Juan Manuel Delgado

Impact of early developmental arsenic exposure on promotor CpG-island methylation of genes involved in neuronal plasticity

Epigenetic mechanisms are crucial to regulate the expression of different genes required for neuronal plasticity. Neurotoxic substances such as arsenic, which induces cognitive deficits in exposed children before any other manifestation of toxicity, could interfere with the epigenetic modulation of neuronal gene expression required for learning and memory. This study assessed in Wistar rats the effects that developmental arsenic exposure had on DNA methylation patterns in hippocampus and frontal cortex. Animals were exposed to arsenic in drinking water (3 and 36ppm) from gestation until 4 months of age, and DNA methylation in brain cells was determined by flow cytometry, immunohistochemistry and methylation-specific polymerase chain reaction (PCR) of the promoter regions of reelin (RELN) and protein phosphatase 1 (PP1) at 1, 2, 3 and 4 months of age. Immunoreactivity to 5 methyl-cytosine was significantly higher in the cortex and hippocampus of exposed animals compared to controls at 1 month, and DNA hypomethylation was observed the following months in the cortex at high arsenic exposure. Furthermore, we observed a significant increase in the non-methylated form of PP1 gene promoter at 2 and 3 months of age, either in cortex or hippocampus. In order to determine whether this exposure level is associated with memory deficits, a behavioral test was performed at the same age points, revealing progressive and dose-dependent deficits of fear memory. Our results demonstrate alterations of the methylation pattern of genes involved in neuronal plasticity in an animal model of memory deficit associated with arsenic exposure.

Decreased nitric oxide markers and morphological changes in the brain of arsenic-exposed rats

Epidemiological studies demonstrate an association between chronic consumption of arsenic contaminated water and cognitive deficits, especially when the exposure takes place during childhood. This study documents structural changes and nitrergic deficits in the striatum of adult female Wistar rats exposed to arsenic in drinking water (3 ppm, approximately 0.4 mg/kg per day) from gestation, throughout lactation and development until the age of 4 months. Kainic acid injected animals (10mg/kg, i.p.) were also analyzed as positive controls of neural cell damage. Morphological characteristics of cells, fiber tracts and axons were analyzed by means of light microscopy as well as immunoreactivity to neuronal nitric oxide synthase (nNOS). As nitrergic markers, nitrite/nitrate concentrations, nNOS levels and expression of nNOS-mRNA were quantified in striatal tissue. Reactive oxygen species (ROS) and lipid peroxidation (LPx) were determined as oxidative stress markers. Arsenic exposure resulted in moderate to severe alterations of thickness, organization, surrounding space and shape of fiber tracts and axons, while cell bodies remained healthy. These anomalies were not accompanied by ROS and/or LPx increases. By contrast, except the expression of nNOS-mRNA, all nitrergic markers including striatal nNOS immunoreactivity presented a significant decrease. These results indicate that arsenic targets the central nitrergic system and disturbs brain structural organization at low exposure levels.

Effects of arsenite on central monoamines and plasmatic levels of adrenocorticotropic hormone (ACTH) in mice

We studied the effects of chronic arsenic exposure on brain monoamines and plasma levels of adrenocorticotropic hormone (ACTH) of mice. After weaning, mice received arsenic (0, 20, 40, 60 or 100 ppm) in drinking water over a period of 9 weeks. Monoamine content was quantified in different brain regions, arsenic was quantified in brain tissue and ACTH levels in plasma. Brain arsenic concentrations up to 200 ng/g showed a significant correlation with exposure levels and produced slight modifications in regional monoamine levels. ACTH plasma levels were significantly associated with norepinephrine (NE) concentrations in the medulla and pons, but not with hypothalamic NE levels. ACTH levels were significantly higher in the group exposed to 20 ppm. Dopamine showed significant dose-related decreases in the hypothalamus. These results show that chronic sodium arsenite exposure produces changes in central monoamines, which are not associated on a dose-dependent basis with major alterations in plasma ACTH.

Decreased arginine methylation and myelin alterations in arsenic exposed rats.

Methylation has an important role in the synthesis of myelin basic protein (MBP), an essential component that confers compactness to myelin, and the correct synthesis and assembling of myelin are fundamental in the development of the central nervous system. Since arsenic metabolism requires a high consumption of S-adenosylmethionine, the main donor of methyl groups in the organism, it has been proposed that arsenic exposure can lead to a demethylation status in the organism comprising DNA and protein hypomethylation. This study documents myelin alterations in brain and changes in levels of methylated arginines in brain and serum of adult female Wistar rats exposed to arsenic (3 and 36 ppm, drinking water) from gestation throughout lactation, development and until 1, 2, 3 and 4 months of age. Morphological characteristics were analyzed by means of light microscopy and methylated arginines were analyzed through HPLC. Arsenic intake resulted in myelin damage reflected as empty spaces in fiber tracts of the exposed animals. The low exposure group (approximately 0.4 mg/kg/day) did not present myelin damage during the first 2 months, only moderate alterations in the third and fourth months. By contrast, animals exposed to 36 ppm (approximately 4 mg/kg/day) showed moderate to severe damage to nerve tracts from the first month of age. These alterations were accompanied by significant lower levels of dimethyl arginine in both exposed groups, as compared with the controls, in the third and fourth months of age and exposure. These data demonstrate that myelin composition is a target of arsenic through interference with arginine methylation, and they suggest that disturbances in nervous transmission through myelinated fibers are an important component of arsenic neurotoxicity.

Effects of arsenic exposure during the pre- and postnatal development on the puberty of female offspring

UNLABELLED The (As) arsenic exposure is a risk factor for causing disturbances in the endocrine organs. OBJECTIVE To evaluate if sub-chronic As exposure during the pre- and postnatal development causes disturbances in the puberty. Moreover, determine adverse effects of As on the ovarian follicle and adrenocortical cell maturation. METHODS Females adult Wistar rats were exposed to sodium arsenite at 3 ppm calculated as As in drinking water from mating, gestation. Following the birth, the female offspring continued exposured to As via lactation. Weaned pups received the same As treatment as mothers, until they were 1-4 months (mo) old. At these ages, blood sampling and tissue harvest were done. The tissues were fixed in situ with 4% paraformaldehyde in phosphate buffer. After the perfusion the ovaries, uterus, adrenal glands were harvested, dissected out, weighted. The ovaries and the adrenal glands were processed to paraffin and sectioned at 5 μM and stained with hematoxylin and eosin for light microscopy. STATISTICAL ANALYSIS Comparisons between groups were made by unpaired t-test or nonparametric Mann-Whitney test as appropriate. RESULTS 100% As treated rats at 1 mo of age were at diestrous stage, with low estradiol E2. As treatment caused disturbances in the morphology of the ovarian cell consisting in DNA damage evidenced by picknotic chromatin, cariorexis, significant cytoplasmic vacuolization and also vasculature damaged. Arrest in follicle maturation was also present. CONCLUSIONS We found that the onset of puberty in the As treated rats was 1 mo delayed since vagina was still closed, the vaginal smear showed that they were at diestrus stage with plasma low E2 levels.

Methyl group balance in brain and liver: role of choline on increased S-adenosyl methionine (SAM) demand by chronic arsenic exposure.

Arsenic toxicity has been related to its interference with one carbon metabolism, where a high demand of S-adenosylmethionine (SAM) for arsenic methylation as well as a failure of its regeneration would compromise the availability of methyl groups for diverse cellular functions. Since exposed animals show disturbances of methylated products such as methylated arginines, myelin and axon membranes, this work investigates whether alterations of SAM, choline and phosphatidylcholine (PC) in the brain of arsenic exposed rats are associated with myelin alterations and myelin basic protein (MBP) immunoreactivity. Also these metabolites, morphologic and biochemical markers of methyl group alterations were analyzed in the liver, the main site of arsenic methylation. In adult, life-long arsenic exposed rats through drinking water (3 ppm), no changes of SAM, choline and PC concentrations where found in the brain, but SAM and PC were severely decreased in liver accompanied by a significant increase of choline. These results suggest that choline plays an important role as methyl donor in arsenic exposure, which could underlie hepatic affections observed when arsenic exposure is combined with other environmental factors. Also, important myelin and nerve fiber alterations, accompanied by a 75% decrease of MBP immunoreactivity were not associated with a SAM deficit in the brain.

Chronic Arsenic Exposure Increases Aβ(1–42) Production and Receptor for Advanced Glycation End Products Expression in Rat Brain

Chronic arsenic exposure during development is associated with alterations of chemical transmission and demyelination, which result in cognitive deficits and peripheral neuropathies. At the cellular level, arsenic toxicity involves increased generation of reactive species that induce severe cellular alterations such as DNA fragmentation, apoptosis, and lipid peroxidation. It has been proposed that arsenic-associated neurodegeneration could evolve to Alzheimer disease in later life.1,2 In this study, the effects of chronic exposure to inorganic arsenic (3 ppm by drinking water) in Wistar rats on the production and elimination of Amyloid-β (Aβ) were evaluated. Male Wistar rats were exposed to 3 ppm of arsenic in drinking water from fetal development until 4 months of age. After behavioral deficits induced by arsenic exposure through contextual fear conditioning were verified, the brains were collected for the determination of total arsenic by inductively coupled plasma-mass spectrometry, the levels of amyloid precursor protein and receptor for advanced glycation end products (RAGE) by Western blot analysis as well as their transcript levels by RT-qPCR, Aβ(1-42) estimation by ELISA assay and the enzymatic activity of β-secretase (BACE1). Our results demonstrate that chronic arsenic exposure induces behavioral deficits accompanied of higher levels of soluble and membranal RAGE and the increase of Aβ(1-42) cleaved. In addition, BACE1 enzymatic activity was increased, while immunoblot assays showed no differences in the low-density lipoprotein receptor-related protein 1 (LRP1) receptor among groups. These results provide evidence of the effects of arsenic exposure on the production of Aβ(1-42) and cerebral amyloid clearance through RAGE in an in vivo model that displays behavioral alterations. This work supports the hypothesis that early exposure to metals may contribute to neurodegeneration associated with amyloid accumulation.