Developmentally Restricted Genetic Determinants of Human Arsenic Metabolism: Association between Urinary Methylated Arsenic and CYT19 Polymorphisms in Children

We report the results of a screen for genetic association with urinary arsenic metabolite levels in three arsenic metabolism candidate genes, PNP, GSTO, and CYT19, in 135 arsenic-exposed subjects from the Yaqui Valley in Sonora, Mexico, who were exposed to drinking water concentrations ranging from 5.5 to 43.3 ppb. We chose 23 polymorphic sites to test in the arsenic-exposed population. Initial phenotypes evaluated included the ratio of urinary inorganic arsenic(III) to inorganic arsenic(V) and the ratio of urinary dimethylarsenic(V) to monomethylarsenic(V) (D:M). In the initial association screening, three polymorphic sites in the CYT19 gene were significantly associated with D:M ratios in the total population. Subsequent analysis of this association revealed that the association signal for the entire population was actually caused by an extremely strong association in only the children (7–11 years of age) between CYT19 genotype and D:M levels. With children removed from the analysis, no significant genetic association was observed in adults (18–79 years). The existence of a strong, developmentally regulated genetic association between CYT19 and arsenic metabolism carries import for both arsenic pharmacogenetics and arsenic toxicology, as well as for public health and governmental regulatory officials

Analyzing Patterns of Community Interest at a Legacy Mining Waste Site to Assess and Inform Environmental Health Literacy Efforts

Understanding a community’s concerns and infor-mational needs is crucial to conducting and improving envi-ronmental health research and literacy initiatives. We hypoth-esized that analysis of community inquiries over time at alegacy mining site would be an effective method for assessingenvironmental health literacy efforts and determining whethercommunity concerns were thoroughly addressed. Through aqualitative analysis, we determined community concerns atthe time of being listed as a Superfund site. We analyzedhow community concerns changed from this starting pointover the subsequent years, and whether: (1) communicationmaterials produced by the U.S. Environmental ProtectionAgency and other media were aligned with community con-cerns; and (2) these changes demonstrated a progression of thecommunity’s understanding resulting from community in-volvement and engaged research efforts. We observed thatwhen the Superfund site was first listed, community memberswere most concerned with USEPA management, remediation,site-specific issues, health effects, and environmental monitor-ing efforts related to air/dust and water. Over the next 5 years,community inquiries shifted significantly to include exposureassessment and reduction methods and issues unrelated to thesite, particularly the local public water supply and home watertreatment systems. Such documentation of community inqui-ries over time at contaminated sites is a novel method to assessenvironmental health literacy efforts and determine whethercommunity concerns were thoroughly addressed.12 month embargo; published online: 21 July 2015This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

P-glycoprotein: Expression and function in normal circulating leukocytes

P-glycoprotein (P-gly) is a well characterized membrane protein, expressed in cancer cells, functioning as an efflux pump. This function confers drug-resistance. P-gly is also expressed in normal tissues such as the liver and kidney. In normal tissues P-gly has restricted expression. In the kidney P-gly is expressed in tubular brush border. This suggests a physiologic role for P-gly. A goal of this work was to determine whether P-gly, if present in leukocytes, followed the cell-type restriction seen in other P-gly positive normal tissues. Assays measuring P-gly included immunofluorescence, immunocytochemistry, and immunoblot analysis. Northern blot analysis and RT-PCR were used to measure mdr1 mRNA. P-gly function was assayed by measuring the verapamil-sensitive retention of rhodamine 123 (rh123). Immunofluorescent staining of leukocytes for lineage and P-gly revealed high levels of P-gly in CD56+ cells. CD8+ cells followed in staining, with CD4+ and CD19+ cells at intermediate levels, and CD14+ and CD15+ cells staining at background. RNA analysis by RT-PCR confirmed the immunofluorescence data, except for CD15+ cells, which demonstrated mdr1 mRNA similar to CD4+ cells. Function assays confirmed the immunofluorescence results, with efficient clearing of dye from CD56+ cells, followed by CD8+, CD4+, and CD19+ cells. CD14+ and CD15+ cells did not demonstrate P-gly function. Immunoblot analysis of membranes and immunocytochemical analysis of CD15+ cells demonstrated P-gly. The high level of functional P-gly observed in CD56+ cells prompted experiments to determine whether P-gly was involved in the CD56+ mediated cytolytic response. Using 4 inhibitors of P-gly mediated efflux, cyclosporine A, PSC 833, R-verapamil, and S-verapamil, NK cells were assayed for cytolytic function. Each compound demonstrated dose-response relationships in inhibiting NK-mediated cytolysis. Each compound also demonstrated a dose-response in inhibition of P-gly mediated efflux, although there was not an exact correlation between efflux inhibition and cytolysis inhibition. Nevertheless, the data in this study demonstrate a relatively high level of P-gly expression in CD56+ and CD8+ cells. In addition, the data support a role for P-gly in the cytolytic function of NK cells, although the point of P-gly involvement in the process of cytolysis remains to be defined

Association of atopy and eczema with polymorphisms in T-cell immunoglobulin domain and mucin domain-IL-2-inducible T-cell kinase gene cluster in chromosome 5 q 33.

International audienceBACKGROUND: The T-cell immunoglobulin domain and mucin domain (TIM) gene family and the gene for IL-2-inducible T-cell kinase (ITK), located in chromosome 5 q 33 and potentially involved in the T-cell proliferation and differentiation, are good candidate genes for allergic diseases. OBJECTIVE: We assessed the role of polymorphisms in the TIM family genes and ITK in atopy, eczema, and asthma. METHODS: Twenty-one polymorphisms in the TIM-ITK gene cluster were genotyped in 564 children enrolled in the Tucson Children's Respiratory Study. Skin prick tests to common allergens were performed at age 6.1 years (n=508), age 10.8 years (n=539), and age 16.6 years (n=424). Asthma and eczema were assessed by questionnaire at these 3 points. Averaged relative risks were estimated. RESULTS: One 15-bp insertion/deletion in exon 4 of TIM 1 was significantly related to atopy and eczema (relative risk associated with carrying at least 1 rare allele=1.24 [1.07--1.45], P=.005; and 1.43 [1.01--2.01], P=.004, respectively). The 3 tested single nucleotide polymorphisms (SNPs) in TIM 3 were significantly related to atopy and eczema. One of them, at position +4259 calculated from the translation start site, predicts a putative change in the amino acid sequence of the protein, and was the most strongly related to atopy (relative risk=1.28 [1.12--1.47]; P=.0003). SNPs in the 5' genomic region in ITK, which show moderate linkage disequilibrium with those in TIM 3, had an independent effect on atopy. None of the polymorphisms studied was related to asthma. CONCLUSION: Our findings support a potential role for SNPs in TIM 1, TIM 3, and ITK, independent of each other, in allergic diseases

Home Water Treatment Habits and Effectiveness in a Rural Arizona Community

Drinking water quality in the United States (US) is among the safest in the world. However, many residents, often in rural areas, rely on unregulated private wells or small municipal utilities for water needs. These utilities may violate the Safe Drinking Water Act contaminant guidelines, often because they lack the required financial resources. Residents may use alternative water sources or install a home water treatment system. Despite increased home water treatment adoption, few studies have examined their use and effectiveness in the US. Our study addresses this knowledge gap by examining home water treatment in a rural Arizona community. Water samples were analyzed for metal(loid)s, and home treatment and demographic data were recorded in 31 homes. Approximately 42% of homes treated their water. Independent of source water quality, residents with higher income (Odds Ratio [OR] = 1.25; 95% Confidence Interval [CI] (1.00–1.64)) and education levels (OR = 1.49; 95%CI (1.12–2.12)) were more likely to treat their water. Some contaminant concentrations were effectively reduced with treatment, while some were not. We conclude that increased educational outreach on contaminant testing and treatment, especially to rural areas with endemic water contamination, would result in a greater public health impact

Epigenetic Regulation of the Cell Type-Specific Gene 14-3-3σ

Epigenetic control participates in processes crucial in mammalian development, such as X-chromosome inactivation, gene imprinting, cell type-specific gene expression. We provide evidence that the p53-inducible gene 14-3-3σ is a new example of a gene important to human cancer, where epigenetic mechanisms participate in the control of normal cell type-specific expression, as well as aberrant gene silencing in cancer cells. Like a previously identified cell type-specific gene maspin, 14-3-3σ is a p53-inducible gene; however, it participates in G2/M arrest in response to DNA-damaging agents. 14-3-3σ expression is restricted to certain epithelial cell types, including breast, prostate, whereas expression is absent in nonepithelial tissues such as fibroblasts, lymphocytes. In this report, we show that in normal cells expressing 14-3-3σ, the 14-3-3σ CpG isl, is unmethylated; associated with acetylated histones, unmethylated histone H3 lysine 9;, an accessible chromatin structure. By contrast, normal cells that do not express 14-3-3σ have a methylated 14-3-3σ CpG isl, with hypoacetylated histones, methylated histone H3 lysine 9, an inaccessible chromatin structure. These findings extend the spectrum of cell typespecific genes controlled partly by normal epigenetic mechanisms, suggest that this subset of genes may represent important targets of epigenetic dysregulation in human cancer