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

Arsenite-induced pseudo-hypoxia results in loss of anchorage-dependent growth in BEAS-2B pulmonary epithelial cells.

Epidemiology studies have established a strong link between lung cancer and arsenic exposure. Currently, the role of disturbed cellular energy metabolism in carcinogenesis is a focus of scientific interest. Hypoxia inducible factor-1 alpha (HIF-1A) is a key regulator of energy metabolism, and it has been found to accumulate during arsenite exposure under oxygen-replete conditions. We modeled arsenic-exposed human pulmonary epithelial cells in vitro with BEAS-2B, a non-malignant lung epithelial cell line. Constant exposure to 1 µM arsenite (As) resulted in the early loss of anchorage-dependent growth, measured by soft agar colony formation, beginning at 6 weeks of exposure. This arsenite exposure resulted in HIF-1A accumulation and increased glycolysis, similar to the physiologic response to hypoxia, but in this case under oxygen-replete conditions. This "pseudo-hypoxia" response was necessary for the maximal acquisition of anchorage-independent growth in arsenite-exposed BEAS-2B. The HIF-1A accumulation and induction in glycolysis was sustained throughout a 52 week course of arsenite exposure in BEAS-2B. There was a time-dependent increase in anchorage-independent growth during the exposure to arsenite. When HIF-1A expression was stably suppressed, arsenite-induced glycolysis was abrogated, and the anchorage-independent growth was reduced. These findings establish that arsenite exerts a hypoxia-mimetic effect, which plays an important role in the subsequent gain of malignancy-associated phenotypes

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

Arsenite-induced phenotypic changes in BEAS-2B.

<p>A) Representative images of soft agar growth over the course of 52 weeks of constant arsenite (1 µM) exposure. B) Colony counts in soft agar. Bars represent mean, 1 standard deviation, from 3 experimental replicates. C) Immunoblot analysis of HIF-1A and E-cadherin (E-cad) in BEAS-2B over the course of 52 weeks of constant arsenite (1 µM) exposure. D) Lactate levels (percent control) in BEAS-2B over the course of 52 weeks of constant arsenite (1 µM) exposure. Absolute lactate production in vector control: 0.733±0.017 µmol/10⁶cells/hr) Bars represent mean +1 standard deviation, from 3 experimental replicates. E) Percentage aneuploid cells in BEAS-2B treated with 1 µM arsenite for 0–52 weeks. Bars represent mean, +1 standard deviation, from 3 experimental replicates. *p<0.05.</p