RESUMEN
The unique DNA packaging of spermatozoa renders them resistant to DNA isolation techniques used for somatic cells, requiring alternative methods that are slow and labor intensive. Here we present a rapid method for isolating high-quality sperm DNA. Isolated human sperm cells were homogenized with 0.2 mm steel beads for 5 min at room temperature in the presence of guanidine thiocyanate lysis buffer supplemented with 50 mM tris(2-carboxyethyl)phosphine (TCEP). Our method yielded >90% high-quality DNA using 3 different commercially available silica-based spin columns. DNA yields did not differ between immediate isolation (2.84 ± 0.04 pg/cell) and isolation after 2 weeks of homogenate storage at room temperature (2.91 ± 0.13 pg/cell). DNA methylation analyses revealed similar methylation levels at both time points for three imprinted loci. Our protocol has many advantages: it is conducted at room temperature; lengthy proteinase K (ProK) digestions are eliminated; the reducing agent, TCEP, is odorless and stable at room temperature; nucleic acids are stabilized, allowing storage of homogenate; and it is adaptable for other mammalian species. Taken together, the benefits of our improved method have important implications for settings where sample processing constraints exist.
Asunto(s)
ADN/aislamiento & purificación , Espermatozoides/química , Animales , Separación Celular , ADN/química , Metilación de ADN , Electroforesis/métodos , Humanos , Masculino , Fosfinas/química , Sustancias Reductoras/química , Dióxido de Silicio/químicaRESUMEN
AIM: In schizophrenia, metabolic syndrome incidence is double that of the general population, with women having a higher incidence. Pharmacogenetically regulated folic acid may be related to this risk. DNA methylation and metabolic syndrome within this group has not been previously studied. METHODS: Metabolic syndrome was evaluated with fasting laboratory measurements, and dietary and lifestyle assessments. Methylation analysis used a peripheral sample for the LINE-1 assay. DNA was also genotyped for MTHFR 677C/T. RESULTS: This analysis included 133 subjects. We found a significant relationship between LINE-1 methylation, and an interaction between MTHFR and gender, controlling for serum folate (p = 0.008). Females with the 677TT genotype had the lowest methylation (56%) compared with the other groups (75%). CONCLUSION: TT genotype females had the lowest methylation, which may explain metabolic syndrome gender differences in schizophrenia. Folate supplementation may be a suggested intervention within schizophrenia; however, additional work is required.
Asunto(s)
Metilación de ADN/genética , Ácido Fólico/metabolismo , Síndrome Metabólico/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo de Nucleótido Simple , Esquizofrenia/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antipsicóticos/administración & dosificación , Antipsicóticos/uso terapéutico , Clorpromazina/administración & dosificación , Clorpromazina/uso terapéutico , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Masculino , Síndrome Metabólico/complicaciones , Síndrome Metabólico/tratamiento farmacológico , Síndrome Metabólico/metabolismo , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Persona de Mediana Edad , Tipificación Molecular , Esquizofrenia/complicaciones , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismo , Factores SexualesRESUMEN
BACKGROUND: Global hypomethylation of DNA is thought to constitute an early event in some cancers and occurs in response to arsenic (As) exposure and/or selenium (Se) deficiency in both in vitro and animal models. In addition, antagonism between As and Se, whereby each reduces toxicity of the other, has been well documented in animal models. Se status may therefore modify the health effects of As in As-exposed populations. OBJECTIVE: The primary objectives of our study were to test the hypothesis that Se deficiency is associated with genomic hypomethylation of lymphocyte DNA and to determine whether Se levels are associated with blood As (bAs) and urinary As (uAs) concentrations in adults exposed to As-contaminated groundwater in Bangladesh. A secondary objective was to explore the relationships between plasma Se and As metabolites. DESIGN: We assessed plasma Se concentrations, As metabolite profiles in blood and urine, and genomic methylation of leukocyte DNA in a cross-sectional study of 287 adults. RESULTS: After adjustment for potential confounders, we observed an inverse association between Se (micrograms per liter) and genomic DNA methylation (disintegrations per minute per 1-µg/L increase in Se): ß = 345.6; 95% confidence interval (CI), 59-632. Se concentrations were inversely associated with total As concentrations (micrograms per liter) in blood (ß = -0.04; 95% CI, -0.08 to -0.01) and urine (ß = -20.1; 95% CI, -29.3 to -10.9). Se levels were negatively associated with the percentage of monomethylarsinic acid (ß = -0.59; 95% CI, -1.04 to -0.13) and positively associated with the percentage of dimethylarsinic acid (ß = 0.53; 95% CI, 0.04 to 1.01) in blood. CONCLUSIONS: Our results suggest that Se is inversely associated with genomic DNA methylation. The underlying mechanisms and implications of this observation are unclear and warrant further investigation. In addition, Se may influence bAs and uAs concentrations, as well as relative proportions of As metabolites in blood.
Asunto(s)
Arsénico/toxicidad , Metilación de ADN/efectos de los fármacos , Leucocitos/efectos de los fármacos , Selenio/sangre , Contaminantes Químicos del Agua/toxicidad , Adolescente , Adulto , Anciano , Arsénico/análisis , Arsénico/sangre , Bangladesh , Estudios Transversales , Interacciones Farmacológicas , Exposición a Riesgos Ambientales , Femenino , Humanos , Leucocitos/metabolismo , Modelos Lineales , Masculino , Persona de Mediana Edad , Selenio/deficiencia , Selenio/orina , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/sangre , Abastecimiento de Agua , Adulto JovenRESUMEN
BACKGROUND: Maternal folate nutritional status and prenatal lead exposure can influence fetal development and subsequent health. The methylenetetrahydrofolate reductase (MTHFR) gene is important for folate metabolism, and 2 common polymorphisms, C677T and A1298C, reduce enzymatic activity; C677T is present at high penetrance in Mexican populations. OBJECTIVE: The objective of this study was to examine potential links between maternal and child MTHFR polymorphisms and child neurodevelopment in a lead-exposed population. DESIGN: Data regarding MTHFR polymorphisms C677T and A1298C, peri- and postnatal lead measures, and Bayley Mental Development Index at 24 mo of age (MDI-24) scores were available for 255 mother-child pairs who participated in the ELEMENT (Early Life Exposures in Mexico to Environmental Toxicants) study during 1994-1995. RESULTS: In covariate-adjusted regression models, maternal MTHFR 677 genotype predicted MDI-24 scores, in which each copy of the maternal MTHFR 677T variant allele was associated with lower MDI-24 scores (beta = -3.52; 95% CI: -6.12, -0.93; P = 0.004). Maternal MTHFR haplotype also predicted MDI-24 scores (mean +/- SE: 93.3 +/- 1.2 for 677C-1298A compared with 89.9 +/- 0.8 for 677T-1298A; P < 0.05). MDI-24 scores were not associated with maternal MTHFR 1298 genotype or child MTHFR genotypes. We did not observe significant MTHFR genotype x lead interactions with respect to any of the subject biomarkers of lead exposure. CONCLUSIONS: The maternal MTHFR 677T allele is an independent predictor of poorer child neurodevelopment at 24 mo. These results suggest that maternal genetic variations in folate metabolism during pregnancy may program offspring neurodevelopment trajectories. Further research is warranted to determine the generalizability of these results across other populations.
Asunto(s)
Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/genética , Discapacidades del Desarrollo/genética , Intoxicación por Plomo/genética , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo Genético , Adolescente , Adulto , Huesos/metabolismo , Preescolar , Discapacidades del Desarrollo/epidemiología , Suplementos Dietéticos , Ingestión de Energía , Femenino , Ligamiento Genético , Genotipo , Haplotipos , Humanos , Plomo/metabolismo , México , Madres , Paridad , Polimorfismo de Nucleótido Simple , Periodo Posparto , Embarazo , Análisis de RegresiónRESUMEN
BACKGROUND: Chronic arsenic exposure currently affects >100 million persons worldwide. Methylation of ingested inorganic arsenic (InAs) to monomethylarsonic (MMAs) and dimethylarsinic (DMAs) acids relies on folate-dependent one-carbon metabolism and facilitates urinary arsenic elimination. OBJECTIVE: We hypothesized that folic acid supplementation to arsenic-exposed Bangladeshi adults would increase arsenic methylation and thereby lower total blood arsenic. DESIGN: In this randomized, double-blind, placebo-controlled trial, we evaluated blood concentrations of total arsenic, InAs, MMAs, and DMAs in 130 participants with low plasma folate (<9 nmol/L) before and after 12 wk of supplementation with folic acid (400 microg/d) or placebo. RESULTS: MMAs in blood was reduced by a mean +/- SE of 22.24 +/- 2.86% in the folic acid supplementation group and by 1.24 +/- 3.59% in the placebe group (P < 0.0001). There was no change in DMAs in blood; DMAs is rapidly excreted in urine as evidenced by an increase in urinary DMAs (P = 0.0099). Total blood arsenic was reduced by 13.62% in the folic acid supplementation group and by 2.49% in the placebo group (P = 0.0199). CONCLUSIONS: Folic acid supplementation to participants with low plasma concentrations of folate lowered blood arsenic concentrations, primarily by decreasing blood MMAs and increasing urinary DMAs. Therapeutic strategies to facilitate arsenic methylation, particularly in populations with folate deficiency or hyperhomocysteinemia or both, may lower blood arsenic concentrations and thereby contribute to the prevention of arsenic-induced illnesses.
Asunto(s)
Arsénico/metabolismo , Suplementos Dietéticos , Ácido Fólico/farmacología , Metilación/efectos de los fármacos , Complejo Vitamínico B/farmacología , Contaminantes Químicos del Agua/metabolismo , Arsénico/sangre , Arsénico/orina , Arsenicales/metabolismo , Arsenicales/orina , Bangladesh/epidemiología , Método Doble Ciego , Exposición a Riesgos Ambientales/efectos adversos , Femenino , Ácido Fólico/administración & dosificación , Ácido Fólico/sangre , Homocisteína/sangre , Humanos , Masculino , Persona de Mediana Edad , Complejo Vitamínico B/administración & dosificación , Complejo Vitamínico B/sangre , Contaminantes Químicos del Agua/sangre , Contaminantes Químicos del Agua/orinaRESUMEN
BACKGROUND: Populations in South and East Asia and many other regions of the world are chronically exposed to arsenic-contaminated drinking water. To various degrees, ingested inorganic arsenic (InAs) is methylated to monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) via folate-dependent one-carbon metabolism; impaired methylation is associated with adverse health outcomes. Consequently, folate nutritional status may influence arsenic methylation and toxicity. OBJECTIVE: The objective of this study was to test the hypothesis that folic acid supplementation of arsenic-exposed adults would increase arsenic methylation. DESIGN: Two hundred adults in a rural region of Bangladesh, previously found to have low plasma concentrations of folate (=9 nmol/L) were enrolled in a randomized, double-blind, placebo-controlled folic acid-supplementation trial. Plasma concentrations of folate and homocysteine and urinary concentrations of arsenic metabolites were analyzed at baseline and after 12 wk of supplementation with folic acid at a dose of 400 microg/d or placebo. RESULTS: The increase in the proportion of total urinary arsenic excreted as DMA in the folic acid group (72% before and 79% after supplementation) was significantly (P < 0.0001) greater than that in the placebo group, as was the reduction in the proportions of total urinary arsenic excreted as MMA (13% and 10%, respectively; P < 0.0001) and as InAs (15% and 11%, respectively; P < 0.001). CONCLUSIONS: These data indicate that folic acid supplementation to participants with low plasma folate enhances arsenic methylation. Because persons whose urine contains low proportions of DMA and high proportions of MMA and InAs have been reported to be at greater risk of skin and bladder cancers and peripheral vascular disease, these results suggest that folic acid supplementation may reduce the risk of arsenic-related health outcomes.