Mammary cancer promotion and MAPK activation associated with consumption of a corn oil-based high-fat diet.

Our earlier work has shown a selective promotional effect on the genesis of mammary carcinomas bearing a wild-type, but not mutant, Ha-ras codon 12 in a 1-methyl-1-nitrosourea (MNU)-induced carcinogenesis model by high-fat diets (Nutr Cancer 23, 283-290, 1995). To test the hypothesis that activation of the mitogen-activated protein kinase (MAPK) pathway is associated with this promotional effect, we compared the in vivo MAPK phosphorylation state of carcinomas from rats consuming a low-fat (5% corn oil, modified AIN-93G) with that from rats consuming a high-fat (25% corn oil) diet. Specifically, 21-day-old female Sprague-Dawley rats were given an intraperitoneal injection of MNU and one week later were randomized to the two diets for six weeks. The number of mammary carcinomas per rat was 68% greater in the high-fat group, and Ha-ras mutation was rare in this short-term model. The levels of the phosphorylated MAPK2 (active) and of proliferating cell nuclear antigen (PCNA) were significantly higher in carcinomas from the high-fat group, and the two parameters were substantially correlated (r2 = 0.43, p < 0.01). The expression level of c-Raf was fourfold higher in the high-fat group but was only modestly associated with MAPK activation (r2 = 0.35, p < 0.05). The levels of the total MAPK1 and MAPK2, guanosine triphosphatase-activating protein, Ha-Ras, and MAPK kinase did not change. These results suggest that an upregulation of c-Raf expression by high fat may in part account for the in vivo MAPK activation, which in turn may enhance cell proliferation and mammary carcinogenesis.

Dissociation of the genotoxic and growth inhibitory effects of selenium.

The effects of forms of selenium compounds that enter the cellular selenium metabolic pathway at different points were investigated in a mouse mammary carcinoma cell line. The goal of these experiments was to determine if the genotoxicity of selenium, defined as its ability to induce DNA single-strand breaks, could be dissociated from activities proposed to account for its cancer inhibitory activity. The results demonstrated that growth inhibition, measured as inhibition of cell proliferation and induction of cell death, was induced by all the forms of selenium evaluated. However, sodium selenite and sodium selenide, which are metabolized predominantly to hydrogen selenide, caused the rapid induction of DNA single-strand breaks as an early event that preceded growth inhibition. Interestingly methylselenocyanate and Se-methylselenocysteine, which are initially metabolized predominantly to methylselenol, induced growth inhibition in the absence of DNA single-strand breakage. Differences in the time course of selenium retention, in the occurrence of membrane damage, and in the induction of morphological changes by selenite versus methylselenocyanate were noted. Collectively, these data indicate that different pathways affecting cell proliferation and cell death are induced depending on whether selenium undergoes metabolism predominantly to hydrogen selenide or to methylselenol.

Selenite induction of DNA strand breaks and apoptosis in mouse leukemic L1210 cells.

The effects of selenite on DNA integrity, cell viability, and long-term proliferative potential of mouse leukemic L1210 cells were examined in this study. Selenite treatment resulted in concentration-dependent increases in DNA single-strand breaks and double-strand breaks, as detected by a modified filter elution assay. A time-course experiment showed that DNA single-strand breaks preceded DNA double-strand breaks. Agarose gel electrophoresis of DNA extracted from selenite-treated cells displayed a nucleosomal fragmentation pattern that is characteristic of apoptotic cell death. The involvement of a Ca2+,Mg(2+)-dependent endonuclease responsible for DNA double-strand fragmentation was implied by the observation that two inhibitors of endonuclease activity, i.e. aurintricarboxylic acid and zinc, blocked selenite-induced DNA double-strand breaks. These inhibitors also prevented selenite-induced cell death as defined by loss of ability to exclude trypan blue dye. Selenite treatment severely impaired the colony-forming ability of cells capable of trypan blue exclusion. The induction of DNA strand breaks and commitment to apoptosis may explain the selenite-mediated growth inhibition and loss of long-term proliferative potential.

Alkaline elution analysis of DNA fragmentation induced during apoptosis.

We report that alkaline elution analysis fails to provide a complete profile of DNA fragmentation induced by some genotoxic agents, particularly if these agents induce apoptotic cell death resulting in fragmentation of DNA into oligonucleosomal length multimers. It was questioned whether these oligonucleosome fragments are responsible for the more rapidly eluting component of DNA that is observed as a biphasic elution profile when cells treated with certain genotoxic agents are subjected to alkaline elution. The results of this study indicate that DNA fragmented during apoptotic cell death is eluted in fractions before alkaline denaturation that are normally discarded. Collection of fractions prior to alkaline denaturation is recommended for complete evaluation of the total spectrum of damage induced by genotoxic agents, especially when the compound is suspected of inducing cell death by apoptosis.