title: Endosulfan decreases cell growth and apoptosis in human HaCaT keratinocytes: partial ROS-dependent ERK1/2 mechanism.
abstract: Endosulfan is an organochlorine insecticide described as a potential carcinogen in humans. This insecticide was recently reported to alter the mitogen-activated protein (MAP) kinase signaling pathways and is suspected to affect cell growth and differentiation in human keratinocytes. This study was designed to assess the mitogenic, apoptogenic, and genotoxic effects of endosulfan on the HaCaT cell line. We first found that 25 microM endosulfan led to persistent extracellular signal-regulated kinase (ERK)1/2 phosphorylation with an accumulation of the phosphorylated form in the nucleus, probably caused by MAP kinase phosphatase (MKP) inhibition. As previously described under sustained ERK1/2 activation, cell growth was decreased: delayed confluency and 35% decrease of BrdU incorporation was demonstrated in endosulfan-treated keratinocytes. In addition, endosulfan has been shown to generate transient reactive oxygen species (ROS), and blocking this oxidative stress by N-acetyl cysteine (NAC) strongly prevented both persistent nuclear ERK1/2 phosphorylation and cell growth decrease. Additional experiments demonstrated that unchanged endosulfan rather than its metabolites has mutagenic effects (Ames positive without S9) and increased DNA strand breaks (Comet assay) in HaCaT cells, via a ROS-dependent mechanism. Therefore, to assess the putative pro-apoptotic response of damaged cells, caspases 3/7 activity and poly(ADP-ribose)-polymerase (PARP) cleavage were measured. The results clearly indicated that endosulfan inhibited both spontaneous and staurosporine-induced apoptosis. Taken together, these findings strongly support that endosulfan induces ROS generation leading to sustained ERK1/2 phosphorylation and decrease in cell growth. Moreover, endosulfan was found to inhibit apoptosis and this could contribute to mutant cell survival and therefore have possible carcinogenic effects.
authors.affiliation: INRA, UMR 1112 Réponses des Organismes aux Stress Environnementaux, Equipe de Toxicologie Cellulaire, Moléculaire et Génomique, Sophia-Antipolis, France. sebantherieu@yahoo.fr
authors.forename: Sebastien
authors.initials: S
authors.lastname: Antherieu
authors.forename: Nathalie
authors.initials: N
authors.lastname: Ledirac
authors.forename: Anne-Pascale
authors.initials: AP
authors.lastname: Luzy
authors.forename: Philippe
authors.initials: P
authors.lastname: Lenormand
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Caron
authors.forename: Roger
authors.initials: R
authors.lastname: Rahmani
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D019925:Cyclin A / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D056741:Cyclin D; D016213:Cyclins / Q000378:metabolism; D004726:Endosulfan / Q000633:toxicity*; D006801:Humans; D007306:Insecticides / Q000633:toxicity; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009153:Mutagens / Q000633:toxicity; D018384:Oxidative Stress; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology
chemical_list: C534699:CCNB1 protein, human; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D056741:Cyclin D; D016213:Cyclins; D007306:Insecticides; D009153:Mutagens; D017382:Reactive Oxygen Species; D019311:Staurosporine; D004726:Endosulfan