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. Author manuscript; available in PMC: 2018 Jan 30.
Published in final edited form as: Birth Defects Res. 2017 Jan 30;109(2):92–98. doi: 10.1002/bdra.23577

Does arsenic increase the risk of neural tube defects among a highly exposed population? A new case-control study in Bangladesh

Maitreyi Mazumdar 1,2,3
PMCID: PMC5388562  NIHMSID: NIHMS817630  PMID: 27801974

Abstract

Background

Neural tube defects are debilitating birth defects that occur when the developing neural plate fails to close in early gestation. Arsenic induces neural tube defects in animal models, but whether environmental arsenic exposure increases risk of neural tube defects in humans is unknown.

Methods

We describe a new case-control study in Bangladesh, a country currently experiencing an epidemic of arsenic poisoning through contaminated drinking water. We plan to understand how arsenic influences risk of neural tube defects in humans through mechanisms that include disruption of maternal glucose and folate metabolism, as well as epigenetic effects. We will also test whether sweat chloride concentration, a potential new biomarker for arsenic toxicity, can be used to identify women at higher risk for having a child affected by neural tube defect. We are collecting dural tissue from cases, obtained at the time of surgical closure of the defect, and believe interrogation of these samples will provide insight into the epigenetic mechanisms by which prenatal arsenic exposure affects the developing nervous system.

Conclusions

These studies explore mechanisms by which arsenic may increase risk of neural tube defects in humans, and use a unique population with high arsenic exposure in order to test hypotheses. If successful, these studies may assist countries with high arsenic exposure such as Bangladesh to identify populations at high risk of neural tube defects, as well as direct development of novel screening strategies for maternal risk.

Keywords: myelomeningocele, neural tube defect, arsenic, environmental health, folatee

BACKGROUND

Neural tube defects are debilitating birth defects characterized by high rates of mortality and lifelong disabilities in surviving children. Neural tube defects occur when the neural plate fails to fold in the first 3 to 4 weeks of gestation, causing death to the fetus or permanent damage to the spinal cord. Despite the success of folic acid-based preventive strategies, neural tube defects remain among the most common birth defects worldwide, and their prevalence varies from 5.3 per 10,000 live births in the United States (Williams and others, 2015) to more than 10 per 1,000 pregnancies in certain areas of China (Li and others, 2006). This variance likely reflects differing contributions from risk factors such as nutritional status, usage of folic acid supplementation and/or folic acid fortification of the food supply, genetic susceptibility, prevalence of obesity and diabetes, and the presence of chemicals in the environment.

Of particular concern is environmental exposure to arsenic, which has become a ubiquitous environmental pollutant over the past several decades and is ranked the number one toxic substance on the U.S. Agency for Toxic Substances and Disease Registry (ATDSR) Priority List of Hazardous Substances (Denny and others, 2013). Individual exposures to arsenic come from various sources including food, water, medicines, and occupational settings. The main source of broader arsenic exposures to human populations is contaminated groundwater, and affected countries include Bangladesh, India, China, Taiwan, Chile, and the United States, among others.

Animal studies have consistently shown arsenic to be a potent teratogen, inducing neural tube defects in several animal models, including mouse (Hill and others, 2008; Hood and Bishop, 1972; Wlodarczyk and others, 1996), rat (Beaudoin, 1974), hamster (Carpenter, 1987), and chick (Peterkova and Puzanova, 1976). Arsenic crosses the placenta and preferentially accumulates in the neuroepithelium of developing hamster, mouse, and monkey embryos (Hanlon and Ferm, 1977). The potential mechanisms of arsenic-induced toxicity in animal models include direct toxicity from reactive oxygen species to the developing neural plate (Han and others, 2011), as well as disruption of glucose metabolism, which subsequently leads to activation of pathways that cause programmed cell death and failure of neural tube closure (Hill and others, 2009; Yang and others, 2013). In mice embryos, arsenic affected the expression of genes association with formation of neural tube defects (Robinson and others, 2011; Wlodarczyk and others, 2006). In addition, animal studies suggest that folate deficiency may exacerbate arsenic toxicity. Mice with specific defects in folate transport had higher rates of neural tube defects after in utero arsenic exposure than wild-type mice similarly exposed (Wlodarczyk and others, 2014). Similarly, mice nullizygous for genes encoding proteins involved in cellular uptake of folate were more susceptible to arsenic-induced neural tube defects (Spiegelstein and others, 2005). However, neither folic acid nor folinic acid supplementation prevented arsenic-induced neural tube defects in Splotch embryos (Gefrides and others, 2002).

Though animal studies generally provide strong support for arsenic's teratogenicity, no study in humans has definitively demonstrated a direct association between arsenic exposure and neural tube defects. In fact, between 1996 and 2001, systematic reviews of the epidemiological literature concluded that arsenic was unlikely to pose a risk to pregnant women or their offspring at levels environmentally relevant to most settings (DeSesso, 2001; DeSesso and others, 1998; Shalat and others, 1996).

Nevertheless, in more recent years an increasing number of reports have suggested that prenatal and early childhood exposures to arsenic may have neurodevelopmental effects on young children (Calderon and others, 2001; Hamadani and others, 2010; Hamadani and others, 2011; Parvez and others, 2011; Rodrigues and others, 2016; von Ehrenstein and others, 2007; Wasserman and others, 2007; Wasserman and others, 2004). This accumulating evidence of arsenic's neurodevelopmental toxicity underlies our interest in understanding whether arsenic contributes specifically to neural tube defect risk, particularly in areas of the world experiencing extremely high levels of arsenic exposure, and leads us to question the mechanisms that might explain these toxic effects.

Here we investigate the hypothesis that arsenic contributes to neural tube defect risk by reviewing two possible pathways that may link arsenic and neural tube defects (Figure 1): 1) arsenic's interaction with folate including folate-mediated epigenetic effects, and 2) arsenic's associations with diabetes, including arsenic's degradation of the cystic fibrosis transmembrane regulator (CFTR), a chloride channel that may provide a mechanism for arsenic-related diabetes. We also describe a new study that will investigate epidemic arsenic exposure and neural tube defect risk in Bangladesh, a country with extremely high levels of arsenic exposure through contaminated drinking water.

Figure 1.

Figure 1

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Conceptual model representing possible mechanisms linking arsenic and neural tube defects. Arrowed lines indicate that data are available from human studies.

Arsenic may affect neural tube defect risk through interactions with folate

Folate status and arsenic metabolism

Epidemiological studies provide evidence that folate status may influence arsenic metabolism. The strongest evidence supporting the role of folate in arsenic comes from a double-blind, placebo-controlled, folic acid supplementation trial conducted in 200 adults in rural Bangladesh. All participants, known to have low plasma folate concentrations at the beginning of the study, were randomly assigned to receive either folic acid supplementation or a placebo. At the end of the trial, the folic acid-supplemented group excreted significantly more urinary arsenic. In addition, blood levels of both total arsenic and the more toxic (and less methylated) species were significantly lower in the folic acid-supplemented group than in the control group (Gamble and others, 2006).

Given the historical links between folic acid and neural tube defects, folate pathway genes have been intensively studied. Positive associations have been reported between specific folate-related gene variants and neural tube defects in several case-control studies (Boyles and others, 2005; Etheredge and others, 2012). A recent search by our group in genome-wide association studies or candidate gene studies identified 14 single nucleotide polymorphisms (SNPs) that were associated with both neural tube defects and arsenic toxicity (Mazumdar and others, 2015d), and this list included genes involved in folate metabolism (MTHRF, MTHFD1, MTR, MTRR), folate transport (SLC19A1), DNA repair (XPD/ERCC2), DNA methylation (DNMT1, DNMT3A, DNMT3B) and arsenic metabolism (AS3MT) (Mazumdar and others, 2015d).

In addition to their similar roles in one-methyl metabolism, arsenic and folate have been shown to interact at the cell membrane through ATP-driven efflux pump families including the multidrug resistance protein (MRP). These protein channels play a role in controlling cellular folate levels. Arsenic induces these channels, causing folate efflux out of the cell, reducing the amount of available folate (Hooijberg and others, 2003). The mechanism is not yet tested in animals or humans, but if arsenic-induced folate efflux were to occur in the developing nervous system, it is biologically plausible that neural tube defects would be more likely to ensue.

Arsenic, folate and DNA methylation

Accumulating evidence from animal studies suggests that DNA methylation, currently the best-understood epigenetic process, plays an important role in the etiology of neural tube defects. For example, mutations in genes that affect DNA methylation result in neural tube defects in mice (Copp and Greene, 2010; Greene and others, 2011; Harris and Juriloff, 2010). Additionally, inactivation of the DNA methyltransferase DNMT3B in mice disrupts DNA methylation and causes multiple birth defects, including neural tube defects (Dunlevy and others, 2006).

An epigenetic model of neural tube defects is further supported by the success of folic acid supplementation programs in the prevention of many cases. Folate is a crucial cofactor in one-carbon metabolism and has an important role in DNA synthesis and replication. In this paradigm, the beneficial effects of folate in neural tube defect prevention might also come from its role as an epigenetic modifier: folate is an essential methyl donor to DNA. Studies in animal models support this paradigm. In a mouse model of neural tube defects, maternal intake of folate prior to conception reverses the proliferation potential of embryonic neural crest stem cells via decreased expression (and increased methylation markers) in promoters of genes involved in neural development (Ichi and others, 2010).

Further, evidence from animal studies has shown that DNA methylation plays an important role in the etiology of neural tube defects directly. For example, mutations in genes that affect DNA methylation result in neural tube defects in mice (Copp and Greene, 2010; Greene and others, 2011; Harris and Juriloff, 2010). Additionally, inactivation of the DNA methyltransferase DNMT3B in mice disrupts DNA methylation and causes multiple birth defects, including neural tube defects (Dunlevy and others, 2006). Aberrant DNA methylation has been linked to neural tube defects in humans as well: among 48 stillborn neural tube defect case subjects in northern China and 49 control subjects who were aborted for nonmedical reasons, DNA methylation analysis from brain tissue showed significantly decreased methylation of genomic DNA and LINE-1 elements among cases compared to controls (Wang and others, 2010). Together these data suggest that our limited access to genomic DNA methylation patterns is a fundamental gap in our knowledge of this developmental disorder and may further reveal how folic acid supplementation leads to its prevention.

It is increasingly recognized that arsenic exposure causes changes in epigenetic gene regulation. The epigenetic effects of arsenic have been an area of intense research since reports in 1997 demonstrated that arsenic causes hypermethylation of the p53 gene (Mass and Wang, 1997). Several mechanisms are proposed to explain how arsenic leads to changes in DNA methylation. Central to these theories is the observation that arsenic inhibits the activity of DNA methyltransferase (DNMT) enzymes. Inhibition of DNMT activity by arsenic appears to cause whole-genome and gene-specific demethylation (Reichard and Puga, 2010) in humans, similar to what is seen in animal models. As discussed earlier, arsenic administration induces neural tube defects in several animal models, and recent studies in chick embryos suggest that arsenic's teratogenic effects involve genomic hypomethylation (Han and others, 2011).

Arsenic may increase neural tube defect risk through dysregulation of maternal glucose metabolism

Two well-established risk factors for neural tube defects are maternal gestational diabetes and prepregnancy obesity (Hendricks and others, 2001; Shaw and others, 2000; Shaw and others, 1996; Soler and others, 1976; Waller and others, 1994; Waller and others, 2007; Watkins and others, 2003; Watkins and others, 1996; Werler and others, 1996). Although mechanisms underlying these risks in humans remain unclear, there is evidence from experimental models that the alteration of glucose homeostasis and hyperglycemia cause excess generation of reactive oxygen species due to mitochondrial dysfunction (Zangen and others, 2002) and activation of programmed cell death, or apoptotic, signaling cascades (Yang and others, 2013).

Accumulating evidence has shown an increase risk of type 2 diabetes in general populations exposed to arsenic (Kuo and others, 2013; Maull and others, 2012; Pan and others, 2013). Insulin insufficiency and impairment in pancreatic islet β-cells is found combined with insulin resistance in T2DM, the most prevalent from of diabetes (Marchetti and others, 2012). In mouse models, low-level chronic exposure to arsenic produces damage to pancreatic β-cells, decreased insulin section and altered glucose homeostasis (Davila-Esqueda and others, 2011). Higher arsenic concentrations caused irreversible damage to β-cells (Liu and others, 2014), reducing the ability to produce insulin. There is evidence that pregnant women also experience increase risk of dysregulated glucose homeostasis with arsenic exposure. For example, maternal arsenic exposure as measured in hair and blood samples was associated with increased risk of impaired glucose tolerance during the second trimester in a cohort exposed to arsenic in Tar Creek, a hazardous waste site in Oklahoma (Ettinger and others, 2009).

Arsenic degradation of CFTR may provide a mechanistic link between arsenic and diabetes

The genetic disease, cystic fibrosis, may provide a model to understand the mechanisms underlying arsenic-related diabetes, and by extension, arsenic's effects on neural tube defect risk. Cystic fibrosis is an autosomal recessive disease most common in people of northern European ancestry and is caused by mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates transmembrane fluid transport in the pancreas, lung, and other organs. The exact cause of diabetes in cystic fibrosis remains elusive, although the destruction of the insulin-secreting pancreatic islets secondary to the obstruction of the pancreatic duct due to defective CFTR has long been considered the underlying cause (Blackman and others, 2009; Lohr and others, 1989). A recent study in cell culture reveals a role of CFTR in glucose-induced electrical activities and insulin secretion in β-cells (Guo and others, 2014), suggesting that CFTR dysfunction is directly involved in the pathogenesis of idiopathic (and not only cystic-fibrosis related) diabetes.

Recent studies in experimental models and in humans demonstrate that arsenic causes destruction or dysfunction of CFTR, suggesting there is a shared mechanism between arsenic toxicity and cystic fibrosis. Studies in killfish (Stanton and others, 2006) and in cell culture (Bomberger and others, 2012) show a dose-dependent decrease in CFTR concentrations after administration of. arsenic. Our recent study in adults (Mazumdar and others, 2015a) was the first in humans to demonstrate CFTR dysfunction, as measured by increased sweat chloride concentration, in the setting of high arsenic exposure. In this study, 100 participants were drawn from a case-control study of arsenic-related skin lesions that took place between 2001 and 2009 in an area of Bangladesh with high levels of arsenic-contaminated groundwater (Breton and others, 2006; Seow and others, 2012). Sweat conductivity was measured for all participants and sweat chloride concentrations were confirmed for participants whose sweat conductivity was elevated. Water and nail arsenic concentrations were higher for participants with abnormal sweat conductivity than for participants with normal/intermediate sweat conductivity, and none of the 11 participants with sweat chloride concentrations in the diagnostic range for cystic fibrosis had a genetic diagnosis of that disease (Mazumdar and others, 2015a). This study demonstrates that CFTR dysfunction is present in individuals exposed to arsenic and provides motivation for exploring whether CFTR degradation is a mechanism for arsenic-related diabetes. If this hypothesis is true, then mothers with glucose dysregulation due to arsenic exposure maybe at higher risk of having pregnancies affected by neural tube defect. In addition, sweat conductivity, an inexpensive and point-of-care biomarker obtained through a noninvasive procedure, may be an effective screening test for arsenic toxicity and possibly also neural tube defect risk.

Taken together, the literature suggests a role for arsenic in neural tube defects, even though no epidemiological study has yet demonstrated a main effect of arsenic. It is likely that further understanding will require consideration of contributing factors, such as folate deficiency, genetic susceptibility and concurrent risk factors for diabetes in order to understand arsenic's role in neural tube defects.

METHODS

A new case-control study in Bangladesh

Study aims, design and recruitment

We will take advantage of a natural, albeit tragic, occurrence in Bangladesh to study whether arsenic is associated with increased risk of neural tube defects. According to survey data from 2000 to 2010, an estimated 35 to 77 million people in Bangladesh have been exposed to arsenic in their drinking water in what has been described as the largest mass poisoning in history (Smith and others, 2000). The most recent survey of wells (2010) reported that 42% of the water samples tested had inorganic arsenic levels exceeding the World Health Organization (WHO) drinking water guideline of 10 μg/L, and 27% had levels that exceeded the Bangladesh guideline of 50 μg/L. In many areas, the arsenic concentrations in drinking water exceeded 1000 μg/L (Chakraborti and others, 2010).

In light of the gaps in our understanding described above, we initiated a large epidemiologic study in 2016 to address the role of environmental arsenic exposure in Bangladesh in neural tube defect risk. This study appears to be the first large-scale epidemiologic study focusing primarily on arsenic exposure and neural tube defect risk, as well as on arsenic's possible effects through maternal folate and glucose metabolism. It uses the case-control design, which provides the most efficient sampling for studies of conditions that are rare or of multifactorial etiology (Hertz-Picciotto and others, 2006).

The study population is sampled from children with myelomeningocele who present to medical facilities in Bangladesh for surgical closure of the defect. All participating children will meet the following criteria: a) below 12 months of age, b) physician-confirmed diagnosis of myelomeningocele, c) mother is available for interviews and d) primary drinking water source of the mother during early pregnancy is known. No further exclusions are made based on genetics, geography, or family history.

Myelomeningocele was the neural tube defect identified most often and most reliably in our pilot studies (Mazumdar and others, 2015b; Mazumdar and others, 2015c) and therefore is the focus of this current study. There is currently no surveillance system in Bangladesh to identify all pregnancies affected by neural tube defect; a surveillance system following all pregnancies might be able to identify a more severe phenotype, such as those who die during gestation or are otherwise undiscovered. We focus on myelomeningocele also because it is increasingly recognized that neural tube defects are not one disorder, but instead a wide array of morphologically distinct malformations (Wallingford and others, 2013). We focus on this one subtype of neural tube defect with the recognition that different contributions of various risk factors may be important among subtypes of neural tube defects.

Control selection

As case ascertainment will be primarily hospital based, potential controls will be drawn from the referring hospital's census records, and matched to cases according to age. Controls will be those with diagnoses believed to be independent of arsenic exposure yet also presented to a neurosurgeon (e.g. trauma, craniosynostosis). To further prevent overmatching on exposure, controls will not necessarily be chosen from the same geographic area as the case.

Data collection protocols

Participation involves assessments of medical co-morbidities and associated anomalies, family histories, a medical examination, biologic specimen collection from child and parents, and environmental specimen collection, specifically drinking water from the well used by the mother in very early pregnancy. Other components include maternal and child medical records review and abstractions.

Community partnerships

A community advisory council will be formed early in the development of this project to maximize participation by parents, clinicians, advocacy organizations and research staff. Feedback from community members has already included plans for campaigns to raise awareness of the benefits of folic acid supplementation and folic acid fortification of foods, suggestions regarding the collection of specimens and information from siblings, and the formation of multidisciplinary spina bifida clinical services in participating hospitals. A scientific advisory board has also been assembled to guide study implementation and interpretation of results.

DISCUSSION

Conclusion

This research project builds on strong preliminary data that suggest arsenic exposure is an important risk factor for neural tube defects. This project will utilize a case-control study design to enroll infants with myelomeningocele to investigate how exposure may contribute to increased risk. Specifically, this study will investigate arsenic's known effects on maternal folate and glucose metabolism. The results of this study may help Bangladesh, a country experiencing an unprecedented epidemic of arsenic poisoning through contaminated drinking water, set policies for neural tube defect surveillance and folic acid supplementation and fortification in high arsenic areas.

Acknowledgments

Grant information and grant numbers: Funding for this study was provided by the National Institutes of Environmental Health Sciences (NIEHS), National Institutes of Health (R01 ES016317). Additional support was provided by the Harvard T.H. Chan School of Public Health NIEHS Center (P30 ES000002) and the Boston Children's Hospital Intellectual and Developmental Disabilities Research Center (P30 HD18655).

Footnotes

Competing Financial Interests Declaration: The author reports that she has no actual and/or potential competing financial interests.

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