Arylsulfatase A pseudodeficiency in Mexico: Enzymatic activity and haplotype analysis.

BACKGROUND: Metachromatic Leukodystrophy (MLD, OMIM 250100) is a neurodegenerative disease caused by mutations in the ARSA gene (OMIM 607574) that lead to deficiency in Arylsulfatase A (ASA). ASA pseudodeficiency (PD-ASA) is a biochemical condition that substantially diminishes ASA activity but is not associated with clinical manifestations. PD-ASA is associated with the c.1055A>G (p.Asn352Ser) (rs2071421) and c.*96A>G (rs6151429) variants, which have an estimated frequency of 2% in the population. OBJECTIVE: To determine the activity of Arylsulfatase A and to identify variants and haplotypes in the ARSA gene in Mexican individuals with pseudodeficiency. METHODS: Two-hundred apparently healthy individuals were included to determine the enzymatic activity of ASA in leukocytes by spectrophotometric analysis, and identification of the PD-ASA alleles was performed by PCR-RFLP assays. Genotypes were confirmed by semi-automated Sanger sequencing. Haplotypes were constructed using Arlequin v.10.04, and linkage disequilibrium analysis was performed with Cube X. RESULTS: The enzymatic activity of ASA was determined to be 1.74-2.09 nmol/mg protein/min and later correlated with genotypes and haplotypes. For the (p.Asn352Ser) variant, we found 126 (0.63) individuals with the AA genotype, 62 with AG (0.31) and 12 with GG (0.06); the frequency of the polymorphic allele was 0.215 (86 alleles, 21.5%), and the variant was in HWE (p = .2484). The variant c.*96A>G was also in HWE (p = .2105): 185 individuals (0.925) with the AA genotype, 14 (0.07) with AG, and 1 (0.005) with (GG), with a frequency of 0.04 (4%) for the polymorphic allele. The inference of haplotypes resulted in 312 (0.78) AA, 72 (0.18) GA, and 16 (0.04) GG haplotypes. The AG haplotype was not found. The variants were found to be in linkage disequilibrium (D' = 1). Of the nine possible diplotypes, AA/AG, AA/GG, and AG/GG were not found, in concordance with the hypothesis that the G allele of c.*96A>G does not occur in the absence of the G allele of c.1055A>G. We found a slight correlation between ASA biochemical activity and variants, mainly due to the G allele of c.*96A>G in either genotypes or haplotypes. CONCLUSIONS: In Northwestern Mexico, the presence of PD-ASA alleles was biochemically and molecularly determined, and the frequencies were found to be in HWE. The frequency of PD-ASA for the North Western Mexican mestizo is 8%.

[Effects of occupational exposure to pesticides on semen quality of workers in an agricultural community of Merida state, Venezuela]. / Efectos de la exposición ocupacional a plaguicidas sobre la calidad del semen en trabajadores de una comunidad agrícola del estado Merida, Venezuela.

Numerous studies report adverse effects of pesticides on male reproductive health. The objectives of this study were to investigate whether there is a relationship between occupational exposure to pesticides and semen quality, and to determine whether chronic exposure to pesticides differentially affects semen quality in men of different ages. A comparative study of 64 farmers and 64 control men was performed. The farmers were interviewed to determine their occupational history and particularly, activities that may involve exposure to pesticides. Semen parameters were evaluated and a comparative analysis of semen variables between exposed and control groups, as well as between age groups: 18-29, 30-37 and 38-60 years was done. Significant alterations of some semen parameters in the exposed group were found, such as: decreases in sperm concentration, slow progressive motility and sperm membrane integrity; at the same time, increases in eosin Y positive and sperm DNA fragmentation index. The results obtained by age groups showed significant differences between exposed and control groups for the parameters of membrane integrity, eosin Y positive and sperm DNA fragmentation index, being the exposed group between 18-29 years that showed the highest altered cases of these parameters. Our results prove that occupational pesticide exposure is associated with alterations in sperm quality, creating a risk to farm workers in their reproductive capacity.

Occupational exposure to organophosphate and carbamate pesticides affects sperm chromatin integrity and reproductive hormone levels among Venezuelan farm workers.

OBJECTIVES: Several reports suggest that chronic pesticide exposure may affect semen quality and male fertility in humans. The objective of this study was to evaluate the association between occupational exposure to organophosphate (OP) and carbamate (CB) pesticides and semen quality, as well as levels of reproductive and thyroid hormones of Venezuelan farm workers. METHODS: Thirty-five healthy men (unexposed group) and 64 male agricultural workers (exposed group) were recruited for clinical evaluation of fertility status. Fresh semen samples were evaluated for sperm quality and analyzed for DNA fragmentation index (DFI) by flow cytometry. Pesticide exposure was assessed by measuring erythrocyte acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BuChE) with a Test-mate ChE field kit. Serum levels of total testosterone (Tt), follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), thyroid stimulating hormone (TSH) and free thyroxine (FT4) were analyzed using enzyme immunoassay kits. RESULTS: Evidence of pesticide exposure was found in 87.5% of farmers based on AChE and BuChE inhibition. Significant increments were observed in sperm DFI with significant decreases in some semen parameters. DFI was negatively correlated with BuChE, sperm concentration, morphology and vitality in these workers. The levels of Tt, PRL, FT4 and TSH appeared to be normal; however, there was a tendency for increased LH and FSH levels in exposed workers. CONCLUSIONS: Our results confirm the potential impact of chronic occupational exposure to OP/CB pesticides on male reproductive function, which may cause damage to sperm chromatin, decrease semen quality and produce alterations in reproductive hormones, leading to adverse reproductive health outcomes.

Efectos de exposición ocupacional a plaguicidas sobre la integridad de la cromatina espermática

Objetivo: Evaluar los efectos de la exposición a plaguicidas organofosforados y carbamatos sobre la integridad de cromatina espermática en trabajadores agrícolas. Métodos: Se evaluaron 64 trabajadores del campo, con edades entre 18 y 55 años, de la comunidad rural de Bailadores, Municipio Rivas Dávila, Estado Mérida, Venezuela, expuestos directamente a plaguicidas. Para el diagnóstico de exposición a plaguicidas, fueron determinados los niveles de las colinesterasas eritrocitaria (AChE) y plasmática (PChE). Para evaluar la fertilidad masculina, a cada trabajador se le realizó seminograma y se les evaluó la integridad de la estructura de cromatina espermática mediante la técnica "Sperm Chromatin Structure Assay" (SCSA). Resultados: El 25% de los trabajadores agrícolas presentó niveles deprimidos de AChE y el 83% con niveles anormales de PChE, con una reducción <75% del nivel normal. El grupo con edades entre 18 y 28 años fue el más afectado. Diferencias significativas fueron obtenidas en los promedios de los niveles de colinesterasas entre los casos normales y alterados, tanto para AChE (3,95±0,42 vs. 2,72±0,16, ρ<0,0001) así como para PChE (2,08±0,25 vs. 1,43±0,32, ρ<0,0001). El 69,7% de los trabajadores con niveles anormales de PChE presentó alteración en el ADN espermático. Se encontró una correlación negativa significativa entre el Índice de Fragmentación de ADN (IDF) espermático y los niveles de PChE (ρ=0,02). Conclusiones: En trabajadores agrícolas expuestos directamente a plaguicidas organofosforados y carbamatos, un alto porcentaje presentó niveles anormalmente deprimidos de PChE, junto con alteración en el ADN espermático. Estos resultados demuestran que los trabajadores se encuentran con alto riesgo de exposición a los efectos tóxicos de plaguicidas, lo cual efectivamente se comprueba con los resultados de análisis de las enzimas colinesterasas y la aplicación de la técnica SCSA para determinar la integridad de la cromatina espermática.

Objective: To evaluate the effects of exposure to organophosphate and carbamate pesticides on the integrity of sperm chromatin in farm workers. Methods: In this study, we evaluated 64 farm workers, aged between 18 and 55 years, from the rural community of Bailadores, Municipality of Rivas Dávila, Mérida State, Venezuela, directly exposed to pesticides. For the diagnosis of pesticide exposure, levels of erythrocyte (AChE) and plasma (PchE) cholinesterases. were determined. To evaluate male fertility, each worker underwent semen analysis and the integrity of sperm chromatin structure was assessed using the "Sperm Chromatin Structure Assay (SCSA) technique. Results: The results of this study showed that 25% of agricultural workers had depressed levels of AChE and a majority, 83%, was found with abnormal levels of PChE. The group with corresponding ages between 18 and 28 years was the most affected. Significant differences were obtained in the average cholinesterase levels between normal and altered cases for both AChE (3.95 ± 0.42 vs. 2.72 ± 0.16, p < 0.0001) and PChE (2.08 ± 0.25 vs. 1.43 ± 0.32, ρ < 0.0001). Among workers with abnormal PChE levels, 69.7% presented alterations of sperm chromatin structure. There was a significant negative correlation between sperm DNA Fragmentation Index (DFI) and the levels of PChE (ρ = 0.02). Conclusions: In agricultural workers directly exposed to organophosphate and carbamate pesticides, a high percentage showed abnormally depressed PChE, along with altered sperm chromatin structure. These results show that farm workers are at high risk of exposure to the toxic effects of pesticides, which were effectively demonstrated with the results of analysis of cholinesterase enzymes and the implementation of the SCSA technique for determining the status of the sperm chromatin structure.

Highlights on ultrastructural pathology of human sperm.

Applications of atomic force microscopy to ultrastructural investigation of human spermatozoa are -discussed, with particular emphasis to their most common pathological alterations, which are recognized to be associated with male infertility. Morphological alterations can be located in the head, neck piece, and/or in the flagellum. The consequences of these defects on infertility-related topics are examined in the light of aberrations caused in varicocele and in other spermatozoa morphological alterations like globozoospermia, oligoasthenospermia, and in semen from patients with HIV syndrome. Special attention is given to the temperature effects on sperm abnormalities. The application of the present approach to pharmacology, namely, the development of male contraceptive methods, is also referred.