cycle arrest in middle age/41 PDs, making them similar to H(2)O(2)-untreated old/58 PDs cells. Although other mechanisms may concur in mediating the effects of H(2)O(2), these results lend support to the concept that oxidative stress may be a key determinant of aging. Measurements of oxidative DNA damage might therefore be exploited as reliable marker of cellular aging.
在WI - 38人二倍体成纤维细胞中，分别在21、41或58个群体倍增（PDs）时研究氧化应激、DNA损伤与衰老之间的因果关系，这三个阶段分别对应年轻、中年或老年成纤维细胞。通过免疫组化检测8 - 羟基 - 2'-脱氧鸟苷（8 - OHdG）加合物或单细胞微凝胶电泳（彗星试验）评估氧化DNA损伤。通过生长速率、衰老相关β - 半乳糖苷酶（SA - β半乳糖苷酶）活性、细胞周期分布和p21表达评估衰老。我们的结果表明：（i）氧化DNA损伤与细胞年龄成正比；（ii）老年/58个PDs细胞中的DNA损伤既反映了急性暴露于亚致死浓度过氧化氢（H₂O₂）诱导的对氧化应激敏感性增加，也反映了修复机制效率降低。我们还表明，长时间暴露于5μM H₂O₂诱导的轻度慢性氧化应激会加速成纤维细胞衰老。事实上，这种处理增加了中年/41个PDs细胞中的8 - OHdG水平、SA - β - 半乳糖苷酶活性和G0/G1细胞周期停滞，使其类似于未用H₂O₂处理的老年/58个PDs细胞。尽管可能有其他机制共同介导H₂O₂的作用，但这些结果支持氧化应激可能是衰老的关键决定因素这一概念。因此，氧化DNA损伤的测量可能被用作细胞衰老的可靠标志物。
