Elsevier

Toxicology Letters

Volume 195, Issue 1, 19 May 2010, Pages 23-30
Toxicology Letters

Role of oxidative stress and MAPK signaling in reference moist smokeless tobacco-induced HOK-16B cell death

https://doi.org/10.1016/j.toxlet.2010.02.020Get rights and content

Abstract

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 3 h 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 μM) were significantly elevated as compared with cell culture medium (4.9 ± 0.6 μM) and the addition of extracellular free iron (14, 30 or 70 μM) to HOK-16B cultures (without STE) caused dose-dependent cell death after 3 h. 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.

Introduction

The use of moist smokeless tobacco (MST) products is increasing in the United States, Norway, and Sweden (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2007). While product usage is often associated with an oral injury at the site of repeated use, such as ulceration, inflammation, and leukoplakia (Johnson et al., 1998, Kallischnigg et al., 2008, Larsson et al., 1991, Roberts, 1997), the mechanism and signaling pathways associated with these oral health effects are not well studied and remain to be fully elucidated. To investigate this oral injury, we and others have examined the acute effect of a reference moist smokeless tobacco extract (STE) on cell death-related processes in cultures of human oral keratinocytes (HOK).

Findings from previous studies indicate that oxidative stress, due to the enhanced production of reactive oxygen species (ROS), reactive nitrogen species, or the depletion of antioxidant systems, plays an important role in STE-induced cell death (Bagchi et al., 1999, Bagchi et al., 2001, Barley et al., 2004, Mangipudy and Vishwanatha, 1999, Samal et al., 2006, Yildiz et al., 1999). Findings from the studies of Bagchi et al., 1999, Bagchi et al., 2001 indicate that exposure of HOK primary cultures to STE for 24 h resulted in enhanced ROS production, lipid peroxidation, and DNA fragmentation leading to apoptotic cell death. In agreement, numerous in vitro studies have implicated oxidative stress in STE-induced toxicity by demonstrating the cytoprotective effect of antioxidants such as superoxide dismutase, catalase, vitamin C, vitamin E, reduced glutathione (GSH) and N-acetyl cysteine (Bagchi et al., 1999, Bagchi et al., 2001, Yildiz et al., 1999). However, the specific constituents in STE which are responsible for this observed oxidative stress and acute cell death have not been identified.

The mechanism(s) by which STE-induced oxidative stress causes HOK cell death remains unknown, as well. Interestingly, recent studies have shown that cigarette smoke extract (CSE) induces cell death in a wide variety of cell types, reportedly through oxidative stress and induction of the mitogen-activated protein kinase (MAPK) pathways (Hoshino et al., 2005, Lee et al., 2008b). CSE was shown to induce human umbilical vein and bronchial epithelial cell death through the activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun NH2-terminal kinase (JNK), and, to a lesser extent, p38 MAPK (Hoshino et al., 2005, Lee et al., 2008b). In fact, the production of ROS and oxidative stress in response to toxic stimuli other than CSE have been shown to activate both JNK and p38 MAPK in multiple cell types. These studies suggest that the activation of MAPK pathways plays an important role in ROS-mediated cell death (Kehrer, 2000, Mitchell et al., 2007).

In the present studies, we investigated the roles of oxidative stress and the MAPK pathways in STE-induced cell death using cultures of HOK-16B cells. We demonstrate that STE causes a dose-dependent induction of cell death and ROS production. This effect was significantly reduced by the addition of antioxidants, an iron chelator, or inhibitors of JNK and p38 MAPK. These novel findings indicate that STE induces cell death in human oral keratinocyte HOK-16B cells, in part, through oxidative stress and the subsequent activation of ASK1 and the JNK1/2 and p38 MAPK pathways.

Section snippets

Cells and reagents

The HOK-16B cell line was generously provided by Dr. N. Park (University of California, Los Angeles, CA). The following were purchased: primary HOK cells (ScienCell Research Laboratories, San Diego, CA); HET-1A cell line (American Type Culture Collection, Manassas, VA); phosphorylated/total-extracellular-signal related kinase (ERK)1/2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); phosphorylated/total-JNK1/2, phosphorylated/total-p38 MAPK antibodies, phosphorylated(Thr-845)/total-ASK1

Cell death induced by STE

Oral mucosal keratinocytes are the first cells exposed to a smokeless tobacco product upon its placement in the oral cavity, and they serve as a physical barrier that protects underlying cells and tissue from potential adverse effects of the product. Damage to these cells has been linked to several oral health effects, including ulceration, inflammation, and leukoplakia (Johnson et al., 1998, Kallischnigg et al., 2008, Larsson et al., 1991, Payne et al., 1998, Roberts, 1997). In fact, oral

Conflict of interest

The authors declare that there are no known conflicts of interest.

Acknowledgements

The authors gratefully acknowledge the contributions to this study and manuscript by George J. Patskan, Barbara K. Zedler and Jeffery S. Edmiston of Altria Client Services. The authors also acknowledge the editorial assistance of Eileen Y. Ivasauskas of Accuwrit Inc.

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