Frank A. Witzmann

An updated two-dimensional gel database of rat liver proteins useful in gene regulation and drug effect studies.

A standard two‐dimensional (2‐D) protein map of Fischer 344 rat liver (F344MST3) is presented, with a tabular listing of more than 1200 protein species. Sodium dodecyl sulfate (SDS) molecular mass and isoelectric point have been established, based on positions of numerous internal standards. This map has been used to connect and compare hundreds of 2‐D gels of rat liver samples from a variety of studies, and forms the nucleus of an expanding database describing rat liver proteins and their regulation by various drugs and toxic agents. An example of such a study, involving regulation of cholesterol synthesis by cholesterol‐lowering drugs and a high‐cholesterol diet, is presented. Since the map has been obtained with a widely used and highly reproducible 2‐D gel system (the Iso‐Dalt® system), it can be directly related to an expanding body of work in other laboratories.

Proteomics: Applications and opportunities in preclinical drug development

Advances in DNA sequencing and the near‐term availability of whole genome sequences for several pharmaceutically relevant organisms promise to dramatically alter the breadth and scale of high‐throughput proteomic studies. The substantial amount of literature is available in the public domain, demonstrate the potential of proteomics in the preclinical phases of pharmaceutical development. Over the next few years, it is anticipated that functional genomics and proteomics will have major impacts on the clinical phases of drug development. Expected benefits are earlier proof‐of‐concept studies in man and increased efficiency of clinical trials through the availability of biologically relevant markers for drug efficacy and safety.

Regional protein alterations in rat kidneys induced by lead exposure.

Lead is a potent neuro‐ and nephrotoxin in humans and a renal carcinogen in rats. Previous studies have detected lead‐induced increases in the activities of specific detoxification enzymes in distinct kidney cell types preceding irreversible renal damage. While preferential susceptibility of the highly vascularized cortex to the effects of lead is clear, lead effects on the medullary region have remained unexplored. The present study was undertaken to investigate the extent to which regional renal protein expression differs and to determine which, if any, regionally distinct protein markers indicative of leads renotoxic mechanism might be detected in kidney cortical and medullary cytosols. We examined protein expression in these two functionally and anatomically distinct regions, and identified several proteins that are differentially expressed in those regions and were significantly altered by lead. Kidney cytosols from rats injected with lead acetate (114 mg/kg, three consecutive daily injections) were separated by two‐dimensional electrophoresis. Lead exposure significantly (P<0.001) altered the abundance (either ↑ or ↓) of 76 proteins in the cortex and only 13 in the medulla. Eleven of the proteins altered in the protein patterns were conclusively identified either by matrix‐assisted laser desorption/ionization mass spectrometry / electrospray ionization‐mass spectrometry (MALDI‐MS/ESI‐MS) analysis of peptide digests, immunological methods, or by gel matching. Several of the cortical proteins altered by lead were unchanged in the medulla while others underwent similar but lesser alterations. These observations reflect the complexity of leads nephrotoxicity and endorse the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicologic paradigms.

Pharmacoproteomics in drug development

ABSTRACTThe field of proteomics is taking on increased significance as the relevance of investigating and understanding protein expression in disease and drug development is appreciated. Recent advances in proteomics have been driven by the availability of numerous annotated whole-genome sequences and a broad range of technological and bioinformatic developments that underscore the complexity of the proteome. This review briefly addresses some of the various technologies that comprise Expression Proteomics and Functional Proteomics, citing examples where these emerging approaches have been applied to pharmacology, toxicology, and the development of drugs.

Proteomic analysis of simulated occupational jet fuel exposure in the lung.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss‐Webster mice exposed 1 h/day for seven days to aerosolized JP‐8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid‐base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP‐8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

Proteomic analysis of the renal effects of simulated occupational jet fuel exposure

We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss‐Webster mice exposed 1 h/day for five days to aerosolized JP‐8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large‐scale, high‐resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP‐8 effect on protein expression in the kidney and provide novel molecular evidence of JP‐8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP‐8 exposed humans.

Toxicity of chemical mixtures:Proteomic analysis of persisting liver and kidney protein alterations induced by repeated exposure of rats to JP‐8 jet fuel vapor

Male Sprague‐Dawley rate were exposed by whole body inhalation to 1000 mg/m3 ± 10% JP‐8 jet fuel vapor or room air control conditions for 6 h/day, 5 days/week for six consecutive weeks. Following a rest period of 82 days rats were sacrificed, and liver and kidney tissues examined by proteomic methods for both total protein abundance and protein charge modification. Kidney and lung samples were solubilized and separated via large scale, high resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Through the use of peptide mass fingerprinting, confirmed by sequence tag analysis, three altered proteins were identified and quantified. Numerical, but not significantly different increases were found in total abundance of lamin A (NCBI Accession No. 1346413) in the liver, and of 10‐formyltetrahydrofolate dehydrogenase (10‐FTHF DH, #1346044) and glutathione‐S‐transferase (GST; #2393724) in the kidneys of vapor‐exposed subjects. Protein charge modification index (CMI) analysis indicated significant alterations (P <u20050.001) in expressed lamin A and 10‐FTHF DH. These persisting changes in liver and kidney proteins are discussed in terms of possible alterations in the functional capacity of exposed subjects.