Sulphur mustard induces time- and concentration-dependent regulation of NO-synthesizing enzymes

Sulphur mustard (SM) is a chemical warfare agent that causes erythema and blistering of the skin with a latency of several hours. Although SM is known for almost 200 years the cellular mechanisms involved in the damaging process are not fully understood. There is evidence that changes in nitric oxide (*NO), reactive oxygen species (ROS) and reactive nitrogen species (RNS) might be involved in the damaging process. Aim: To find out more about the pathophysiology of SM, we investigated the initial formation of biochemical markers of nitrosative and oxidative stress as well as activation (translocation from plasma-membrane) and upregulation of eNOS and iNOS, respectively. Methods: Human immortalized keratinocytes (HaCaT cell line) were exposed to SM (100 mu M or 300 mu M) for 30 min. Cells were fixed after 1 h, 3 h or 6 h of incubation in SM-free medium and immunostained. Live cell experiments were performed using the NO-sensitive dye DAF2-DA. In order to assess cell viability after BH4 supplementation, we analyzed apoptosis using CDD-ELISA. Results: SM significantly increased biochemical markers of nitrosative and oxidative stress already I h after exposure. Moreover, the NO producing enzymes eNOS and iNOS showed concentration- and time-dependent changes in their activation or expression levels. Initially, live cell imaging experiments could not confirm NO production after SM exposure. Only when cells were supplemented with tetrahydrobiopterine, stable NO production was detectable. Apoptotic activity was increased due to SM exposure and could be reduced after BH4 treatment. Conclusion: Our data point towards concentration- and time-dependent formation of iNOS and activation of eNOS due to translocation from plasma-membrane. Live cell experiments yielded first indications of catalytic decoupling of NOS that could be reversed by supplementation with tetrahydrobiopterin (BH4). Addition of BH4 1 h after SM exposure significantly decreased apoptosis compared to the unsupplemented control.