Superfund Research Program
Integrated Response to Toxic Perturbation
Metabolomic approaches to characterizing toxic responses. In the first of two approaches, Dr. Rice and his research team compare peaks between a treated sample and untreated control using LC-Time of Flight Mass Spectrometry (LC-TOF) to obtain a global fingerprint of exposure. Exemplified using cells treated with arsenic, the researchers found that such analyses resulted in data sets too large for their computers to handle. Thus they effectively used the LC to compress the sample so that the detector was not overwhelmed and ion suppression did not occur. The Core binned the mass units at high resolution and computationally eliminated the LC component. This simplifies the data set while the high resolution TOF gives a unique mass for each compound. Using PCA to find differences between the samples, the researchers determined which peaks changed dramatically during the exposure. Although some practitioners of metabolomics stop here, they proceed to identify the compound responsible for the peak and develop highly quantitative assays. Because of small sample sizes, the researchers use a combination largely of MS technologies. The long term goal is to combine analytes from many exposures to different toxicants so as to distinguish changes above background, despite variation in age, sex, nutrition, etc., to yield a highly quantitative and annotated toxicology analyte collection. The second approach is to target a metabolic pathway or metabolite class to develop a quantitative and well characterized multi analyte procedure. The Core has targeted the tryptophan pathway for neurobiology, the eicosanoid or regulatory lipid pathway for inflammation, and the vitamin D pathway for osteoporosis and cancer. These metabolomic approaches to the arachidonic acid cascade, for example, are being used to characterize several inflammatory models.
Proteomic approach to differentiated cells as toxic targets. Use of hair and nail as indicators of deleterious exposures to cells of the skin and, by extension, the rest of the body rely on detection of trace chemical residues. An alternate type of indicator, perturbation of cellular function, would be greatly assisted by knowing the protein complement so that changes in gene expression could be inferred. Advances in mass spectrometry now permit identification of proteins in moderately complex mixtures after tryptic digestion. An application of this approach to the hair shaft revealed the presence of H350 identifiable proteins. Most of these were previously not known to be found in hair because extensive protein cross-linking prevented their disaggregation and isolation. A similar approach has identified an initial H70 proteins in the nail plate and a similar number in the cross-linked envelope of mature epidermal corneocytes. The results point to sets of proteins that are distinct for each keratinocyte lineage and that overlap in expression among the three compartments. This information promises to be of importance for understanding differential gene regulation among different keratinocyte lineages and its perturbation by toxic exposures. Another project involves characterization of the murine renal cell proteome. The Core has mapped and identified many proteins of the papilla and cortex isolated from mouse kidneys, finding numerous ones, some unique to the kidney, that are involved in specific kidney functions, including the excretion of nephrotoxins. This is important to understand how the kidney responds to and compensates for high local nephrotoxin concentrations. In the process, Core researchers have developed and optimized a new MALDI matrix (HABA) that gives better ionization and higher sequence coverage of peptides.
DNA microarray approach to characterize keratinocyte immortalization. To help understand how toxic agents, acting as carcinogens, may induce immortalization in target cells, an early step in tumorigenesis, a DNA microarray study was performed on keratinocyte strains from two unrelated skin samples that underwent spontaneous immortalization. The basic defect in the cells appeared to be naturally elevated telomerase activity that extended the cell lifespan until rare variants evaded senescence and took over the population. An improved protocol for analysis of DNA microarray data permitted detection of 707 transcriptional changes, among them reduced expression of the p16Ink4A tumor suppressor. Telomerase catalytic subunit mRNA was greatly elevated in the cells of low passage compared to normal cells, and the level increased dramatically with passage in concert with increased colony forming ability. The data raise the possibility of an unusual, tumor prone natural phenotype that could be screened in the human population. Cells with this property are likely sensitive to environmental insults leading to a carcinogenic outcome.