Toxicology Letters 209 (2012) 161 165

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Toxicology Letters

jou rn al h om epage: www.elsev ier .com

Ethano wiphosph in

Angel Le dinTodd A. Ta Facultad de F exicob The Universit erque,

a r t i c l

Article history:Received 30 NReceived in re17 December 2Accepted 19 DAvailable onlin

Keywords:Breast cancerEthanolROSOxidative stressEGFRCYP2E1

ancencerer in

rema), incere is

100 m resu

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 treatmentin CYP2E1 overexpressing cells. These studies support a hypothesis that EtOH might increase humanmammary cell activation, via an EGFR-dependent signaling mechanism associated with oxidative stress.

1. Introdu

Previousreactive oxreceptor (EGcells by suanisms (Buassociated w(Mill et al., quinones d1,6-benzo(aBPQ and 3,ROS, increanormal groincrease EGleading to pholipase C2009).

Recent edence that

CorresponE-mail add

0378-4274/$ doi:10.1016/j. 2011 Elsevier Ireland Ltd. All rights reserved.

ction

studies by our laboratories have demonstrated thatygen species (ROS) activate epidermal growth factorFR) signaling pathways in human mammary epithelialperoxide and hydrogen peroxide-dependent mech-rdick et al., 2003). Activation of EGFR signaling isith tumor promotion and progression in epithelial cells

2009). We have previously worked with redox-cyclingerived from benzo(a)pyrene (BaP), which included the)pyrene quinone and 3,6-benzo(a)pyrene quinone (1,6-6-BPQ). These quinones have been found to generatese mammary cell proliferation and replace the need forwth factors such as EGF, in long term cultures. BPQsFR tyrosine phosphorylation on several phosphositesdownstream cell signaling pathways including phos- and several STAT pathways (Rodrguez-Fragoso et al.,

pidemiological studies have provided convincing evi- ethanol (EtOH) consumption is associated with an

ding author. Tel.: +1 505 272 0920.ress: sburchiel@salud.unm.edu (L. Rodrguez-Fragoso).

increased risk of breast cancer in women (Chen et al., 2011). Themechanism(s) whereby EtOH increases breast cancer risk is stillunclear. Because EtOH can be metabolized by several differentpathways, one of which is associated with a microsomal enzymeoxidizing system (MEOS) and CYP2E1 in liver resulting in the for-mation of superoxide anion (Cederbaum et al., 2009), we wereinterested in determining whether CYP2E1 metabolism of EtOH caninduce ROS formation in human mammary epithelial cells. There-fore, we examined the possibility that CYP2E1-related metabolismof ethanol may form sufcient amounts of superoxide to activatethe EGFR in human mammary epithelial cells. The results of thesestudies show that ethanol increases oxidative stress and EGFR tyro-sine phosphorylation in MCF-10A overexpressing CYP2E1.

2. Materials and methods

2.1. Chemicals

All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indi-cated. 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 anhydroustissue culture grade dimethyl sulfoxide (DMSO). The nal concentration of DMSO inall experiments was 0.1%. 5-(and-6)-chloromethyl-2 ,7-dichlorodihydrouoresceindiacetate, acetyl ester, CM-H2DCFDA, *mixed isomers* (DCF) was purchased fromInvitrogen (Carlsbad, CA).

see front matter 2011 Elsevier Ireland Ltd. All rights reserved.toxlet.2011.12.009l-induced oxidative stress is associatedorylation in MCF-10A cells overexpress

n-Buitimeaa,b, Lourdes Rodrguez-Fragosoa,, Frehompsonb, Scott W. Burchielb

armacia, Universidad Autnoma del Estado de Morelos, Cuernavaca, Morelos 62209, My of New Mexico, College of Pharmacy, Department of Pharmaceutical Sciences, Albuqu

e i n f o

ovember 2011vised form011ecember 2011e 28 December 2011

a b s t r a c t

Breast cancer is the most common cworldwide. The etiology of breast caestablished risk factor for breast canccarcinogenic activity in breast tissueproduce reactive oxygen species (ROSstudies, we investigated whether thMCF-10A cells. We found that 30 andoverexpressing cells. Based on these/ locate / tox le t

th EGF receptorg CYP2E1

e T. Lauerb, Harmony Bowlesb,

NM 87131, United States

r and the second leading cause of cancer-related mortality is very diverse and ethanol (EtOH) consumption is a well-

women. However, the mechanism by which EtOH exerts itsins unknown. CYP2E1 is known to metabolize ethanol and

luding superoxide in epithelial cells. Therefore, in the present an increase in ROS following overexpression of CYP2E1 inM EtOH increased ROS levels after 2 h treatment in CYP2E1

lts and our previous studies with ROS-producing chemicals,

162 A. Len-Buitimea et al. / Toxicology Letters 209 (2012) 161 165

2.2. Antibodies

Antibody against CYP2E1 was purchased from Abcam (Cambridge, MA). Anti-bodies against EGFR, actin, tubulin and horse radish peroxidase (HRP)-conjugatedgoat were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-body against HTechnology (Bgen/Biosource

2.3. Cell cultu

MCF-10A cmammary epicoated (PureCCorning, NY, U10% CO2 and 3MCF-10A cellsof several toxicmammary epigin of the mosta variety of tyand thus possresponse to di34 (and higheSFIHE with 2% was added. EtOand 100 mM etrations of EtOplates. We estfore these are twas removed aand insulin (Sthen incubatedat 1 M 3,6-BPmaintained instudies. For althat could be o

2.4. Western b

Lysates w8, 150 mM so100, 2 g/mL a(PMSF), 1 mMand phosphatascraping cells tein concentra(BioRad Labor(Pierce, Rockfdenaturing SDtrotransferredwashed with 14 C in TBS wibrane was thenusing the manin TBST and thRT using the mA chemiluminnal visualizatquantied usin

2.5. CYP2E1 tr

A MCF-10Astable transfeccontained the increased CYPfor CYP2E1 prbackbone vectas a control. C

2.6. Assessme

MCF-10A acells and incuthen treated wfor 2 h under twere centrifuganalyzed on a

xpresesenc

Westent re

as co

R imm

tes w mM EM PMoche Droteinitatio

for 1) at es an). TheyacryembraBST ausingn incut RT usrol. A

the of eaester

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f the oftwaarianof

A. Len-Buitimea et al. / Toxicology Letters 209 (2012) 161 165 163

Fig. 2. Quantication of ROS levels in CYP2E1 transfected and untransfected MCF-10A cells treated with ethanol. Cells were incubated with 5 M dichlorouoresceindiacetate for 30 min and then treated with 30 and 100 mM ethanol for 2 h. Cells wereanalyzed on a FacScan ow cytometer. Values are from a representative experimentrepeated at least twice and are the mean of three replicates S.E.M. *p < 0.05 ascompared to control. *Signicantly different from MCF-10A (p < 0.05), #signicantlydifferent from 1.2 BB, and &signicantly different from DMSO 4.1 2E1 (p < 0.05).

levels demonstrated by 88 and 84 channel MCF shifts, respectivelyas compared to the 1.2 BB cells (p < 0.05). We observed differencesin the MCF for MCF-10A cells treated with 30 mM or 100 mM of4 and 63 ctreated cellincreased inhave low extransfectionan increase

3.3. Ethanocells

Becausesuch as 1,6lation in Mcan be an Rodrguez-Factivate EGshows the a

CYP2E1 transfected 4.1 2E1 cells and backbone transfected (1.2BB). Untransfected cells showed an increase in pY1086 EGFR phos-phorylation treated with 10 ng/mL EGF and 1 M 3,6-BPQ, 5.6-foldand 4.8-fold respectively (p < 0.05). CYP2E1 transfected cells (4.12E1 cells) demonstrated an increase in the level of phosphory-lation of pY1086 EGFR when cells were treated for 18 h with1 M 3,6-BPQ (used as a positive control based on the ndings ofRodrguez-Fragoso et al., 2009, 20). Treatment with 30 mM ethanolproduced a 1.2-fold increase in pY1086 phosphorylation (similar to3,6-BPQ), and 100 mM ethanol produced a 3-fold increase, as com-pared to untreated cells and backbone vector controls (p < 0.05).These results demonstrate that CYP2E1-generated ROS are able toactivate the EGFR signaling pathway, as previously described forB(a)P-quinones (Rodrguez-Fragoso et al., 2009).

4. Discussion

Breast cancer is the most common cancer and the second lead-ing cause of cancer-related mortality among American women(Draper, 2006). The etiology of breast cancer is very diverse andEtOH consumption is a well-established risk factor for breast can-cer in women (Rohan et al., 2000; Boffeta and Hashibe, 2006;Smith-Warner et al., 1998; Hamajima et al., 2002; Singletary andGapstur, 2011). However the mechanisms through which this agentis involved in the development of breast cancer is not fully elu-cidated. Acute and chronic EtOH consumption has the ability to

lipidoducion ode (Hincluked cellu

owthas bend aerousmave bFR (Cumorays aetast

Fig. 3. EtOH aEGFR antibodyphosphorylatiphosphorylatihannels, respectively as compared with 0.1% DMSO-s (p < 0.05). The positive control, 1 M 1,6-BPQ, was also

4.1 2E1 cells. These results show that MCF-10A cellspression levels of CYP2E1 that can form ROS, but that

leads to an increase in baseline levels of ROS as well asd response to ethanol and 1,6-BPQ.

l induces pY1086 EGFR phosphorylation in MCF-10A

we have previously shown that ROS-generating agents,- and 3,6-BPQs, increase EGFR tyrosine phosphory-CF-10A cell which increases cell proliferation and

indication of tumor promotion (Burdick et al., 2003;ragoso et al., 2009), we wanted to see if ethanol could

FR in CYP2E1 transfected (4.1 2E1) MCF-10A cells. Fig. 3nalysis of the pY1086 EGFR phosphorylation status in

induceROS prformatperoxieases, are linof intraand gr

It hdants aof numthe plaROS hathe EGulate tpathwand mnd BPQs stimulate the pY1086 EGFR phosphorylation in CYP2E1 transfected and untra and Protein A/G PLUS-Agarose for EGFR immunoprecipitation. Samples were then sepon using and anti-pY1086 EGFR. Non-stimulated and EGF-stimulated A431 cell lysates won levels were quantitated using digital imaging as compared to controls (DMSO) of the s peroxidation, protein and DNA adducts and increasetion (Wright et al., 1999; Poschl and Seitz, 2004). Thef ROS such as superoxide anion (O2) and hydrogen2O2) causes oxidative injury leading to various dis-ding cancer. Changes in the intracellular redox statusto pathologic processes that are related to alterationslar signaling pathways associated with protein kinases

factor receptor activation (Wu, 2006).en reported that several agents such as radiation, oxi-lkylating agents induce ligand-independent activations receptor tyrosine kinases at the cytoplasmic side of

membrane, including the EGFR (Knebel et al., 1996).een included among intracellular signal transducers forhiarugi and Buricchi, 2007). EGFR has the ability to stim-

growth and progression by activating several signalingssociated with cell proliferation, angiogenesis, invasion,asis (Mill et al., 2009).

nsfected MCF-10A cells. 350 g of lysate was incubated with anti-arated by SDS-PAGE and subject to Western blot analysis for EGFRere included as negative and positive control, respectively. Relativeame cell type.

164 A. Len-Buitimea et al. / Toxicology Letters 209 (2012) 161 165

The role of CYP2E1 in the metabolism of xenobiotics in mam-mary gland, including ethanol, has not been well dened. It hasbeen reported that CYP2E1 is induced in the liver by the presenceof high concentrations of ethanol or after chronic intake (Lieberand DeCarlireports in tethanol in hsion of CYP2treated for ently in ext

Since wstable transstrate that othe ROS propression of following ECederbaum(2011) usedcells not extreatment wROS only ingested thatCYP2E1 promary neurocultures expan effect thaADH/CYP2Estrated thatIn the presCYP2E1, MCVector contROS levels Therefore, o

ROS havsengers thawith tumorreceptors (Zhave shownformation ihave shown(agents thaEGFR transaliferation (Bshowed thaincreased Ewith severa

We havvarious xenelements inboth 3,6-BPgenes (HMO(NQO1, NQ(PAH) metatain EGFR as(Burchiel etcells had a EGFR phospresults suggof EGFR couMCF-10A ceand presenxenobiotic ractivate difwith BPQs.

In this oxidative st

phosphorylation in MCF10A cells treated with EtOH. The relevanceof the phosphorylation of pY1086 is that it facilitates the bind-ing of the SH2 domains of growth factor receptor-bound protein2 (GRB2). This binding results in mitogen activated protein kinase

) act (Jorverexed tummand 1. Exetab

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J., Ramhanism. Med., R.N., ermalRes. 2A., Rahyrosints. EM, 1970; Guengerich et al., 1991). However, there are nohe literature showing that CYP2E1 can be induced byuman mammary cells. Our results showed that expres-E1 protein was not induced when MCF-10A cells were18 h suggesting that this enzyme may respond differ-rahepatic tissues after an acute ethanol exposure.e did not see any CYP2E1 induction, we developed afection of CYP2E1 into MCF-10A cells in order to demon-verexpression of CYP2E1 correlates with an increase induction. It has been previously reported that overex-CYP2E1 protein and the production of ROS is increasedtOH exposure (Roychowdhury et al., 2009; Wu and, 2010; Cederbaum et al., 2009). Chandrasekaran et al.

cells expressing ADH/CYP2E1 proteins (VL-17A) andpressing CYP2E1 protein (HepG2) in order to show thatith 100 mM ethanol for 72 h increased the levels of

cells expressing CYP2E1 protein. These results sug- ROS production was dependent on the expression oftein. The same effect has also been observed in pri-nal cultures (Haorah et al., 2008). Primary neuronalosed to 17.5 mM ethanol increased ROS levels by 68%,t was inhibited by the pretreatment with an inhibitor of1 (4-methylpyrazole). Thus, these results also demon-

ROS production was dependent on CYP2E1 expression.ent study we demonstrated that cells overexpressingF-10A cells transfected with the pIRES2-DsRed-Expressaining the human CYP2E1 cDNA, showed an increase inwhen they were exposed to 30 and 100 mM ethanol.ur results are consistent with previous reports.e been described as molecules that act as second mes-t are able to activate signaling pathways associated

promotion through transactivation of growth factorima and Kalousova, 2005). Yusuf and Frenkel (2010)

that an oxidative environment promotes tumor trans-n MCF-10A cells. Previous studies in our laboratories

that exposure of MCF-10A cells to 1,6- and 3,6-BPQst increase ROS levels through redox cycling), inducedctivation and caused an increase in mammary cell pro-urdick et al., 2003). Rodrguez-Fragoso et al. (2009)t MCF-10A cells exposed to 1 M 1,6- and 3,6-BPQsGFR phosphorylation in several phosphosites associatedl signaling pathways leading to cell proliferation.e evaluated the effect of BPQs on the activation ofobiotic response elements and anti-oxidant response

MCF-10A cells. Our previous results have shown thatQ and 1,6-BPQ induced oxidative stress associatedX1, GCLC, GCLM, and SLC7A11), phase 2 enzyme genesO2, and ALDH3A1), polycyclic aromatic hydrocarbonbolizing genes (CYP1B1, EPHX1, and AKR1C1), and cer-sociated genes (EGFR, IER3, ING1, SQSTM1 and TRIM16)

al., 2007). Here, we show that CYP2E1 overexpressinghigher amount of ROS and an increase in the pY1086horylation as compared to non-transfected cell. Theseest that the increase of ROS levels and phosphorylationld be associated with an overexpression of CYP2E1 inlls. According to previous results from our laboratory

t data we suggest that EtOH might be able to activateesponse elements, to induce EGFR associated genes andferent phosphosites in EGFR in MCF-10A cells, as seen

study we investigated whether CYP2E1-dependentress was associated with an increase of pY1086 EGFR

(MAPKerationcells oincreas

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Knebel, tor tagenivation, a signaling pathway associated with cell prolif-issen et al., 2003). Our results demonstrate that MCF10Apressing CYP2E1 exposed to 30 and 100 mM ethanol

he EGFR phosphorylation at phosphosite pY1086.ary, our results show that EtOH increases oxidativeactivation of EGFR in MCF-10A cells overexpressingperimental data clearly support the hypothesis thatolism via CYP2E1 may transactivate EGFR and activate

cell signaling in MCF-10A cells. Additional studies arelucidate the signaling pathways that are activated afterorylation of pY1086 EGFR and its relation to cellularssociated with tumor promotion.

interest statement

ors declare that they have no nancial or other conictso declare for the studies conducted in this manuscript,conclusions that have been reached.

gments

udies were supported in part by NIH RO1 ES-07259 and CONACYT Fellowship to Angel Len-Buitimea number

shibe, M., 2006. Alcohol and cancer. Lancet Oncol. 7, 149156., Thompson, T.A., Lauer, F.T., Oprea, T.I., 2007. Activation of dioxinlement (DRE)-associated geneses by benzo(a)pyrene 3,6-quinone andyrene 1,6-quinone in MCF-10A human mammary epithelial cells. Tox-

Pharmacol. 221, 203214.Davis, J.W., Liu, J.K., Hudson, L.G., Shi, H., Monske, M.L., Burchiel, S.W.,zo(a)pyrene quinones increase cell proliferation, generate reactiveecies, and transactivate the epidermal growth factor receptor in breastcells. Cancer Res. 63, 78257833..I., Lu, Y., Wu, D., 2009. Role of oxidative stress in alcohol-induced liverh. Toxicol. 83, 519548 (review).an, K., Swaminathan, K., Kumar, S.M., Chatterjee, S., Clemens, D.L.,11. Elevated glutathione level does not protect against chronic alco-ted apoptosis in recombinant human hepatoma cell line VL-17Aessing alcohol metabolizing enzymes alcohol dehydrogenase ande P450 2E1. Toxicol. In Vitro 25, 969978.sner, B., Hankinson, S.E., Colditz, G.A., Willett, W.C., 2011. Moderatensumption during adult life, drinking patterns, and breast cancer risk., 18841890.uricchi, F., 2007. Protein tyrosine phosphorylation and reversible

two cross-talking posttranslational modications. Antioxid. Redox23.., Burdick, A.D., Lauer, F.T., Burchiel, S.W., 2003. The aryl hydro-ceptor antagonist, 3-methoxy-4-nitroavone, attenuates 2,3,7,8-odibenzo-p-dioxin-dependent regulation of growth factor signalingosis in the MCF-10A cell line. Toxicol. Appl. Pharmacol. 188, 4249.6. Breast cancer, trends, risks, treatments and, effects. AAHN J. 54,

P., Kim, D.H., Iwasaki, M., 1991. Role of human cytochrome P450IIE1 inion of many low molecular weight cancer suspects. Chem. Res. Toxicol.9., Hirose, K., Tajima, K., et al., 2002. Alcohol, tobacco and breastllaborative reanalysis of individual data from 53 epidemiologicalcluding 58 515 women with breast cancer and 95 067 women withoute. Br. J. Cancer 87, 12341245.irez, S.H., Floreani, N., Gorantla, S., Morsey, B., Persidsky, Y., 2008.

of alcohol-induced oxidative stress and neuronal injury. Free Radic. 45, 15421550.Walker, F., Pouliot, N., Garrett, T.P., Ward, C.W., Burgess, A.W., 2003.

growth factor receptor: mechanisms of activation and signaling. Exp.84 (1), 3153.msdorf, H.J., Ullrich, A., Herrlich, P., 1996. Dephosphorylation of recep-e kinase as target of regulation by radiation, oxidants or alkylatingBO J. 15, 53145325.

A. Len-Buitimea et al. / Toxicology Letters 209 (2012) 161 165 165

Lieber, C.S., DeCarli, L.M., 1970. Hepatic microsomal ethanol-oxidizing systems:in vitro characteristics and adaptive properties in vivo. J. Biol. Chem. 245,25052512.

Mill, C.P., Chester, J.A., Riese, D.J., 2009. EGFR may couple moderate alcohol con-sumption to increased breast cancer risk. Breast Cancer (Lond.) 1, 3138.

Poschl, G., Seitz, H.K., 2004. Alcohol and cancer. Alcohol Alcohol. 39, 155165.Rodrguez-Fragoso, L., Melendez, K., Hudson, L.G., Lauer, F.T., Burchiel, S.W., 2009.

EGF-receptor phosphorylation and downstream signaling are activated bybenzo[a]pyrene 3,6-quinone and benzo[a]pyrene 1,6-quinone in human mam-mary epithelial cells. Toxicol. Appl. Pharmacol. 235, 321328.

Rohan, T., Jain, M., Howe, G., Miller, A., 2000. Alcohol consumption and risk of breastcancer: a cohort study. Cancer Causes Control. 11, 239247.

Roychowdhury, S., McMullen, M.R., Pritchard, M.T., Li, W., Salomon, R.G., Nagy,L.E., 2009. Formation of gamma-ketoaldehyde-protein adducts during ethanol-induced liver injury in mice. Free Radic. Biol. Med. 47, 15261538.

Singletary, K.W., Gapstur, S.M., 2011. Alcohol and breast cancer: review of epi-demiologic and experimental evidence and potential mechanisms. JAMA 286,21432151.

Smith-Warner, S.A., Spiegelman, D., Yaun, S.S., Van den Brandt, P.A., Folsom, A.R.,Goldbohm, R.A., Graham, S., Holmberg, L., Howe, G.R., Marshall, J.R., Miller,

A.B., Potter, J.D., Speizer, F.E., Willett, W.C., Wolk, A., Hunter, D.J., 1998. Alco-hol and breast cancer in women: a pooled analysis of cohort studies. JAMA 279,535540.

Soule, H.D., Maloney, T.M., Wolman, S.R., Petersen Jr., W.D., Brenz, R., McGrath, C.M.,Russ, J., Pauley, R.J., Jones, R.F., Brooks, S.C., 1990. Isolation and characteriza-tion of a spontaneously immortalized human breast epithelial cell line, MCF-10.Cancer Res. 50, 60756086.

Wright, M.R., McManaman, L.J., Repine, E.J., 1999. Alcohol-induced breast cancer: aproposed mechanism. Free Radic. Biol. Med. 26, 348354.

Wu, D., 2006. Alcohol-induced oxidative stress and cell responses. J. Gastroenterol.Hepatol. 21, S26S29.

Wu, D., Cederbaum, A., 2010. Activation of ASK-1 and downstream MAP kinases incytochrome P4502E1 potentiates tumor necrosis factor alpha liver injury. FreeRadic. Biol. Med. 49, 348360.

Yusuf, R., Frenkel, K., 2010. Morphologic transformation of human breastepithelial cells MCF-10A: dependence on an oxidative microenvironmentand estrogen/epidermal growth factor receptors. Cancer Cell Int. 10,3046.

Zima, T., Kalousova, M., 2005. Oxidative stress and signaling transduction pathwaysin alcoholic liver disease. Alcohol Clin. Exp. Res. 29, 110S115S.

Ethanol-induced oxidative stress is associated with EGF receptor phosphorylation in MCF-10A cells overexpressing CYP2E11 Introduction2 Materials and methods2.1 Chemicals2.2 Antibodies2.3 Cell culture and treatment2.4 Western blot analysis2.5 CYP2E1 transfection2.6 Assessment of intracellular ROS by DCF2.7 EGFR immunoprecipitation and Western blot analysis2.8 Statistical analysis

3 Results3.1 Expression of CYP2E1 protein in MCF-10A cells3.2 Stable transfection of CYP2E1 into MCF-10A cells results in an increase of EtOH-induced oxidative stress3.3 Ethanol induces pY1086 EGFR phosphorylation in MCF-10A cells

4 DiscussionConflict of interest statementAcknowledgmentsReferences