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[2-14C]2-amino-1-methyl-phenlimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 μg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, 90% of the administered [14CPhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine- N2-glucuronide (N-hydroxy-PhIP-N2-glucuronide, accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen. [ABSTRACT FROM PUBLISHER]

Brain-on-a-chip systems are designed to simulate brain activity using traditional in vitro cell culture on an engineered platform. It is a noninvasive tool to screen new drugs, evaluate toxicants, and elucidate disease mechanisms. However, successful recapitulation of brain function on these systems is dependent on the complexity of the cell culture. In this study, we increased cellular complexity of traditional (simple) neuronal cultures by co-culturing with astrocytes and oligodendrocyte precursor cells (complex culture). We evaluated and compared neuronal activity (e.g., network formation and maturation), cellular composition in long-term culture, and the transcriptome of the two cultures. Compared to simple cultures, neurons from complex co-cultures exhibited earlier synapse and network development and maturation, which was supported by localized synaptophysin expression, up-regulation of genes involved in mature neuronal processes, and synchronized neural network activity. Also, mature oligodendrocytes and reactive astrocytes were only detected in complex cultures upon transcriptomic analysis of age-matched cultures. Functionally, the GABA antagonist bicuculline had a greater influence on bursting activity in complex versus simple cultures. Collectively, the cellular complexity of brain-on-a-chip systems intrinsically develops cell type-specific phenotypes relevant to the brain while accelerating the maturation of neuronal networks, important features underdeveloped in traditional cultures.

Background: The emergence of three-dimensional (3D) cell culture in neural tissue engineering has significantly elevated the complexity and relevance of in vitro systems. This is due in large part to the incorporation of biomaterials to impart structural dimensionality on the neuronal cultures. However, a comprehensive understanding of how key seeding parameters affect changes in cell distribution and viability remain unreported., New Method: In this study, we systematically evaluated permutations in seeding conditions (i.e., cell concentration and atmospheric CO 2 levels) to understand how these affect key parameters in 3D culture characterization (i.e., cell health and distribution). Primary rat cortical neurons (i.e., 2 × 10 6 , 4 × 10 6 , and 1 × 10 7 cells/mL) were entrapped in collagen blended with ECM proteins (ECM-Collagen) and exposed to atmospheric CO 2 (i.e., 0 vs 5% CO 2 ) during fibrillogenesis., Results: At 14 days in vitro (DIV), cell distribution within the hydrogel was dependent on cell concentration and atmospheric CO 2 during fibrillogenesis. A uniform distribution of cells was observed in cultures with 2 × 10 6 and 4 × 10 6 cells/mL in the presence of 5% CO 2 , while a heterogeneous distribution was observed in cultures with 1 × 10 7 cells/mL or in the absence of CO 2 . Furthermore, increased cell concentration was proportional to the rise in cell death at 14 DIV, although cells remain viable >30 DIV., Comparison With Existing Methods: ECM-Collagen gels have been shown to increase cell viability of neurons long-term., Conclusion: In using ECM-collagen gels, we highlight the importance of optimizing seeding parameters and thorough 3D culture characterization to understand the neurophysiological responses of these 3D systems., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The brain's extracellular matrix (ECM) is a macromolecular network composed of glycosaminoglycans, proteoglycans, glycoproteins, and fibrous proteins. In vitro studies often use purified ECM proteins for cell culture coatings, however these may not represent the molecular complexity and heterogeneity of the brain's ECM. To address this, we compared neural network activity (over 30 days in vitro) from primary neurons co-cultured with glia grown on ECM coatings from decellularized brain tissue (bECM) or MaxGel, a non-tissue-specific ECM. Cells were grown on a multi-electrode array (MEA) to enable noninvasive long-term interrogation of neuronal networks. In general, the presence of ECM accelerated the formation of networks without affecting the inherent network properties. However, specific features of network activity were dependent on the type of ECM: bECM enhanced network activity over a greater region of the MEA whereas MaxGel increased network burst rate associated with robust synaptophysin expression. These differences in network activity were not attributable to cellular composition, glial proliferation, or astrocyte phenotypes, which remained constant across experimental conditions. Collectively, the addition of ECM to neuronal cultures represents a reliable method to accelerate the development of mature neuronal networks, providing a means to enhance throughput for routine evaluation of neurotoxins and novel therapeutics.

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.

Prevailing commercialized cardiac platforms for in vitro drug development utilize planar microelectrode arrays to map action potentials, or impedance sensing to record contraction in real time, but cannot record both functions on the same chip with high spatial resolution. Here we report a novel cardiac platform that can record cardiac tissue adhesion, electrophysiology, and contractility on the same chip. The platform integrates two independent yet interpenetrating sensor arrays: a microelectrode array for field potential readouts and an interdigitated electrode array for impedance readouts. Together, these arrays provide real-time, non-invasive data acquisition of both cardiac electrophysiology and contractility under physiological conditions and under drug stimuli. Human induced pluripotent stem cell-derived cardiomyocytes were cultured as a model system, and used to validate the platform with an excitation-contraction decoupling chemical. Preliminary data using the platform to investigate the effect of the drug norepinephrine are combined with computational efforts. This platform provides a quantitative and predictive assay system that can potentially be used for comprehensive assessment of cardiac toxicity earlier in the drug discovery process.

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.

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 © 2013 John Wiley & Sons, Ltd.)

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® herbal tonic is a complex mixture of eight herbal extracts used by cancer patients because of anecdotal evidence that it can treat or prevent disease. In this study four experimental groups of female MMTV-Neu mice were left untreated or treated with 3% Flor-Essence® in utero, from birth until 5 weeks of age, or throughout their lifetime. Palpable mammary tumor incidence and body weight was determined weekly for each group. The mice were sacrificed at 28 weeks of age and mammary tumors were enumerated to determine average tumor incidence and multiplicity for each group. Female mice exposed to Flor-Essence® herbal tonic in utero weighed significantly more than the control group (p < 0.001). The average tumor incidence and tumor multiplicity in the experimental mice treated with Flor-Essence® herbal tonic did not differ from the control animals. Flor-Essence® does not inhibit mammary tumor incidence or mammary tumor multiplicity in MMTV-Neu transgenic mice. Flor-Essence® exposure in utero causes increased body weight in experimental animals. This conclusion challenges widely available anecdotal information as well as the hopes of the consumer that this product will inhibit or suppress tumor development.

Characterizing the molecular contents of individual cells is critical for understanding fundamental mechanisms of biological processes. Imaging mass spectrometry (IMS) of biological systems has been steadily gaining popularity for its ability to create precise chemical images of biological samples, thereby revealing new biological insights and improving understanding of disease. In order to acquire mass spectral images from single cells that contain relevant molecular information, samples must be prepared such that cell-culture components, especially salts, are eliminated from the cell surface and that the cell contents are accessible to the mass spectrometer. We have demonstrated a cellular preparation technique for IMS that preserves the basic morphology of cultured cells, allows mass spectrometric chemical profiling of cytosol, and removes the majority of the interfering species derived from the cellular growth medium. Using this protocol, we achieve high-quality, reproducible IMS images from three diverse cell types: MCF7 human breast cancer cells, Madin-Darby canine kidney (MDCK) cells, and NIH/3T3 mouse fibroblasts. This preparation method allows rapid and routine IMS analysis of cultured cells, making possible a wide variety of experiments to further scientific understanding of molecular processes within individual cells.

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.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is utilized to examine the mass spectra and fragmentation patterns of seven isomeric monosaccharides. Multivariate statistical analysis techniques, including principal component analysis (PCA), allow discrimination of the extremely similar mass spectra of stereoisomers. Furthermore, PCA identifies those fragment peaks that vary significantly between spectra. Heavy isotope studies confirm that these peaks are indeed sugar fragments, allow identification of the fragments, and provide clues to the fragmentation pathways. Excellent reproducibility is shown by multiple experiments performed over time and on separate samples. This study demonstrates the combined selectivity and discrimination power of TOF-SIMS and PCA and suggests new applications of the technique including differentiation of subtle chemical changes in biological samples that may provide insights into cellular processes, disease progress, and disease diagnosis.

Background: People diagnosed with cancer often self-administer complementary and alternative medicines (CAMs) to supplement their conventional treatments, improve health, or prevent recurrence. Flor-Essence and Essiac Herbal Tonics are commercially available complex mixtures of herbal extracts sold as dietary supplements and used by cancer patients based on anecdotal evidence that they can treat or prevent disease. In this study, we evaluated Flor-Essence and Essiac for their effects on the growth of human tumor cells in culture., Methods: The effect of Flor-Essence and Essiac((R)) herbal tonics on cell proliferation was tested in MCF-7, MDA-MB-436, MDA-MB-231, and T47D cancer cells isolated from human breast tumors. Estrogen receptor (ER) dependent activation of a luciferase reporter construct was tested in MCF-7 cells. Specific binding to the ER was tested using an ICI 182,780 competition assay., Results: Flor-Essence and Essiac herbal tonics at 1%, 2%, 4% and 8% stimulated cell proliferation relative to untreated controls in both estrogen receptor positive (MCF-7 and T47D) and estrogen receptor negative (MDA-MB-231 and MDA-MB-436) cell lines. Exposure to the tonics also produced a dose-dependent increase in ER dependent luciferase activity in MCF-7 cells. A 10(-7) M concentration of ICI 182,780 inhibited the induction of ER dependent luciferase activity by Flor-Essence and Essiac, but did not affect cell proliferation., Conclusion: Flor-Essence and Essiac Herbal Tonics can stimulate the growth of human breast cancer cells through ER mediated as well as ER independent mechanisms of action.

We use time-of-flight secondary ion mass spectrometry (TOF-SIMS) to image and classify individual cells on the basis of their characteristic mass spectra. Using statistical data reduction on the large data sets generated during TOF-SIMS analysis, similar biological materials can be differentiated on the basis of a combination of small changes in protein expression, metabolic activity and cell structure. We apply this powerful technique to image and differentiate three carcinoma-derived human breast cancer cell lines (MCF-7, T47D, and MDA-MB-231). In homogenized cells, we show the ability to differentiate the cell types as well as cellular compartments (cytosol, nuclear, and membrane). These studies illustrate the capacity of TOF-SIMS to characterize individual cells by chemical composition, which could ultimately be applied to detect and identify single aberrant cells within a normal cell population. Ultimately, we anticipate characterizing rare chemical changes that may provide clues to single cell progression within carcinogenic and metastatic pathways.

Many carcinogens have been shown to cause tissue specific tumors in animal models. The mechanism for this specificity has not been fully elucidated and is usually attributed to differences in organ metabolism. For heterocyclic amines, potent carcinogens that are formed in well-done meat, the ability to either bind to the estrogen receptor and activate or inhibit an estrogenic response will have a major impact on carcinogenicity. Here, we describe our work with the human estrogen receptor alpha (ERalpha), the mutagenic/carcinogenic heterocyclic amines PhIP, MeIQx, and IFP, and the hydroxylated metabolite of PhIP, N2-hydroxy-PhIP. We demonstrate both by computational docking and NMR analysis that PhIP binds with the ligand binding domain (LBD). This binding competes with estradiol (E2) in the native E2 binding cavity of the receptor. In vitro assays show that PhIP, in contrast to the other heterocyclic amines, increases cell proliferation in MCF-7 human breast cancer cells and activates the ERalpha receptor. We also find that other heterocyclic amines and N2-hydroxy-PhIP inhibit ERalpha activation. We propose that the mechanism for the tissue-specific carcinogenicity seen in the rat breast tumors and the presumptive human breast cancer associated with the consumption of well-done meat maybe mediated by this receptor activation.

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 < or = 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.

Carcinogenic heterocyclic amines are produced from overcooked foods and are highly mutagenic in most short-term test systems. One of the most abundant of these amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces breast, colon and prostate tumors in rats. Human dietary epidemiology studies suggest a strong correlation between either meat consumption or well-done muscle meat consumption and cancers of the colon, breast, stomach, lung and esophagus. For over 20 years our laboratory has helped define the human exposure to these dietary carcinogens. In this report we describe how various environmental exposures may modulate the risk from exposure to heterocyclic amines, especially PhIP. To assess the impact of foods on PhIP metabolism in humans, we developed an LC/MS/MS method to analyze the four major PhIP urinary metabolites following the consumption of a single portion of grilled chicken. Adding broccoli to the volunteers' diet altered the kinetics of PhIP metabolism. At the cellular level we have found that PhIP itself stimulates a significant estrogenic response in MCF-7 cells, but even more interestingly, co-incubation of the cells with herbal teas appear to enhance the response. Numerous environmental chemicals found in food or the atmosphere can impact the exposure, metabolism, and cell proliferation response of heterocyclic amines.

We devised an assay to quantify the metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human urine following a single exposure to well-cooked meat. Our method uses LC/MS/MS to detect four metabolites and four deuterated internal standard peaks in a single chromatographic run. N2-OH-PhIP-N2-glucuronide was the most abundant urinary metabolite excreted by the 12 individuals who participated in our study. N2-PhIP glucuronide was the second most abundant metabolite for 8 of the 12 volunteers. The stability of PhIP metabolism over time was studied in three of the volunteers who repeated the assay eight times over a 2.5 year-period. PhIP metabolite excretion varied in each subject over time, although the rate of excretion was more constant. Our results suggest that quantifying PhIP metabolites should make future studies of individual susceptibility and dietary interventions possible.

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.

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.

Heterocyclic amines produced from overcooked foods are extremely mutagenic in numerous in vitro and in vivo test systems. One of these mutagens, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces breast tumors in rats and has been implicated in dietary epidemiology studies as raising the risk of breast cancer in humans. Efforts in our laboratory and others have centered on defining the exposure to PhIP and other dietary mutagens derived from cooked food. We accomplish this by analyzing the foods with a series of solid-phase extractions and HPLC. We have developed an LC/MS/MS method to analyze the four major human PhIP metabolites (sulfates and glucuronides) following a single meal containing 27 microg of cooking-produced PhIP in 200 g of grilled meat. Although the intake of PhIP was similar for each of eight women, the total amount excreted in the urine and the metabolite profiles differed among the subjects. It appears that adsorption (digestion) from the meat matrix, other foods in the diet, and genetic differences in metabolism may contribute to the variation. The four major metabolites that can be routinely assayed in the urine are N(2)-OH-PhIP-N(2)-glucuronide, PhIP-N(2)-glucuronide, 4'-PhIP-glucuronide, and N(2)-OH-PhIP-N3-glucuronide. This work is suited to investigate individual exposure and risk, especially for breast cancer, from these potent dietary mutagens.

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 method's sensitivity after real-life exposures.

Many studies suggest that mutagenic/carcinogenic chemicals in the diet, like 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), may play a role in human cancer initiation. We have developed a method to quantify PhIP metabolites in human urine and have applied it to samples from female volunteers who had eaten a meal of cooked chicken. For this analysis, urine samples (5 ml) were spiked with a deuterium-labeled internal standard, adsorbed to a macroporous polymeric column and then eluted with methanol. After a solvent exchange to 0.01 M HCl, the urine extracts were passed through a filter, applied to a benzenesulfonic acid column, washed with methanol/acid and eluted with ammonium acetate and concentrated on a C(18) column. The metabolites were eluted from the C(18) column and quantified by LC/MS/MS. In our studies of human PhIP metabolism, eight volunteers were fed 200 g of cooked chicken containing a total of 27 microg PhIP. Urine samples were collected for 24 h after the meal, in 6 h aliquots. Although no metabolites could be found in urine collected from volunteers before eating the chicken, four major human PhIP metabolites, N:(2)-OH-PhIP-N:(2)-glucuronide, PhIP-N:(2)-glucuronide, 4'-PhIP-sulfate and N:(2)-OH-PhIP-N:3-glucuronide, were found in the urine after the chicken meal. The volunteers in the study excreted 4-53% of the ingested PhIP dose in the urine. The rate of metabolite excretion varied among the subjects, however, in all of the subjects the majority of the metabolites were excreted in the first 12 h. Very little metabolite was detected in the urine after 18 h. In humans, N:(2)-OH-PhIP-N:(2) glucuronide is the most abundant urinary metabolite, followed by PhIP-N:(2)-glucuronide. The variation seen in the total amount, excretion time and metabolite ratios with our method suggests that individual digestion, metabolism and/or other components of the diet may influence the absorption and amounts of metabolic products produced from PhIP.

To better understand the interactions of the pathways of activation and detoxification on the metabolism of the putative carcinogen, PhIP, we administered a dose of 70-84 microg [2-14C] PhIP (17.5 [microCi 14C) 48-72 h before scheduled colon surgery. Blood and urine collected for the next 48-72 h was evaluated by linear accelerator mass spectroscopy (AMS) and scintillation counting LC-MS to identify specific PhIP metabolites. The thermostable phenol sulfotransferase (SULT1A1) phenotype was correlated with the 4'-PhIP-SO4 levels in the urine at 0-4 h (R = 0.86, P = 0.059). The CYP1A2 activity had a negative correlation with PhIP serum levels at 1 h (R = 0.94, P = 0.06) and a positive correlation with urine N-OH-PhIP levels at 0-4 h (R = 0.85, P = 0.15). This low level radioisotope method of determining the influence of phenotype on metabolism will significantly improve our understanding of the interrelationships of these pathways and provide a critical foundation for the development of individual risk assessment.

[2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine ([14C]PhIP), a putative human carcinogenic heterocyclic amine found in well-done cooked meat, was administered orally to three colon cancer patients undergoing a partial colonectomy. Forty-eight to seventy-two hours prior to surgery, subjects received a 70-84 microg dose of 14C. Urine and blood were analyzed by HPLC for PhIP and PhIP metabolites. Metabolites were identified based on HPLC co-elution with authentic PhIP metabolite standards, mass spectral analysis and susceptibility to enzymatic cleavage. In two subjects, approximately 90% of the administered [14C]PhIP dose was eliminated in the urine, whereas in the other, only 50% of the dose was found in the urine. One subject excreted three times more radioactivity in the first 4 h than did the others. Twelve radioactive peaks associated with PhIP were detected in the urine samples. The relative amount of each metabolite varied by subject, and the amounts of each metabolite within subjects changed over time. In all three subjects the most abundant urinary metabolite was identified as 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine-N2-glucuron ide (N-hydroxy-PhIP-N2-glucuronide), accounting for 47-60% of the recovered counts in 24 h. PhIP accounted for <1% of the excreted radiolabel in all three patients. Other metabolites detected in the urine at significant amounts were 4-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate, N-hydroxy-PhIP-N3-glucuronide and PhIP-N2-glucuronide. In the plasma, N-hydroxy-PhIP-N2-glucuronide accounted for 60, 18 and 20% of the recovered plasma radioactivity at 1 h post PhIP dose in subjects 1, 2 and 3 respectively. Plasma PhIP was 56-17% of the recovered dose at 1 h post exposure. The relatively high concentration of N-hydroxy-PhIP-N2-glucuronide and the fact that it is an indicator of bioactivation make this metabolite a potential biomarker for PhIP exposure and activation. Determining the relative differences in PhIP metabolites among individuals will indicate metabolic differences that may predict individual susceptibility to carcinogenic risk from this suspected dietary carcinogen.

Cyclin-dependent kinase 5 (CDK5) is the 34 kDa catalytic subunit of a recently characterized neuronal cdc2-like protein kinase which appears to be involved in regulation of the neurocytoskeleton. Using the rat postdecapitative model, the effect of brain ischemia on histone H1 and tau protein CDK5 phosphorylating activity was examined. Histone H1 kinase activity increased in both cytosolic and particulate fractions of the hippocampus and neocortex after 5 min and 15 min of ischemia, then declined to control levels. CDK5 tau protein phosphorylating activity increased after 15 min ischemia; however, no electrophoretic shifts or changes in radiodensity of the tau bands were observed autoradiographically. On Western blot analysis, the CDK5 protein band did not change after 25 min ischemia, despite the increase and subsequent decline in enzyme activity. These data demonstrate a postischemic increase in CDK5 activity, an associated increase in CDK5 tau phosphorylating activity and a decline in activity in the absence of massive proteolysis. CDK5 appears to play a role in the events associated with neuronal response to ischemic injury.

Iron chelation, known to block progression through the cell cycle, was examined for effects on the activity and subunit levels of the cyclin-dependent protein kinases (cdk). Treatment of asynchronous MDA-MB-453 cells with the iron chelators mimosine or desferrioxamine (DFO) for 24 h stopped cell division, but did not produce a single, synchronous block. DNA content analysis demonstrated that although a majority of the cells were blocked in G1 (87.3%), an unexpectedly large fraction of the cells were blocked in S phase (11.5%). Western blot analysis of the treated lysates demonstrated the presence of cyclin B, confirming that part of the cell population was blocked in S phase. After release from mimosine treatment, 84% of the cell population remained in G1 up to 8 h. Treating breast cancer cells with 400 microM mimosine for 24 h inhibited cyclin E- and cyclin A-associated kinase activity by 85% or more, although immunoblots using anti-cyclin A, cyclin E, cdc2, and cdk2 antibodies showed that these key subunits were still present in the cells at pretreatment levels. Interestingly, Western blot analysis also demonstrated that iron chelation decreased the protein levels of the cyclin D and cdk4 subunits as compared to control and produced a change in retinoblastoma protein phosphorylation. These results indicate that iron deprivation effects the activity and protein levels of the cyclin-dependent kinases, and ultimately, the pathways that control cell division.

Mimosine is a toxic nonprotein amino acid that is a major constituent of the tropical legumes Leucaena and Mimosa. Mimosine has been shown to cause acute and chronic toxicosis in livestock fed from forage containing these plants. Recently, mimosine has been demonstrated to reversibly block cell cycle progression in mammalian cells in culture. In this study, we compared the effects of mimosine to desferrioxamine (DFO), a well-characterized iron chelator, and found that both chemicals similarly altered cell cycle progression in MDA-MB-453 human breast cancer cells. Mimosine (400 microM) and DFO (150 microM) both reduced DNA synthesis by greater than 90% of control within 4 hr of treatment, and suppressed total proline-directed protein kinase activity to less than 10% of control after 16 hr treatment. These effects were antagonized by the addition of iron as ferrous sulfate (250 microM), which is bound to transferrin and imported into the cell via transferrin receptor endocytosis, or as hemin (100 microM), which passes through the cell membrane and releases iron into the cytosol. After 24 hr treatment with the chelators, a large portion of the available transferrin receptors moved to the cell surface, indicating that the cells were iron-starved. Our data demonstrate that mimosine, through iron chelation, blocks cell cycle progression in MDA-MB-453 human breast cancer cells.