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