Superfund Research Program

Integrated Response to Toxic Perturbation

Project Leader: Robert H. Rice
Co-Investigators: Alan R. Buckpitt, J. Bruce German, Dietmar Kültz
Grant Number: P42ES004699
Funding Period: 2000-2010

Project-Specific Links

Final Progress Reports

Year:   2009  2004 

Progress during the past year has focused on proteomic approaches to understanding toxic response in three major tissue targets. In addition, analysis of samples from the Salton Sea, a site accumulating hazardous agents, has been initiated as part of the effort devoted to the kidney.

1. Lung. A key issue in evaluating the possible human health consequences of exposure to environmental chemicals involves extrapolating between animal models or cultured cells to the intact human. Part of the effort concerns discovery of potential peptide markers eliminated in the urine that are associated with exposure to metabolically activated lung toxicants. For example, ¹⁴C- or ²H- labeled naphthalene are administered followed by collection of urine for 24 or 48 hrs. Although the most abundant metabolites are water soluble derivatives reported in earlier studies, a number of less abundant entities have been isolated. These include 2 labeled proteins/protein fragments of 15 and 35 kDa and a number of labeled peptides from HPLC with molecular masses between 400 and 900 Da. These presumed peptides are radioactive (from those animals treated with ¹⁴C) and have the expected 6, 7 or 8 Da shifts in both parent and daughter ions in the mass spectrum from those animals treated with a mixture of ¹H/²H-naphthalene. Six small peptides containing cysteine, lysine and/or histidine have been reacted with naphthalene oxide or naphthoquinones (naphthalene diol epoxide is being prepared as well) to study the behavior of these different reactive metabolites with nucleophilic amino acids and to act as a training set for fragmentation patterns on the mass spectra. This work shows the potential for monitoring adducted urinary peptides as a biomonitor of exposure.
2. Skin and appendages. In cooperation with the USEPA, researchers have analyzed the cross-linked proteome of the nail plate and compared it to hair shaft from individuals from Mongolia exposed to high or low concentrations of arsenic in their drinking water. Samples from unexposed individuals permitted a detailed comparison of the prominent proteins in hair versus nail. The hair samples did not display significant effects on the proteome of arsenic exposure. By contrast, samples of nail did exhibit effects of arsenic exposure on the levels of several proteins. The results raise the possibility of using nail samples to distinguish among individuals who are sensitive or insensitive to effects of arsenic.
3. Kidney. Researchers have established an LC/MS/MS workflow for protein identification and also a workflow for MALDI imaging. The MALDI imaging approach was applied to mouse kidney and enabled them to identify proteins that are primarily expressed in the inner medulla versus those that are primarily expressed in the cortex. They also identified proteins that are overrepresented in mouse inner medulla, in particular during urine concentration. Many of these proteins have chaperone and anti-oxidant functions illustrating that the corresponding biochemical pathways are critical for the ability of renal inner medullary cells to tolerate extremely high toxicant and salt concentrations that occur as a result of urine concentration in this part of the kidney.

Site. Researchers have started work on the Salton Sea (in partnership with the United States Geological Survey). The Salton Sea is a sink for many toxicants, including triclocarban (TCC) and triclosan (TCS) as well as pesticides and heavy metals. Effects of TCC and TCS on tilapia and Daphnia proteomes have been analyzed to identify the mechanistic basis of their toxicity. One MS student (Stephanie Chan) focused her thesis on TCC and TCS effects on Daphnia proteins and a PhD student (Alison Gardell) and undergraduate student (Katie Ridenour) investigated effects of TCC and TCS on the ability of tilapia to induce organic osmolyte synthesis (via myo-inositol phosphate synthase) in tilapia gill epithelium during salinity challenge. These studies are ongoing.