Arsenic exposure and human blood DNA methylation and hydroxymethylation profiles in two diverse populations from Bangladesh and Spain.

BACKGROUND: Associations of arsenic (As) with the sum of 5-mC and 5-hmC levels have been reported; however, As exposure-related differences of the separated 5-mC and 5-hmC markers have rarely been studied. METHODS: In this study, we evaluated the association of arsenic exposure biomarkers and 5-mC and 5-hmC in 30 healthy men (43-55 years) from the Aragon Workers Health Study (AWHS) (Spain) and 31 healthy men (31-50 years) from the Folic Acid and Creatinine Trial (FACT) (Bangladesh). We conducted 5-mC and 5-hmC profiling using Infinium MethylationEPIC arrays, on paired standard and modified (ox-BS in AWHS and TAB in FACT) bisulfite converted blood DNA samples. RESULTS: The median for the sum of urine inorganic and methylated As species (ΣAs) (µg/L) was 12.5 for AWHS and 89.6 for FACT. The median of blood As (µg/L) was 8.8 for AWHS and 10.2 for FACT. At a statistical significance p-value cut-off of 0.01, the differentially methylated (DMP) and hydroxymethylated (DHP) positions were mostly located in different genomic sites. Several DMPs and DHPs were consistently found in AWHS and FACT both for urine ΣAs and blood models, being of special interest those attributed to the DIP2C gene. Three DMPs (annotated to CLEC12A) for AWHS and one DHP (annotated to NPLOC4) for FACT remained statistically significant after false discovery rate (FDR) correction. Pathways related to chronic diseases including cardiovascular, cancer and neurological were enriched. CONCLUSIONS: While we identified common 5-hmC and 5-mC signatures in two populations exposed to varying levels of inorganic As, differences in As-related epigenetic sites across the study populations may additionally reflect low and high As-specific associations. This work contributes a deeper understanding of potential epigenetic dysregulations of As. However, further research is needed to confirm biological consequences associated with DIP2C epigenetic regulation and to investigate the role of 5-hmC and 5-mC separately in As-induced health disorders at different exposure levels.

Retinal ganglion cell topography and spatial resolving power of eye in the native chicken of Bangladesh / Topografía de células ganglionares de la retina y poder de resolución espacial del ojo en el pollo nativo de Bangladesh

SUMMARY: The distribution of retinal ganglion cells (RGCs) was observed in the retinal wholemount of native chicken (Gallus gallus domestricus) of Bangladesh by using light microscopy. We considered five different anatomic regions (central, nasal, temporal, dorsal, and ventral) of Nissl stained wholemount, and the RGCs were counted, plotted, and measured accordingly. The average area of the retina was 431.75 mm2 while the total number of ganglion cells was 2124431 on average. Only the central area of the retina was the peak density (10400 cells/mm2) area, signifying the acute visual area, whilst the maximum spatial resolving power was 11 cycles/degree. The overall concentration of RGCs gradually declined towards the periphery but the size of cells generally decreased towards centrally. The size of ganglion cell was not uniform (12 to 180 µm2), specifically the central retina, just above the optic disc was packed with tiny-sized cells. The number, topographic distribution, and size of RGCs in native chicken signified their domesticated or terrestrial characters, uneven visual acuteness, and possibly only the central retina was the area for fine vision as the function of RGCs.

RESUMEN: En este studio se observó la distribución de las células ganglionares en la retina (CGR) de pollo nativo (Gallus gallus domesticus) de Bangladesh mediante el uso de microscopía óptica. Consideramos cinco regiones anatómicas diferentes (central, nasal, temporal, dorsal y ventral). Las muestras de CGR se tiñeron con Nissl, posteriormente, se midieron y contó el número de células totales. El área promedio de la retina fue de 431,75 mm2, mientras que el promedio del número total de células ganglionares fue de 2124431. El área central de la retina fue el área de densidad máxima (10400 células / mm2), señalando el área visual aguda, mientras que el poder de resolución espacial máximo fue de 11 ciclos / grado. La concentración general de CGR disminuyó gradualmente hacia la periferia, sin embargo, el tamaño de las células disminuyó hacia el centro. El tamaño de las CGR no fue uniforme (12 a 180 mm2), específicamente en la retina central, por encima del disco óptico, aumentaron significativamente las células pequeñas. El número, la distribución topográfica y el tamaño de las CGR en pollos nativos determinaron las características domésticas o terrestres, agudeza visual desigual y, posiblemente, la función de las CGR, en la retina central era el área de visión fina.

Anatomical features of some bones of the forelimbs of lions (Panthera leo) / Características anatómicas de algunos huesos de los miembros torácicos de leones (Panthera leo)

SUMMARY: We studied the bones of forelimb of four adult lions (Panthera leo) of both sexes to record the gross anatomical and morphometrical features of the scapula, humerus, radius and ulna. We observed some unique anatomical features that will be helpful for radiographic interpretation and forensic investigations. The lateral surface of scapula was unequally divided into supraspinous (fossa supraspinata) and infraspinous fossa (fossa infraspinata) by a well developed spine (spina scapulae). The acromion process was subdivided into suprahamate process (processus suprahamatus)and hamate process (processus hamatus); the later one was over hanged the glenoid cavity (cavitas glenoidalis), but the supraglenoid tubercle (tuberculum supraglenoidalis) was absent. The shaft (diaphysis) of humerus was compressed craniocaudally in proximal part, rounded to oval in middle part and compressed mediolaterally in distal part. A long, narrow supracondyloid foramen was found at distal limb just above the medial epicondyle (epicondylus medialis) which didn't connect the radial fossa (fossa radialis) with the olecranon fossa (fossa olecrani). The radius and ulna were twin bones where radius was articulated craniolateral to the ulna proximally and craniomedial to the ulna distally. However, the ulna was the longest bone in the forelimb of lion. The olecranon tuberosity of this bone had three prominences - two were cranially, whereas the caudal one was the largest and rounded. Distally projected styloid processes (processus styloideus) were found in the distal limb of both radius and ulna.

RESUMEN: Estudiamos los huesos de las miembros torácicos de cuatro leones adultos (Panthera leo) de ambos sexos para registrar las características anatómicas y morfométricas macroscópicas de la escápula, el húmero, el radio y la ulna. Se observaron algunas características anatómicas únicas que serán útiles para la interpretación radiográfica y las investigaciones forenses. La superficie lateral de la escápula se dividió de manera desigual en fosa supraespinosa y fosa infraspinosa por una columna bien desarrollada (espina de la escápula). El proceso del acromion se subdividió en proceso suprahamato (processus suprahamatus) y proceso hamato (processus hamatus); el tubérculo supraglenoideo (tuberculum supraglenoidalis) estaba ausente. La diáfisis (diafisis) del húmero estaba comprimida craneocaudalmente en la parte proximal, redondeada a ovalada en la parte media y comprimida mediolateralmente en la parte distal. Se encontró un foramen supracondileo largo y estrecho en la extremidad distal, por encima del epicóndilo medial (epicondylus medilaris) que no conectaba la fosa radial (fosa radial) con la fosa olecraneana (fossa olecrani). El radio y la ulna eran huesos idénticos en los que el radio se articulaba craneolateral a la ulna proximalmente, y craneomedial a la ulna distalmente. Sin embargo, la ulna era el hueso más largo del miembro torácico del león. La tuberosidad del olécranon de este hueso tenía tres prominencias: dos eran craneales, mientras que la caudal era la más grande y redondeada. Se encontraron procesos estiloides proyectados distalmente (processus styloideus) en la extremidades distales del radio y la ulna.

Association between body mass index and arsenic methylation in three studies of Bangladeshi adults and adolescents.

BACKGROUND: Water-borne arsenic (As) exposure is a global health problem. Once ingested, inorganic As (iAs) is methylated to mono-methyl (MMA) and dimethyl (DMA) arsenicals via one-carbon metabolism (OCM). People with higher relative percentage of MMA (MMA%) in urine (inefficient As methylation), have been shown to have a higher risk of cardiovascular disease and several cancers but appear to have a lower risk of diabetes and obesity in populations from the US, Mexico, and Taiwan. It is unknown if this opposite pattern with obesity is present in Bangladesh, a country with lower adiposity and higher As exposure in drinking water. OBJECTIVE: To characterize the association between body mass index (BMI) and As methylation in Bangladeshi adults and adolescents participating in the Folic Acid and Creatine Trial (FACT); Folate and Oxidative Stress (FOX) study; and Metals, Arsenic, and Nutrition in Adolescents Study (MANAS). METHODS: Arsenic species (iAs, MMA, DMA) were measured in urine and blood. Height and weight were measured to calculate BMI. The associations between concurrent BMI with urine and blood As species were analyzed using linear regression models, adjusting for nutrients involved in OCM such as choline. In FACT, we also evaluated the prospective association between weight change and As species. RESULTS: Mean BMIs were 19.2/20.4, 19.8/21.0, and 17.7/18.7 kg/m2 in males/females in FACT, FOX, and MANAS, respectively. BMI was associated with As species in female but not in male participants. In females, after adjustment for total urine As, age, and plasma folate, the adjusted mean differences (95% confidence) in urinary MMA% and DMA% for a 5 kg/m2 difference in BMI were -1.21 (-1.96, -0.45) and 2.47 (1.13, 3.81), respectively in FACT, -0.66 (-1.56, 0.25) and 1.43 (-0.23, 3.09) in FOX, and -0.59 (-1.19, 0.02) and 1.58 (-0.15, 3.30) in MANAS. The associations were attenuated after adjustment for choline. Similar associations were observed with blood As species. In FACT, a 1-kg of weight increase over 2 to 10 (mean 5.4) years in males/females was prospectively associated with mean DMA% that was 0.16%/0.19% higher. DISCUSSION: BMI was negatively associated with MMA% and positively associated with %DMA in females but not males in Bangladesh; associations were attenuated after plasma choline adjustment. These findings may be related to the role of body fat on estrogen levels that can influence one-carbon metabolism, e.g. by increasing choline synthesis. Research is needed to determine whether the associations between BMI and As species are causal and their influence on As-related health outcomes.

Betaine and choline status modify the effects of folic acid and creatine supplementation on arsenic methylation in a randomized controlled trial of Bangladeshi adults.

PURPOSE: Methylation of ingested inorganic arsenic (InAs) to monomethyl- (MMAs) and dimethyl-arsenical species (DMAs) facilitates urinary arsenic elimination. Folate and creatine supplementation influenced arsenic methylation in a randomized controlled trial. Here, we examine if baseline status of one-carbon metabolism nutrients (folate, choline, betaine, and vitamin B12) modified the effects of FA and creatine supplementation on changes in homocysteine, guanidinoacetate (GAA), total blood arsenic, and urinary arsenic metabolite proportions and indices. METHODS: Study participants (N = 622) received 400 or 800 µg FA, 3 g creatine, 400 µg FA + 3 g creatine, or placebo daily for 12 weeks. RESULTS: Relative to placebo, FA supplementation was associated with greater mean increases in %DMAs among participants with betaine concentrations below the median than those with levels above the median (FDR < 0.05). 400 µg FA/day was associated with a greater decrease in homocysteine among participants with plasma folate concentrations below, compared with those above, the median (FDR < 0.03). Creatine treatment was associated with a significant decrease in %MMAs among participants with choline concentrations below the median (P = 0.04), but not among participants above the median (P = 0.94); this effect did not significantly differ between strata (P = 0.10). CONCLUSIONS: Effects of FA and creatine supplementation on arsenic methylation capacity were greater among individuals with low betaine and choline status, respectively. The efficacy of FA and creatine interventions to facilitate arsenic methylation may be modified by choline and betaine nutritional status. CLINICAL TRIAL REGISTRATION: Clinical Trial Registry Identifier: NCT01050556, U.S. National Library of Medicine, https://clinicaltrials.gov ; registered January 15, 2010.

Association of Arsenic Exposure with Whole Blood DNA Methylation: An Epigenome-Wide Study of Bangladeshi Adults.

BACKGROUND: Arsenic exposure affects [Formula: see text] people worldwide, including [Formula: see text] in Bangladesh. Arsenic exposure increases the risk of cancer and other chronic diseases, and one potential mechanism of arsenic toxicity is epigenetic dysregulation. OBJECTIVE: We assessed associations between arsenic exposure and genome-wide DNA methylation measured at baseline among 396 Bangladeshi adults participating in the Health Effects of Arsenic Longitudinal Study (HEALS) who were exposed by drinking naturally contaminated well water. METHODS: Methylation in whole blood DNA was measured at [Formula: see text] using the Illumina InfiniumMethylationEPIC (EPIC) array. To assess associations between arsenic exposure and CpG methylation, we used linear regression models adjusted for covariates and surrogate variables (SVs) (capturing unknown technical and biologic factors). We attempted replication and conducted a meta-analysis using an independent dataset of [Formula: see text] from 400 Bangladeshi individuals with arsenical skin lesions. RESULTS: We identified 34 CpGs associated with [Formula: see text] creatinine-adjusted urinary arsenic [[Formula: see text]]. Sixteen of these CpGs annotated to the [Formula: see text] array, and 10 associations were replicated ([Formula: see text]). The top two CpGs annotated upstream of the ABR gene (cg01912040, cg10003262 ). All urinary arsenic-associated CpGs were also associated with arsenic concentration measured in drinking water ([Formula: see text]). Meta-analysis ([Formula: see text] samples) identified 221 urinary arsenic-associated CpGs ([Formula: see text]). The arsenic-associated CpGs from the meta-analysis were enriched in non-CpG islands and shores ([Formula: see text]) and depleted in promoter regions ([Formula: see text]). Among the arsenic-associated CpGs ([Formula: see text]), we observed significant enrichment of genes annotating to the reactive oxygen species pathway, inflammatory response, and tumor necrosis factor [Formula: see text] ([Formula: see text]) signaling via nuclear factor kappa-B ([Formula: see text]) hallmarks ([Formula: see text]). CONCLUSIONS: The novel and replicable associations between arsenic exposure and DNA methylation at specific CpGs observed in this work suggest that epigenetic alterations should be further investigated as potential mediators in arsenic toxicity and as biomarkers of exposure and effect in exposed populations. https://doi.org/10.1289/EHP3849.

A missense variant in FTCD is associated with arsenic metabolism and toxicity phenotypes in Bangladesh.

Inorganic arsenic (iAs) is a carcinogen, and exposure to iAs via food and water is a global public health problem. iAs-contaminated drinking water alone affects >100 million people worldwide, including ~50 million in Bangladesh. Once absorbed into the blood stream, most iAs is converted to mono-methylated (MMA) and then di-methylated (DMA) forms, facilitating excretion in urine. Arsenic metabolism efficiency varies among individuals, in part due to genetic variation near AS3MT (arsenite methyltransferase; 10q24.32). To identify additional arsenic metabolism loci, we measured protein-coding variants across the human exome for 1,660 Bangladeshi individuals participating in the Health Effects of Arsenic Longitudinal Study (HEALS). Among the 19,992 coding variants analyzed exome-wide, the minor allele (A) of rs61735836 (p.Val101Met) in exon 3 of FTCD (formiminotransferase cyclodeaminase) was associated with increased urinary iAs% (P = 8x10-13), increased MMA% (P = 2x10-16) and decreased DMA% (P = 6x10-23). Among 2,401 individuals with arsenic-induced skin lesions (an indicator of arsenic toxicity and cancer risk) and 2,472 controls, carrying the low-efficiency A allele (frequency = 7%) was associated with increased skin lesion risk (odds ratio = 1.35; P = 1x10-5). rs61735836 is in weak linkage disequilibrium with all nearby variants. The high-efficiency/major allele (G/Valine) is human-specific and eliminates a start codon at the first 5´-proximal Kozak sequence in FTCD, suggesting selection against an alternative translation start site. FTCD is critical for catabolism of histidine, a process that generates one-carbon units that can enter the one-carbon/folate cycle, which provides methyl groups for arsenic metabolism. In our study population, FTCD and AS3MT SNPs together explain ~10% of the variation in DMA% and support a causal effect of arsenic metabolism efficiency on arsenic toxicity (i.e., skin lesions). In summary, this work identifies a coding variant in FTCD associated with arsenic metabolism efficiency, providing new evidence supporting the established link between one-carbon/folate metabolism and arsenic toxicity.

The association between telomere length and mortality in Bangladesh.

Telomeres are tandem repeat sequences at the end of chromosomes that bind proteins to protect chromosome ends. Telomeres shorten with age, and shorter leukocyte telomere length (TL) has been associated with overall mortality in numerous studies. However, this association has not been tested in populations outside of Europe and the U.S. We assessed the association between TL and subsequent mortality using data on 744 mortality cases and 761 age-/sex-matched controls sampled from >27,000 participants from three longitudinal Bangladeshi cohorts: Health Effects of Arsenic Longitudinal Study (HEALS), HEALS Expansion (HEALS-E), and Bangladesh Vitamin E and Selenium Trial (BEST). We used conditional logistic regression to estimate odds ratios (ORs) for the association between a standardized TL variable and overall mortality, as well as mortality from chronic diseases, respiratory diseases, circulatory diseases, and cancer. In HEALS and BEST, we observed an association between shorter TL and increased overall mortality (P=0.03 and P=0.03), mortality from chronic disease (P=0.01 and P=0.03) and mortality from circulatory disease (P=0.03 and P=0.04). Results from pooled analyses of all cohorts were consistent with HEALS and BEST. This is the first study demonstrating an association between short TL and increased mortality in a population of non-European ancestry.

Sex-Specific Associations between One-Carbon Metabolism Indices and Posttranslational Histone Modifications in Arsenic-Exposed Bangladeshi Adults.

BACKGROUND: Posttranslational histone modifications (PTHMs) are altered by arsenic, an environmental carcinogen. PTHMs are also influenced by nutritional methyl donors involved in one-carbon metabolism (OCM), which may protect against epigenetic dysregulation. METHODS: We measured global levels of three PTHMs, which are dysregulated in cancers (H3K36me2, H3K36me3, H3K79me2), in peripheral blood mononuclear cells (PBMC) from 324 participants enrolled in the Folic Acid and Creatine Trial, a randomized trial in arsenic-exposed Bangladeshi adults. Sex-specific associations between several blood OCM indices (folate, vitamin B12, choline, betaine, homocysteine) and PTHMs were examined at baseline using regression models, adjusted for multiple tests by controlling for the false discovery rate (PFDR). We also evaluated the effects of folic acid supplementation (400 µg/d for 12 weeks), compared with placebo, on PTHMs. RESULTS: Associations between choline and H3K36me2 and between vitamin B12 and H3K79me2 differed significantly by sex (Pdiff < 0.01 and <0.05, respectively). Among men, plasma choline was positively associated with H3K36me2 (PFDR < 0.05), and among women, plasma vitamin B12 was positively associated with H3K79me2 (PFDR < 0.01). Folic acid supplementation did not alter any of the PTHMs examined (PFDR = 0.80). CONCLUSIONS: OCM indices may influence PTHMs in a sex-dependent manner, and folic acid supplementation, at this dose and duration, does not alter PTHMs in PBMCs. IMPACT: This is the first study to examine the influences of OCM indices on PTHMs in a population that may have increased susceptibility to cancer development due to widespread exposure to arsenic-contaminated drinking water and a high prevalence of hyperhomocysteinemia. Cancer Epidemiol Biomarkers Prev; 26(2); 261-9. ©2016 AACR.

Supplementation with Folic Acid, but Not Creatine, Increases Plasma Betaine, Decreases Plasma Dimethylglycine, and Prevents a Decrease in Plasma Choline in Arsenic-Exposed Bangladeshi Adults.

BACKGROUND: Folic acid (FA) supplementation facilitates urinary excretion of arsenic, a human carcinogen. A better understanding of interactions between one-carbon metabolism intermediates may improve the ability to design nutrition interventions that further facilitate arsenic excretion. OBJECTIVE: The objective was to determine if FA and/or creatine supplementation increase choline and betaine and decrease dimethylglycine (DMG). METHODS: We conducted a secondary analysis of the Folic Acid and Creatine Trial, a randomized trial in arsenic-exposed Bangladeshi adults (n = 605, aged 24-55 y, 50.3% male) who received arsenic-removal water filters. We examined treatment effects of FA and/or creatine supplementation on plasma choline, betaine, and DMG concentrations, measured by LC-tandem mass spectrometry at baseline and at week 12. Group comparisons were between 1) 400 and 800 µg FA/d (FA400 and FA800, respectively) compared with placebo, 2) creatine (3 g/d) compared with placebo, and 3) creatine plus FA400 compared with FA400. RESULTS: Choline decreased in the placebo group (-6.6%; 95% CI: -10.2%, -2.9%) but did not change in the FA groups (FA400: 2.5%; 95% CI: -0.9%, 6.1%; FA800: 1.4%; 95% CI: -2.5%, 5.5%; P < 0.05). Betaine did not change in the placebo group (-3.5%; 95% CI: -9.3%, 2.6%) but increased in the FA groups (FA400: 14.1%; 95% CI: 9.4%, 19.0%; FA800: 13.0%; 95% CI: 7.2%, 19.1%; P < 0.01). The decrease in DMG was greater in the FA groups (FA400: -26.7%; 95% CI: -30.9%, -22.2%; FA800: -27.8%; 95% CI: -31.8%, -23.4%) than in the placebo group (-12.3%; 95% CI: -18.1%, -6.2%; P < 0.01). The percentage change in choline, betaine, and DMG did not differ between creatine treatment arms and their respective reference groups. CONCLUSION: Supplementation for 12 wk with FA, but not creatine, increases plasma betaine, decreases plasma DMG, and prevents a decrease in plasma choline in arsenic-exposed Bangladeshi adults. This trial was registered at clinicaltrials.gov as NCT01050556.

Low-Dose Creatine Supplementation Lowers Plasma Guanidinoacetate, but Not Plasma Homocysteine, in a Double-Blind, Randomized, Placebo-Controlled Trial.

BACKGROUND: Creatine synthesis from guanidinoacetate consumes ~50% of s-adenosylmethionine (SAM)-derived methyl groups, accounting for an equivalent proportion of s-adenosylhomocysteine (SAH) and total homocysteine (tHcys) synthesis. Dietary creatine inhibits the synthesis of guanidinoacetate, thereby lowering plasma tHcys in rats. OBJECTIVE: We tested the hypotheses that creatine supplementation lowers plasma guanidinoacetate, increases blood SAM, lowers blood SAH, and lowers plasma tHcys. METHODS: Bangladeshi adults were randomly assigned to receive 1 of 4 treatments for 12 wk: placebo (n = 101), 3 g/d creatine (Cr; n = 101), 400 µg/d folic acid (FA; n = 153), or 3 g/d creatine plus 400 µg/d folic acid (Cr+FA; n = 103). The outcomes of plasma guanidinoacetate and tHcys, as well as whole blood SAM and SAH, were analyzed at baseline and week 12 by HPLC. Treatment effects of creatine supplementation were examined with the use of the group comparisons of Cr vs. placebo and Cr+FA vs. FA. RESULTS: Plasma guanidinoacetate declined by 10.6% (95% CI: 4.9, 15.9) in the Cr group while increasing nonsignificantly in the placebo group (3.7%; 95% CI: -0.8, 8.5) (Pgroup difference = 0.0002). Similarly, plasma guanidinoacetate declined by 9.0% (95% CI: 3.4, 14.2) in the Cr+FA group while increasing in the FA group (7.0%; 95% CI: 2.0, 12.2) (Pgroup difference < 0.0001). Plasma tHcys declined by 23.4% (95% CI: 19.5, 27.1) and 21.0% (95% CI: 16.4, 25.2) in the FA and Cr+FA groups, respectively (Pgroup difference = 0.41), with no significant changes in the placebo or Cr groups (Pgroup difference = 0.35). A decrease in guanidinoacetate over time was associated with a decrease in tHcys over time in the Cr+FA group (ß = 0.30; 95% CI: 0.17, 0.43; P < 0.0001). CONCLUSIONS: Our findings indicate that whereas creatine supplementation downregulates endogenous creatine synthesis, this may not on average lower plasma tHcys in humans. However, tHcys did decrease in those participants who experienced a decline in plasma guanidinoacetate while receiving creatine plus folic acid supplementation. This trial was registered at clinicaltrials.gov as NCT01050556.