Elsevier

Toxicology Letters

Volume 209, Issue 2, 7 March 2012, Pages 161-165
Toxicology Letters

Ethanol-induced oxidative stress is associated with EGF receptor phosphorylation in MCF-10A cells overexpressing CYP2E1

https://doi.org/10.1016/j.toxlet.2011.12.009Get rights and content

Abstract

Breast cancer is the most common cancer and the second leading cause of cancer-related mortality worldwide. The etiology of breast cancer is very diverse and ethanol (EtOH) consumption is a well-established risk factor for breast cancer in women. However, the mechanism by which EtOH exerts its carcinogenic activity in breast tissue remains unknown. CYP2E1 is known to metabolize ethanol and produce reactive oxygen species (ROS), including superoxide in epithelial cells. Therefore, in the present studies, we investigated whether there is an increase in ROS following overexpression of CYP2E1 in MCF-10A cells. We found that 30 and 100 mM EtOH increased ROS levels after 2 h treatment in CYP2E1 overexpressing cells. Based on these results and our previous studies with ROS-producing chemicals, we also examined epidermal growth factor receptor (EGFR) activation following exposure to ethanol. We found that there was an increase in phosphorylation of pY1086 EGFR after 18 h EtOH treatment in CYP2E1 overexpressing cells. These studies support a hypothesis that EtOH might increase human mammary cell activation, via an EGFR-dependent signaling mechanism associated with oxidative stress.

Highlights

CYP2E1 overexpression in human mammary epithelial cells results in formation of ROS. ► EtOH increased EGFR activation in cells expressing high levels of CYP2E1. ► Individuals with high CYP2E1 activity may be at risk for EtOH-induced breast cancer.

Introduction

Previous studies by our laboratories have demonstrated that reactive oxygen species (ROS) activate epidermal growth factor receptor (EGFR) signaling pathways in human mammary epithelial cells by superoxide and hydrogen peroxide-dependent mechanisms (Burdick et al., 2003). Activation of EGFR signaling is associated with tumor promotion and progression in epithelial cells (Mill et al., 2009). We have previously worked with redox-cycling quinones derived from benzo(a)pyrene (BaP), which included the 1,6-benzo(a)pyrene quinone and 3,6-benzo(a)pyrene quinone (1,6-BPQ and 3,6-BPQ). These quinones have been found to generate ROS, increase mammary cell proliferation and replace the need for normal growth factors such as EGF, in long term cultures. BPQs increase EGFR tyrosine phosphorylation on several phosphosites leading to downstream cell signaling pathways including phospholipase Cγ and several STAT pathways (Rodríguez-Fragoso et al., 2009).

Recent epidemiological studies have provided convincing evidence that ethanol (EtOH) consumption is associated with an increased risk of breast cancer in women (Chen et al., 2011). The mechanism(s) whereby EtOH increases breast cancer risk is still unclear. Because EtOH can be metabolized by several different pathways, one of which is associated with a microsomal enzyme oxidizing system (MEOS) and CYP2E1 in liver resulting in the formation of superoxide anion (Cederbaum et al., 2009), we were interested in determining whether CYP2E1 metabolism of EtOH can induce ROS formation in human mammary epithelial cells. Therefore, we examined the possibility that CYP2E1-related metabolism of ethanol may form sufficient amounts of superoxide to activate the EGFR in human mammary epithelial cells. The results of these studies show that ethanol increases oxidative stress and EGFR tyrosine phosphorylation in MCF-10A overexpressing CYP2E1.

Section snippets

Chemicals

All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated. 1,6-BPQ and 3,6-BPQ were purchased from the Midwest Research Institute (Kansas City, MO) at >99% purity and maintained as stock solutions in anhydrous tissue culture grade dimethyl sulfoxide (DMSO). The final concentration of DMSO in all experiments was 0.1%. 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester, CM-H2DCFDA, *mixed isomers* (DCF) was purchased from Invitrogen (Carlsbad,

Expression of CYP2E1 protein in MCF-10A cells

Fig. 1 shows the protein expression of CYP2E1 protein in ethanol-treated MCF-10A cells. MCF-10A cells were found to express low levels of CYP2E1, and these levels were not changed following an 18 h exposure to 10, 30 and 100 mM ethanol (Fig. 1). Because the levels of CYP2E1 protein was found to be quite low in MCF-10A as compared to normal human mammary epithelial cells (data not shown), we decided to develop a stable transfectant of MCF-10A cells overexpressing CYP2E1.

Stable transfection of CYP2E1 into MCF-10A cells results in an increase of EtOH-induced oxidative stress

ROS levels from CYP2E1

Discussion

Breast cancer is the most common cancer and the second leading cause of cancer-related mortality among American women (Draper, 2006). The etiology of breast cancer is very diverse and EtOH consumption is a well-established risk factor for breast cancer in women (Rohan et al., 2000, Boffeta and Hashibe, 2006, Smith-Warner et al., 1998, Hamajima et al., 2002, Singletary and Gapstur, 2011). However the mechanisms through which this agent is involved in the development of breast cancer is not fully

Conflict of interest statement

The authors declare that they have no financial or other conflicts of interest to declare for the studies conducted in this manuscript, nor for the conclusions that have been reached.

Acknowledgments

These studies were supported in part by NIH RO1 ES-07259 and by a Mexico CONACYT Fellowship to Angel León-Buitimea number 21416.

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