Elsevier

Toxicology Letters

Volume 224, Issue 2, 13 January 2014, Pages 264-271
Toxicology Letters

Exposure to ethanol and nicotine induces stress responses in human placental BeWo cells

https://doi.org/10.1016/j.toxlet.2013.10.032Get rights and content

Highlights

  • Nicotine reduces proliferation of BeWo cells without affecting cell viability.

  • Nicotine increases the expression of ER-stress related GRP78/BiP protein.

  • Nicotine and ethanol increase ROS production.

  • Nicotine and ethanol induce changes in stress response-related MAPK proteins.

Abstract

Human placental trophoblastic cancer BeWo cells can be used as a model of placental trophoblasts. We found that combined exposure to relevant exposure concentrations of ethanol (2‰) and nicotine (15 μM) induces an increase in the amount of reactive oxygen species (ROS). Neither ethanol or nicotine alone, nor their combination affected cell viability. However, nicotine decreased cell proliferation, both alone and combined with ethanol. Nicotine increased the expression of the endoplasmic reticulum (ER)-stress related protein GRP78/BiP, but not another marker of ER-stress, IRE1α. We also studied the effects of nicotine and/or ethanol on phosphorylation and expression of three mitogen-activated protein kinases (MAPKs), i.e. JNK, p38 and ERK1/2. Nicotine decreased the phosphorylation of JNK and also had similar effect on total amount of this protein. Phosphorylation and expression of p38 were increased 1.7- and 1.6-fold, respectively, by nicotine alone, and 1.9- and 2.1-fold by the combined treatment. Some increase (1.8-fold) was also seen in the phosphorylation of ERK2 at 48 h, in cells exposed to both ethanol and nicotine. This study shows that ethanol and nicotine, which harm the development of fetus may induce both oxidative and ER stress responses in human placental trophoblastic cells, implicating these mechanisms in their fetotoxic effects.

Introduction

Ethanol and nicotine are toxic compounds, which disturb fetal development (for recent reviews, see e.g. Bruin et al., 2010, Ornoy and Ergaz, 2010). Nevertheless, many pregnant women both smoke and use alcohol simultaneously (Leonardson and Loudenburg, 2003, Walker et al., 2011, Jones et al., 2013). Combined effects of ethanol and nicotine at the molecular level in human placenta are not well known. Because normal placental function is critical for fetal development, disturbance of placental signaling pathways by xenobiotics may disrupt normal fetal development that can, at worst, lead to miscarriage (Gundogan et al., 2010, Liu et al., 2011).

Alcohol-related mental retardation is one of the worst consequences of drinking during pregnancy (West and Blake, 2005). In addition, fetal exposure to alcohol is associated with a multitude of other adverse long-term outcomes (Jones et al., 2013). Fetal alcohol spectrum disorder (FASD) includes a variety of harmful effects, such as learning disabilities (May et al., 2009, Senecky et al., 2009). The mechanisms of FASD are not known, but oxidative stress may be involved in its development (for a recent review, see Brocardo et al., 2011). Oxidative stress may be involved also in toxic effects of nicotine as shown e.g. by Crowley-Weber et al. (2003) in human HCT-116 colon adenocarcinoma cells. In addition to being addictive (Le Houezec, 1998, Nutt et al., 2007), nicotine can affect various cellular processes associated with carcinogenesis. Nicotine increases cell proliferation and prevents DNA damage-induced apoptosis in epithelial and endothelial cells (for reviews, see Catassi et al., 2008, Egleton et al., 2008, Arias et al., 2009). Nicotine can also induce angiogenesis (for a review, see Lee and Cooke, 2012) and emerging evidence suggests that nicotine promotes tumor growth (Grozio et al., 2007) and metastasis (Petros et al., 2012).

Human placenta expresses many proteins that are activated under chemical and other stress situations. These include p38, JNK and ERK1/2, which represent three well-characterized subfamilies of mitogen-activated protein kinases (MAPKs). MAPKs are involved in various cellular processes such as proliferation and differentiation (for an extensive review, see Cargnello and Roux, 2011) including differentiation of human trophoblasts (for a review, see Vaillancourt et al., 2009). They are also activated in oxidative stress and play important roles in inhibiting the oncogenic potential of reactive oxygen species (ROS) by inducing apoptosis (for reviews, see McCubrey et al., 2006, Runchel et al., 2011). In human placental explants, several stress-induced factors which are associated with pre-eclampsia, such as angiotensin II, hypoxia and inflammatory cytokines, increase phosphorylation of p38 and JNK (Xiong et al., 2013). Luo et al. (2011) observed similar activation of p38 in pre-eclamptic human placentas. Boronkai et al. (2009) showed that in JAr cell line originating from choriocarcinoma hydrogen peroxide-induced oxidative stress increased phosphorylation of JNK, but not p38 and ERK1/2. In another human trophoblast cell line, immortalized HTR-8/SVneo cells, hydrogen peroxide and also cadmium have been shown to phosphorylate all three MAPK subfamilies (Valbonesi et al., 2008). Oxidative stress may be associated with endoplasmic reticulum (ER) stress (for reviews, see Rath and Haller, 2011, Bando, 2012).

ER-stress is associated with unfolded and misfolded proteins. Correct protein folding is essential for proper function of proteins in the regulation of cellular processes, such as cell survival and death. Cells have efficient mechanisms to prevent misfolding of proteins. One of these mechanisms is activation of glucose-regulated protein 78 (GRP78/BiP), which functions as a chaperone to catalyze protein folding. ER-stress-induced unfolded protein response is regulated by three signaling pathways: protein kinase-like endoplasmic reticulum kinase (PERK), inositol requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF6) pathways (for a review, see Healy et al., 2009, Hetz, 2012). GRP78/BiP is involved in all of these pathways. In addition, the amount of GRP78/BiP protein is increased in many cancer types (for reviews, see Fu and Lee, 2006, Healy et al., 2009) and it has been linked to malignant transformation in epithelial tumors (Huang et al., 2012) with prognostic significance at least in prostate cancer (Daneshmand et al., 2007).

Ethanol can disrupt cell signaling by causing ER-stress in liver (for a review, see Kaplowitz and Ji, 2006, Kojima et al., 2010) and pancreas (for a review, see Pandol et al., 2010). Ke et al. (2011) showed that ethanol also activates several signaling pathways associated with ER stress, including GRP78/BiP, IRE1α, ATF6 and PERK, in developing mouse brain. As to nicotine it has been studied much less in this respect. In the few studies published so far it has been shown that nicotine can also induce ER stress. Nicotine induces ER stress in human periodontal ligament cells (Lee et al., 2012b) and nicotine-induced ER-stress is also supported by the finding that nicotine activates the promoter of GRP78/BiP (Crowley-Weber et al., 2003). There are no studies available in which the effects of ethanol or nicotine on MAPKs or ER-stress in human placenta or placental cells have been studied.

Human fetal exposure and/or effects of xenobiotics in vivo in placenta can be studied in rare occasions. One of these is related to medication judged to be beneficial during pregnancy. Information can also be gained after chemical accidents. However, experimental studies of xenobiotics in vivo in placenta or fetus are naturally impossible due to ethical reasons. Previously we have studied the effect of ethanol on nicotine transfer across human placenta by using human placental perfusion (Veid and Karttunen et al., 2011). Both nicotine and ethanol crossed human placenta easily. Ethanol did not have any effects on nicotine transfer. In addition, transport of ethanol across the placenta resembles passive transport (Mørck et al., 2010, Veid et al., 2011; Mose et al., 2012). Also human placental cell lines offer a relevant model for reproductive toxicology studies. In this study, we have used human trophoplastic cancer (BeWo) cells, which have similar morphological properties and express many of the same enzymes and transporters as normal placental trophoblasts. They express e.g. breast cancer resistant protein ABCG2/BCRP (Vähäkangas and Myllynen, 2009, Vähäkangas et al., 2011). In addition, human placental trophoblasts are from the same origin as the fetus itself. However, BeWo cells do not form a confluent monolayer and they also seem to lack another important transporter protein, ABCB1/P-gp (Evseenko et al., 2006).

Our overall hypothesis is that in addition to direct fetal effects, nicotine and ethanol can also disturb fetal development via adverse effects on the function of human placenta. Using human choriocarcinoma BeWo cells we studied whether ethanol and nicotine induce stress responses in human placental cells. To our knowledge it has not been studied before whether nicotine and/or ethanol induce oxidative or ER-stress in placental cells.

Section snippets

Chemicals

Nicotine was purchased from Sigma–Aldrich, St. Louis, MO, USA. Ethanol was from Altia Corporation, Finland. The antibody for p53 was from Novocastra Laboratories Ltd., UK and all the other primary antibodies were from Cell Signaling Technology (USA). Anti-mouse antibody was from Amersham (UK) and anti-rabbit antibody from Calbiochem (Germany). Non-essential amino acids, sodium-pyruvate, penicillin-streptomycin (10 000 U/ml + 10 000 U/ml), fetal bovine serum (FBS) and Dulbecco's Phosphate Buffered

Reactive oxygen species, cell number and viability

Only the combination of ethanol (2‰) and nicotine (15 μM) increased the formation of reactive oxygen species (ROS) at 24 and 48 h (p < 0.01), whereas at 72 h the effect was not statistically significant (Fig. 2). At 48 h, the increased ROS production caused by combined exposure to ethanol and nicotine may be even synergistic. Ethanol or nicotine alone did not increase ROS production statistically significantly at any studied time points. Nicotine decreased the relative cell number (RCN, percentage of

Discussion

We have shown for the first time that nicotine increases the expression of GRP78/BiP in human trophoblastic BeWo cells. Because GRP78/BiP is induced in ER-stress and regarded as a clear ER-stress marker (for a review, see Li and Lee, 2006), our finding suggests that nicotine can cause ER-stress in human placenta. In our earlier placental perfusion study (Veid and Karttunen et al., 2011) nicotine, which basically goes through human placenta by passive diffusion, however, was retained to some

Conflict of interest statement

None of the authors have any conflicts of interest.

Acknowledgements

The authors appreciate the excellent technical assistance of senior technician Virpi Koponen and wish to thank PhD-student Heta Salo. We also appreciate the help of Dr. Marjo Huovinen, PhD. This work was financially supported by Toxicology section of FinPharma Doctoral Program.

References (69)

  • J. Lee et al.

    Nicotine and pathological angiogenesis

    Life Sci.

    (2012)
  • W.K. Lee et al.

    ERK1/2-dependent bestrophin-3 expression prevents ER-stress-induced cell death in renal epithelial cells by reducing CHOP

    Biochim. Biophys. Acta

    (2012)
  • G.R. Leonardson et al.

    Risk factors for alcohol use during pregnancy in a multistate area

    Neurotoxicol. Teratol.

    (2003)
  • T.J. Mørck et al.

    Placental transport and in vitro effects of Bisphenol A

    Reprod. Toxicol.

    (2010)
  • T. Mose et al.

    Meta-analysis of data from human ex vivo placental perfusion studies on genotoxic and immunotoxic agents within the Integrated European Project NewGeneris

    Placenta

    (2012)
  • D. Nutt et al.

    Development of a rational scale to assess the harm of drugs of potential misuse

    Lancet

    (2007)
  • A.X. Schönthal

    Pharmacological targeting of endoplasmic reticulum stress signalling in cancer

    Biochem. Pharmacol.

    (2013)
  • M. Tampio et al.

    Benzo(a)pyrene increases phosphorylation of p53 at serine 392 in relation to p53 induction and cell death in MCF-7 cells

    Toxicol. Lett.

    (2008)
  • S.E. Thomas et al.

    p53 and translation attenuation regulate distinct cell cycle checkpoints during endoplasmic reticulum (ER) stress

    J. Biol. Chem.

    (2013)
  • K.H. Vähäkangas et al.

    The significance of ABC transporters in human placenta for the exposure of the fetus to xenobiotics

  • P. Valbonesi et al.

    Effects of cadmium on MAPK signalling pathways and HSP70 expression in a human trophoblast cell line

    Placenta

    (2008)
  • J. Veid et al.

    Acute effects of ethanol on the transfer of nicotine and two dietary carcinogens in human placental perfusion

    Toxicol. Lett.

    (2011)
  • A.A. Alfadda et al.

    Reactive oxygen species in health and disease

    J. Biomed. Biotechnol.

    (2012)
  • Y. Bando

    The functional role of stress proteins in ER stress mediated cell death

    Anat. Sci. Int.

    (2012)
  • A. Boronkai et al.

    Effects of pituitary adenylate cyclase activating polypeptide on the survival and signal transduction pathways in human choriocarcinoma cells

    Ann. N. Y. Acad. Sci.

    (2009)
  • J.W. Brewer et al.

    Mammalian unfolded protein response inhibits cyclin D1 translation and cell-cycle progression

    Proc. Natl. Acad. Sci. U. S. A.

    (1999)
  • J.W. Brewer et al.

    PERK mediates cell-cycle exit during the mammalian unfolded protein response

    Proc. Natl. Acad. Sci. U. S. A.

    (2000)
  • P.S. Brocardo et al.

    The role of oxidative stress in fetal alcohol spectrum disorders

    Brain Res. Rev.

    (2011)
  • J.E. Bruin et al.

    Long-term consequences of fetal and neonatal nicotine exposure: a critical review

    Toxicol. Sci.

    (2010)
  • A. Cardinale et al.

    Nicotine: specific role in angiogenesis, proliferation and apoptosis

    Crit. Rev. Toxicol.

    (2012)
  • M. Cargnello et al.

    Activation and function of the MAPKs and their substrates, the MAPK-activated protein kinases

    Microbiol. Mol. Biol. Rev.

    (2011)
  • D.A. Evseenko et al.

    ABC drug transporter expression and functional activity in trophoblast-like cell lines and differentiating primary trophoblast

    Am. J. Physiol. Regul. Integr. Comp. Physiol.

    (2006)
  • Y. Fu et al.

    Glucose regulated proteins in cancer progression, drug resistance and immunotherapy

    Cancer Biol. Ther.

    (2006)
  • A. Grozio et al.

    Nicotine, lung and cancer

    Anticancer Agents Med. Chem.

    (2007)
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