Superfund Research Program
Integrated Response to Toxic Perturbation
Work during the past year has focused on proteomic approaches to understanding toxic response in three major tissue targets of toxic agents.Kidney
Using 2D gel analysis and MALDI-Tof/ Tof mass spectrometry (MS), the research team has identified ~160 proteins of mouse kidney that are differentially expressed (>2 fold difference) in the renal papilla relative to the cortex. The researchers hypothesize that these proteins represent key nodes of intracellular networks of stress signaling and of effect or mechanisms that confer extreme stress tolerance to the renal papilla. These proteins are currently being analyzed further using bioinformatic network modeling. Region-specific expression of some of these proteins has been confirmed with Western blot analysis and immunohistochemistry. The investigators from this project have also developed a new quantitative workflow and set up the necessary infrastructure and obtained necessary permits and clearances for utilizing tilapia larvae and HEK293 (human) and mIMCD3 (mouse) kidney cell lines for mechanistic studies of arsenic toxicity, singly and in combination with other relevant environmental toxins of interest to Superfund.Lung
The research team has extended its work focusing on posttranslational modifications of protein thiols in pulmonary epithelial cells to compare changes in thiol status of individual proteins in response to the non cytotoxic glutathione depletor, diethylmaleate with the response to naphthalene, a cytotoxic glutathione depletor. Naphthalene and diethylmaleate caused significant changes in the sulfhydryl status of 82 proteins total compared to control (p<0.05). Administration of diethylmaleate resulted in the oxidation of greater numbers of proteins to a greater extent than those recovered from murine airway epithelial samples after naphthalene. However, several proteins containing reactive thiols are oxidized to a greater extent by naphthalene compared to diethylmaleate including those involved in transcriptional regulation (zinc finger proteins), protein synthesis/folding (ribosomal proteins, serine peptidase inhibitor), cell growth/cycle control, and apoptosis (Suv39h2 Histone-lysine N-methyltransferase, Bcl2-associated transcription factor 1). The researchers conclude that specific proteins/protein pathways are disrupted by oxidation/arylation in response to naphthalene that are less affected by nontoxic doses of diethylmaleate.Skin
Pursuant to determining the proteome of human hair shaft last year, the project leaders have now performed similar analyses on human nail plate and epidermal callus. Using a streamlined approach that permits much faster data gathering, they have identified roughly 100 proteins in each case. Little overlap among the proteins in hair and epidermis was detected, but nail exhibited considerable overlap with hair and epidermis. The results provide a new perspective on the cross-linking process by which epidermis and its appendages are stabilized, emphasizing its efficiency, and provide a foundation for assessing changes induced by toxic exposures, genetic disease and their interaction.