Toxicoproteomics--the next step in the evolution of environmental biomarkers.

The scientific community has become increasingly concerned about the potential adverse health effects to humans and wildlife resulting from environmental exposure to persistent industrial, pharmaceutical, and natural chemicals with estrogenic, androgenic, or thyroid-disrupting properties (Colborn et al., 1993; Waring and Harris, 2005). To assess exposure to these endocrine-disrupting chemicals (EDCs), researchers have employed a variety of molecular biomarkers. A high-quality biomarker of a specific chemical class or specific mechanism of action should have the following attributes: (1) the biomarker should be inducible or repressible, (2) the measured response should be specific to chemicals within that class, (3) the response should have sufficient sensitivity for routine detection, (4) the biomarker should be highly accurate and reproducible among experiments within a laboratory and among different laboratories and animal models, and (5) the biomarker should be quantifiable so that degree of risk can be estimated. One of the most utilized biomarkers of EDC exposure is the egg yolk precursor protein vitellogenin (Vtg), which is highly expressed in the liver and plasma of oviparous animals in response to 17bestradiol. Induction of plasma Vtg in fish has been routinely used as a biomarker of xenoestrogen exposure, both in controlled laboratory settings and in field studies (Sumpter and Jobling, 1995), and is currently being incorporated as a standard tier 1 bioassay within the U.S. Environmental Protection Agency Endocrine Disruptor Screening Program. Other examples of biomarkers of estrogenicity include in vitro assessment of MCF-7 cell proliferation, estrogen receptor binding and transactivation, and in vivo assessment of endometrial thickness in mammals. Recent technological advances in the areas of genomics, proteomics, and metabolomics have provided researchers with new tools for developing biomarkers, specifically indicators that reflect both chemical exposure and the subsequent biological effect. This paradigm shift is particularly relevant in that traditional, single end point bioassays for xenoestrogens, such as the Vtg assay, provide little insight about the physiological consequences of exposure, particularly when attempting to estimate potential adverse human health effects. These new ‘‘omics’’ disciplines apply high-throughput methodologies in which changes in expression of hundreds to thousands of genes, proteins, or metabolites are assessed simultaneously. A direct comparison of expression values obtained for a control versus an altered condition reveals a set of biomarkers indicative of that altered state. This exposure ‘‘fingerprint’’ can then be used as a tool for classifying chemical exposures and predicting mode of action (Hamadeh et al., 2002). The use of toxicogenomic and toxicoproteomic approaches to biomarker discovery can be widely applied to both environmental and clinical exposure scenarios, such as environmental exposure to xenoestrogens in wastewater effluent or altered gene or metabolite expression in response to disease. The greatest advances to date in the application of ‘‘omics’’ technologies to EDC biomarker development have been in the field of toxicogenomics. Both commercial and custom DNA microarray platforms have been utilized to evaluate changes in gene expression in response to a number of known toxicants, including xenoestrogens, in both mammalian and aquatic animal models (Benninghoff and Williams, 2006; Brown et al., 2004; Larkin et al., 2003; Moens et al., 2006; Naciff et al., 2002; Wang et al., 2004). In these studies, gene sets that were responsive to estrogen exposure were identified, and the biological processes associated with these transcriptional profiles were evaluated to gain insight into the network of biological processes responding to the chemicals in question. While gene expression profiling has proven to be useful in the development of biomarkers of EDC exposure, one must recognize that changes in rates of gene transcription do not necessarily correlate with protein expression or protein activity 1 For correspondence via fax: (541) 737-7966. E-mail: benninga@science. oregonstate.edu.