Emma S. Calderón-Aranda

Urinary Trivalent Methylated Arsenic Species in a Population Chronically Exposed to Inorganic Arsenic

Chronic exposure to inorganic arsenic (iAs) has been associated with increased risk of various forms of cancer and of noncancerous diseases. Metabolic conversions of iAs that yield highly toxic and genotoxic methylarsonite (MAsIII) and dimethylarsinite (DMAsIII) may play a significant role in determining the extent and character of toxic and cancer-promoting effects of iAs exposure. In this study we examined the relationship between urinary profiles of MAsIII and DMAsIII and skin lesion markers of iAs toxicity in individuals exposed to iAs in drinking water. The study subjects were recruited among the residents of an endemic region of central Mexico. Drinking-water reservoirs in this region are heavily contaminated with iAs. Previous studies carried out in the local populations have found an increased incidence of pathologies, primarily skin lesions, that are characteristic of arseniasis. The goal of this study was to investigate the urinary profiles for the trivalent and pentavalent As metabolites in both high- and low-iAs–exposed subjects. Notably, methylated trivalent arsenicals were detected in 98% of analyzed urine samples. On average, the major metabolite, DMAsIII, represented 49% of total urinary As, followed by DMAsV (23.7%), iAsV (8.6%), iAsIII (8.5%), MAsIII (7.4%), and MAsV (2.8%). More important, the average MAsIII concentration was significantly higher in the urine of exposed individuals with skin lesions compared with those who drank iAs-contaminated water but had no skin lesions. These data suggest that urinary levels of MAsIII, the most toxic species among identified metabolites of iAs, may serve as an indicator to identify individuals with increased susceptibility to toxic and cancer-promoting effects of arseniasis.

Assessment of lymphocyte subpopulations and cytokine secretion in children exposed to arsenic

Exposure of several human populations to arsenic has been associated with a high incidence of detrimental dermatological and carcinogenic effects. To date, studies examining the immunotoxic effects of arsenic in humans, and specifically in children, are lacking. Therefore, we evaluated several parameters of immunological status in a group of children exposed to arsenic through their drinking water. Peripheral blood mononuclear cells (PBMCs) of 90 children (6 to 10 years old) were collected. Proportions of lymphocyte subpopulations, PBMC mitogenic proliferative response, and urinary arsenic levels were evaluated. Increased urine arsenic levels were associated with a reduced proliferative response to phytohemaglutinin (PHA) stimulation (P=0.005), CD4 subpopulation proportion (P=0.092), CD4/CD8 ratio (P=0.056), and IL‐2 secretion levels (P=0.003). Increased arsenic exposure was also associated with an increase in GM‐CSF secretion by mononucleated cells (P=0.000). We did not observe changes in CD8, B, or NK cell proportions, nor did we observe changes in the secretion of IL‐4, IL‐10, or IFN‐γ by PHA‐activated PBMCs. These data indicate that arsenic exposure could alter the activation processes of T cells, such that an immunosuppression status that favors opportunistic infections and carcinogenesis is produced together with increased GM‐CSF secretion that may be associated with chronic inflammation.

Metals in urine and peripheral arterial disease

Exposure to metals may promote atherosclerosis. Blood cadmium and lead were associated with peripheral arterial disease (PAD) in the 1999–2000 National Health and Nutrition Examination Survey (NHANES). In the present study we evaluated the association between urinary levels of cadmium, lead, barium, cobalt, cesium, molybdenum, antimony, thallium, and tungsten with PAD in a cross-sectional analysis of 790 participants ≥40 years of age in NHANES 1999–2000. PAD was defined as a blood pressure ankle brachial index < 0.9 in at least one leg. Metals were measured in casual (spot) urine specimens by inductively coupled plasma–mass spectrometry. After multivariable adjustment, subjects with PAD had 36% higher levels of cadmium in urine and 49% higher levels of tungsten compared with noncases. The adjusted odds ratio for PAD comparing the 75th to the 25th percentile of the cadmium distribution was 3.05 [95% confidence interval (CI), 0.97 to 9.58]; that for tungsten was 2.25 (95% CI, 0.97 to 5.24). PAD risk increased sharply at low levels of antimony and remained elevated beyond 0.1 μg/L. PAD was not associated with other metals. In conclusion, urinary cadmium, tungsten, and possibly antimony were associated with PAD in a representative sample of the U.S. population. For cadmium, these results strengthen previous findings using blood cadmium as a biomarker, and they support its role in atherosclerosis. For tungsten and antimony, these results need to be interpreted cautiously in the context of an exploratory analysis but deserve further study. Other metals in urine were not associated with PAD at the levels found in the general population.

Biomonitoring of metal in children living in a mine tailings zone in Southern Mexico: A pilot study.

Accumulation of metals in soil represents a health risk for individuals living near mining areas, especially for children who have a higher susceptibility to metal related diseases. The Taxco mining district in Southern Mexico was one of the largest Mexican metal producers of silver and gold, among other metals. The aim of this pilot study was to evaluate metal exposure on children aged 6-11 years living in and around the Taxco mine tailings zone. Lead in blood (PbB) was measured by graphite furnace atomic absorption spectrophotometry (AAS). Urine arsenic (AsU) was measured by hydride generation AAS, urinary Hg (HgU) by flow injection cold vapor atomic absorption, and urinary concentration of other metals such as chromium (Cr), nickel (Ni), cadmium (Cd), barium (Ba), cobalt (Co), copper (Cu), zinc (Zn), manganese (Mn), molybdenum (Mo), strontium (Sr), and iron (Fe) were determined by inductively coupled plasma optical emission spectrometry. Fifty samples were analyzed for PbB, AsU, and HgU, and 35 samples for the other metals. The mean concentration+/-SD for each metal was: PbB, 9.4+/-3.3 microg/dL; NiU, 75.4+/-30.7 microg/L; BaU, 18.4+/-4.1 microg/L; MnU, 5.2+/-0.7 microg/L; CuU, 29.6+/-6.8 microg/L; AsU, 16.5+/-8.3 microg/L; HgU, 0.7+/-0.86 microg/L; CdU, 4.7+/-2.7 microg/L; CrU, 15.1+/-4.45 microg/L; CoU, 18.3+/-9.7 microg/L; SrU, 49.2+/-30.7 microg/L; ZnU, 628.4+/-438.9 microg/L; FeU, 30.5+/-17.7 microg/L; and MoU, 52.1+/-29.3 microg/L. Results of this exploratory study show that children residing in the mining area of Taxco were environmentally exposed to several metals and a high percentage of these children had levels of Ni, Ba, Mn, Cr, Co, Cd, As, Hg, and Pb above reference values. Thus, further studies are needed to assess the effects of simultaneous exposure to toxic metals in children residing in mining areas.

Role of nitric oxide produced by iNOS through NF-κB pathway in migration of cerebellar granule neurons induced by Lipopolysaccharide.

Inflammatory stimulus during development increases the risk for adverse neurologic outcome. One possible mechanism is disrupting neuronal migration. Using lipopolysaccharide (LPS)-treatment to assess inflammatory stimulus on neuronal migration of cerebellar granule neurons, we previously found that LPS-activation increased the neuronal migration. The precise mechanisms behind these effects have not been investigated. Independently, it was shown that nitric oxide (NO(•-)) regulates neuronal migration during development, that NO(•-) is produced by inducible nitric oxide synthase (iNOS) in response to LPS through the activation of nuclear factor (NF)-κB, and that LPS induce the expression of genes under the transcriptional control of NF-κB in primary cultures from developing mouse cerebellum. To investigate the relationship between these events, we used this culture model to study the role of NO(•-) produced by iNOS through NF-κB signaling pathway, in the effect of LPS on neuron migration. LPS increased NO(•-) production, iNOS protein levels and NF-κB nuclear levels; concomitantly with NO(•-) production, LPS increased the neuronal migration as compared to non stimulated cultures. The necessary roles of the NO(•-) and iNOS were demonstrated by chelating of NO(•-) with hemoglobin and the inhibition of iNOS by 1400W. Each of these treatments reduced neuronal migration induced by LPS. The role of NF-κB was showed by using the inhibitor JSH-23, which decreased NO(•-) production and neuronal migration in LPS activated cultures. These results suggest that neuronal migration during development is susceptible to be modified by pro-inflammatory stimulus such as LPS through intracellular pathways associated with their receptors.

Arsenic alters monocyte superoxide anion and nitric oxide production in environmentally exposed children

Arsenic (As) exposure has been associated with alterations in the immune system, studies in experimental models and adults have shown that these effects involve macrophage function; however, limited information is available on what type of effects could be induced in children. The aim of this study was to evaluate effects of As exposure, through the association of inorganic As (iAs) and its metabolites [monomethylated arsenic (MMA) and dimethylated arsenic (DMA)] with basal levels of nitric oxide (NO(-)) and superoxide anion (O(2)(-)), in peripheral blood mononuclear cells (PBMC) and monocytes, and NO(-) and O(2)(-) produced by activated monocytes. Hence, a cross-sectional study was conducted in 87 children (6-10 years old) who had been environmentally exposed to As through drinking water. Levels of urinary As species (iAs, MMA and DMA) were determined by hydride generation atomic absorption spectrometry, total As (tAs) represents the sum of iAs and its species; tAs urine levels ranged from 12.3 to 1411 microg/g creatinine. Using multiple linear regression models, iAs presented a positive and statistical association with basal NO(-) in PBMC (beta=0.0048, p=0.049) and monocytes (beta=0.0044, p=0.044), while basal O(2)(-) had a significant positive association with DMA (beta=0.0025, p=0.046). In activated monocytes, O(2)(-) showed a statistical and positive association with iAs (beta=0.0108, p=0.023), MMA (beta=0.0066, p=0.022), DMA (beta=0.0018, p=0.015), and tAs (beta=0.0013, p=0.015). We conclude that As exposure in the studied children was positively associated with basal levels of NO(-) and O(2)(-) in PBMC and monocytes, suggesting that As induces oxidative stress in circulating blood cells. Additionally, this study showed a positive association of O(2)(-) production with iAs and its metabolites in stimulated monocytes, supporting previous data that suggests that these cells, and particularly the O(2)(-) activation pathway, are relevant targets for As toxicity.

Immunization with A91 peptide or copolymer-1 reduces the production of nitric oxide and inducible nitric oxide synthase gene expression after spinal cord injury

Immunization with neurally derived peptides (INDP) boosts the action of an autoreactive immune response that has been shown to induce neuroprotection in several neurodegenerative diseases, especially after spinal cord (SC) injury. This strategy provides an environment that promotes neuronal survival and tissue preservation. The mechanisms by which this autoreactive response exerts its protective effects is not totally understood at the moment. A recent study showed that INDP reduces lipid peroxidation. Lipid peroxidation is a neurodegenerative phenomenon caused by the increased production of reactive nitrogen species such as nitric oxide (NO). It is possible that INDP could be interfering with NO production. To test this hypothesis, we examined the effect of INDP on the amount of NO produced by glial cells when cocultured with autoreactive T cells. We also evaluated the amount of NO and the expression of the inducible form of nitric oxide synthase (iNOS) at the injury site of SC‐injured animals. The neural‐derived peptides A91 and Cop‐1 were used to immunize mice and rats with SC injury. In vitro studies showed that INDP significantly reduces the production of NO by glial cells. This observation was substantiated by in vivo experiments demonstrating that INDP decreases the amount of NO and iNOS gene expression at the site of injury. The present study provides substantial evidence on the inhibitory effect of INDP on NO production, helpingour understanding of the mechanisms through which protective autoimmunity promotes neuroprotection.

Environmental exposure to lead and mercury in Mexican children: a real health problem

Exposure to lead (Pb) and mercury (Hg) remains a world public health problem, particularly for young children in developing countries. In Mexico, the main sources of exposure to Pb and Hg are wastes from human activities that increase the natural sources of these metals. Pb and Hg are highly toxic during development and maturation periods of the central nervous system (CNS); these effects are associated with the risk for neurodegenerative diseases. Mexico has numerous exposure sources to Pb and Hg; nevertheless, information on exposure in children is limited, particularly for Hg. Therefore, we conducted a review of the studies performed in children exposed to Pb and Hg. Data presented support that an important proportion of Mexican children have Pb levels above values associated with dangerous effects. On the other hand, studies on Hg-exposure are scarce, so we need more studies to estimate the magnitude of the problem and to determine exposure levels in Mexican children. Available data support the urgent need for coordinated actions among researchers, and health and environmental government authorities to implement education and nutritional campaigns, as well as to decrease exposure and effects of Pb and Hg. In addition, there must be a priority for the implementation of educational campaigns directed to the general population, but with emphasis in parents, education staff and health care providers to decrease both the risk of exposure of children to Pb and Hg and the effects of the exposure to these metals.

A Permethrin/Allethrin Mixture Induces Genotoxicity and Cytotoxicity in Human Peripheral Blood Lymphocytes

Two pyrethroids, permethrin and allethrin, are often combined for large-scale use in public health programs to control vector-borne diseases. In this study, the genotoxic potential of a commercial formulation of permethrin and allethrin was examined using cultured human peripheral blood lymphocytes (PBL). Genotoxicity was evaluated using the cytokinesis-block micronucleus cytome (CBMN cyt) assay by measuring the frequency of micronuclei (MN), nuclear division index (NDI), formation of nucleoplasmic bridges (NPB) and nuclear buds (NBUD), as well as apoptotic and necrotic cells. Human PBL were treated with different concentrations of a permethrin/allethrin mixture (1/0.01, 5/0.07, and 10/0.14 μg/ml) for 24 or 36 h. The highest concentration (10/0.14 μg/ml) of permethrin/allethrin mixture significantly increased MN frequency and percent apoptotic cells after incubations for 24 or 36 h. The NDI was markedly decreased in response to treatment with 5/0.07 or 10/0.14 μg/ml permethrin/allethrin for both 24 and 36 h. Exposure to the permethrin/allethrin mixture did not significantly alter formation of NBUD, NPB, or percent necrotic cells. The MN frequency was significantly correlated with the number of apoptotic and necrotic cells but inversely correlated with NDI. Data demonstrated that a mixture of permethrin and allethrin induced concentration- and time-dependent cytotoxic and genotoxic damage to human PBL in vitro.

p53 Expression in circulating lymphocytes of non-melanoma skin cancer patients from an arsenic contaminated region in Mexico. A pilot study

Arsenic is a common environmental toxicant and epidemiological studies associate arsenic exposure with various pathologic disorders and several types of cancer. Skin cancers are the most common arsenic-induced neoplasias and the prevalence of skin lesions has been reported to be significantly elevated in individuals exposed to arsenic via drinking water in México. Being lymphocytes the main cells used for human monitoring, we evaluated the expression of p53 protein in the lymphocytes from 44 healthy individuals and 19 samples from individuals living in a chronic arsenicism endemic region. Of the latter group, 12 individuals had non-melanoma skin cancer and 9 of them expressed p53 in the circulating lymphocytes, whereas only one of the 7 non-cancer arsenic exposed individuals expressed it. In the healthy non-arsenic exposed group only one from 44 individuals expressed the protein. These results suggest a clear relationship between non-melanoma skin cancer and p53 expression in circulating lymphocytes. p53 expression in circulating lymphocytes should be evaluated as a potential biomarker of effect or susceptibility.