Ethanol cytotoxic effect on trophoblast cells
Graphical abstract
Introduction
Chronic alcohol consumption can cause damage to several organs, resulting in certain disturbances (Benassi-Evans and Fenech, 2011). Most importantly, prenatal chronic exposure to ethanol can produce severe deleterious effects on neurodevelopment. The exact mechanism of this damage is unknown and it depends on gestational age of the exposed fetus and the level and characteristics of sustained consumption. There are different hypotheses and placental damage due to ethanol is one of them. One of the health consequences of alcohol consumption in pregnant women is Fetal Alcohol Syndrome (FAS), a condition induced by the exposure of the developing embryo to ethanol (Jones and Smith, 1973). The clinical features of FAS can be broadly divided into: morphological malformations, especially craniofacial defects, central nervous system impairment, neuropsychological traits, and growth retardation (Moore et al., 2007, Rostand et al., 1990). Offsprings of mothers who drink heavily during pregnancy can develop FAS with all the symptoms described above, but in some cases there is no physical or morphological evidence of prenatal alcohol effects at birth, which is explained by variations in the extent of exposure and continuity (Gemma et al., 2006). On the other hand, current estimates suggest that at least 9.1/1000 of the pediatric population has Fetal Alcohol Syndrome Disorder (FASD) (Sampson et al., 1997).
Of the three clinical features of FAS, growth retardation is the most prevalent (Greene et al., 1991, Hellstrom et al., 1996, Pennington et al., 1983). Specifically, intrauterine growth retardation has been partly associated with a placenta that cannot fulfill all its functions during pregnancy (Gundogan et al., 2008, Salihu et al., 2010). Since placenta acts as a barrier to protect the fetus from toxic chemicals coming from the maternal circulation, there is a possible accumulation of these compounds which can induce changes in placental cells (Kuczkowski, 2007, Ortigosa et al., 2012). It has been shown previously that placental function can be altered by ethanol exposure (Burd et al., 2012).
Furthermore, considering other cell types, previous studies have described apoptotic induction in neurons located in the developing brain (Luo, 2012). It has been also described that prenatal ethanol exposure causes damage to endocrine and hypothalamic neurons (Hellemans et al., 2008, Sarkar et al., 2007) and that, upon ethanol exposure during fetal development, a large number of these neurons undergo cell death by apoptotic pathways (Chen et al., 2006).
Apoptotic in vitro studies should include viable cell counts to see how the total number of cells varies. Assays such as Trypan Blue dye exclusion method and total protein concentration measurement are common (Rodriguez-Gonzalez et al., 2013). The necrosis levels should be quantified as well by calculating the amount of cells which died by plasma membrane ruptures. It is therefore common to use intracellular markers, such as lactate dehydrogenase (LDH), which can be released to the medium due to this cause (Krysko et al., 2008). Finally, programmed cell death markers, such as histone H2AX phosphorylation (P-H2AX) (Svetlova et al., 2010), caspase-3 activation (Porter and Janicke, 1999) and poly-ADP-ribose polymerase 1 cleavage (PARP-1) (Soldani and Scovassi, 2002) should be determined.
In this sense, prenatal ethanol exposure disrupts placental function, possibly causing intrauterine growth retardation (Gundogan et al., 2008, Salihu et al., 2010). By the other hand, it is also known that ethanol can cause apoptosis in several types of neurons (Chen et al., 2006, Hellemans et al., 2008, Luo, 2012, Sarkar et al., 2007). Knowing ethanol's cytotoxic effects on trophoblast cells, may allow to extrapolate the consequences to other cell types which underwent the same alcohol exposure pattern.
The aim of this study was to determine the cytotoxic effects of sustained ethanol exposure in an in vitro system of human trophoblast cells as a surrogate marker of damage due to prenatal exposure to ethanol.
Section snippets
Cell culture
The aim was to use specifically placental cells previously used in the literature. Human placental choriocarcinoma cell line was purchased from the American Type Culture Collection (ATCC): JEG3 (HTB-36, Manassas, USA). Cells were maintained in Minimum Essential Media (MEM) supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 20 mM l-glutamine, 10 mM sodium pyruvate, 100 mg/mL streptomycin and 100 U/mL penicillin; all get from Gibco, Montreal, CA. Cell cultures were maintained at 37 °C in humidified
Viable cell count and total protein concentration
Exposed trophoblasts showed a reduction in viable cell count compared to the control (Fig. 1(1)). Moreover, this decrease was observed at both periods: group A decreased 25.95% at beginning and 36.39% at end, while group B showed reductions of 59.77% at beginning and 55.66% at end. All values were significant vs. the group C (p < 0.05) (Fig. 1(1)). Total protein concentration levels were also decreasing relative to control at both periods (Fig. 1(2)). Group A values decreased 17.76% at beginning
Discussion
Exposed groups showed a decrease in viable cell count, in total protein concentration and in LDH activity. Furthermore, apoptotic markers such as P-H2AX, caspase-3 and PARP-1 were detected. These in vitro results demonstrate cytotoxic damage by activating apoptotic pathways in JEG3 ethanol exposed cells. It was also found that there is a dose-dependent relationship between the degree of cell damage and the ethanol dosage received.
The decrease in viable cells, measured by cell count and total
Conclusion
In conclusion, sustained ethanol exposure causes cytotoxicity in JEG3 trophoblast cells by activating apoptotic pathways as a result of DNA damage. This activation of apoptosis represents an induction of cellular damage that can be related in part to the previously described functional alterations in placenta. Moreover, the results of our study may offer an explanation of the consequences found in other tissues as well, such as neurons of the CNS.
Funding sources
This study was supported by Grants from Fondo de Investigaciones Sanitarias (FIS) (PI10/02593), from the Instituto Carlos III (Madrid, Spain), and Red de Salud Materno-Infantil y del Desarrollo (SAMID) (RD12/0026/0003) from the Instituto Carlos III (Spain), intramural funding of the Neuroscience Program at IMIM (Institut Hospital del Mar d’Investigacions Mèdiques) and partially supported by Generalitat de Catalunya (Spain) AGAUR (2009SGR1388).
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors want to thank J. Klein for excellent language editing service and to J. Yelamos for providing PARP-1 antibody and his technical support.
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These authors contributed equally to this work.