title: 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.
abstract: 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 Ser(1177), Thr(495) and Ser(116) 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.
authors.affiliation: Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany. Electronic address: Boelck@dshs-koeln.de.
authors.forename: Birgit
authors.initials: B
authors.lastname: Bölck
authors.affiliation: Department of Histology, Faculty of Medicine, Tanta University, Tanta, Egypt.
authors.forename: Marwa
authors.initials: M
authors.lastname: Ibrahim
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany; Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Steinritz
authors.affiliation: Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Morguet
authors.affiliation: Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Dühr
authors.affiliation: Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Suhr
authors.affiliation: Bundeswehr Institute of Medical Occupational and Environmental Safety, Berlin, Germany.
authors.forename: Juxian
authors.initials: J
authors.lastname: Lu-Hesselmann
authors.affiliation: Department of Molecular and Cellular Sport Medicine, German Sport University Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Bloch
mesh_terms: D001564:Benzo(a)pyrene / Q000633:toxicity*; D015415:Biomarkers / Q000378:metabolism; D002273:Carcinogens / Q000633:toxicity*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D015237:Dinoprost / Q000031:analogs & derivatives / Q000378:metabolism; D005609:Free Radicals / Q000378:metabolism; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D018384:Oxidative Stress; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D015415:Biomarkers; D002273:Carcinogens; D005609:Free Radicals; D006575:Heterocyclic Compounds, 3-Ring; C548887:MK 2206; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; C075750:8-epi-prostaglandin F2alpha; D009569:Nitric Oxide; D001564:Benzo(a)pyrene; C002744:3-nitrotyrosine; D014443:Tyrosine; D015237:Dinoprost; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D011065:Poly(ADP-ribose) Polymerases; D051057:Proto-Oncogene Proteins c-akt; D020928:Mitogen-Activated Protein Kinases; D053148:Caspase 3
keywords: Apoptosis; Benzo[a]pyrene; HaCaT; Keratinocytes; Nitrosative stress; Oxidative stress