Effects of the WD on redox active compound levels

To investigate responses to oxygen stress after 6 mo of WD feeding, we Paloma Picasso Loving Heart bracelet colon homogenates of 6 mice from both dietary groups for concentrations of major intracellular redox buffers (Table 3). The cellular redox status is maintained by a balance between intracellular reduced sulfhydryls and oxidized disulfides. Enhanced oxygen stress was evidenced by decreased cysteine:cystine and GSH:GSSG ratios and a 50% decrease in endogenous methionine, a precursor to cysteine. Because mice synthesize ascorbate, the increased levels of ascorbic acid in the WD group may represent a compensatory response to oxidative stress.

The WD is an accepted risk factor for colorectal cancer development in humans (2). However, WD consumption is accompanied by other lifestyle factors; thus, identification of molecular mechanism(s) underlying carcinogenic effects of the diet is difficult. A rodent WD, high in fat and relatively low in calcium and cholecalciferol content, uniquely induces colonic preneoplastic changes in wild-type C57B1/6 mice (4) and with additional folate and methionine depletion, adenoma and colon cancer formation within 12-24 mo (5). Our studies show that the WD induces oxidative stress and alters immune responses in the colon of wild-type mice long before tumors occur. Additionally, pathway analysis of colonic transcriptome changes identified other biologically important and related networks altered by the WD, including lipid and xenobiotic metabolism.

The WD-induced colonic transcriptome Return to Tiffany Double Heart Pendant were based upon microarray analysis in 8 mice per dietary group (at 3 and 6 mo) validated by qPCR. The whole colon wall was used, which did not permit separation of effects on epithelial cells, leukocytes, or other cellular components but allowed evaluation of macrophage numbers. The WD, containing 4-fold more lipid than control diets, induced persistent regulation of fatty acid and sterol metabolism and upregulated soluble and mitochondrial HMG CoA reductase, Ianosterol synthase, and squalene epoxidase genes. Major stimulation of lipid biosynthesis and metabolism suggest that the WD directly affected the colonic epithelial transcriptome.

WD feeding led to Nrf2-associated oxidative stress responses in the colon. Nrf2, a redox-sensitive basic leucine zipper transcription factor, regulates gene expression through interactions with the antioxidant-response element (ARE) found in the promoter regions of many cellular defense genes (23). During oxidative stress, Nrf2 activates ARE-dependent transcription of phase II and antioxidant defense enzymes and controls expression of -γ-glutamylcysteine synthetase, the rate-limiting enzyme for GSH synthesis. Thus, Nrf2-ARE pathway activation is an efficient mechanism increasing cellular detoxification and antioxidant capacity (23-25). Nrf2-knockout mice are more susceptible to chemically induced colitis and carcinogenesis than their corresponding litter mates, Cupcake charm and chain the importance of Nrf2 in maintaining colonic mucosal integrity (26). Canonical pathway analysis and GSEA showed induction of AREregulated cytoprotective genes by the WD, implying increased colonic production of reactive oxygen species (ROS) and responses to oxidative stress mediated presumably by the Nrf2-ARE pathway.