Ethanol cytotoxic effect on trophoblast cells

Highlights

• Sustained ethanol exposure causes cytotoxicity in JEG3 trophoblast cells.

• The mechanism is by activating apoptotic pathways as a result of DNA damage.

• This activation of apoptosis represents an induction of cellular damage.

• This damage can be related in part to functional alterations in placenta.

• These results may explain the consequences in other tissues, such as neurons.

Abstract

Prenatal ethanol exposure may cause both, altered fetal neurodevelopment and impaired placental function. These disturbances can lead to growth retardation, which is one of the most prevalent features in Fetal Alcohol Syndrome (FAS). It is not known whether there is a specific pattern of cytotoxicity caused by ethanol that can be extrapolated to other cell types. The aim of this study was to determine the cytotoxic effects caused by sustained exposure of trophoblast cells to ethanol. The cytotoxic effect of sustained exposure to standard doses of ethanol on an in vitro human trophoblast cell line, JEG3, was examined. Viable cell count by exclusion method, total protein concentration, lactate dehydrogenase (LDH) activity and activation of apoptotic markers (P-H2AX, caspase-3 and PARP-1) were determined. Sustained exposure to ethanol decreased viable cell count and total protein concentration. LDH activity did not increased in exposed cells but apoptotic markers were detected. In addition, there was a dose-dependent relationship between ethanol concentration and apoptotic pathways activation. Sustained ethanol exposure causes cellular cytotoxicity by apoptotic pathways induction as a result of DNA damage. This apoptotic induction may partially explain the altered function of placental cells and the damage previously detected in other tissues.

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.