Blanca Sánchez-Ramírez

Cytotoxicity of functionalized carbon nanotubes in J774A macrophages

Cytotoxicity of carbon nanotubes (CNTs) is a prime concern for its use as antigen carriers. Here we evaluated the cytotoxic effect of unpurified (UP-CNTs), purified (P-CNTs), fluorescein isothiocyanate-functionalized (FITC-CNTs), and Entamoeba histolytica 220-kDa lectin-functionalized CNTs (L220-CNTs) in J774A macrophage (MOs) cell line. Cell viability and apoptosis were analyzed by MTT and TUNEL assays, respectively. Cyclooxygenase-2 (COX-2) was analyzed by reverse transcription-polymerase chain reaction. Cytotoxicity at 6.0 mg/L was higher with UP-CNTs > P-CNTs > FITC-CNTs, showing a decrease in cell viability and an increase in apoptosis. In contrast, MOs interacted with L220-CNTs showed an increase in cell viability without signs of apoptosis. Although UP-CNTs and P-CNTs exhibited COX-2 induction with 6.0 mg/L, functionalized CNTs were able to induce COX-2 at concentrations as low as 0.06 mg/L. These results suggest that functionalization decreases toxicity, and that L220-CNTs may be an excellent candidate for the production of a nanovaccine against amebiasis.

Chronic Exposure to Arsenic and Markers of Cardiometabolic Risk: A Cross-Sectional Study in Chihuahua, Mexico

Background Exposure to arsenic (As) concentrations in drinking water > 150 μg/L has been associated with risk of diabetes and cardiovascular disease, but little is known about the effects of lower exposures. Objective This study aimed to examine whether moderate As exposure, or indicators of individual As metabolism at these levels of exposure, are associated with cardiometabolic risk. Methods We analyzed cross-sectional associations between arsenic exposure and multiple markers of cardiometabolic risk using drinking-water As measurements and urinary As species data obtained from 1,160 adults in Chihuahua, Mexico, who were recruited in 2008–2013. Fasting blood glucose and lipid levels, the results of an oral glucose tolerance test, and blood pressure were used to characterize cardiometabolic risk. Multivariable logistic, multinomial, and linear regression were used to assess associations between cardiometabolic outcomes and water As or the sum of inorganic and methylated As species in urine. Results After multivariable adjustment, concentrations in the second quartile of water As (25.5 to < 47.9 μg/L) and concentrations of total speciated urinary As (< 55.8 μg/L) below the median were significantly associated with elevated triglycerides, high total cholesterol, and diabetes. However, moderate water and urinary As levels were also positively associated with HDL cholesterol. Associations between arsenic exposure and both dysglycemia and triglyceridemia were higher among individuals with higher proportions of dimethylarsenic in urine. Conclusions Moderate exposure to As may increase cardiometabolic risk, particularly in individuals with high proportions of urinary dimethylarsenic. In this cohort, As exposure was associated with several markers of increased cardiometabolic risk (diabetes, triglyceridemia, and cholesterolemia), but exposure was also associated with higher rather than lower HDL cholesterol. Citation Mendez MA, González-Horta C, Sánchez-Ramírez B, Ballinas-Casarrubias L, Hernández Cerón R, Viniegra Morales D, Baeza Terrazas FA, Ishida MC, Gutiérrez-Torres DS, Saunders RJ, Drobná Z, Fry RC, Buse JB, Loomis D, García-Vargas GG, Del Razo LM, Stýblo M. 2016. Chronic exposure to arsenic and markers of cardiometabolic risk: a cross-sectional study in Chihuahua, Mexico. Environ Health Perspect 124:104–111; http://dx.doi.org/10.1289/ehp.1408742

Associations between Arsenic Species in Exfoliated Urothelial Cells and Prevalence of Diabetes among Residents of Chihuahua, Mexico

Background: A growing number of studies link chronic exposure to inorganic arsenic (iAs) with the risk of diabetes. Many of these studies assessed iAs exposure by measuring arsenic (As) species in urine. However, this approach has been criticized because of uncertainties associated with renal function and urine dilution in diabetic individuals. Objectives: Our goal was to examine associations between the prevalence of diabetes and concentrations of As species in exfoliated urothelial cells (EUC) as an alternative to the measures of As in urine. Methods: We measured concentrations of trivalent and pentavalent iAs methyl-As (MAs) and dimethyl-As (DMAs) species in EUC from 374 residents of Chihuahua, Mexico, who were exposed to iAs in drinking water. We used fasting plasma glucose, glucose tolerance tests, and self-reported diabetes diagnoses or medication to identify diabetic participants. Associations between As species in EUC and diabetes were estimated using logistic and linear regression, adjusting for age, sex, and body mass index. Results: Interquartile-range increases in trivalent, but not pentavalent, As species in EUC were positively and significantly associated with diabetes, with ORs of 1.57 (95% CI: 1.19, 2.07) for iAsIII, 1.63 (1.24, 2.15) for MAsIII, and 1.31 (0.96, 1.84) for DMAsIII. DMAs/MAs and DMAs/iAs ratios were negatively associated with diabetes (OR = 0.62; 95% CI: 0.47, 0.83 and OR = 0.72; 95% CI: 0.55, 0.96, respectively). Conclusions: Our data suggest that uncertainties associated with measures of As species in urine may be avoided by using As species in EUC as markers of iAs exposure and metabolism. Our results provide additional support to previous findings suggesting that trivalent As species may be responsible for associations between diabetes and chronic iAs exposure. Citation: Currier JM, Ishida MC, González-Horta C, Sánchez-Ramírez B, Ballinas-Casarrubias L, Gutiérrez-Torres DS, Hernández Cerón R, Viniegra Morales D, Baeza Terrazas FA, Del Razo LM, García-Vargas GG, Saunders RJ, Drobná Z, Fry RC, Matoušek T, Buse JB, Mendez MA, Loomis D, Stýblo M. 2014. Associations between arsenic species in exfoliated urothelial cells and prevalence of diabetes among residents of Chihuahua, Mexico. Environ Health Perspect 122:1088–1094; http://dx.doi.org/10.1289/ehp.1307756

A Concurrent Exposure to Arsenic and Fluoride from Drinking Water in Chihuahua, Mexico

Inorganic arsenic (iAs) and fluoride (F−) are naturally occurring drinking water contaminants. However, co-exposure to these contaminants and its effects on human health are understudied. The goal of this study was examined exposures to iAs and F− in Chihuahua, Mexico, where exposure to iAs in drinking water has been associated with adverse health effects. All 1119 eligible Chihuahua residents (>18 years) provided a sample of drinking water and spot urine samples. iAs and F− concentrations in water samples ranged from 0.1 to 419.8 µg As/L and from 0.05 to 11.8 mg F−/L. Urinary arsenic (U-tAs) and urinary F− (U-F−) levels ranged from 0.5 to 467.9 ng As/mL and from 0.1 to 14.4 µg F−/mL. A strong positive correlation was found between iAs and F− concentrations in drinking water (rs = 0.741). Similarly, U-tAs levels correlated positively with U-F− concentrations (rs = 0.633). These results show that Chihuahua residents exposed to high iAs concentrations in drinking water are also exposed to high levels of F−, raising questions about possible contribution of F− exposure to the adverse effects that have so far been attributed only to iAs exposure. Thus, investigation of possible interactions between iAs and F− exposures and its related health risks deserves immediate attention.

Metabolomic Characteristics of Arsenic-Associated Diabetes in a Prospective Cohort in Chihuahua, Mexico

Chronic exposure to inorganic arsenic (iAs) has been linked to an increased risk of diabetes, yet the specific disease phenotype and underlying mechanisms are poorly understood. In the present study we set out to identify iAs exposure-associated metabolites with altered abundance in nondiabetic and diabetic individuals in an effort to understand the relationship between exposure, metabolomic response, and disease status. A nested study design was used to profile metabolomic shifts in urine and plasma collected from 90 diabetic and 86 nondiabetic individuals matched for varying iAs concentrations in drinking water, body mass index, age, and sex. Diabetes diagnosis was based on measures of fasting plasma glucose and 2-h blood glucose. Multivariable models were used to identify metabolites with altered abundance associated with iAs exposure among diabetic and nondiabetic individuals. A total of 132 metabolites were identified to shift in urine or plasma in response to iAs exposure characterized by the sum of iAs metabolites in urine (U-tAs). Although many metabolites were altered in both diabetic and nondiabetic 35 subjects, diabetic individuals displayed a unique response to iAs exposure with 59 altered metabolites including those that play a role in tricarboxylic acid cycle and amino acid metabolism. Taken together, these data highlight the broad impact of iAs exposure on the human metabolome, and demonstrate some specificity of the metabolomic response between diabetic and nondiabetic individuals. These data may provide novel insights into the mechanisms and phenotype of diabetes associated with iAs exposure.

Effects of exposure to pesticides during pregnancy on placental maturity and weight of newborns: A cross-sectional pilot study in women from the Chihuahua State, Mexico

It is known that pesticides cross the placental barrier and can cause alterations in the development of placental structures resulting in adverse effects in reproduction. The objectives of this study were to investigate the effects of pesticide exposure during pregnancy on placental maturity and to evaluate the relationship between placental maturity, gestational age and birth weight. We collected the placentas from singleton pregnancies from women exposed (n = 9) and non-exposed (n = 45 full-term and n = 31 preterm) to pesticides as evaluated geographically, by questionnaire and by acetylcholinesterase levels. Placental morphometry from the central and peripheral regions was examined by microscopy and staining with hematoxylin and eosin. The placental maturity index (PMI) was estimated by dividing the number of epithelial plates in terminal villi to their thickness in 1 mm2 of the placental parenchyma. Gestational age, birth weight and the following characteristics of the mother were also recorded: pre-pregnancy body mass index, weight gain during pregnancy and hemoglobin concentrations. Birth weight and the gestational age were correlated with PMI (r = .54 and r = .44, respectively; p < .01). Pesticide exposure was associated with a higher PMI (beta = 7.38, p = .01) after adjusting by variables related to placental maturity. In conclusion, the results suggest a relationship between prenatal exposure to pesticides and placental maturity and may potentially affect the nutrient transport from the mother to the fetus.

Altered expressions of MMP-2, MMP-9, and TIMP-2 in placentas from women exposed to lead

Experimental studies have shown that prenatal exposure to lead (Pb) produces morphological changes related to extracellular matrix remodelling. To analyse whether the matrix metalloproteinases (MMPs), particularly MMP-2, MMP-9, and the tissue inhibitor of metalloproteinases-2 (TIMP-2), are associated with morphological alterations found in placentas, the expression of these enzymes was evaluated by immunohistochemical and image analyses in placentas of women with histories of environmental exposure to Pb. The median maternal concentration of Pb in blood was 4.68 µg/dL ( x ˉ = 5.85 ± 6.48 µg/dL). Significant differences related to the exposure to Pb were not detected in newborn or placenta weight. MMP-2, MMP-9, and TIMP-2 were expressed in the syncytiotrophoblast layer of placental villi. A significant increase in both MMP-2 and MMP-9 was observed in placentas of women with concentrations of Pb in blood ≥4.68 µg/dL (p = 0.01 and 0.03 for MMP-2 and MMP-9, respectively) and decrease in TIMP-2 expression (p = 0.01) resulted in a significant increase in MMP-2/TIMP-2 ratio (p < 0.01). Increased expression of MMPs may be induced to aid in repairing placental tissue damaged by the exposure to Pb and that TIMP-2 decreases its expression to permit tissue repair. Increased expression of MMPs may be important to consider as a mechanism for generating placental abnormalities and in the induction of preterm delivery or abortion.

Identification of Novel Gene Targets and Putative Regulators of Arsenic-Associated DNA Methylation in Human Urothelial Cells and Bladder Cancer

There is strong epidemiologic evidence linking chronic exposure to inorganic arsenic (iAs) to myriad adverse health effects, including cancer of the bladder. We set out to identify DNA methylation patterns associated with arsenic and its metabolites in exfoliated urothelial cells (EUCs) that originate primarily from the urinary bladder, one of the targets of arsenic-induced carcinogenesis. Genome-wide, gene-specific promoter DNA methylation levels were assessed in EUCs from 46 residents of Chihuahua, Mexico, and the relationship was examined between promoter methylation profiles and the intracellular concentrations of total arsenic and arsenic species. A set of 49 differentially methylated genes was identified with increased promoter methylation associated with EUC tAs, iAs, and/or monomethylated As (MMAs) enriched for their roles in metabolic disease and cancer. Notably, no genes had differential methylation associated with EUC dimethylated As (DMAs), suggesting that DMAs may influence DNA methylation-mediated urothelial cell responses to a lesser extent than iAs or MMAs. Further analysis showed that 22 of the 49 arsenic-associated genes (45%) are also differentially methylated in bladder cancer tissue identified using The Cancer Genome Atlas repository. Both the arsenic- and cancer-associated genes are enriched for the binding sites of common transcription factors known to play roles in carcinogenesis, demonstrating a novel potential mechanistic link between iAs exposure and bladder cancer.

In vitro assessment of the genotoxicity of ethyl paraoxon in newborns and adults

This in vitro experiment measured the genotoxic effects of ethyl paraoxon, the active metabolite of ethyl parathion. To assess genotoxicity, we used the micronuclei (MN) technique by blocking cytokinesis, and the ‘comet’ assay. We cultured peripheral blood samples from healthy adults and umbilical cord blood samples from four clinically healthy newborns to identify the frequency of MN. After 48 hours, we added the following ethyl paraoxon concentrations to the cultures: 0.0, 0.075, 0.100, 0.160, and 0.200 μg/mL. For the comet assay, following Singhs technique, we treated the blood samples for 2 hours with similar doses of the metabolite. The comet assay results, at a concentration of 0.075 μg/mL, showed that ethyl paraoxon causes a greater DNA migration that followed a dose-response pattern, a greater intensity being observed in lymphocytes from newborns. A comparison of the treatment and control groups indicated that only the 0.200 μg/mL concentration produced a slight increase in MN. In conclusion, our study identified primary DNA damage due to ethyl paraoxon, with a major effect on newborn lymphocytes, as well as an effect on the frequency of MN in the study groups at high concentrations only.

Cytotoxicity of carbon nanotubes on J774 macrophages is a purification-dependent effect

The cytotoxicity of the carbon nanotubes (CNTs) is an important factor for the manufacture of nanovaccines. The aim of this work was to evaluate the relationship of the purification method of CNTs in cellular toxicity using macrophages (MOs) from the J774 cell line. Viability test was performed with MTT assays at 24 h of exposure at concentrations of 0.06, 0.6, and 6 mg/L of unpurified (UP-CNTs) or purified (P-CNTs) CNTs by two different methods: (1) reflux with 3M HNO3 and (2) sonication in H2SO4/HNO3. Characterization and COOH content of CNTs was performed using scanning electron microscopy, raman spectroscopy, and titration with NaHCO3. P-CNTs1 had lengths >100 µm and 2.76% COOH content, while P-CNTs2 had lengths >1 µm and 7% COOH content. This last particle showed a lower toxic effect. The results suggest that the lenght and COOH content are important factors in the toxicity of the CNTs.