Contrasting roles of dietary selenium and selenoproteins in chemically induced hepatocarcinogenesis

Abstract

Selenium (Se) has long been known for its cancer prevention properties, but the molecular basis remains unclear. The principal questions in assessing the effect of dietary Se in cancer are whether selenoproteins, small molecule selenocompounds, or both, are involved, and under which conditions and genotypes Se may be protective. In this study, we examined diethylnitrosamine-induced hepatocarcinogenesis in mice lacking a subset of selenoproteins due to expression of a mutant selenocysteine tRNA gene (Trsp (A37G) mice). To uncouple the effects of selenocompounds and selenoproteins, these animals were examined at several levels of dietary Se. Our analysis revealed that tumorigenesis in Trsp (A37G) mice maintained on the adequate Se diet was increased. However, in the control, wild-type mice, both Se deficiency and high Se levels protected against tumorigenesis. We further found that the Se-deficient diet induced severe neurological phenotypes in Trsp A37G mice. Surprisingly, a similar phenotype could be induced in these mice at high dietary Se intake. Overall, our results show a complex role of Se in chemically induced hepatocarcinogenesis, which involves interaction among selenoproteins, selenocompounds and toxins, and depends on genotype and background of the animals.

Fig. 1.

Characterization of Trsp ^A37G transgenic mice and experimental design of the study. (A) FVB (control) and Trsp ^A37G mice were metabolically labeled with ⁷⁵Se by intraperitoneal injection, and 48h later sacrified and their tissues collected and labeled selenoproteins analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by PhosphorImager analysis (left panel) and immunoprecipitation with antibodies specific for indicated selenoproteins (right panel). (B) Experimental design and timing of DEN injection and tumor analysis.

Fig. 2.

Survival of Trsp ^A37G mice maintained on Se diets. FVB (wild-type) and Trsp ^A37G mice were maintained on indicated Se diets and injected with DEN. The graphs show survival of animals during the course of the experiment.

Fig. 3.

Expression of GFAP is significantly reduced in the brains of Trsp ^A37G transgenic mice. (A) GFAP expression in somatosensory cortex. Astrocytes in white matter, along the meninges, and around blood vessels are normally expressing GFAP. In Trsp ^A37G mice, including those fed with low Se and high Se diets, GFAP expression is reduced, although astrogliosis and increased GFAP are normally associated with neurodegenerative disease. (B) In the hippocampal formation, high GFAP expression is normal. In Trsp ^A37G mice, GFAP expression is clearly reduced irrespective of Se content in the diet.

Fig. 4.

Stress-related selenoproteins and liver tumor development. Mice were maintained on the 0.4 p.p.m. Se diet and subjected to DEN injection. (A) Incidence of liver lesions, including abscesses, necrosis, adenomas, carcinomas and fatty nodules in FVB and Trsp ^A37G mice. Numbers of tumor-bearing animals and a total number of animals in the group (in parentheses) are shown above the bars. (B) Representative images of livers dissected from FVB and Trsp ^A37 mice. Visible liver lesions are indicated with arrows. (C) Representative images of liver sections from tumor and surrounding normal tissues, stained with Ki67 antibodies. Ki67-containing cells are visualized by the more darkly stained cells. (D) Quantification of Ki67 immunostaining. Four animals per group were analyzed and three fields of view per section were averaged. (E) Western blot analysis of selenoproteins in livers and kidneys of FVB and Trsp ^A37G mice. Samples in different lanes represent different animals.

Fig. 5.

Both Se-deficient and Se-enriched diets protect mice from tumor development. Mice were maintained on Se diets as indicated in the figure. (A) Tumor incidence (percent of mice developing tumors) in FVB mice on Se deficient and 0.4 and 2.25 p.p.m. Se supplemented diets. Number of tumor-bearing animals and a total number of animals in the group (in parentheses) are indicated above the bars. (B) Representative images of liver sections stained with Ki67 antibodies show Ki67-containing cells are visualized by the more darkly stained cells. (C) Quantification of Ki67 immunostaining. Four mice per group were analyzed and three fields of view per section were averaged. (D) Western blot analysis of selenoproteins in livers of FVB mice on the indicated Se diets. Samples in different lanes represent different animals.

Fig. 6.

Regulation of stress-related protein expression by dietary Se, selenoprotein deficiency and background of mice. Mice were maintained on Se diets as indicated in the figure. (A) Expression in TR1 and GSTA1 in Trsp ^A37G mice and wild-type controls (FVB and C57BL6/129 backgrounds) in mice treated with DEN as indicated. (B) Expression of glutathione reductase (GR), carbonyl reductase 3 (CBR3), carbonic anhydrase 3 (CA III) and catalase (CAT) in Trsp ^A37G and control (FVB) mice treated with DEN. (C) Expression of the same proteins in FVB and C57BL6/129 mice treated with DEN.