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 billions 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 have proposed 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 focuses on developing mouse models to assess the role of selenium and selenoproteins in cancer prevention and development. In vivo carcinogenesis studies involving selenoprotein 15 (Sep15) knockout mice were undertaken to confirm our findings with cells in culture on the role of this selenoprotein in colon cancer. As reported last year, we initially examined the formation of chemically-induced aberrant crypt foci (ACF) in the colon of Sep15 mice compared to heterozygous and wild type litter mate controls. In addition, we examined the influence of dietary selenium on the formation of ACF in these three groups of mice. Interestingly, we found that that the incidence of ACF formation was dramatically reduced in Sep15 deficient mice and this finding was independent of selenium levels administered in the diet. Microarray and protein expression analyses showed that the expression of guanylate binding protein 1 (GBP-1) was significantly up-regulated in Sep15 knockout mice, and since this protein has a role in inflammation process, this observation suggests an involvement of inflammation-regulated genes. Thus, the possibility exists that the B-catenin/WNT signaling pathway may be involved due to the involvement of this pathway in inflammation. Sep15 may therefore play a stimulatory role (possible as an oncogene) in cancer etiology in colonic tissue, whereas the possible links to GBP-1 are being further examined. These results were recently submitted for publication. Because of the suspected link between Sep15 and GBP-1, we are investigating inflammation-induced tumorigenesis in Sep15 knockout mice. We have also examined the effect of acetaminophen (APAP) on mice encoding a TR1 knockout in liver as APAP hepatotoxicity (APAP-H) is the most common cause of drug-induced liver failure throughout the world. Since the depletion of glutathione (GSH) plays a major role in APAP liver damage and much of the underlying metabolism of GSH in liver damage has been elucidated, we began a study two years ago to assess the role of TR1, also a major antioxidant in mammalian cells, in APAP-H, and also examine the interrelationship of this selenoenzyme with the glutathione system. Initially, liver-specific TR1 knockout mice were treated with a hepatotoxic dose of APAP. Surprisingly, we observed that TR1-deficient mice were resistant to APAP toxicity as histology and liver enzymes used for demonstrating hepatotoxicity were not significantly different from untreated mice. Control, treated mice, however, showed extensive liver damage. The results strongly suggest that TR1 knockout mice are primed for xenobiotic detoxification primarily through NRF2 activation. The findings provide new insights into APAP-induced liver damage and suggest new therapeutic targets for prevention and/or amelioration of drug-induced liver damage. In the last year, we completed this study by demonstrating that TR1 knockout mice showed no decrease in GSH levels after APAP treatment and were primed for detoxification of APAP as indicated by increased expression and protein levels of glutathione-S transferases (GSTs) and the ATP-binding cassette transporters, ABCC3 and ABCC4, which likely contribute to the conjugation and rapid expulsion from hepatocytes. This was also evident by the increased production of urinary CYS-APAP (produced from GSH-APAP) in the TR1-deficient mice. However, and likely most intriguing, knockout mice showed potent induction of c-JUN and c-FOS following APAP exposure similar to wild-type counterparts, and exhibited phosphorylation of JNK, a key initiator of hepatic necrosis and apoptosis. Another study that has now been completed and published during this fiscal year involved an examination of the role of TR1 in liver cancer, wherein we previously exposed liver TR1 knockout mice to the liver carcinogen, diethylnitrosamine, (DEN). We found that the incidence of tumor development in liver-specific TR1 knockout mice was much higher than control mice, wherein 90% of the knockout mice had tumors compared to 16% of control mice. The TR1-dependent effect was observed independent of sex, and, in control mice, tumorigenesis did not affect the expression of TR1. We observed up-regulation of another selenoenzyme, glutathione peroxidase 2, and components of the glutathione system, including those that generate reduced glutathione. Interestingly, the study showed that TR1 protected against chemically induced hepatocarcinogenesis via the control of the cellular redox state, whereas its role in promoting this type of cancer is minimal. In fact, the role of TR1 in liver appears only to involve one of protecting hepatocytes from malignancy that is unlike other organs we have examined, wherein TR1 has a split personality in both protecting and promoting cancer. We have previously shown that selenoproteins play a role in proper macrophage function by targeting the removal of the selenocysteine tRNA gene, and thus all selenoproteins, in macrophage, that TR1 is up-regulated in macrophage upon stimulation with lipopolysaccharide (LPS) and that this stimulation is dependent upon p38 signaling. We began a study on examining the role of TR1 in macrophage function by developing and characterizing a macrophage-specific TR1 knockout mouse and found that this mouse had no overt phenotype. However, in the last year, we assessed the alterations in thioredoxin-dependent inflammatory signaling and showed, by 75Se-labeling during LPS stimulation, that TR1 is the only LPS-inducible selenoprotein in macrophages. TR1 induction occurred at the transcriptional level and was dependent on the intracellular signaling pathways mediated by p38 MAP kinase and IkB kinase. Macrophage-specific ablation of TR1 in mice also resulted in a drastic decrease in the expression of VSIG4, a B7 family protein known to suppress T cell activation. These results revealed TR1 as both a regulator and a regulated target in the macrophage gene expression network, and suggest a link between selenium metabolism and immune signaling. We have also completed and published another study wherein we established a breast selenoprotein conditional knockout mouse model using loxP-Cre technology to target the removal of the selenocysteine tRNA gene for elucidating the role of selenoproteins in breast cancer. Mice were administered a breast cancer carcinogen, 7,12-dimethylbenzylbenz[a]antracene. 55.0% of the selenoproteinless mice developed mammary tumors and exhibited significantly shorter survival than the corresponding control mice, wherein only 36.4% developed tumors. The results provided evidence that mice not expressing selenoproteins in breast epithelial cells are much more likely to develop carcinogen-induced mammary tumors and that selenoproteins provide a protection against carcinogen-induced mammary cancer.
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