Toxic metals (lead, cadmium, mercury and arsenic) are widely found in our environment. Humans are exposed to these metals from numerous sources, including contaminated air, water, soil and food. Recent studies indicate that transition metals act as catalysts in the oxidative reactions of biological macromolecules therefore the toxicities associated with these metals might be due to oxidative tissue damage. Redox-active metals, such as iron, copper and chromium, undergo redox cycling whereas redox-inactive metals, such as lead, cadmium, mercury and others deplete cells' major antioxidants, particularly thiol-containing antioxidants and enzymes. Either redox-active or redox-inactive metals may cause an increase in production of reactive oxygen species (ROS) such as hydroxyl radical (HO.), superoxide radical (O2.-) or hydrogen peroxide (H2O2). Enhanced generation of ROS can overwhelm cells' intrinsic antioxidant defenses, and result in a condition known as "oxidative stress". Cells under oxidative stress display various dysfunctions due to lesions caused by ROS to lipids, proteins and DNA. Consequently, it is suggested that metal-induced oxidative stress in cells can be partially responsible for the toxic effects of heavy metals. Several studies are underway to determine the effect of antioxidant supplementation following heavy metal exposure. Data suggest that antioxidants may play an important role in abating some hazards of heavy metals. In order to prove the importance of using antioxidants in heavy metal poisoning, pertinent biochemical mechanisms for metal-induced oxidative stress should be reviewed.

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In vitro studies demonstrate that the hydroxyl radical converts L-phenylalanine into m-tyrosine, an unnatural isomer of L-tyrosine. Quantification of m-tyrosine has been widely used as an index of oxidative damage in tissue proteins. However, the possibility that m-tyrosine might be generated oxidatively from free L-phenylalanine that could subsequently be incorporated into proteins as an L-tyrosine analogue has received little attention. In the present study, we demonstrate that free m-tyrosine is toxic to cultured CHO (Chinese-hamster ovary) cells. We readily detected radiolabelled material in proteins isolated from CHO cells that had been incubated with m-[14C]tyrosine, suggesting that the oxygenated amino acid was taken up and incorporated into cellular proteins. m-Tyrosine was detected by co-elution with authentic material on HPLC and by tandem mass spectrometric analysis in acid hydrolysates of proteins isolated from CHO cells exposed to m-tyrosine, indicating that free m-tyrosine was incorporated intact rather than being metabolized to other products that were subsequently incorporated into proteins. Incorporation of m-tyrosine into cellular proteins was sensitive to inhibition by cycloheximide, suggesting that protein synthesis was involved. Protein synthesis using a cell-free transcription/translation system showed that m-tyrosine was incorporated into proteins in vitro by a mechanism that may involve L-phenylalanine-tRNA synthetase. Collectively, these observations indicate that m-tyrosine is toxic to cells by a pathway that may involve incorporation of the oxidized amino acid into proteins. Thus misincorporation of free oxidized amino acids during protein synthesis may represent an alternative mechanism for oxidative stress and tissue injury during aging and disease.

During the last decade there has been growing interest in physical-chemical oxidation processes and the behavior of free radicals in living systems. Radicals are known as intermediate species in a variety of biochemical reactions. Numerous techniques, assays and biomarkers have been used to measure reactive oxygen and nitrogen species (ROS and RNS), and to examine oxidative stress. However, many of these assays are not entirely satisfactory or are used inappropriately. The purpose of this chapter is to review current EPR (Electron Paramagnetic Resonance) spectroscopy methods for measuring ROS, RNS, and their secondary products, and to discuss the strengths and limitations of specific methodological approaches.

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