The use of smokeless tobacco products is often associated with an oral injury at the site of repeated use. To further our understanding of this injury process, the effect of reference moist smokeless tobacco extract (STE) on cell death, oxidative stress, and MAPK signaling in a human oral keratinocyte cell line, HOK-16B, was investigated. STE caused dose-dependent cell death and reactive oxygen species (ROS) production within 30 min to 3h of exposure. This same insult enhanced the activity of ERK1/2, JNK1/2, p38 MAPK and ASK1, an upstream activator of JNK1/2 and p38 MAPK. Inhibition of JNK1/2 and to a lesser extent p38 MAPK, but not ERK1/2, suppressed STE-induced cell death. Pretreatment with antioxidants and an iron chelator, deferoxamine suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Interestingly, extracellular free iron levels in STE (29.4+/-0.5 microM) were significantly elevated as compared with cell culture medium (4.9+/-0.6 microM) and the addition of extracellular free iron (14, 30 or 70 microM) to HOK-16B cultures (without STE) caused dose-dependent cell death after 3h. Thus, acute exposure to STE leads to HOK-16B cell death in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.
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