Introduction

proteins fractionated on a two-dimensional gel, robotic excision of proteins from the gel, and identification of the proteins by mass spectrometry.12

Examples of experimental designs using proteomics are as follows: Proteins are identified and compared in samples from animals treated or not treated with a test material; proteins are identified whose expression changes after exposure to the test material which may be involved in a toxic response to the test material in the target tissue. A similar experiment could be carried out with normal or diseased tissue, identifying proteins that are involved in etiology or progression of a particular disease.

Proteomics is viewed as a sister technology to genomics and there is a strong synergy between the two approaches. However, in some cases gene expression is abnormal in a diseased tissue, but comparable change is not observed at the protein level, and protein expression in the diseased tissue correlates poorly with gene expression. This is especially true when a xeno-biotic chemical interferes with protein synthesis and/or posttranslational modification of a protein. Therefore, the information that can be derived about a pathological process from gene expression is less complete than the information that can be derived from proteomics. Proteomics has the potential to reveal the full phenotypic picture of secreted proteins.

Proteomic analysis of body fluids can be of particular value in identifying potential noninvasive biomarkers.34 This capability is enhanced by the ability to remove high abundance proteins from the gel such as albumin, IgG, haptoglobulin, and transferrin. When an immunoaffinity-based enrichment technique is used to remove these proteins, hundreds of proteins can be identified that would not have been detected due to strong signals from the high abundance proteins.

When using any novel technology, scientific rigor is paramount. It is crucial to have a sound scientific protocol based on good toxicological principles for generating samples.5 There is also a need for a comprehensive and careful analysis of the results. These principles will be demonstrated in the experiments described below, in which the proteome of the rat was studied in animals dosed with the antibiotic gentamicin.3 The proteomics analysis was compared to more conventional techniques including clinical chemistry, haematology, and histopathology, and the proteomics approach compared favorably. The study was designed to answer the following questions:

1. Can a dose response be identified in the proteome?

2. Is proteomic analysis more sensitive than classical endpoints?

3. Is the drug-induced expression of up-regulated or down-regulated proteins reversed after a drug-free withdrawal phase?

4. Can protein markers of toxicity be identified?

5. Can additional mechanistic information be acquired?

The following protocol was designed to answer some of these questions. The protocol also took into account available information concerning renal toxicity of gentamicin.

Table 23.1 Seven Days of Treatment with Gentamicin Sulphate Followed by 14-day recovery period

Route of administration:

intravenous

Dose levels:

0, 0.1, 1.0, 10.0, 40.0, 60.0 mg/kg/day

Group size:

10 male rats per treated group

20 male rats in control group

Blood and urine samples:

2, 3, and 8 days

Blood Parameters:

BUN and creatinine

Urine parameters:

NAG, GGT, volume, and specific gravity

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