Differences in urinary biomolecules following ovariectomy, and the impact of dietary polyphenols (grapeseed extract [GSE]) in rodents were examined by nanoLC‐tandem mass spectrometry, and the impact of dietary polyphenols (grapeseed extract [GSE]). Urines from sham‐ovariectomy (OVX) and ovariectomized ((OVX) rats were analyzed, where both groups were given GSE, thus, any differences were due to the OVX. In positive mode LC‐MS/MS, 5,589 peaks were resolved with unique retention times. Of these, 12% were significantly different in abundance (p<0.05), with 2% of this subgroup (p<0.001). Conclusive identification of significant peaks required both the METLIN software on the Scripps XCMS server (Tautenhahn et al., Anal. Chem, 2012) and the Human Metabolome Database (Wishart et al., Nucleic Acids Res, 2007). Experiments are ongoing to continue identification of peaks using MS/MS, as well as to populate the appropriate databases with conjugated forms of polyphenols to enable deeper analysis. In TARGETED metabolomics analysis, we determined that glucuronidated grapeseed polyphenols were increased in the urines of OVX rats, indicating that OVX increased the amount or activity of the relevant glucuronosyltransferases. This is the first metabolomics analysis of urinary biomolecules in a model of menopause, and of effects and fate of dietary polyphenols.Grant Funding Source: NIH/NCCAM R21‐AT00480531

Abstract Steroid hormones modulate expression of enzymes that metabolize xenobiotics, including dietary supplements. Half of the human population undergoes menopause, yet the effect of this age-related loss of ovarian steroid hormones on the metabolism of dietary supplements has yet to be determined. Grape seed extract (GSE) is a dietary supplement comprised of monomeric and oligomeric catechins and has health benefits in models of age-related diseases. We hypothesized that surgically-induced loss of ovarian hormones would increase methylation, glucuronidation, and/or sulfation of the grape seed polyphenols (+)-catechin and (–)-epicatechin. Fourteen-week-old spontaneously hypertensive rats (SHRs) were ovariectomized (OVX) or sham-OVX. At 17 weeks of age, SHRs were gavaged with vehicle (water) or GSE (300 mg/kg body weight) once daily for 6 days. Urinary excretion of (+)-catechin, (–)-epicatechin, and their metabolites was analyzed by liquid chromatography-mass spectrometry. Although total urinary output of (+)-catechin, (–)-epicatechin, and their methylated metabolites was unaffected by OVX, the amounts of (+)-catechin, (–)-epicatechin and their methylated metabolites that were not conjugated with glucuronic acid or sulfate were lowered by OVX. Specifically, urine from OVX SHRs administered GSE contained 30% higher proportions (91.8% vs. 62.3%) of glucuronidated (+)-catechin and (–)-epicatechin and glucuronidated methyl (+)-catechin and methyl (–)-epicatechin than urine from sham-OVX SHRs. However, there were no differences in urinary levels of total methylated or sulfated catechins in OVX SHRs. This is the first quantitative characterization of metabolites of grape seed polyphenols in a model of menopause; it indicates that ovariectomy causes either an increase in expression and/or activity of select uridine 5′-diphospho-glucuronosyltransferase(s).

The selection of foods to eat is a complex interplay of vision, taste, smell, and texture. In addition to micro- and macronutrients, plant-based foods also contain several classes of phytochemicals. In many cases, the phytochemicals account for the various colors of foods. Although aesthetically pleasing, the color of foods may mislead consumers as to their phytochemical content, which is particularly true with regard to polyphenols. Polyphenols are a broad class of compounds with antioxidant and other health benefits. Human vision is limited to a small window (390-765 nm) of the electromagnetic spectrum. Many important phytochemicals (e.g., vitamin C) have no absorbance in this range. Therefore, the human eye cannot directly judge the vitamin C content of foods. Being able to see in the ultraviolet range allows bees to locate the pollen-rich region of flowers, whereas pit vipers locate their prey by being able to "see" them in the infrared range. Assessing the impact of phytochemicals on human health depends on several factors. Colorless phytochemicals in unprocessed foods may be lost during the cooking process because no visual guide exists to ensure their retention. The molecular structures of phytochemicals influence the extent to which they are altered by cooking processes and the methods by which they are absorbed from the gastrointestinal tract. Extensive metabolism by phase I/II enzymes and by the gut microbiome may also create compounds that the eye is never allowed to appreciate.

To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2-dimensional-difference gel electrophoresis (2D-DIGE), we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 nonredundant proteins. 2D-DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 nonredundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by Western blot analysis and immunohistochemistry (IHC). Thus, the 2D-DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies.

Bioactive compounds in botanicals may be beneficial in preventing age-related neurodegenerative diseases, but for many compounds conventional methods may be inadequate to detect if these compounds cross the blood brain barrier or to track the pharmacokinetics in the brain. By combining a number of unique technologies it has been possible to utilize the power of AMS to study the pharmacokinetics of bioactive compounds in the brain at very low concentrations. (14)C-labeled compounds can be biosynthesized by plant cell suspension cultures co-incubated with radioisotopically-labeled sucrose and isolated and separated into a series of bioactive fractions.To study the pharmacokinetics and tissue distribution of (14)C labeled plant polyphenols, rats were implanted with jugular catheters, subcutaneous ultrafiltration probes and brain microdialysis probes. Labeled fractions were dosed orally. Interstitial fluid (ISF) and brain microdialysate samples were taken in tandem with blood samples. It was often possible to determine (14)C in blood and ISF with a β-counter. However, brain microdialysate samples (14)C levels on the order of 10(7) atoms/sample required AMS technology. The Brain Microdialysate(AUC)/Serum(AUC) ranged from .021- to .029, with the higher values for the glycoside fractions. By using AMS in combination with traditional methods, it is possible to study uptake by blood, distribution to ISF and determine the amount of a dose which can reach the brain and follow the pharmacokinetics in the brain.

Grape polyphenols confer potential health benefits, including prevention of neurodegenerative diseases. To determine the absorption and tissue distribution of the complex grape polyphenol mixture, (14)C-labeled polyphenols were biosynthesized by grape cell suspension cultures, during co-incubation with radioisotopically labeled sucrose, and fractionated into polyphenolic subfractions. The pharmacokinetics and distribution of grape polyphenols into blood, brain, and peripheral interstitial fluid were determined by tracking the (14)C label. The blood peak (14)C concentration of the fractions ranged from 15 minutes to 4 hours. Absorption and tissue distribution varied greatly between fractions. Concentrations in interstitial fluid were lower than in blood. The amount of residual label in the brain at 24 hours ranged from 0.1% to 1.7% of the dose, depending on the fraction. (14)C label found in the brain tissue and brain microdialysate indicated that grape polyphenols or their metabolites are able to cross the blood-brain barrier. Using (14)C-labeled plant polyphenols it is possible to track the compounds or their metabolic products into any tissue and determine distribution patterns in spite of low concentrations. A central question regarding the potential role of dietary polyphenolics in neurodegenerative research is whether they are bioavailable in the brain. Our observations indicate that some grape-derived polyphenolics do reach the brain, which suggests their potential value for applications in neurodegenerative disorders.

Cell culture models of oncogenesis that use cellular reprogramming to generate a neoplastic cell from a normal cell provide one of the few opportunities to study the early stages of breast cancer development. Human mammary epithelial cells (HMECs) were induced to undergo a neoplastic transformation using defined genetic elements to generate transformed HMECs (THMECs). To identify proteins that displayed significantly different levels of abundance at three consecutive time points in oncogenesis over an 80 day period, protein extracts were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). Nine proteins were found to be significantly different in abundance: keratin 1, keratin 7, heat shock protein 4A-like, t-complex protein 1, stathmin, gelsolin, FK506 binding protein 5, ribosomal protein P0, and maspin. Keratin 7 and maspin displayed a linear down-regulation over 80 days. All of these proteins have been shown to be involved in the maintenance of a metastatic state including cytoskeletal modifications and motility. We conclude that, following neoplastic induction, THMECs display an early and progressive increase in metastatic potential. Further investigations into the function and regulatory mechanisms of these proteins will provide an unparalleled understanding of the initial states through which a breast cancer cell transitions following acquisition of the genetic abnormalities required for oncogenesis.

This summary addresses the progress and limitations of existing research on the physiologic properties of the isoflavone daidzein metabolite equol. Previous research demonstrating that physiological equol is its S-enantiomer has led to the preparation of S-(-)equol–enriched products formed by the bacterial fermentation of soy germ. Although this product has interesting properties as described in this workshop, the following important issues must be addressed: 1) the product should be evaluated against a preparation containing an equal amount of pure S-(-)equol to determine whether other components resulting from the fermentation are contributing to the physiological effects; 2) evaluation of the cellular mechanisms of S-(-)equol using cell culture methods should be conducted at concentrations consistent with those encountered physiologically (in the nmol/L range) and in several cell lines representing a target tissue; and 3) in follow-up studies in animal models and in human clinical trials, standardized preparations of S-(-)equol should be made available. Research opportunities now exist to determine whether equol′s apparent effects on menopausal symptoms (hot flashes, sleep disturbances, bone health) in equol producers can be extended to equol nonproducers. It will be important to ensure that such research is not complicated by cultural differences, differences in lifetime exposure to soy products, experimental techniques, and other variables. Further areas of research that would benefit from the availability of S-(-)equol preparations include its use in skin care (either as an antioxidant or as an estrogen receptor agonist) and in the treatment of brain injury as well as postmenopausal cognitive decline. J. Nutr. 140: 1390S–1394S, 2010.

The Purdue-UAB Botanicals Research Center for Age Related Disease uses multidisciplinary and innovative technologies to investigate the bioavailability of bioactive polyphenolic constituents from botanicals and their relationship to human health. Many age-related diseases are associated with oxidative stress and tissue damage. One of the research goals of the Purdue-UAB Center is to investigate the bioavailability of bioactive natural compounds from a complex botanical mixture to the organ affected by the disease, determine the uptake and metabolism of these compounds, and relate these data to a protective mechanism. Equally important is to screen commercially available botanicals for their safety and efficacy. The central aims of the Center include the investigation of botanicals and their relationship to bone antiresorptive capacity, cognitive function, vascular effects, and cancer prevention.

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CORats are shown to be more susceptible to the development of mammary carcinomas when they are exposed to DMBA, during the 30-50 day-old (pubertal) period. By contrast, rats when exposed to genistein, during the early stages of mammary development (prenatal, neonatal, or pre-pubertal periods), have a reduced risk of chemical-induced mammary cancer. In the present study, we show that the abundance and activity of TCA cycle enzymes such as succinate dehydrogenase-B, fumarase and NAD+ isocitrate dehydrogenase in the mammary gland of Sprague-Dawley rats are significantly lower during puberty (day 50) compared to the prepubertal (day 21) period. Moreover, there was a subsequent activation of HIF-1\#945; in the epithelial cells of mammary gland during pubertal period. On the contrary, the abundance of these enzymes was significantly higher in resveratrol and genistein treated rats on day 50 and day 21 than the untreated control animals. These results indicate that, during puberty, the mammary gland microenvironment exists in a \#8220;pseudo-hypoxic\#8221; condition, which may potentially make it more vulnerable to tumor initiation by carcinogens. Restoration of TCA cycle enzyme levels and subsequent biochemical changes in the mammary gland may be one of the mechanisms by which dietary polyphenols reduce the risk of chemical-induced mammary cancer. Further studies will help to develop new approaches to breast cancer prevention, timely implementation of prevention strategies successfully in high-risk groups, and elucidate the mechanism of mammary carcinogenesis.Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 13.

Exposure to altered microgravity during space travel induces changes in the brain and these are reflected in many of the physical behavior seen in the astronauts. The vulnerability of the brain to microgravity stress has been reviewed and reported. Identifying microgravity-induced changes in the brain proteome may aid in understanding the impact of the microgravity environment on brain function. In our previous study we have reported changes in specific proteins under simulated microgravity in the hippocampus using proteomics approach. In the present study the profiling of the hypothalamus region in the brain was studied as a step towards exploring the effect of microgravity in this region of the brain. Hypothalamus is the critical region in the brain that strictly controls the pituitary gland that in turn is responsible for the secretion of important hormones. Here we report a 2-dimensional gel electrophoretic analysis of the mouse hypothalamus in response to simulated microgravity. Lowered glutathione and differences in abundance expression of seven proteins were detected in the hypothalamus of mice exposed to microgravity. These changes included decreased superoxide dismutase-2 (SOD-2) and increased malate dehydrogenase and peroxiredoxin-6, reflecting reduction of the antioxidant system in the hypothalamus. Taken together the results reported here indicate that oxidative imbalance occurred in the hypothalamus in response to simulated microgravity.

In rat models of mammary carcinogenesis, the highest incidence of carcinomas occurs when the animals are exposed to carcinogens during puberty (typically postnatal day 50). Gene expression analysis of rat mammary gland in Sprague‐Dawley rats revealed a consistent down‐regulation of Krebs cycle genes in tissues from 50‐day old animals relative to 21‐day animals. This result was confirmed at the protein level by Western blot analysis and assessment of specific activity of selected Krebs cycle enzymes; succinate dehydrogenase (SDH)‐B, fumarase (FH), NAD+ isocitrate dehydrogenase (IDH3A) were significantly reduced at late puberty (day 50) compared with prepubertal (day 21) in the mammary gland. A nanoLC‐multiple reaction ion monitoring‐mass spectrometry method was developed that measures each of these proteins in a single assay in tissue and mitochondrial preparations. The results reported here support the gene expression data obtained by microarray analysis and suggest that the increased vulnerability of the rat mammary gland at late puberty to breast cancer‐inducing carcinogens is correlated with an overall down‐regulation of aerobic metabolism. Supported by NCI U54 CA100949.

Proanthocyanidin rich plant extracts derived from grape seed extract (GSE), hawthorn and cranberry are on markets for their preventive effects against cardiovascular diseases and uroinfections in woman. However, the importance of these health beneficial effects of these botanicals remains elusive due to incomplete understanding of uptake, metabolism and bioavailability of proanthocyanidins in vivo. In the present study rats were given GSE orally (300mg/kg, twice a day) and blood and urine were collected over a 24h period. Monomeric catechins and their methylated metabolites, and proanthocyanidins up to trimers were detected in blood samples treated with GSE using LC-MS/MS operating in the multiple reaction monitoring (MRM) mode. A new tetramethylated metabolite of dimeric proanthocyanidin (m/z 633) in GSE-treated urine was tentatively identified. Using LC-MS/MS, (+)-catechin and (−)-epicatechin were identified in the brain conclusively. These data suggested that GSE catechins cross the blood brain barrier and may be responsible for the neuroprotective effects of GSE.

MS, with or without pre-analysis peptide fractionation, can be used to decipher the residues on proteins where oxidative modifications caused by peroxynitrite, singlet oxygen or electrophilic lipids have occurred. Peroxynitrite nitrates tyrosine and tryptophan residues on the surface of actin. Singlet oxygen, formed by the interaction of UVA light with tryptophan, can oxidize neighbouring cysteine, histidine, methionine, tyrosine and tryptophan residues. Dose-response inactivation by 4HNE (4-hydroxynonenal) of hBAT (human bile acid CoA:amino acid N-acyltransferase) and CKBB (cytosolic brain isoform of creatine kinase) is associated with site-specific modifications. FT-ICR (Fourier-transform ion cyclotron resonance)-MS using nanoLC (nano-liquid chromatography)-ESI (electrospray ionization)-MS or direct-infusion ESI-MS with gas-phase fractionation identified 14 4HNE adducts on hBAT and 17 on CKBB respectively. At 4HNE concentrations in the physiological range, one member of the catalytic triad of hBAT (His362) was modified; for CKBB, although all four residues in the active site that were modifiable by 4HNE were ultimately modified, only one, Cys283, occurred at physiological concentrations of 4HNE. These results suggest that future in vivo studies should carefully assess the critical sites that are modified rather than using antibodies that do not distinguish between different modified sites.

2725 Initiation of chemical- and radiation-induced carcinogenesis in the rat mammary depends on the age of the animal. In this study we have examined the changes in gene expression in the mammary gland at two critical stages of mammary gland development, at the time of weaning at 21 days of age prior to onset of puberty and at 50 days of age, the time of maximum sensitivity to 7,12-dimethylbenz[a]anthracene (DMBA). The experiment and the analysis were carefully constructed to avoid unintended bias and yield reproducible results. Animals were fed a polyphenol-free, semi-purified AIN76A diet. The fourth right abdominal mammary gland was rapidly dissected from eight animals at days 21 and 50 and frozen in liquid N2. Rigorous quality control, normalization, and linear models analyses were conducted. The correlation (r 2 ) among the biological samples was over 0.98. Representative significantly changed genes were validated by quantitative RT-PCR. As a test of the veracity of the results obtained, the entire experiment was replicated in different animals raised and analyzed one year after the first experiment. Despite the time separation, the results of the two experiments were highly concordant. RESULTS : Microarray data analyses demonstrated that progression to day 50 resulted in a significant shift in expression of 11616 genes with a p-value 2 = 0.76, p 90% of genes), fatty acid synthesis (>80% of genes), sterol/cholesterol biosynthesis (>70% of genes) and portions of glycolysis at 50 days of age. A number of these pathway and individual level changes were also observed in DIGE experiments in additional biologically distinct samples. These changes in expression lead to a marked shift in metabolism favoring the formation of fatty acids and sterols and a decrease in aerobic generation of ATP and critical intermediates needed for the synthesis and repair of macromolecules during a period of rapid cell proliferation.