The slow pace of discovery of new protein biomarkers of toxicity or “toxicity signature protein/peptides” is partially due to the lack of high-throughput and high-sensitivity biomarker devices. Traditional proteomics platforms have been very useful, but there is a general consensus among scientists that significant improvements are needed to further our research into the understanding of injurious agents and how they affect biological systems during toxicity and disease. Some of the drawbacks of individual technologies have prompted investigators to use complementary multiple proteomics platforms that can bring more data to bear upon toxicological problems.
In the last few years, there has been a greater appreciation by the scientific community of proteomics platforms that use antibody or other affinity reagents to selectively capture a single or a closely related group of chemical substances. The tandem or on-line coupling of highly selective antibody capture agents with the high-resolving power of capillary electrophoresis is gaining recognition as a powerful analytical tool for the enrichment and quantitation of ultralow-abundance analytes in complex matrices. This development will have a significant impact on the identification and characterization of many putative biomarkers and on biomedical research in general.
Immunoaffinity capillary electrophoresis (IACE) technology is rapidly emerging as the most promising method for the analysis of low-abundance biomarkers. Its power comes from a three-step procedure: 1) bioselective adsorption and 2) subsequent recovery of compounds from an immobilized affinity ligand followed by 3) separation of the enriched compounds. This technology is highly suited to automation and can be engineered as a multiplex instrument capable of routinely performing hundreds of assays per day. Furthermore, a significant enhancement in sensitivity can be achieved for the purified and enriched affinity targeted analytes. Thus, a compound that exists in a complex biological matrix at a concentration far below its usual limit of detection is easily brought to well within its range of quantitation.
This article describes a high-throughput, high-sensitivity immunoaffinity capillary electrophoresis instrument used as a biomarker analyzer in toxicoproteomics research.
There are numerous chemical and biological substances that have the potential to act as toxins in the body. The potentially poisonous substances can be introduced into the body by several means, and their origins are primarily derived from pharmaceutical or biopharmaceutical drug products; foods, beverages, or nutritional products; environmental hazards; or acquired through exposure to viruses, bacteria, insects, animals, etc.1–5 Toxic substances are sometimes ingested by accident. However, in many cases toxicity is produced by an overdose of drugs in suicide attempts, illicit drug consumption, medication errors, or intentional poisoning.6–10 Intentional poisoning occurs through chemical or biological criminal activity or terrorism. 9,10 Furthermore, drugs that are prescribed or over-the-counter may also produce adverse drug reactions. The poisonous chemical or biological substance can act as a toxin by directly interacting with an important cellular component, and as a consequence blocking a crucial biological process or pathway, or the substance may be converted to a metabolic product that can by itself exert the toxic effect.11
Another source of toxicity can also be generated internally in the body as a change in the homeostasis process (such as autoimmune diseases, generally considered a multifactorial etiology, including genetic and environmental factors),12 or as a product of a physical injury (such as trauma or severe burn).13 Under these conditions, a disregulated immune system can be formed, triggering a cascade of cellular and biochemical changes and the production of certain proinflammatory biological substances and/or antibodies directed to some protein constituents of the same person. These antibodies directed against a person’s own body are called autoantibodies.12
Proteins/peptides that change homeostasis may become toxic substances having either a “mild effect” or a “killer effect” in a number of diseases. Trauma, for instance, due to massive injury leads to activation of nearly all components of the immune system. It activates the neuroendocrine system, and local tissue destruction and accumulation of toxic by-products of metabolic respiration lead to release of mediators that are responsible for the proinflammatory response.13 Among the proinflammatory mediators produced are the large peptides cytokines and chemokines. It is expected that proteomics studies will identify new protein/peptide biomarkers or “toxicity signature proteins/ peptides” related to many toxicity events.
Biomarkers are used ubiquitously as indicators of biological health. Biomarkers or molecular signatures are defined as cellular, biochemical, or genetic changes by which a normal or an abnormal biological process can be recognized or monitored. Biomarkers are measurable in biological fluids, tissues, and/or cells.14–16 The application of proteomics has generated a heightened sense of optimism in many established scientific disciplines of basic biology, medicine, toxicology, and pharmaceutical development.17–20 In the case of diseases caused by toxins, there is an emerging discipline of toxicology named toxicoproteomics that associates the presence of certain protein/peptide biomarkers in biological fluids, tissues, and/or cells as a response of the body to the exposure of certain chemical or biological substances.21,22
Although toxicoproteomics was initiated under the umbrella of toxicogenomics and proteomics, it has emerged as its own discipline. A number of reviews have been reported in this field, and many toxicologists are using several proteomics platforms to further understand how specific exposures to chemical and biological substances alter protein expression, protein behavior, and host response to cause injury and disease.23–25
Toxicoproteomics is a parallel approach in identifying biomarkers and complements the findings already in progress in the areas of toxicogenomics and toxicometabolomics. Researchers are pursuing strategies of conducting parallel DNA and proteomics analyses on the same tissues from each toxicogenomics study.26 The goal is to find new protein/peptide biomarkers, or find co- and post-translational modifications of existing proteins/peptides affected by toxicants. The appearance of new or modified protein/peptide biomarkers in biological fluids, tissues, or cells may reveal early-stage organ toxicity and disease.
Currently, a major effort is being undertaken to find biomarkers to toxicity in serum and plasma proteomes of blood. These toxicoproteomics biomarkers can uniquely reveal signs of specific organ toxicity or pathology from the peptides and proteins passively leaked or actively secreted during dysfunction. 26 However, toxic biomarkers can also be found in other biological fluids.27
Benefits of early detection of toxic biomarkers
If discovered at an early stage, many diseases are potentially curable by pharmaceutical drugs, surgery, alternative medicine, or a combination of these. The goal of modern medicine is to identify criteria that can be used to assess a particular disease or a toxic episode long before symptoms become obvious. Several studies have uncovered an association between the characterization and quantification of certain biomarkers and the diagnostic and/or prognosis of a disease or toxicological state. The earlier we begin to understand the activation of the molecular machinery that actually triggers the homeostatic imbalance, the more accurately we can detect and diagnose a particular disease.
The efficacy and toxicity of most drug therapies vary widely among individuals due to a number of factors including age, environmental exposure, genetic predisposition, and metabolic abnormality.28–30 In the United States, more than 100,000 patients die, and 2.2 million are injured annually from adverse drug reactions.28,31 Adverse drug reaction is defined as an undesirable clinical manifestation resulting from the administration of a particular drug. Adverse drug reactions are ranked as the fourth leading cause of death in the U.S. The health-care cost of drug-related morbidity and mortality in the U.S. is estimated to be in the vicinity of $180 billion annually.32 In developing new drugs, the identification and characterization of toxicoproteomics biomarkers could be used in clinical trials to identify potential individual drug responses and to ascertain the origin of proteomics characteristics, and in turn, genetic signatures.
Fatal reactions are almost always unpredictable due to the complex underlying immunological mechanisms. Judicious management of these reactions requires a proper understanding of the various immunological phenomena in their pathomechanism. In certain cases, drugs or their toxic reactive metabolites act as haptens and render some cells antigenic by binding to their surface. This interaction can trigger a drug-specific T-cell activation and the production of inflammatory cytokines.33
Figure 1 - Categorization of cytokines into prodisease development cytokines and antidisease development cytokines, based on their effect on certain disease animal models and from data obtained on inflammatory diseases affecting humans. a) The balance between proinflammatory cytokines is crucial for lesion development, and imbalance is colloquially said to exacerbate disease, as in the case of atherosclerosis. b) Overview of the cytokines with consistent antiatherogenic effects (left circle); proatherogenic effects (right circle); or variable, dual function (intersection). c) A cardiovascular diabetologic approach to the 29 known interleukins. The anti-inflammatory or protective or “good” interleukins are represented in the left circle. The aloof or “indifferent” interleukins are in the middle circle. The proinflammatory or noxious or “bad” interleukins are in the right circle. IL-3 is the “ambiguous” interleukin and is depicted in the lower half-circle. (Figure modified from Refs. 35 and 36.)
The production of inflammatory cytokines in humans often results in altered drug responses and increased toxicities, which have major implications in inflammation and infection when the capacity of the liver and other organs to handle drugs is severely compromised. Identification and characterization of some key inflammatory cytokines in biological fluids and tissue biopsies are definitively helping in the understanding of the changes that occur in important organs or tissues, and may help to predict at an early stage of medication the severity and complications to be produced by certain therapies. For example, serum levels of IFN-γ, IL-6, TNF-α, and IL-5 were increased significantly in carbamazepine-induced hypersensitivity syndrome.34 Cellular regulation by cytokines depends on the circumstances under which the cell is living at a particular time. Measurement of single cytokines may reveal one part of the story. Therefore, the identification and quantification of a panel of biomarkers is recommended in each toxic or disease state.
In the case of atherosclerosis, inflammation has emerged as a major driving force of atherosclerotic lesion development. Proinflammatory cytokines of the interleukin category are considered to be key players in this chronic inflammatory disease.35,36 The presence of interleukins and their receptors has been demonstrated in atheromatous tissue, and the serum levels of several of these cytokines have been found to correlate positively with (coronary) arterial disease and its sequelae. More refined analysis of local vascular inflammation and the cytokines expressed in atherosclerotic plaques revealed that there is a balance between proinflammatory and anti-inflammatory cytokines and that this balance is crucial for lesion development (see Figure 1 and Refs. 35 and 36).