Detection of key enzymes, free radical reaction products and activated signaling molecules as biomarkers of cell damage induced by benzo[a]pyrene in human keratinocytes

- Benzo[a]pyrene (BaP) treatment enhanced the oxidative and nitrosative stress.
- BaP increased eNOS phosphorylation at serine1177.
- BaP treatment significantly enhanced NO and ROS production after 5 min of exposure.
- 1 μM BaP treatment enhanced activated caspase-3 and led to PARP cleavage.
- BaP-induced increase of Akt phosphorylation was completely suppressed by MK-2206.

Benzo[a]pyrene (BaP) is a known carcinogenic and cell damaging agent. The underlying cell damaging pathomechanisms have not been totally revealed. Especially BaP-related induction of oxidative and nitrosative stress has not been previously investigated in detail. The presented study investigated these effects in order to elucidate the pathomechanism and as well to identify potential biological markers that may indicate a BaP exposure. Human immortalized keratinocytes (HaCaT cells) were exposed to BaP (1 μM) for either 5 min or 6 h, respectively. BaP-induced cellular damage was evaluated by immunocytochemistry analysis of multiple signaling 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).Our results show that BaP exposure significantly enhanced NO and ROS productions in HaCaT cells. BaP led to eNOS-phosphorylation at Ser1177, Thr495 and Ser116 residues. Using specific inhibitors, we found that the Erk1/2 pathways seemed to have strong impact on eNOS phosphorylation. In addition, BaP-induced apoptosis was observed by caspase-3 activation and PARP cleavage.Our results suggest that BaP mediates its toxic effect in keratinocytes through oxidative and nitrosative stress which is accompanied by complex changes of eNOS phosphorylation and changes of Akt and MAPK pathways.