Michael Q. Kemp

Cyclooxygenase-2 Promoter Activation by the Aromatic Hydrocarbon Receptor in Breast Cancer MCF-7 Cells: Repressive Effects of Conjugated Linoleic Acid

Abstract The role of the aromatic hydrocarbon receptor (AhR) in transcriptional regulation of the human cyclooxygenase-2 (COX-2) gene remains elusive. We report that the AhR-ligands benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced transcription activity of COX-2 in breast cancer MCF-7 cells. The TCDD-dependent activation of the COX-2 promoter was abrogated by mutation of 2 xenobiotic response elements (XREs) = CGTG). We found that TCDD stimulated the binding of the AhR to COX-2 and cytochrome P4501A1 (CYP1A1) oligonucleotides containing consensus XREs. Conversely, the cotreatment with TCDD plus a mixture of conjugated linoleic acid (CLA) or selected CLA isomers prevented (CLAmix = t10,c12-CLA > c9,t11-CLA) the induction of transcription from the COX-2 promoter. The TCDD-induced binding of the AhR to COX-2 and CYP1A1 oligonucleotides was repressed by cotreatment with CLA (t10,c12-CLA > c9,t11-CLA), and the AhR antagonists, 3-methoxy-4-naphthoflavone, and resveratrol. We conclude that the AhR may be a suitable target for prophylactic strategies that target COX-2 expression.

Spanish Black Radish (Raphanus Sativus L. Var. niger) Diet Enhances Clearance of DMBA and Diminishes Toxic Effects on Bone Marrow Progenitor Cells

Vegetables of the Cruciferae family contain high levels of glucosinolates, metabolites of which are believed to enhance detoxification. Spanish black radishes (SBR) contain 4× more glucosinolates than other crucifers. This study examined whether feeding mice a diet containing 20% SBR for 2 wk could enhance metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) and inhibit DMBA-mediated bone marrow toxicity. Expression of Phase I and II detoxification enzymes was significantly greater for mice fed SBR than control diet. Six hours after DMBA administration, the blood levels of DMBA in mice fed the SBR diet were significantly lower than mice fed a control diet. DMBA reduced bone marrow cells in mice fed control diet to a significantly greater extent than mice fed the SBR diet. Colony forming assays demonstrated that mice on the SBR diet had 1) less reduction in lymphoid CFU-preB progenitor cells, 2) greater recovery of CFU-preB progenitor cells at 168 h, and 3) less reduction of CFU-GM progenitor cells at 6 h. Therefore, mice fed a 20% SBR diet for 2 wk had greater expression of detoxification enzymes, faster metabolism of DMBA, and a reduction in DMBA-induced bone marrow toxicity. Overall, these results support the hypothesis that glucosinolates in SBR are protective against acute toxicity.

Survival and Diversity of Human Homologous Dietary MicroRNAs in Conventionally Cooked Top Sirloin and Dried Bovine Tissue Extracts

Dietary microRNAs (miRNAs), notably those found in milk, are currently being investigated for their potential to elicit biological effects via canonical binding to human messenger RNA targets once ingested. Besides milk, beef and other bovine tissue-derived ingredients could also be a relevant source of potentially bioactive dietary miRNAs. In this study, we characterized the human homologous miRNA profiles in food-grade, bovine-sourced sirloin, heart and adrenal tissue (raw, cooked, and pasteurized, freeze-dried extracts) via deep-sequencing and quantitative reverse transcription PCR (RT-qPCR). A total of 198 human homologous miRNAs were detected at 10 or more normalized reads in all replicates (n = 3) of at least one preparation method. Tissue origin rather than preparation method was the major differentiating factor of miRNA profiles, and adrenal-based miRNA profiles were the most distinct. The ten most prevalent miRNAs in each tissue represented 71–93% of the total normalized counts for all annotated miRNAs. In cooked sirloin, the most abundant miRNAs were miR-10b-5p, (48.8% of total annotated miRNA reads) along with the muscle-specific miR-1 (24.1%) and miR-206 (4.8%). In dried heart extracts, miR-1 (17.0%), miR-100-5p (16.1%) and miR-99a-5p (11.0%) gave the highest normalized read counts. In dried adrenal extracts, miR-10b-5p (71.2%) was the most prominent followed by miR-143-3p (7.1%) and 146b-5p (3.7%). Sequencing results for five detected and two undetected miRNAs were successfully validated by RT-qPCR. We conclude that edible, bovine tissues contain unique profiles of human homologous dietary miRNAs that survive heat-based preparation methods.

Role of Dietary Xenobiotics-Gene Interactions in Carcinogenesis: Protective Effects of Nutritional Factors

In this paper, we discuss how interactions between dietary xenobiotics and nutrients influence cancer risk by modulating overlapping biochemical pathways leading to repression of tumor suppressor genes, activation of tumor promoters, or both. Epidemiologic studies in humans suggested that diet is an important vehicle of exposure to various xenobiotics, including polycyclic aromatic hydrocarbons (PAHs), dioxins, and chlorinated hydrocarbons (CHs). The activation of the aromatic hydrocarbon receptor (AhR) pathway by PAHs and dioxins stimulates the expression of several genes including cytochrome P450s, which metabolize PAHs to highly mutagenic compounds that cause fixation of mutations in the p53 gene and repress the expression of the tumor suppressor gene, BRCA-1. Conversely, PAHs and dioxins have been shown to activate the expression of cyclooxygenase-2 (COX-2), whose protein product participates in the production of reactive PAH-metabolites and synthesis of proinflammatory prostaglandins (PGs). The carcinogenicity of CHs has been attributed to their ability to activate the expression of oncogenes including c-myc, and fatty acid activation of the peroxisome proliferator-activated receptor-α (PPARα). Dietary components that may protect against the activation of the AhR pathway include flavonoids, which comprise a large family of dietary phenolic phytochemicals found in fruits and vegetables. Nutrients such as dietary fatty acids may differentially influence the risk of cancer by inducing (linoleic and arachidonic acid) or repressing (conjugated linoleic acid (CLA); n-3PUFA) the expression of COX- 2. Finally, we present original findings produced in our laboratory documenting the protective effects of natural (genestein) and synthetic (α-naphthoflavone) flavonoids against PAH-induced changes in gene expression.