Elsevier

Toxicology in Vitro

Volume 27, Issue 5, August 2013, Pages 1503-1512
Toxicology in Vitro

Perfluorooctane sulfonate (PFOS) induced embryotoxicity and disruption of cardiogenesis

https://doi.org/10.1016/j.tiv.2013.03.014Get rights and content

Highlights

  • Embryotoxicity evaluation of PFOS was performed using four endpoints.

  • The disruption of the BMP signaling pathway by PFOS, including key cardiac markers was observed firstly in cell model.

  • PFOS could disturb the expression of Nkx2.5 and Myl4 specifically and persistently.

  • It was PFOS rather than PFOS induced ROS that contributed to the disruption of cardiac differentiation.

Abstract

Prenatal exposure to perfluorooctane sulfonate (PFOS) is correlated with birth defects and adverse health effects. However, the mechanisms remain largely unknown. In current study, the embryonic stem cell test (EST) was performed to evaluate the embryotoxicity of PFOS, and embryonic stem cells (ESCs)-derived cardiomyocytes were used as a model of the early stages of heart development to determine the developmental toxicity of PFOS. One validated endpoint and three molecular endpoints were observed to ensure accurate evaluation of toxicity. According to the criteria of the EST, PFOS was classified as weak embryotoxic. In addition, a cascade of genes related to normal cardiac development was examined at three different time points to monitor cardiogenesis. We found that PFOS significantly interfered with gene expression during cardiogenesis, especially on Nkx2.5 and Myl4. Further, PFOS reduced ATP production in ESCs-derived cardiomyocytes, together with PFOS induced apoptosis, could explain the reduction in beating ability. PFOS-induced reactive oxygen species (ROS) accumulated within cells, which was accompanied by an interfering expression of apoptosis-related genes, ultimately leading to apoptosis. In conclusion, PFOS altered the expression of crucial genes, reduced ATP production, induced ROS, and stimulated apoptosis during the early stages of cardiogenesis; these effects may result in poor developmental outcomes.

Introduction

Perfluorooctane sulfonate (PFOS), a degradation product of many perfluorinated compounds, has been widely used as an industrial material in food packagings, textiles, and water-resistant paint (Fromme et al., 2009). It can be distributed within ecosystems and has been detected in soil, water, air, animals, and even the human body (Inoue et al., 2004, Liu et al., 2011, Raymer et al., 2011). PFOS induces several types of adverse effects on aquatic animals, mammals, and human, including impairment of the cardiovascular system (Huang et al., 2011, van Dartel et al., 2010); disruption of the reproductive system (Chan et al., 2011, Stein et al., 2009); interference with immunological functions (Mollenhauer et al., 2011); and damage to hepatic functions (Elcombe et al., 2012). A physiological based pharmacokinetic model (PBPK) shows that PFOS can barely be excreted via feces or urine and can pass through the placental membrane and migrate into mammary glands with high transport efficiency (Loccisano et al., 2011). The concentration of PFOS detected in the serum of mothers and infants was found to be nearly identical (Liu et al., 2011).

As previously reported, the order of bioaccumulation for PFOS in biological organs is greatest for the liver, followed by the heart, kidneys, and lungs (Cui et al., 2008). Cardiac development is one of the earliest organs formed during embryogenesis. It is such an accurate process that any abnormality during this period could result in the malformation of the organ, and worse, could result in embryo lethal. Because the heart is the second greatest target organ of PFOS, and because PFOS can be distributed through the placental membrane, we hypothesized that PFOS would induce cardiac toxicity as early as cardiogenesis. Currently, little is known with regards to the cardiac toxicity of PFOS. Up to date, no epidemiological investigation on the relationship between PFOS exposure and cardiovascular diseases was done. In aquatic models, such as zebrafish and medaka, PFOS-induced abnormalities have been observed, including an increase in the sinus venosus–bulbus arteriosus distance, an alteration of heart rate and a decrease in hatch time and hatch rate (Huang et al., 2011, Shi et al., 2008). Due to a short growth period, it is easy to monitor the developmental processes within aquatic models. However, due to interspecies differences, the results are difficult to translate to mammals. Commonly, developmental toxicity was most observed in mammals with sacrificing embryos or pups.

Therefore, an alternative method, named embryonic stem cells test (EST), was established and validated by the European Center for the Validation of Alternative Methods Registry (ECVAM) following the 3R’s rule, namely reduction, refinement and replacement. Two cell lines, mouse embryonic fibroblasts (MEFs) and embryonic stem cells (ESCs) were used in this test and, 50% inhibition of cells growth (IC50) of MEFs and ESCs, as well as 50% inhibition of ESCs differentiation (ID50) were determined for the EST evaluation. Based on the beating rate of ESCs derived cardiomyocytes, the classification was carried out. Besides, three molecular endpoints were added in current study, including expression of myosin heavy chain (MHC), myosin light chain (MLC) and cardiac troponin T (cTnT), due to their importance in the structural formation or conduction of cardiomyocytes (England and Loughna, 2012, Huang et al., 2007). In addition, based on bone morphogenetic proteins (BMPs) signaling, a set of myocardial specific transcription factors were selected to monitor the process of cardiogenesis when cells exposed to PFOS.

Reactive oxygen species (ROS) play dual roles in many signaling pathways or cellular functions. In ESCs, on one hand, a certain level of ROS is required to undergo cardiogenesis, and is essential for cardiac differentiation and myocardial beating (Crespo et al., 2010). On the other hand, ROS resulted in oxidative stress could affect normal cellular function, damage nucleic acids, and thus been implicated in carcinogenesis (Eriksen et al., 2010). Whether PFOS could induce ROS in ESCs derived cardiomyocytes, and what kind of role ROS played during the cardiogenesis were studied in current study as a supplementary explanation for the embryotoxicity.

Overall, the aims of current study were to evaluate the embryotoxicity of PFOS, to investigate plausible reasons for failures in differentiation, to monitor whether the myocardial networks in the process of early differentiation could be disturbed by PFOS and to tell the difference between PFOS and PFOS induced ROS in the interference.

Section snippets

Culture and differentiation of ESCs

ESC line R1, which was derived from 129 mouse strain was used in current study. ESCs were cultivated in an undifferentiated state on a mitomycin C inactivated feeder layer of C57BL/6 mouse strain derived mouse embryonic fibroblasts. ESC medium contained KnockOut Dulbecco’s modified Eagle’s medium (KO DMEM, Gibco, USA) supplemented with 15% fetal calf serum (Gibco, USA), 0.1 mM beta-mercaptoethanol (Gibco, USA), 2 mM l-glutamine (Gibco, USA), 0.1 mM non-essential amino acids and 1000 U/ml leukemia

Evaluation of PFOS-induced embryotoxicity

As shown in Fig. 1A, the viability of all groups, except for 15 μg/ml PFOS group, was different from the control group. The values of IC50 MEFs and IC50 ESCs were calculated respectively. Fig. 1B revealed that the MLC-positive cells were located within the EB center; it was coincident with the beating area determined by light microscopy, therefore the beating area was considered as ESCs cardiomyocytes. Fig. 1C revealed the contract positive rate of ESCs derived cardiomyocyte decreased

Discussions

The EST was developed to reduce the use of animals for experimental and other scientific purposes. Calculations based on three parameters were performed and compared, thereafter, the embryotoxicity potential of chemical was categorized as non-embryotoxic, weak embryotoxic, and strong embryotoxic, according to the classification criteria (Scholz et al., 1999, Seiler and Spielmann, 2011). Applying this special prediction model, three molecular endpoints with important functions in cardiomyocytes

Conflict of interest

The authors declare that there is no conflict of interest associated with the present study.

Acknowledgments

This study was supported by Shanghai Science & Technology Development Foundation (12140901000) and 12th Five-Year Plan of National Science and Technology Support (2012BAK17B10).

References (37)

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    Therefore, PFASs may be one category of embryonic development disorders and caused apoptosis during differentiation. Previously, Cheng et al. (2013) reported the toxicological effects of PFOS on cardiomyocytes differentiated from mouse embryonic stem cells in 3D cultures. During the early stages of cardiogenesis, PFOS contributes to adverse developmental outcomes by altering key gene expression, reducing ATP production, inducing excess reactive oxygen species (ROS), and stimulating apoptosis.

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Co-corresponding author. Address: Room 710, No. 5 Building, No. 280 South Chongqing Road, Shanghai 200025, PR China. Tel.: +86 02163846590 776607; fax: +86 02164667346.

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