Selenium and Selenoprotein Deficiencies Induce Widespread Pyogranuloma Formation in Mice, while High Levels of Dietary Selenium Decrease Liver Tumor Size Driven by TGFα

Changes in dietary selenium and selenoprotein status may influence both anti- and pro-cancer pathways, making the outcome of interventions different from one study to another. To characterize such outcomes in a defined setting, we undertook a controlled hepatocarcinogenesis study involving varying levels of dietary selenium and altered selenoprotein status using mice carrying a mutant (A37G) selenocysteine tRNA transgene ($Trsp^{tG37}$) and/or a cancer driver TGFα transgene. The use of $Trsp^{tG37}$ altered selenoprotein expression in a selenoprotein and tissue specific manner and, at sufficient dietary selenium levels, separate the effect of diet and selenoprotein status. Mice were maintained on diets deficient in selenium (0.02 ppm selenium) or supplemented with 0.1, 0.4 or 2.25 ppm selenium or 30 ppm triphenylselenonium chloride (TPSC), a non-metabolized selenium compound. $Trsp^{tG37}$ transgenic and TGFα/$Trsp^{tG37}$ bi-transgenic mice subjected to selenium-deficient or TPSC diets developed a neurological phenotype associated with early morbidity and mortality prior to hepatocarcinoma development. Pathology analyses revealed widespread disseminated pyogranulomatous inflammation. Pyogranulomas occurred in liver, lungs, heart, spleen, small and large intestine, and mesenteric lymph nodes in these transgenic and bi-transgenic mice. The incidence of liver tumors was significantly increased in mice carrying the TGFα transgene, while dietary selenium and selenoprotein status did not affect tumor number and multiplicity. However, adenoma and carcinoma size and area were smaller in TGFα transgenic mice that were fed 0.4 and 2.25 versus 0.1 ppm of selenium. Thus, selenium and selenoprotein deficiencies led to widespread pyogranuloma formation, while high selenium levels inhibited the size of TGFα–induced liver tumors

Selenium and Selenoprotein Deficiencies Induce Widespread Pyogranuloma Formation in Mice, while High Levels of Dietary Selenium Decrease Liver Tumor Size Driven by TGFα

Changes in dietary selenium and selenoprotein status may influence both anti- and pro-cancer pathways, making the outcome of interventions different from one study to another. To characterize such outcomes in a defined setting, we undertook a controlled hepatocarcinogenesis study involving varying levels of dietary selenium and altered selenoprotein status using mice carrying a mutant (A37G) selenocysteine tRNA transgene (Trsp[superscript tG37]) and/or a cancer driver TGFα transgene. The use of Trsp[superscript tG37] altered selenoprotein expression in a selenoprotein and tissue specific manner and, at sufficient dietary selenium levels, separate the effect of diet and selenoprotein status. Mice were maintained on diets deficient in selenium (0.02 ppm selenium) or supplemented with 0.1, 0.4 or 2.25 ppm selenium or 30 ppm triphenylselenonium chloride (TPSC), a non-metabolized selenium compound. Trsp[superscript tG37] transgenic and TGFα/Trsp[superscript tG37] bi-transgenic mice subjected to selenium-deficient or TPSC diets developed a neurological phenotype associated with early morbidity and mortality prior to hepatocarcinoma development. Pathology analyses revealed widespread disseminated pyogranulomatous inflammation. Pyogranulomas occurred in liver, lungs, heart, spleen, small and large intestine, and mesenteric lymph nodes in these transgenic and bi-transgenic mice. The incidence of liver tumors was significantly increased in mice carrying the TGFα transgene, while dietary selenium and selenoprotein status did not affect tumor number and multiplicity. However, adenoma and carcinoma size and area were smaller in TGF alpha transgenic mice that were fed 0.4 and 2.25 versus 0.1 ppm of selenium. Thus, selenium and selenoprotein deficiencies led to widespread pyogranuloma formation, while high selenium levels inhibited the size of TGFα-induced liver tumors

Multi-organ pyogranulomatous/granulomatous inflammation^a.

a<p>Incidence of pyogranulomatous inflammation in mice maintained on the Torula yeast based diet. Animals were sacrificed at 120 days and various tissues examined for the occurrence of pyogranulomatous/granulomatous inflammation. Pyogranuloma incidence of groups for each organ were compared using Fisher’s Exact test (results given in text).</p>b<p>Values shown represent incidence of pyogranulomatous inflammation/number of mice used in the study and the value in parenthesis is the percent incidence.</p

Labeling of selenoproteins in liver.

<p>Mice were labeled with ⁷⁵Se, protein extracts prepared, aliquots of the extracts electrophoresed on denaturing gels, the developed gels stained with Coomassie Blue to use as a loading control (lower panel) and ⁷⁵Se-labeled selenoproteins detected using a PhosphorImager (upper panel). Molecular weights of proteins are indicated on the left of the panels and selenoprotein identity by arrows on the right of the upper panel. Details of the procedures are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057389#s2" target="_blank">Materials and Methods</a>.</p

Levels and distributions of the Sec tRNA^[Ser]Sec isoforms in liver of mice^a.

a<p>Bulk tRNA was isolated from liver of the four mouse lines (column 1) that were maintained on different diets (column 2), the tRNA aminoacylated with ³H-serine, the resulting labeled seryl-tRNA fractionated on a RPC-5 column, the percent of the i⁶A⁻and wild type seryl-tRNA^[Ser]Sec isoforms within the total seryl-tRNA^[Ser]Sec population determined (columns 3 and 4, respectively), the distributions of mcm⁵U and mcm⁵Um assessed as the percent of wild type mcm⁵U and mcm⁵Um seryl-tRNA^[Ser]Sec isoforms within the total seryl-tRNA population (columns 5 and 6, respectively) and ratio of mcm⁵Um to mcm⁵U determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057389#s2" target="_blank">Materials and Methods</a>.</p

Area and size of liver tumors^a.

a<p>Tumor number (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057389#pone-0057389-t003" target="_blank">Table 3</a>), size, and area were assessed at necropsy and compared using least square means procedures (see text). Tumor numbers are shown as raw means ± standard deviations. The sizes of approximately 15% of tumors were not measured, including all tumors for Group 4.</p>b<p>Tumor area and size are shown as geometric means ± standard deviations.</p>c<p>NA, no size measure for tumors available.</p

Pyogranulomatous inflammation of the mesenteric lymph node.

<p>Cross section showing confluent pyogranulomatous inflammation (PG) with caseous necrotic centers (arrows) (shown at at 4X magnification). Details of processing and staining of the tissue are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057389#s2" target="_blank">Materials and Methods</a>.</p