Antioxidant foods and ingredients are an important component of the food industry. In the past, antioxidants were used primarily to control oxidation and retard spoilage, but today many are used because of putative health benefits. However, the traditional message that oxidative stress, which involves the production of reactive oxygen species (ROS), is the basis for chronic diseases and aging is being reexamined. Accumulating evidence suggests that ROS exert essential metabolic functions and that removal of too many ROS can upset cell signaling pathways and actually increase the risk of chronic disease. It is imperative that the food industry be aware of progress in this field to present the science relative to foods in a forthright and clear manner. This may mean reexamining the health implications of adding large amounts of antioxidants to foods.

Ferritins, an ancient family of protein nanocages, concentrate iron in iron-oxy minerals for iron-protein biosynthesis and protection against oxy radical damage. Of the two genetic mechanisms that regulate rates of ferritin-L synthesis, DNA transcription and mRNA translation, more is known about mRNA regulation where iron targets complexes of an mRNA structure, the iron-responsive element (IRE) sequence, and ferritin IRE repressors (iron regulatory proteins 1 and 2). Neither the integration of mRNA and DNA regulation nor the ferritin-L DNA promoter are well studied. We now report the combined effects of DNA transcription and mRNA translation regulation of ferritin-L synthesis. First, the promoter of human ferritin-L, encoding the animal-specific subunit associated with human diseases, was identified, and contained an overlapping Maf recognition element (MARE) and antioxidant responsive element (ARE) that was positively regulated by tert-butylhydroquinone, sulforaphane, and hemin with responses comparable to thioredoxin reductase (ARE regulator) or quinone reductase (MARE͞ARE regulator). Iron, a poor regulator of the ferritin-L promoter, was 800 times less effective than sulforaphane. Combining the ferritin-L MARE͞ARE and IRE produced a response to hemin that was 3-fold greater than the sum of responses of the MARE͞ARE or IRE alone. Regulation of ferritin-L by a MARE͞ARE DNA sequence emphasizes the importance of ferritin-L in oxidative stress that complements the mRNA regulation in iron stress. Combining DNA and mRNA mechanisms of regulation, as for ferritin-L, illustrates the advantages of using two types of genetic targets to achieve sensitive responses to multiple signals.antioxidant responsive element ͉ Maf recognition element ͉ oxygen ͉ iron responsive element ͉ combinatorial regulation

Traditionally, mouse humanization studies have used human fecal transfer to germ-free animals. This practice requires gnotobiotic facilities and is restricted to gnotobiotic mouse lines, which limits humanized mouse research. We have developed a generalizable method to humanize non germ-free mice using antibiotic treatment and human fecal transfer. The method involves depleting resident intestinal microbiota with broad-spectrum antibiotics, introducing human microbiota from frozen fecal samples by weekly gavage, and maintaining mice in HEPA-filtered microisolator cages. Pyrosequencing cecal microbiota 16S rRNA genes showed that recipient mice adopt a humanized microbiota profile analogous to their human donors, and distinct from mice treated with only antibiotics (no fecal transfer) or untreated control mice. In the humanized mice, 75% of the sequence mass was observed in their respective human donor and conversely, 68% of the donor sequence mass was recovered in the recipient mice. Principal component analyses of GC- and HPLC-separated cecal metabolites were performed to determine effects of transplanted microbiota on the metabolome. Cecal metabolite profiles of mice treated with only antibiotics (no fecal transfer) and control mice were dissimilar from each other and from humanized mice. Metabolite profiles for mice humanized from different donor samples clustered near each other, yet were sufficiently distinct that separate clusters were apparent for each donor. Also, cecal concentrations of 57 metabolites were significantly different between humanization treatments. These data demonstrate that our protocol can be used to humanize non germ-free mice and is sufficiently robust to generate metabolomic differences between mice humanized from different human donors.

Controlling iron/oxygen chemistry in biology depends on multiple genes, regulatory messenger RNA (mRNA) structures, signaling pathways and protein catalysts. Ferritin, a protein nanocage around an iron/oxy mineral, centralizes the control. Complementary DNA (antioxidant responsive element/Maf recognition element) and mRNA (iron responsive element) responses regulate ferritin synthesis rates. Multiple iron-protein interactions control iron and oxygen substrate movement through the protein cage, from dynamic gated pores to catalytic sites related to di-iron oxygenase cofactor sites. Maxi-ferritins concentrate iron for the bio-synthesis of iron/heme proteins, trapping oxygen; bacterial mini-ferritins, DNA protection during starvation proteins, reverse the substrate roles, destroying oxidants, trapping iron and protecting DNA. Ferritin is nature's unique and conserved approach to controlled, safe use of iron and oxygen, with protein synthesis in animals adjusted by dual, genetic DNA and mRNA sequences that selectively respond to iron or oxidant signals and link ferritin to proteins of iron, oxygen and antioxidant metabolism.

Selenium (Se) from high-Se garlic reduces the incidence of chemically induced mammary tumors, and Se from high-Se broccoli reduces colon cancer. However, the ability of Se from high-Se broccoli to protect against mammary cancer has not been tested. Also, the sprout form of broccoli contains many secondary plant compounds that are known to reduce cancer risk, but the anticarcinogenic activity of broccoli sprouts has not been investigated. The present studies examined the ability of high-Se broccoli or high-Se broccoli sprouts to protect against chemically induced mammary or colon cancer. In one experiment, Sprague--Dawley rats that consumed diets containing 3.0 microg of Se/g supplied as high-Se broccoli had significantly fewer mammary tumors than rats fed 0.1 microg of Se as selenite with or without the addition of regular broccoli. In the second experiment, Fisher F-344 rats fed 2.0 microg of Se/g of diet supplied as either high-Se broccoli florets or high-Se broccoli sprouts had significantly fewer aberrant colon crypts than rats fed 0.1 or 2 microg of Se/g of diet supplied as selenite with or without the addition of low-Se broccoli. These data demonstrate that the cancer-protective effect of Se in high-Se broccoli extends to mammary cancer and the protective forms of broccoli against colon cancer include high-Se broccoli sprouts.

Ferritin gene transcription is regulated by heme as is ferritin mRNA translation, which is mediated by the well studied mRNA⅐IRE/IRP protein complex. The heme-sensitive DNA sequence in ferritin genes is the maf recognition/antioxidant response element present in several other genes that are induced by heme and repressed by Bach1. We now report that chromatin immunoprecipitated with Bach1 antiserum contains ferritin DNA sequences. In addition, overexpression of Bach1 protein in the transfected cells decreased ferritin expression, indicating insufficient endogenous Bach1 for full repression; decreasing Bach1 with antisense RNA increased ferritin expression. Thioredoxin reductase1, a gene that also contains a maf recognition/ antioxidant response element but is less studied, responded similarly to ferritin, as did the positive controls heme oxygenase1 and NADP(H) quinone (oxido) reductase1. Bach1-DNA promoter interactions in cells were confirmed in vitro with soluble, recombinant Bach1 protein and revealed a quantitative range of Bach1/DNA stabilities: ferritin L ϳ ferritin H ϳ ␤-globin, ␤-globin ϳ 2-fold >heme oxygenase1 ‫؍‬ quinone reductase ␤-globin ϳ 4-fold >thioredoxin reductase1. Such results indicate the possibility that modulation of cellular Bach1 concentrations will have variable effects among the genes coordinately regulated by maf recognition/antioxidant response elements in iron/oxygen/antioxidant metabolism.Genes encoding proteins that manage proteins of iron and oxygen traffic and metabolism are at the nexus of chemical reactions that are both critical and dangerous to life. The iron porphyrin complex, heme, has emerged as a key signal for iron and oxygen metabolism genes, including ferritin L (ftl) 3 (1) and ferritin H (fth) (2). Transcriptional regulation of NADP(H) quinone (oxido) reductase (qr) (3), heme oxygenase1 (ho1) (4), and ␤-globin (5) by heme requires the maf recognition/antioxidant response element (MARE/ARE), a conserved regulatory sequence found in the promoter or enhancer, and the heme binding transcriptional repressor Bach1.Both ftl and fth contain heme-responsive canonical MARE/ ARE promoter sequences (1, 2). The role of Bach1 in hemeregulated ferritin transcription is not known. By contrast, the role of IRP1 and IRP2 in heme-regulated ftl and fth mRNA translation is known (6 -9). The translational mechanism uses IRP1 and IRP2 to coordinate fth and ftl mRNA regulation with that of several other mRNAs important in iron and oxygen homeostasis by binding to iron-responsive elements (IRE) in each of the mRNAs. The IRE is a specific three-dimensional loop-helix-loop-helix structure (10) in the noncoding regions of the mRNAs (11-16). Specific interactions between the iron regulatory proteins IRP1 and IRP2 and the different IREs in the mRNAs create a natural, combinatorial array of RNA⅐protein complexes (17). In the case of ferritin, when IRP1 or 2 are bound to the mRNA the ability of eukaryotic initiation factor 4F to recruit translational machinery to the mRNA is compromised and translat...

We previously reported the in vitro and in vivo induction of thioredoxin reductase (TR) by sulforaphane (SF) purified from broccoli. The present study was designed to determine whether this induction is mediated by putative antioxidant response elements (ARE) found in the promoter. Luciferase reporter constructs were built using the TR promoter sequence. Sulforaphane, tert-butylhydroquinone and beta-napthoflavone, as well as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), increased luciferase activity in HepG2 cells transfected with the reporter construct (P < 0.0001). Quinone reductase (QR) is an enzyme with a well-characterized ARE, and QR reporter constructs built as positive controls showed similar patterns of induction. Mutation of the core sequence of a putative ARE in the TR promoter drastically decreased inducibility by SF, but mutations in nonconsensus areas of the ARE and outside of the ARE did not affect inducibility. Results from electrophoretic mobility shift assay analysis corroborated mutated reporter gene findings. Induction by TPA was not affected by mutation of the putative ARE. Se plus SF, and SF alone were equally effective for induction of TR reporter luciferase activity (P < 0.0001); Se alone had no effect. Se and SF independently increased TR activity (P < 0.0001) and when combined, increased TR activity synergistically (P = 0.036). These data suggest that TR is transcriptionally regulated by electrophilic compounds via an ARE in the 5' region of the gene, and that this mechanism is unrelated to the established Se-dependent induction of selenoproteins.

Multiple components of broccoli, such as sulforaphane (Sf) and phenolic acids, may inhibit cancer. Additionally, broccoli can accumulate selenium (Se), and Se has been demonstrated to reduce the risk of cancer. Studies were conducted to determine whether enhancement of broccoli with Se would produce a plant with superior health benefits. Although increasing the concentration of Se in broccoli from <1.0 to >800 microg/g resulted in inhibition of colon cancer in rats, it also decreased the Sf content by >80% and inhibited production of most phenolic acids. The inclusion of Se-enriched broccoli in the diet of rats induced the activity of the selenoprotein thioredoxin reductase beyond the maximum activity induced by Se alone. These results emphasize the complex interactions of bioactive chemicals in a food; attempts to maximize one component may affect accumulation of another, and consumption of high amounts of multiple bioactive compounds may result in unexpected metabolic interactions within the body.