In this study, hES cells were differentiated into neuronal lineages and exposed to either 0, 0.1 or 0.3% ethanol at three developmental stages, EBs, neural progenitors and neurons, to model the effects of ethanol exposure on early human brain development. This in vitro model was coupled with metabolomics to test the hypothesis that ethanol exposure induces statistically significant changes to endogenous small molecules. We also sought to define specific small molecules and biochemical pathways that are altered in alcohol-induced developmental neurotoxicity, leading to FASD. Neural differentiation of hES cells is an important model to study the earliest effects of alcohol on human development given that the timing of neuronal and glial differentiation from hES cells is similar to lineage allocation in vivo (Zhang, 2006
). Alcohol exposure during embryogenesis and early neural differentiation, a period when pregnancy may be unknown, leads to a higher incidence of craniofacial defects and mental disabilities (Ernhart et al., 1987
; Guerri, 2002
). There is a significant need to identify the biochemical pathways that play a causal role in alcohol-induced developmental neurotoxicity. Our study confirms that the exposure of hES cell-derivatives to ethanol results in significant changes to the abundance of human endogenous metabolites.
No significant features were shared between the three cell types following ethanol treatment. This likely reflects the disparate maturation states of these differentiating cells. Approximately half of the features showed similar responses at both ethanol concentrations, although the magnitude of change did not always achieve statistical significance. Other features, such as T4 and kynurenine, were increased at 0.1% ethanol but had decreased abundance at 0.3%. This differential response likely reflects the cells’ adaptive responses to the higher ethanol level over the four-day culture period (e.g. Buck and Harris, 1991
; Yakovleva et al. 2011
). This interpretation is endorsed by the finding that 0.1% ethanol treatment produced appreciably more significant features (154) than did 0.3% ethanol (112), of which 15 were significant in both exposures. Many individuals with FASD experience chronic alcohol exposure and biomarkers that reflect those adaptive mechanisms are clinically relevant.
Embryoid bodies are comprised of the three primary germ layers and thus model the earliest stages of differentiation. We identified statistically significant changes in multiple developmentally important metabolites between 0, 0.1% and 0.3% ethanol-treated EBs. The abundance of 5’-methylthioadenosine (MTA) was significantly decreased in 0.3% ethanol-treated EBs compared to untreated controls. MTA is produced in all mammalian cells during the synthesis of polyamines putrescine, spermidine and spermine from decarboxylated S-adenosylmethionine (SAM), and its levels directly reflect the rates of polyamine synthesis (Avila et al., 2004
; Sufrin et al., 1989
). Inhibition of polyamine synthesis impedes embryo growth (Fozard et al. 1980
; Mendez 1989
) whereas exogenous polyamines protect against embryotoxicants (Chirino-Galindo et al., 2009
). Both acute and chronic ethanol exposure suppress polyamine synthesis and its dysregulation was identified as a potential mechanism contributing to FASD (Desiderio et al., 1987
; Sessa and Perin, 1997
; Shibley et al., 1995
; Thadani et al., 1977
). The decline in MTA is consistent with those reports and thus secreted MTA may be a useful biomarker for alcohol’s toxicity. MTA also supplies the sulfur atom for methionine synthesis within the methionine salvage pathway. Reductions in MTA might also contribute to the depleted methionine and SAM levels observed in chronic ethanol consumption (Finkelstein et al., 1974
; Walcher and Miller, 2008
). Importantly, because MTA is produced from SAM, its alteration may reflect a larger disruption of methyl group homeostasis within ethanol-exposed cells, an interpretation affirmed by the increased folic acid and polyglutamate content found in ethanol-treated EBs and neural progenitors (Supplemental Table 1 and 2
). Methyl group supplements in the form of choline, betaine and SAM can prevent or reverse ethanol’s developmental toxicity both in vitro and in vivo (Seyoum and Persaud, 1994
; Thomas et al. 2009
). To our knowledge there are no previous reports specifically linking MTA to ethanol exposure or FASD. Its metabolic links with polyamine and methyl metabolism make MTA an excellent potential biomarker in addition to offering mechanistic insights into FASD.
We additionally confirmed thyroxine as a putative biomarker of prenatal ethanol exposure during the embryogenesis stage. Thyroxine abundance was significantly increased in 0.1% ethanol-treated EBs compared to untreated controls. Thyroxine (T4) is one of the two hormones known as thyroid hormone secreted by the thyroid gland. In these cultures T4 likely originated from the serum in the culture medium. Thyroxine signaling through its nuclear receptors is critical for normal central nervous system development and contributes to multiple processes including cell migration, dendrite/axon outgrowth and synaptogenesis. A precise balance of thyroid hormone is necessary for proper brain development and both deficiency and hyperthyroidism can cause significant and irreversible brain dysfunction (Bernal and Nunez, 1995
; Lauder, 1977
; Pharoah et al., 1971
). Our studies show a 24% increase in exogenous thyroxine levels, suggesting an ethanol-induced perturbation to thyroxine metabolism and/or its uptake by the early embryo. This is consistent with prior demonstrations of altered thryroid hormone metabolism in animal models and infants with FASD (Cudd et al. 2002
; Hannigan and Bellisario 1990
; Kornguth et al. 1979
). In rat models, prenatal alcohol exposure (PAE) also alters the levels of iodothyronine deiodinase III and thyroid hormone receptor alpha-1 (Shukla et al., 2010
, 2011). Thus the significant increase in thyroxine detected herein suggests that ethanol exposure is likely to impair proper thyroid hormone metabolism in the early embryo, corroborating a proposed mechanistic pathway that ethanol may produce neurodevelopmental disorders by negatively affecting maternal-fetal hormonal homeostasis. Our study is the first to indicate a differential uptake or processing of maternal thyroxine by human embryos as a result of PAE. It is plausible to speculate that this detrimental effect may be mediated by functional impairment of the thyroid hormone-specific transporters Oatp1c1 (organic anion transporting polypeptide 1c1), monocarboxylate transporter (MCT) 8 and MCT10. Mutations to MCT8 actually increase the extracellular abundance of thyroxine in vivo, producing severe psychomotor retardation (Visser et al. 2008
; Grijota-Martinez et al., 2011
). There are currently no published studies examining the direct effects of ethanol exposure on thyroxine-specific transporters, which will be a focus of future studies.
Ethanol exposure at two different stages of neurogenesis produced statistically significant changes to tryptophan metabolism. These changes are summarized in . These differences were not attributed to trivial effects of ethanol upon differentiation states, as the cellular type distribution between treatments did not differ. Tryptophan is metabolized primarily along the kynurenine pathway and kynurenine levels were increased in neural progenitors treated with 0.1% ethanol, as were the levels of the tryptamine catabolite indoleacetaldehyde in neurons exposed to 0.1% ethanol. Dysregulation of tryptophan metabolism is reported in several models of alcohol exposure. Kynurenine levels are increased in human plasma following acute ethanol consumption (Badawy et al. 2009
) and tryptophan levels are significantly reduced in the plasma, brain and liver of rat pups following gestational ethanol exposure (Lin et al. 1990). Serum levels of kynurenine are also perturbed in several cognitive disorders such as Down syndrome, bipolar disorder and postpartum depression but had not been previously described in FASD. Kynurenine has additionally been identified in hES cells treated with valproate, a known inducer of neurodevelopmental defects (Cezar et al. 2007
). Kynurenine was recently reported to activate the aryl hydrocarbon xenobiotic receptor (AhR) with an apparent Kd ~4 µM (Optiz et al. 2011). AhR activation adversely affects brain development and behavior (Latchney et al. 2011
; Seo et al. 1999
). Our finding of elevated Kyn in ethanol-treated neural progenitors is a novel insight into the mechanisms underlying FASD, and confers this metabolite a translational role as a candidate biomarker of FASD.
Figure 7 Summary of ethanol-induced changes in tryptophan metabolism in human ES cells. Black font indicates metabolites that are detected in media of ethanol-treated human EBs, neural precursors, and neurons. Asterisk indicates those metabolites whose levels (more ...)
Our study is the first to indicate a direct effect of ethanol exposure on the human endogenous metabolite IAA. Tryptophan is critical for normal neurogenesis as the precursor to the neurotransmitter serotonin (5HT) and its availability has a direct effect on 5HT synthesis (Fernstrom and Wurtman, 1971
). Development of the serotonergic system occurs earlier than other neurotransmitter systems and its sensitivity to disruption by PAE is well documented (Druse et al., 1991
; Maier et al., 1996
; Sari et al., 2010
). Elevated IAA decreased the activity of tryptophan hydroxylase, the rate-limiting enzyme in serotonin synthesis, by as much as 33% (Nilsson and Tottmar, 1987
). Thus, elevations of kynurenine and IAA detected here could be biomarkers for a metabolic diversion of tryptophan away from serotonin synthesis and toward these alternate metabolic pathways and may explain the reduced tryptophan and 5HT content of the ethanol-exposed fetal brain (Maier et al., 1996
; Sari et al., 2010
). In fact, an increase in the IAA-related 5-hydroxylindoleacetic acid (5-HIAA) was measured in the urine of rats chronically exposed to ethanol (Bonner et al., 1993
) and IAA itself is amenable to detection in urine, which renders it a translational application in biofluids. Alternatively, we note that kynurenine also lies along the synthetic pathway of nicotinamide from its tryptophan precursor. Ethanol oxidation places a high requirement for niacin and NADPH reducing equivalents, and one possibility is that ethanol’s effects on cellular redox potential might be driving these changes in tryptophan metabolites. Whether one or both of these hypotheses underlie increased kynurenine levels in ethanol-exposed neural progenitors, serving as the cause of serotonergic dysfunction, will be elucidated in future mechanistic studies. Nonetheless, it is noteworthy that significant perturbations to the tryptophan pathway and its metabolites were consistent across different timelines of neural differentiation, neural precursors and neurons.
In addition to the four confirmed metabolites with marked changes resulting from ethanol exposure, we identified multiple unknown compounds with statistically significant changes in abundance. Further experiments are required to determine the chemical formula of such compounds by ion fragmentation and other analytical platforms such as NMR. The fact that statistically significant small molecules are not annotated in public databases does not preclude their potential application as candidate diagnostic biomarkers for FASD. Given the highly sensitive, high resolution nature of the mass spectrometric detection system employed in this study, a measurable endpoint (exact mass) is determined which can be used for continued chemical structure determinations. An important limitation to the lack of a chemical annotation is that these unannotated small molecules cannot be mapped to a biochemical pathway for mechanistic elucidation.
No changes in the overall cell differentiation ability of hES cells were measured in response to 0.1% or 0.3% ethanol (), at least for the culture conditions employed in this study and for the relatively short ethanol exposure period. There was a non-significant trend for an increased percentage of neurons expressing synaptophysin and SV2A following the ethanol treatment of mature neurons. Together with the multiple changes in the levels of tryptophan metabolites, these data may indicate an overall trend of increased synaptogenesis in response to ethanol exposure. In a finding that seemed counterintuitive, we found modest non-significant decreases in apoptosis frequency following ethanol treatment of neural precursors and neurons. Prock and Miranda (2007)
reported a similar resistance to apoptosis in ethanol-treated mouse neural progenitors, a population substantially similar to those studied herein. Our findings endorse their interpretation that apoptosis in response to ethanol may be a differentiation state-specific response. Further study is required to understand why this population is resistant to apoptosis upon exposure to ethanol.
In conclusion, exposing hES cell-derived EBs, neural progenitors and neurons to ethanol identified marked changes in biochemical pathways and metabolites critical for proper neurodevelopment. Our findings not only provide novel candidate diagnostic biomarkers for FASD, but also corroborate independent studies that have proposed alterations to methionine, methyl group, thyroid hormone and tryptophan metabolism as potential mechanisms underlying FASD. Although some of these metabolites, i.e. kynurenine, have been described as putative biomarkers for other neurodevelopmental disorders, it is the integration of several of these endogenous small molecules from independent biochemical pathways, that may be used as a specific fingerprint test set or specific fingerprint for the diagnosis of FASD in biofluids. Metabolomics is able to measure simultaneous changes to multiple unrelated biochemical pathways, providing not only a biochemical signature specific to FASD but also mechanistic insight into the effects of PAE on early human embryogenesis. The identification of differential biomarkers in our in vitro model prior to analysis of biofluids allows for a more targeted approach in an in vivo model, which has multiple confounding factors, i.e. maternal-fetal interactions. Biofluids are complex mixtures of systemic byproducts influenced by both endogenous and exogenous factors e.g., genetics, diet and environment. Using our strategy, we can design specific analytical protocols to examine biofluids based on our confirmed metabolites. Our future efforts will examine whether molecules detected here are subject to similar changes in in vivo models and children diagnosed with FASD. The biochemical pathways revealed by metabolomics of hES cell derivatives may also present new targets for the identification and development of novel therapies for FASD.