Title: Nutrition, Arsenic, Metals, and Cognitive Function in Adolescents
Description: Background: Environmental exposure to inorganic arsenic (InAs) is a considerable worldwide problem, and over 57 million people in Bangladesh have been chronically exposed to arsenic-contaminated drinking water. Ingested inorganic arsenic (InAs) undergoes hepatic methylation generating monomethyl- (MMAs) and dimethyl- (DMAs) arsenic species in a process that facilitates urinary As (uAs) elimination. Of these three metabolites (InAs, MMA and DMA), MMA the most toxic, InAs is the second most toxic, and DMA is the least toxic. Consequently, increased MMAs is associated with increased risk of As-related adverse health outcomes. One-carbon metabolism (OCM), the biochemical pathway that provides methyl groups for As methylation, is influenced by folate and B12. A growing body of research, including cell-culture, animal-model, and epidemiological studies, have demonstrated the role of OCM-related micronutrients in As methylation. While folate supplementation is known to increase As methylation and lowers blood As (bAs) in adults, little data is available for adolescents.

OCM also supports nucleotide and amino acid synthesis, particularly during periods of rapid growth, such as adolescence. In Bangladesh, deficiencies in folate and vitamin B12 are widespread. These micronutrients are essential for OCM and As methylation, and they are also critical for neural development, since they are necessary for the synthesis of neurotransmitters and myelin, and critical for generation of S-adenosyl-methionine (SAM) via OCM. Deficiencies in folate and B12 have been shown to negatively influence cognitive function in adults and children, but this has not been adequately characterized in adolescents. Individuals living in Bangladesh, facing As exposure and nutritional deficiency, also experience environmental exposure to elevated levels of cadmium (Cd), manganese (Mn), and lead (Pb). These metals have been linked to adverse neurocognitive outcomes in adults and children, though their effects on adolescents are not yet fully characterized. Additionally, previous studies have linked selenium (Se) levels to protective effects against toxicity of these other metals, as Se is an essential nutrient.

Metal mixtures are also understudied in adolescents, and more research is needed. Objectives: Firstly, the research presented in this dissertation will examine the previously published evidence that nutritional status and nutritional interventions can influence the metabolism and toxicity of As, with a primary focus on folate. Secondly, the associations between OCM-related micronutrients and As methylation in Bangladeshi adolescents chronically exposed to As-contaminated drinking water will be studied. Thirdly, this dissertation will investigate the associations between folate and B12 nutritional status, homocysteine, and cognitive function as measured by two different test instruments in Bangladeshi adolescents. Finally, the associations between mixed metals exposure and cognitive function in Bangladeshi adolescents will be examined. Methods: The Metals, Arsenic, & Nutrition in Adolescents study (MANAs) is a cross-sectional study of 738 Bangladeshi adolescents aged 14-16 years, whose parents were enrolled in our group’s previous Health Effects of Arsenic Longitudinal Study (HEALS).

Venous blood samples were collected from adolescent participants for measurement of plasma folate, red blood cell (RBC) folate, plasma B12, plasma homocysteine (Hcys), blood As (bAs), blood cadmium (bCd), blood manganese (bMn), blood lead (bPb), and blood selenium (bSe). Urine samples were collected for measurement of urinary arsenic and urinary arsenic metabolites (InAs, MMA, and DMA) expressed as a percentage of total urinary As: %InAs, %MMAs, %DMAs. Additionally, participants completed a modified version of the Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV) and an abbreviated version of the Cambridge Neuropsychological Test Automated Battery (CANTAB). Associations between predictors and outcomes were assessed using linear regression analyses. Associations for the mixture of metals were also examined via Bayesian Kernel Machine Regression (BKMR), which assessed the effects of the metals mixture in addition to examining the effects each individual metal component. Results: In the linear regression analyses examining associations between nutritional status and arsenic methylation profiles controlling for water As and BMI, in girls we observed that RBC folate was inversely associated with bAs, plasma B12 was inversely associated with uAs, and, somewhat unexpectedly, plasma Hcys was inversely associated with %MMA.

Among boys, we saw that plasma folate was inversely associated with %InAs and positively associated with %DMA, RBC folate was inversely associated with %InAs and positively associated with %MMA, while Hcys was positively associated with %InAs. In linear regression analyses examining the associations between nutritional status and cognitive function controlling for covariates, we found no significant associations between nutritional status and cognitive function as measured by WISC. For cognitive function as measured by CANTAB, we observed positive associations between plasma folate and spatial recognition memory, and between plasma B12 and spatial working memory. In our investigation of the associations between metal exposures and cognitive outcomes, linear regression analysis revealed negative associations between exposure to As and Mn and spatial working memory. Negative associations were also seen between bCd and Spatial Recognition Memory, and between bPb and Delayed Match to Sample.

Finally, a positive association was seen between bSe and Spatial Span Length. Our BKMR results showed no overall effect of the mixture but further characterized the associations for individual metals within the mixture. BKMR analyses indicate that bPb has a negative association to Delayed Match to Sample, and that there are positive associations between bSe and Planning, Reaction Time, and Spatial Span. BKMR also showed higher concentrations of bCd to be negatively associated with Spatial Recognition Memory. Posterior inclusion probability consistently rated Se, which has a protective effect, as the most influential component of the mixture. Conclusions: These findings suggest that associations between OCM nutritional status, blood arsenic, and distribution of urinary As metabolites in adolescents are similar to previously reported observations in adults and in children. The inverse association between Hcys and %MMA in girls is unexpected since Hcys is known to be an indicator of impaired OCM and low folate/B12 in adults.

These findings also suggest that associations between nutritional status and cognitive function in adolescents are consistent with previously reported associations in adults and children. The observation of positive associations between folate and spatial recognition memory and between B12 and spatial working memory are consistent with findings in animal models, children, and adults, all of which link deficiencies in these two micronutrients to memory deficits. The findings of the metals investigation are also consistent with previously reported observations in adults and children, and these results also suggest agreement between linear regression and BKMR analysis of the mixed metal exposure, with the BKMR further demonstrating associations seen in the linear regression analysis. Generally, Se had a protective effect for cognitive outcomes, whereas Mn and As were linked to poorer working memory, and Cd and Pb were linked to poorer visual recognition and memory. BKMR reinforced and further characterized results of the linear regression analyses.

Overall, these results indicate that the associations between OCM-related micronutrients, arsenic methylation, metal exposure, and cognitive function in adolescents are generally similar to prior findings in adults and children. However, additional studies are needed to evaluate the impact of OCM and As methylation on As-related adverse health outcomes (such as cancer and cardiovascular disease) in people exposed to As during adolescence. These results also suggest that further investigation into the associations between nutritional status and measures of cognitive function in adolescents is merited, and that further exploration of homocysteine’s role in adolescent physiology is needed. Most importantly, these findings suggest that both nutritional interventions involving B12 and folate supplementation and metal exposure mitigation efforts may have a positive impact on overall health and well-being for individuals facing these environmental exposures. In the long term, interventions to reduce exposure to metals and nutritional deficiency, and interventions to attenuate the effects of these exposures have the potential to influence lifelong cognitive function, possibly influencing professional achievement and economic outcomes in regions having a high prevalence of nutritional deficiencies, arsenic exposure, and environmental exposure to metals mixtures.