Kristen S. Kulp

NFkappaB selectivity of estrogen receptor ligands revealed by comparative crystallographic analyses

Our understanding of how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems that are common to steroid hormone receptors are circumvented by mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the structure of an apo steroid receptor that reveals a ligand-accessible channel allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFkappaB-selective signaling through the estrogen receptor, thus revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define structural features of signaling specificity that would not be apparent in a single structure.

Factors affecting human heterocyclic amine intake and the metabolism of PhIP

We are working to understand possible human health effects from exposure to heterocyclic amines that are formed in meat during cooking. Laboratory-cooked beef, pork, and chicken are capable of producing tens of nanograms of MeIQx, IFP, and PhIP per gram of meat and smaller amounts of other heteroyclic amines. Well-done restaurant-cooked beef, pork, and chicken may contain PhIP and IFP at concentrations as high as tens of nanograms per gram and MeIQx at levels up to 3 ng/g. Although well-done chicken breast prepared in the laboratory may contain large amounts of PhIP, a survey of flame-grilled meat samples cooked in private homes showed PhIP levels in beef steak and chicken breast are not significantly different (P=0.36). The extremely high PhIP levels reported in some studies of grilled chicken are not seen in home-cooked samples.Many studies suggest individuals may have varying susceptibility to carcinogens and that diet may influence metabolism, thus affecting cancer susceptibility. To understand the human metabolism of PhIP, we examined urinary metabolites of PhIP in volunteers following a single well-done meat exposure. Using solid-phase extraction and LC/MS/MS, we quantified four major PhIP metabolites in human urine. In addition to investigating individual variation, we examined the interaction of PhIP with a potentially chemopreventive food. In a preliminary study of the effect of broccoli on PhIP metabolism, we fed chicken to six volunteers before and after eating steamed broccoli daily for 3 days. Preliminary results suggest that broccoli, which contains isothiocyanates shown to induce Phases I and II metabolism in vitro, may affect both the rate of metabolite excretion and the metabolic products of a dietary carcinogen. This newly developed methodology will allow us to assess prevention strategies that reduce the possible risks associated with PhIP exposure.

Preparation of Single Cells for Imaging/Profiling Mass Spectrometry

Characterizing chemical changes within individual cells is important for determining fundamental mechanisms of biological processes that will lead to new biological insights and improved disease understanding. Analyzing biological systems with imaging and profiling mass spectrometry (MS) has gained popularity in recent years as a method for creating chemical maps of biological samples. To obtain mass spectra that provide relevant molecular information about individual cells, samples must be prepared so that salts and other cell culture components are removed from the cell surface and that the cell contents are rendered accessible to the desorption beam. We have designed a cellular preparation protocol for imaging/profiling MS that removes the majority of the interfering species derived from the cellular growth medium, preserves the basic morphology of the cells, and allows chemical profiling of the diffusible elements of the cytosol. Using this method, we are able to reproducibly analyze cells from three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation technique makes possible routine imaging/profiling MS analysis of individual cultured cells, allowing for understanding of molecular processes within individual cells.

An in vitro model system to predict the bioaccessibility of heterocyclic amines from a cooked meat matrix

To understand the impact of variation in digestion parameters on the release of heterocyclic amines naturally formed during cooking, we developed and characterized a model system to assess the effect of amylase, pepsin, and pancreatin on digestion of well-done chicken. The amounts of MeIQx, DiMeIQx, IFP, and PhIP in the liquid portion of the digestate were compared to levels in the undigested meat to determine the percentage released (accessible fraction). Incubating the meat with amylase and pepsin did not change the accessibility of HAs when compared to incubation with water alone. In contrast, increasing amounts of pancreatin increased the accessibility up to 6.4-fold. Comparing the amounts of the HAs in the liquid to the solid fraction showed that there was more MeIQx, DiMeIQx, and IFP in the liquid fraction. In contrast, PhIP was equally divided between the solid and liquid fractions. For all four compounds, increasing the doneness of the meat decreased the amount of the compound accessible from the meat matrix. Our data suggest that bioaccessability of HAs may vary according to the polarity of the individual HAs and also may depend upon the doneness of the meat. These results may have important ramifications for human feeding studies, which assume that the total amount of each HA in the meat matrix is equally bioavailable.

Liquid chromatography-tandem mass spectrometry method of urine analysis for determining human variation in carcinogen metabolism.

We developed a solid-phase extraction LC-MS-MS method for the analysis of the four major metabolites of PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) in human urine after a meal of well-done chicken. Ten volunteers each ate either 150 or 200 g of well-done chicken breast containing 9-21 microg of PhIP. Among the individual volunteers there is 8-fold variation in the total amount of metabolites and 20-fold variation in the relative amounts of individual metabolites, showing individual differences in carcinogen metabolism. PhIP metabolites were also detected in urine from a subject consuming chicken in a restaurant meal, demonstrating the methods sensitivity after real-life exposures.

Prostate cancer invasion and metastasis: insights from mining genomic data

Prostate cancer (PCa) is the second most commonly diagnosed malignancy in men in the Western world and the second leading cause of cancer-related deaths among men worldwide. Although most cancers have the potential to metastasize under appropriate conditions, PCa favors the skeleton as a primary site of metastasis, suggesting that the bone microenvironment is conducive to its growth. PCa metastasis proceeds through a complex series of molecular events that include angiogenesis at the site of the original tumor, local migration within the primary site, intravasation into the blood stream, survival within the circulation, extravasation of the tumor cells to the target organ and colonization of those cells within the new site. In turn, each one of these steps involves a complicated chain of events that utilize multiple protein-protein interactions, protein signaling cascades and transcriptional changes. Despite the urgent need to improve current biomarkers for diagnosis, prognosis and drug resistance, advances have been slow. Global gene expression methods such as gene microarrays and RNA sequencing enable the study of thousands of genes simultaneously and allow scientists to examine molecular pathways of cancer pathogenesis. In this review, we summarize the current literature that explored high-throughput transcriptome analysis toward the advancement of biomarker discovery for PCa. Novel biomarkers are strongly needed to enable more accurate detection of PCa, improve prediction of tumor aggressiveness and facilitate the discovery of new therapeutic targets for tailored medicine. Promising molecular markers identified from gene expression profiling studies include HPN, CLU1, WT1, WNT5A, AURKA and SPARC.

DNA Isolation and Sample Preparation for Quantification of Adduct Levels by Accelerator Mass Spectrometry

Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.

D-Lactate production as a function of glucose metabolism in Saccharomyces cerevisiae

Methylglyoxal, a reactive, toxic dicarbonyl, is generated by the spontaneous degradation of glycolytic intermediates. Methylglyoxal can form covalent adducts with cellular macromolecules, potentially disrupting cellular function. We performed experiments using the model organism Saccharomyces cerevisiae, grown in media containing low, moderate and high glucose concentrations, to determine the relationship between glucose consumption and methylglyoxal metabolism. Normal growth experiments and glutathione depletion experiments showed that metabolism of methylglyoxal by log‐phase yeast cultured aerobically occurred primarily through the glyoxalase pathway. Growth in high‐glucose media resulted in increased generation of the methylglyoxal metabolite d‐lactate and overall lower efficiency of glucose utilization as measured by growth rates. Cells grown in high‐glucose media maintained higher glucose uptake flux than cells grown in moderate‐glucose or low‐glucose media. Computational modelling showed that increased glucose consumption may impair catabolism of triose phosphates as a result of an altered NAD+:NADH ratio. Copyright

Flor-Essence herbal tonic does not inhibit mammary tumor development in Sprague Dawley rats.

Background: Women who are diagnosed with breast cancer often self-administer complementary and alternative medicines to augment their conventional treatments, improve health, or prevent recurrence. Flor-Essence® tonic is a complex mixture of herbal extracts used by cancer patients because of anecdotal evidence that it can treat or prevent disease. Methods: Female Sprague-Dawley rats were given water or exposed to 3 or 6% Flor-Essence® beginning at 1 day of age. Mammary tumors were induced with a single oral 40 mg/kg/bw dose of dimethyl-benz[a]anthracene at 50 days of age and sacrificed at 23 weeks. Rats were maintained on AIN-76A diet. Results: Control rats had palpable mammary tumor incidence of 51.0% at 19 weeks of age compared to 65.0 and 59.4% for the 3 and 6% Flor-Essence® groups respectively. Overall, no significant difference in time until first palpable tumor was detected among any of the groups. At necropsy, mammary tumor incidence was 82.5% for controls compared to 90.0 and 97.3% for rats consuming 3 and 6% Flor-Essence®, respectively. Mean mammary tumor multiplicity (±SES) for the controls was 2.8 (±0.5) and statistically different from the 3 or 6% Flor-Essence® groups with 5.2 (±0.7), and 4.8 (±0.6), respectively (p £ 0.01). As expected, the majority of isolated tumors were diagnosed as adenocarcinomas. Conclusions: Flor-Essence® can promote mammary tumor development in the Sprague-Dawley rat model. This observation is contrary to widely available anecdotal evidence as well as the desire of the consumer that this commercially available herbal tonic will suppress and/or inhibit tumor growth

Maternal exposure to an environmentally relevant dose of triclocarban results in perinatal exposure and potential alterations in offspring development in the mouse model

Triclocarban (TCC) is among the top 10 most commonly detected wastewater contaminants in both concentration and frequency. Its presence in water, as well as its propensity to bioaccumulate, has raised numerous questions about potential endocrine and developmental effects. Here, we investigated whether exposure to an environmentally relevant concentration of TCC could result in transfer from mother to offspring in CD-1 mice during gestation and lactation using accelerator mass spectrometry (AMS). 14C-TCC (100 nM) was administered to dams through drinking water up to gestation day 18, or from birth to post-natal day 10. AMS was used to quantify 14C-concentrations in offspring and dams after exposure. We demonstrated that TCC does effectively transfer from mother to offspring, both trans-placentally and via lactation. TCC-related compounds were detected in the tissues of offspring with significantly higher concentrations in the brain, heart and fat. In addition to transfer from mother to offspring, exposed offspring were heavier in weight than unexposed controls demonstrating an 11% and 8.5% increase in body weight for females and males, respectively. Quantitative real-time polymerase chain reaction (qPCR) was used to examine changes in gene expression in liver and adipose tissue in exposed offspring. qPCR suggested alterations in genes involved in lipid metabolism in exposed female offspring, which was consistent with the observed increased fat pad weights and hepatic triglycerides. This study represents the first report to quantify the transfer of an environmentally relevant concentration of TCC from mother to offspring in the mouse model and evaluate bio-distribution after exposure using AMS. Our findings suggest that early-life exposure to TCC may interfere with lipid metabolism and could have implications for human health.