Grantee Research Project Results

To explore arsenic-induced alterations, we exposed confluent bronchial airway epithelial cell cultures (16HBE) to 30, 60 or 290 ppb arsenite and monitored wound healing, activity of metalloproteinases and associated intracellular Ca²⁺ concentration ([Ca²⁺]_i) changes. Following a 24 exposure to arsenic, the ability of the cells to undergo mechanical wound repair was inhibited by arsenic exposures of 60 ppb and greater. This was accompanied by an increase in the levels and activity of MMP-9.  Mechanical wounding results in a coordinated increase in [Ca²⁺]_i among neighboring cells (calcium wave).  Arsenic at 60 ppb resulted in a slight decrease in the number of cells involved in the calcium wave.  However, the difference was not significant.  Because wound-induced calcium signaling is blocked by apyrase, we also tested whether arsenite treated cells displayed altered responses to purinergic agonists.  Treatment with 60 ppb arsenite for 24 hours resulted in a significant reduction in the number of cells responding to ATP stimulation.  Over 50% of untreated cells responded with increased [Ca²⁺]_i after stimulation with 1 mM ATP.  60 ppb arsenite treatment reduced the percentage of responding cells to 20%.  While over 30% of cells responded to 500 nM ATP stimulation, this was reduced to 4% in arsenic treated cells.  Taken together, these data indicate that environmental levels of arsenic can reduce purinergic dependent [Ca²⁺]_i signaling and wound healing in human bronchial epithelial cells. 

 

We have previously shown that arsenic delays the ability of cells to form monolayers.  To test if arsenic altered functional tight junctions, we repeated the experiments from above, with cells grown on nuclepore filter supports and directly measured transepithelial resistance (TER) in the presence or absence of arsenic. Arsenic added at the time of plating greatly reduced the ability of the cells to form a functional monolayer.  This was apparent even at 60 ppb.  If cells were first allowed to form a tight monolayer prior to addition of arsenic, high levels of arsenic were still capable of reducing the epithelial resistance.  Cells exposed under these conditions showed alterations in the levels and types of expression of tight junctional proteins, occuldin and claudins.

 

We have analyzed altered protein expression in the lung lining fluid and airway epithelial cells of mice exposed to arsenic in their drinking water for up to 4 weeks.  Soluble proteins in the lung lining fluid were obtained through bronchoalveolar lavage (BAL).  Proteins were identified using 2-D gel electrophoresis (N=3) or multidimensional protein identification technology (MUDPIT) (N=2) coupled with mass spectrometry (MS). A total of 44 proteins were identified.  Proteins that were seen to be present in the BAL of control animals while absent in the treated animals include:  glutathione-S-transferase omega-1 (GST-omega-1), contraspin, apolipoprotein A-I and A-IV, receptor for advanced glycation end products (RAGE) and alpha-1-antitrypsin (AAT). Proteins up regulated by arsenic included enolase-1 and peroxiredoxin-6  Using Western Blot analysis, we have shown that levels of RAGE in the BAL decreases as a function of arsenic treatment in mice treated with arsenic in their drinking water.   Previous investigators have identified GST omega-1 as an important arsenic metabolizing enzyme.  The function of RAGE in chronic inflammatory disease, wound healing and cancer has been previously reported.  In addition, alpha-1-antitrypsin levels were also affected by arsenic treatment. We have also used a selective airway epithelial cell digestion to isolate and analyze altered proteins.  Using MUDPIT analysis we have identified over 150 proteins changed by arsenic.  We have also analyzed the most likely cell functions and disease states that are associated with the altered proteins from BAL and from the cell fractions.  Using a curated analysis system (MetaCore) we have identified cell motility and alteration in wound repair as the most likely affected cellular functions and disease.  This is consistent with our in vitro data.   In order to verify wound healing as a site of action of arsenic, we have examined the effect of arsenic on the time for recovery from epithelial damage caused by exposure to naphthalene.  We have found that while normal recoevery from naphthalene exposure occurs within 2 weeks of exposure, damage in animals that had been exposed to arsenic was still present after 2 weeks.  These data indicate that asenic can affect wound healing in an animal model. 

 

Because of the importance of MMPs, RAGE and alpha-1antrypsin in chronic inflammatory diseases we investigated arsenic-induced alterations in these proteins in human induced-sputum.  Samples were collected from 56 individuals living in Ajo, Arizona (tap water arsenic ~ 20 ppb) and from Tucson, Arizona (tap water arsenic ~ 5 ppb).  First morning void urine was also collected and arsenic speciation analysis was performed.  MMP-2, MMP-9, TIMP-1, RAGE and AAT levels in sputum were determined using commercially available ELISA kits.  Regression analysis demonstrated a significant positive correlation between sputum MMP-9/TIMP-1 ratios (p=0.005) and total urinary inorganic arsenic.  Sputum levels of RAGE (p=0.016) and AAT (p-0.028) were also significantly negatively correlated with levels of total urinary arsenic.  This is a similar response to that seen in our in vitro and animal models.  Therefore, use of in vitro and animal models combined with proteomics approaches provide an unbiased determination of important biomarkers that may be related to human disease. Most recently, in a separate, higher exposed population (65 ppb) we have found that serum MMP-9 appears to be positively correlated with arsenic exposures. 

 

• Arsenic alters wound repair, both in vitro and in vivo
• Altered calcium signaling, especially through purinergic receptors
• Altered epithelial junctional proteins and function

·         Elevated MMP-9 levels in human populations exposed to arsenic

·         Alterations in calcium signaling after exposure to arsenic

·         Altered expression of junctional proteins after arsenic exposure, in vitro and in vivo

·         Decreased airway epithelial repair in mice following chronic arsenic exposure