benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and 5-chloro-2-methyl-4-isothiazolin-3-one

A 3:1 combination of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) is widely used to preserve cosmetic products. We show here that CMI/MI induced apoptosis in normal human keratinocytes (NHK) as at low concentrations (0.001-0.05% documented by subdiploid DNA content and phosphatidylserine exposure, while at the highest concentration (0.1% as supplied, 15 p.p.m.) the response was necrosis. Various molecular events accompanied the cytotoxic effects of CMI/MI. Generation of ROS and hyperpolarization of mitochondrial transmembrane potential (DeltaPsim) were early events, followed by increased Fas expression and activation of caspase-8, and then activation of caspase-3 and -9. The drop in DeltaPsim occurred only later in the cell death pathway, when NHK showed signs of apoptosis. Pretreatment of cells for 2 h with the redox-active agent N-acetyl-L-cysteine conferred complete protection against the CMI/MI-induced cytotoxic effects, DeltaPsim loss, and apoptosis. The pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F blocked the CMI/MI-induced apoptosis without preventing ROS generation and the drop in DeltaPsim. These results indicate that the generation of ROS plays an important part in mediating apoptosis and necrosis associated with CMI/MI treatment. This new aspect of the in vitro toxicity of CMI/MI may provide important information about the relationship between the preservative's in vitro apoptotic activity and its in vivo toxicity.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Cell Membrane; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Drug Combinations; Enzyme Activation; fas Receptor; Humans; Keratinocytes; Membrane Potentials; Mitochondria; Necrosis; Oxidative Stress; Preservatives, Pharmaceutical; Reactive Oxygen Species; Reference Values; Thiazoles