Selenium is an essential micronutrient in the diet of humans and other mammals. Many health benefits have been attributed to selenium that include preventing various forms of cancer (e.g., colon, prostate, lung and liver cancers), heart disease and other cardiovascular and muscle disorders. Numerous human clinical trials have been undertaken in recent years to assess the role of this element in cancer prevention, delaying the progression of AIDS, etc., at a cost of hundreds of millions of dollars, but little is known about the mechanism of how selenium acts at the metabolic level in mammals to exert these many health benefits. We proposed several years ago that the health benefits of selenium are due largely to its presence in selenoproteins as the selenium-containing amino acid, selenocysteine (Sec). Our program therefore focused on developing mouse models to assess the role of all selenium-containing proteins, of two subclasses of selenium-containing proteins, designated housekeeping and stress-related selenoproteins, and on individual selenoproteins in preventing and promoting cancer and in mammalian development. In the past year, we completed and published, or completed and submitted for publication, the following collaborative studies: 1) the role of two individual selenoproteins, thioredoxin reductase 1 (TR1) and glutathione peroxidase 4 (GPx4), in skin development with Dr. Stuart Yuspa's group. The targeted removal of the GPx4 gene in keratinocytes altered epidermal differentiation and disturbed hair follicle morphogenesis, while TR1 loss had no apparent phenotypic affect; 2) the role of the Sec insertion sequence element protein, designated SPB2, was confirmed to be essential in embryonic development and in the incorporation of Sec into selenoproteins with Dr. Ulrich Schweizer's group; and two additional studies showing that reduced selenoprotein expression in brain tissue caused cerebellar hypoplasia and coordination defects in mice as a result of striatal neuronal loss; 3) the role of hypomethylation induced in mammalian cells resulted in the loss of the Um34 Sec tRNA isoform and consequently down-regulation of stress-related selenoprotein expression with Dr. Diane Handy's group; and 4) the double knockdown of TR1 and the 15-kDa selenoprotein, Sep15, in mouse colon cancer cells reversed the anticancer effects that we previously found with the single knockdown of either of these genes with Dr. Petra Tsuji suggesting that Sep15 and TR1 participate in competing or interfering regulatory pathways in colon cancer cells. One ongoing collaboration we have is with Dr. Krishna Chatterjee on assessing the effect of a C-G mutation at position 65 in Sec tRNA on selenoprotein synthesis in an eight year old male. The patient suffers from fatigue, muscle weakness and abdominal discomfort. The available evidence suggested that this mutation results in Sec tRNA failing to synthesize Um34 and thus the patient cannot make stress-related selenoproteins. We are further confirming these observations. In addition, we had previously found that the knockout of the Sec tRNA gene, Trsp, resulted in the total loss of selenoprotein synthesis in mouse liver, but the animals survived for several months. Surprisingly, the knockout of GPx4 in liver resulted in death within the first 24-48 hours after birth, while knockout of TR1 did not cause any apparent phenotype. However, in the knockout of Trsp in liver that resulted in the total loss of selenoprotein expression, both GPx4 and TR1 were synthesized as a result of amino acid replacement of Sec with another amino acid . We are presently determining the amino acid that is present in place of Sec in non-selenium-containing GPx4 and TR1 proteins and whether these proteins have a role in liver metabolism. We are also examining the antioxidant proteins in liver and lung tumors and comparing them to the corresponding surrounding normal tissues and to each other; and we undertook this project because cancer cells are known to suffer from oxidative stress and must have enriched antioxidants and/or antioxidant systems to combat the burdens of oxidative stress, Thus far, we have found differences in the thioredoxin and glutathione systems between liver and lung cancer tissues and differences in the antioxidant enzymes, superoxide dismutase 1 and catalase. Thioredoxin reductase 1 (TR1) levels were elevated in both malignant tissues compared to surrounding normal tissues as assessed by western blotting, but their enzymatic activities were similar suggesting amino acid replacement of the Sec moiety. We are currently identifying the amino acid at the Sec site in TR1 in both liver and lung tumors.