Kehe K, Raithel K, Kreppel H, Jochum M, Worek F, Thiermann H
Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstr. 11, 80937 Munich, Germany.
Arch Toxicol. 2008 Jul;82(7):461-70. doi: 10.1007/s00204-007-0265-7. Epub 2007 Nov 29.
Sulfur mustard (SM) is a bifunctional alkylating agent. Its primary toxic consequence is severe skin damage with blisters, occurring after skin contact. These vesicant properties of SM have been linked to cell death of proliferating keratinocytes in the basal layer of the skin. Catalytic activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP-1) has been demonstrated to be a major event in response to high levels of DNA damage, and PARP-1 activation may be part of apoptotic signaling. In other contexts, overstimulation of PARP-1 triggers necrotic cell death because of rapid consumption of its substrate, beta-nicotinamide adenine dinucleotide (NAD+) and the consequent depletion of ATP. These findings prompted us to evaluate whether SM induces apoptosis in keratinocytes like HaCaT cells and to determine whether blocking of PARP enzyme activity with 3-aminobenzamide (3AB) can influence the mode of cell death. HaCaT cells were exposed to SM (10-1,000 microM; 30 min) and then cultivated in SM-free medium with or without 3AB for up to 48 h. This treatment resulted in a time and SM dose-dependent increase of apoptotic cell death characterized by PARP-1 cleavage and DNA fragmentation during the experimental period. After just 45 min of exposure to 1 mM SM, we observed a significant increase in PARP-1 activity in HaCaT cells. About 6 h after exposure, intracellular ATP levels were diminished by 22%, which seemed to be completely prevented by the addition of 3AB directly after exposure. However, 18 h later, this 3AB effect on the SM concentration-dependent loss of ATP was no longer detectable. Interestingly, the effect of SM on total cell viability was not changed by 3AB. However, the mode of cell death was influenced by 3AB exhibiting an increase of apoptotic cells and a concomitant decrease of necrotic HaCaT cells during the first 24 h after SM exposure. Our results indicate that SM concentrations of 1 mM or higher induce a prominent PARP activation leading to ATP depletion and necrosis. In contrast, lower concentrations of SM cause minor PARP activation and, especially, PARP-1 cleavage by caspase 3 without ATP depletion. Because ATP is required for apoptosis, we suggest that ATP acts as an early molecular switch from apoptotic to necrotic modes of SM-induced cell death, at least at high concentrations (> or =1 mM). Thus, the observed early proapoptotic effect of 3AB at lower SM concentrations may point to the influence of ATP-independent cell-death regulating mechanisms.
硫芥（SM）是一种双功能烷基化剂。其主要毒性后果是皮肤接触后出现严重的皮肤损伤并伴有水泡。SM的这些糜烂性特性与皮肤基底层增殖性角质形成细胞的死亡有关。核酶聚（ADP - 核糖）聚合酶（PARP - 1）的催化激活已被证明是对高水平DNA损伤作出反应的一个主要事件，并且PARP - 1激活可能是凋亡信号传导的一部分。在其他情况下，PARP - 1的过度刺激会触发坏死性细胞死亡，因为其底物β - 烟酰胺腺嘌呤二核苷酸（NAD +）被快速消耗，进而导致ATP耗竭。这些发现促使我们评估SM是否能诱导如HaCaT细胞等角质形成细胞发生凋亡，并确定用3 - 氨基苯甲酰胺（3AB）阻断PARP酶活性是否会影响细胞死亡方式。将HaCaT细胞暴露于SM（10 - 1000 microM；30分钟），然后在含有或不含有3AB的无SM培养基中培养长达48小时。这种处理导致在实验期间以PARP - 1裂解和DNA片段化为特征的凋亡细胞死亡呈时间和SM剂量依赖性增加。在暴露于1 mM SM仅45分钟后，我们观察到HaCaT细胞中PARP - 1活性显著增加。暴露约6小时后，细胞内ATP水平降低了22%，而在暴露后直接添加3AB似乎完全阻止了这种情况。然而，18小时后，不再能检测到3AB对SM浓度依赖性ATP损失的这种影响。有趣的是，3AB并未改变SM对总细胞活力的影响。然而，细胞死亡方式受到3AB的影响，在SM暴露后的最初24小时内，凋亡细胞增加，坏死的HaCaT细胞相应减少。我们的结果表明，1 mM或更高浓度的SM会诱导显著的PARP激活，导致ATP耗竭和坏死。相比之下，较低浓度的SM会引起轻微的PARP激活，特别是通过caspase 3导致PARP - 1裂解，且不会导致ATP耗竭。因为凋亡需要ATP，我们认为至少在高浓度（≥1 mM）时，ATP作为SM诱导的细胞死亡从凋亡模式向坏死模式转变的早期分子开关。因此，在较低SM浓度下观察到的3AB早期促凋亡作用可能指向与ATP无关的细胞死亡调节机制的影响。
