Molecular Biomarkers & Diagnosis

Detection of Free Radical Reaction Products and Activated Signalling Molecules as Biomarkers of Cell Damage in Human Keratinocytes upon Lead Exposure

Abstract

Birgit Bölck, Marwa Ibrahim, Juxian Lu-Hesselmann, Dirk Steinritz, Frank Suhr and Wilhelm Bloch

Lead (Pb) is one of the most important environmental pollutant metals accumulating in the atmosphere, water, foods, and in organisms living in contaminated areas. Skin is one of the main targets of Pb toxicity based on its ability of direct penetration upon exposure. The underlying cell damaging pathomechanisms have not been revealed in detail. Herein, we focus on Pb-induced oxidative and nitrosative stress that has not been previously thoroughly investigated. We investigated these effects in order to elucidate the pathomechanisms and as well to identify potential biological markers that may indicate Pb toxicity. Human immortalized keratinocytes (HaCaT cells) were exposed to Pb (100 μM) either for 5 minutes or 6 hours. Pb-induced cellular damage was evaluated by immunocytochemistry analysis of multiple signalling cascades, e.g. apoptosis, Akt, MAPK, NOS, nitrotyrosine and 8-isoprostane formation, detection of nitrosative stress using Diaminofluorescein (DAF-FM) and oxidative stress using 3'-(p aminophenyl) fluorescein (APF). We found that Pb exposure resulted in significantly enhanced NO and ROS production in HaCaT cells. Pb led to enhanced eNOS-phosphorylation at Ser1177, and Ser116 residues but not Thr495. AKT phosphorylation but not MAP kinases were enhanced by Pb In addition, Pb induced apoptosis as shown by Caspase-3 activation and PARP cleavage. Our results suggest that Pb mediates its toxic effect in keratinocytes through oxidative and nitrosative stress which is accompanied by differential changes of eNOS phosphorylation and apoptosis. These data significantly contribute to understanding of underlying mechanisms of Pb-induced cellular damage.