Superfund Research Program
Arsenic as an Endocrine Disrupter
Project Leaders: Joshua W. Hamilton (Marine Biological Laboratory), Joshua W. Hamilton (Marine Biological Laboratory)
Grant Number: P42ES007373
Funding Period: 1995-2014
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Progress Reports
Year: 2013 2012 2011 2010 2009 2008 2007 2006 2005 2004 2003 2002 2001 2000 1999 1998 1997 1996 1995
Aims
The goal of this project is to understand the molecular basis for the ability of arsenic to act as a potent endocrine disruptor, and to determine the pathophysiological consequences of such endocrine disruption.
Studies and results
The research team previously demonstrated that arsenic has potent effects on a wide array of nuclear hormone receptors and their ability to regulate gene expression. This includes the receptors for all five major steroid hormone classes (i.e., estrogen, testosterone, progesterone, glucocorticoids, mineralocorticoids) and several type 2 receptor hormones (retinoic acid, thyroid hormone, PPAR agonists). In order to better understand the mechanism, the researchers focused on the glucocorticoid receptor (GR) and the effects of arsenic to increase hormone-activated transcription at extremely low doses while suppressing such transcription at slightly higher but still non-cytotoxic doses. The research team constructed a GR molecule with a green fluorescent protein (GFP) tag and stably expressed this construct in a cell line that is hormone-responsive. Using confocal microscopy and digital quantitation, the researchers were able to demonstrate that arsenic significantly decreases the amount and rate of GR entry into the nucleus following sub-saturating hormone stimulation, correlating directly with the decrease in transcriptional activation and mRNA expression by target genes. These confocal observations were confirmed by Western blotting and other biochemical measures. This suggests that the nuclear translocation machinery that these nuclear receptors share may be the actual target for these arsenic effects rather than the receptor itself, which is likely why arsenic is able to affect in a similar manner so many different receptors that share little or no structural homology. The researchers are currently completing a manuscript that summarizes this work. Another paper was submitted that demonstrates that the monomethylated form of arsenic disrupts GR binding to its DNA recognition element, whereas inorganic arsenic does not, suggesting a role for arsenic metabolism and speciation in nuclear hormone receptor alterations.
The research team also completed studies aimed at understanding the effects of arsenic on the PPAR receptors and the role of such effects in arsenic-mediated dysregulation of lipid metabolism. The researchers have demonstrated that arsenic has profound effects on the ability of undifferentiated cells to differentiate into mature adipocytes and to accumulate lipid droplets, as well as changes in other measures of lipid metabolism in response to normal hormone signaling, suggesting that one consequence of arsenic exposure and endocrine disruption at the hormone receptor level. This correlates with their recent studies demonstrating that arsenic has profound effects on growth and development in mice. The researchers have submitted a manuscript describing these cell culture and molecular studies.
The research team also completed studies examining the effects of 10 ppb arsenic in drinking water (the current US EPA Maximum Contaminant Level, MCL) on mothers and their offspring in pregnant mice. The mothers had significant changes in their blood triglyceride levels and profiles, as well as changes in triglycerides in their breast milk. They also demonstrated severe hepatosteatosis, or fatty liver, in response to this low-level arsenic exposure. As a consequence, their pups were born with significant growth and development deficits from in utero only, nursing only, or in utero plus nursing period arsenic exposure via the mother. Cross-fostering of arsenic-exposed pups with control moms largely compensated for these deficits in male offspring but not in females, suggesting a gender difference in these early developmental exposures. This work was recently published in PLoSONE. Studies in non-pregnant adult females and adult males demonstrated effects of arsenic on white adipose tissue accumulation, further correlating these in vivo effects with our cell culture studies suggesting endocrine disruption via adipocytes.
Significance
These studies are significant because they demonstrate that very low levels of arsenic, relevant to the US population, cause significant endocrine disruption in model cell culture systems and animal models, resulting in significant pathophysiological responses that are directly related to endocrine disruption of specific hormone-mediated pathways. Furthermore, studies of pregnant mice who were exposed to arsenic at the current US drinking water standard that is considered "safe" indicate that arsenic may cause profound effects on in utero and early childhood development, which may result in later in life health effects in both the mothers and their offspring.
Plans
In 2013, the research team will complete studies proposed in the application funded from 2008-2013, and will continue studies presented as preliminary data in the renewal application examining the low dose effects (10 ppb) of arsenite on glucose and fat metabolism in neonates and mothers in a unique mouse model.