Fetal alcohol spectrum disorders constitute a constellation of phenotypes caused by exposure to ethanol during early development (e.g., Glass et al., 2023). Ethanol exposure has been shown to have teratogenic effects on the developing central nervous system (CNS), which can have life-long impacts for the exposed individual (Chokroborty-Hoque et al., 2014; Glass et al., 2023). Although the teratogenic effects of ethanol have been known for at least half a century and despite publicity campaigns, the incidence of FASD remains as high as 5 in every 100 live births (May et al., 2018, 2021). This has led to a focus on identifying treatments and therapeutics that can ameliorate ethanol’s teratogenic effects.

One of the most effective treatments, to date, has been choline supplementation. Choline is an essential nutrient that has several functions within the cell, influencing synthesis of phospholipids in cell membranes, synthesis of the neurotransmitter acetylcholine, and acting as a methyl donor in a number of cellular processes (Wallace et al., 2020). In animal models, choline has been shown to mitigate many, but not all, of the effects of developmental ethanol exposure during development ranging from brain to behavioral changes (Thomas et al., 2000, 2009, 2010; Monk et al., 2012; Bottom et al., 2020; Baker et al., 2022; Ernst et al., 2022; Naik et al., 2022). Choline is effective whether administered during prenatal alcohol or after birth (Thomas et al., 2009, 2010). Initial clinical studies have provided promising results that choline may be an effective treatment for at least some of ethanol’s teratogenic effects, particularly as they relate to the CNS (Jacobson et al., 2018; Wozniak et al., 2020; Warton et al., 2021; Ernst et al., 2022; Gimbel et al., 2022). In fact, choline deficiency has been shown to exacerbate ethanol’s teratogenic effects (Idrus et al., 2017).

Although choline continues to show promise as an effective intervention, it is critical to understand factors that can influence choline’s efficacy. One factor that has been proposed to be important is genetics. Two lines of evidence provide support for the role of genetics in choline’s efficacy. First, a study by Fischer and colleagues examined the relationship between the level of choline and its metabolite betaine in breast milk, and the amount of choline consumed in women with differing genetic backgrounds. Their results showed that polymorphisms in two choline-related enzymes, phosphatidylethanolamine-N-methyltransferase (PEMT) and methylenetetrahydrofolate dehydrogenase 1 (MTHFR), had a significant effect on either the basal levels of choline or on changes in the levels in breast milk following choline supplementation (Fischer et al., 2010a,b; Corbin and Zeisel, 2012). Second, a recent study examined children with FASD that were given choline supplementation or placebo and tested for tasks that assessed learning and memory functions along with concomitant evaluation of single nucleotide polymorphisms (SNPs) in various choline-metabolizing genes. The results demonstrated a significant association between SNPs in the gene for the choline transporter gene solute carrier family 44 member 1 (SLC44A1) and the ability to perform the tasks (Smith et al., 2021). The current study is designed to further evaluate the role of genetics in choline’s efficacy in ameliorating ethanol-induced cell death early in the development of the CNS.

Mouse models are frequently used to study the genetic pathways involved in FASD and possible candidates for the genetic variation. The BXD recombinant inbred (RI) panel is the largest mouse RI mapping panel, derived by crossing B6 and D2 mouse strains (Peirce et al., 2004). BXD recombinant inbred strains have often been used to study the genetics and epigenetics of alcoholism, since they show significant differences in their preference for ethanol (Cunningham, 1995; Philip et al., 2010). Additionally, a study by Downing et al. (2012) examined morphological phenotypes (such as abnormalities in the digits and kidney) across BXD strains following prenatal alcohol exposure and found differential sensitivity in these strains. Previous work in our lab identified strains of BXD mice that were more susceptible to ethanol’s induction of cell death in the neural tube (“high cell death strains”) and those that were less susceptible (“low cell death strains”). We examined three BXD strains that have been identified as high cell death strains in the present study: BXD2, BXD51, and BXD73 (although there was low cell death in the brainstem) (Theberge et al., 2019). Additionally, the parental B6 strain was also a high cell death strain and was thus also evaluated in the current study.

In the present study, we examined these 4 high cell death strains to address the following. (1) Previous work has shown that choline can ameliorate ethanol-induced cell death (Wang and Bieberich, 2010) and this study was undertaken to confirm the previous report in vivo. (2) Previous work in rats and sheep has suggested that, at certain doses and phenotypes examined, choline itself can be teratogenic (Goeke et al., 2018; Carugati et al., 2022) and this study was undertaken to determine if differing genetic backgrounds showed evidence of baseline teratogenicity due to choline. (3) Choline may improve performance on some, but not all, behavioral outcomes, so we examined two brain regions to assess choline’s efficacy across brain regions. (4) We evaluated two doses of choline, a moderate dose (100 mg/kg body weight) and a high dose (250 mg/kg body weight) to determine if one dose was more or less effective. (5) We evaluated the effects of the genetic background by determining if the effects were equivalent across embryos of differing genetic backgrounds.

The present study demonstrates that choline was effective in ameliorating ethanol-induced cell death in the early developing neural tube. This is consistent with the study by Wang and Bieberich (2010) that demonstrated that choline treatment prevented ethanol-induced cell death at the same age in neural crest derived cell cultures. The ability of choline to reduce apoptosis in the face of other insults, such as folate deficiency, has also been shown at older ages (Craciunescu et al., 2003), suggesting that choline may protect against cell death at multiple developmental periods. This study provides additional validation for the use of choline as a potential treatment for ethanol-induced teratogenesis and demonstrates that even in early prenatal development of the CNS, choline can mitigate ethanol-related neuropathology.

It is interesting to note that both doses of choline were shown to be effective in ameliorating the cell death induced by ethanol (see Figures 2, 3). In the present study, both 100 mg/kg and 250 mg/kg doses of choline were used, and both of these doses have been used in previous studies and have been shown to be effective in different paradigms in ameliorating a range of phenotypes that are both behavioral and morphological (Thomas et al., 2000, 2009, 2010; Monk et al., 2012; Baker et al., 2022; Ernst et al., 2022; Naik et al., 2022). However, it is interesting to note that the low dose of choline was more consistent than the high dose in ameliorating ethanol-induced cell death, providing a potential cautionary warning about high doses.

Equally as important, these results show little evidence for enhanced ethanol-induced cell death caused by choline treatment. This is shown both by the lack of effect of choline in the control animals and the lack of additional cell death in the ethanol-exposed embryos that also received choline. Our findings align with the majority of previous studies which have shown that choline is safe for the developing embryo (Thomas et al., 2009, 2010; Kwan et al., 2021; Baker et al., 2022) although there have been a few exceptions, particularly at high doses such as 100 mM (Goeke et al., 2018; Carugati et al., 2022).

One of the important findings of this study is that there are strain differences in choline’s ability to ameliorate ethanol-induced cell death but only at the higher dose. This adds to the growing literature that suggests that genetics play an important role in choline’s efficacy (e.g., Smith et al., 2021). This is consistent with previous studies showing that allelic differences in various enzymes in the choline metabolic pathway can impact its efficacy (Fischer et al., 2010a,b). Further research is needed to assess whether these allelic differences are present within these BXD RI strains. Moreover, these results suggest that, at low doses, the effect of genetics is less apparent which could have important implications for its use in human populations.

These studies show the efficacy of choline but do not provide clues as to the mechanism of choline’s protection. Choline has numerous effects but three are most likely to act within the CNS. First, choline is part of the pathway for synthesis of the neurotransmitter acetylcholine (ACh). At older ages, it is likely that this is a relevant mechanism for choline’s efficacy. However, at this early developmental stage, it is unclear how much ACh is present (Schambra et al., 1989; Abreu-Villaca et al., 2011) and therefore this mechanism is unlikely to be important at this age. Second, it is a component of phospholipid synthesis for cell membrane components. Ethanol can affect membrane composition (e.g., Naik and Ramadoss, 2023) and while it is unclear whether this is the mechanism of choline’s efficacy, it is a strong potential mechanism. Third, it is a methyl donor that is essential for methylation which is an important epigenetic modification. Several studies have shown that ethanol exposure during development can alter DNA methylation and choline can modify ethanol’s effects (Balaraman et al., 2017; Sarkar et al., 2019). Previously, we examined several epigenetic modifications across multiple brain regions in neonatal mice given ethanol 7 h prior to tissue collection (Schaffner et al., 2020). The results demonstrated that there were both altered DNA methylation and altered gene expression in this short timeframe, suggesting that altered methylation of either DNA or histones could be a significant mechanism for choline’s protective actions with the early neural tube.

Our results indicate that whatever the mechanism(s) of neural protection is/are, it needs to help explain the complex nature of the protective effects: the strain-, dose-, and region-specific differences seem amongst the four strains that were analyzed in this study. Data from human clinical trial work using choline has discovered allelic differences in key choline utilization genes, cellular choline transporter gene solute carrier family 44 member 1 (SLC44A1) (Smith et al., 2021), methylenetetrahydrofolate dehydrogenase 1 (MTHFR, also known as Mthfd1 in the mouse) (Fischer et al., 2010a) and phosphatidylethanolamine-N-methyltransferase (PEMT) (Fischer et al., 2010a,b) that alter choline related phenotypes. To speak to the polymorphisms found in humans using a mouse model, it would be interesting to examine SNPs in reference populations like the BXD RI lines to see if a gene of interest can have a phenotypic impact. For example, in the BXD population there are two alleles of the mthfd1 gene that segregate the BXD51 and BXD2 lines as distinct from the BXD73 line. Therefore, the BXD population can be used to examine polymorphisms in other genes as they relate to choline’s ability to reduce ethanol-induced cell death.

The dose of ethanol used in the present study was a relatively high one. The rationale for this is three-fold. First, as the purpose of the present study was to evaluate the efficacy of choline in reducing ethanol-induced cell death, this dose was chosen because it has been shown to more reliably cause cell death with similar dosages used by ourselves and others (Young et al., 2005; Young and Olney, 2006; Goldowitz et al., 2014; Theberge et al., 2019; Boschen et al., 2021). Second, the alcohol exposure used in the present study models binge ethanol consumption which is the most common drinking pattern in women of child-bearing age and is characterized as episodes of high ethanol consumption in a short time window (Kanny et al., 2018; Bohm et al., 2021). Finally, the high dose used in the present study is to achieve effectiveness as small animals, including mice, metabolize ethanol at a faster rate than humans (Cederbaum, 2012).

Importantly, there are several limitations to the present study. First, the sex of the embryos remains unknown and there could be differential effects based on sex (Kwan et al., 2017). This is an important consideration and will be done in the future. Second, this study examined the acute effects of choline on mitigating ethanol-induced cell death. It will be important to determine if the effects of choline translate into long-term amelioration of ethanol-related cell loss. Third, only two doses of choline were examined and it is possible that additional differences would be detected at either higher or lower doses of choline. The current data suggest that a threshold effect was achieved at 100 mg/kg in all strains, but it would critical to know if genetics influences the minimal choline level needed, given variation in choline intake and need among clinical populations. Fourth, there could be differences in bioavailability of choline to the embryos and whether or not alcohol impacts this bioavailability. Currently, this interesting phenomenon has not been examined, but will be evaluated in future studies.

In the present study, we confirm that choline is effective in ameliorating ethanol-induced cell death in the developing neural tube but further demonstrate that there are strain differences in these effects. Moreover, the differential strain effects are most pronounced at the high dose suggesting that the lowest effective dose would likely be the most beneficial and the least likely to have untoward other effects. Currently, the mechanism behind either choline’s protective effects or the molecular pathways that underlie the genetic differences are unknown. However, because the liver is an essential organ in metabolizing choline and is the first organ to encounter choline after absorption, the potential for differences in liver metabolism of choline across strains exists and this may prove to be important for understanding genetic differences in humans (Smith et al., 2021). Future research will be needed to address this issue as well as to gain a better understanding of how choline protects against ethanol’s teratogenic actions.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by Institutional Animal Care and Use Committee at the University of Tennessee Health Science Centre. The study was conducted in accordance with the local legislation and institutional requirements.

KH, JT, and DG contributed to conception and design of the study. KH, DG, and FX contributed to the generation of all data in the manuscript and analyses of the data. KH, FX, and DG wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.

This research was funded by National Institute on Alcohol Abuse and Alcoholism R01AA023508 (KH, DG), University of Tennessee Health Science Center CORNET Award (KH), and the BC Children’s Hospital Research Institute (DG).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.