Reference moist smokeless tobacco‐induced oxidative stress in human oral keratinocyte cell lines leads to cell death via JNK and p38 MAPK signaling

Abstract

The use of moist smokeless tobacco products is associated with acute oral injuries such as leukoplakia. Unfortunately, there is a limited understanding of the mechanisms and signaling pathways associated with these injuries. The present studies investigate the effects of a Reference 1S3 moist smokeless tobacco extract (STE) on oral injury, oxidative stress and MAPK signaling in multiple human oral keratinocyte cell lines utilizing cytospin slide analysis to observe alterations in cell morphology, DCF‐DA fluorescence, and western blotting techniques. STE caused dose‐dependent keratinocyte cell death within 30 minutes to 3 h of exposure. STE 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 or p38 MAPK, but not ERK1/2, suppressed STE‐induced cell death. STE promoted the generation of reactive oxygen species (ROS). The anti‐oxidants, Trolox and L‐ascorbic acid, suppressed ROS production, ASK1, JNK1/2 and p38 MAPK activation, and reduced cell death after STE exposure. Thus, reference STE leads to cell death in human oral keratinocyte cell lines in part through oxidative stress via activation of ASK1 and the JNK1/2 and p38 MAPK pathways.Research supported by Altria Client Services, 601 East Jackson St. Richmond, VA 23219 U.S.A.