Controlled placement of multiple CNS cell populations to create complex neuronal cultures.

In vitro brain-on-a-chip platforms hold promise in many areas including: drug discovery, evaluating effects of toxicants and pathogens, and disease modelling. A more accurate recapitulation of the intricate organization of the brain in vivo may require a complex in vitro system including organization of multiple neuronal cell types in an anatomically-relevant manner. Most approaches for compartmentalizing or segregating multiple cell types on microfabricated substrates use either permanent physical surface features or chemical surface functionalization. This study describes a removable insert that successfully deposits neurons from different brain areas onto discrete regions of a microelectrode array (MEA) surface, achieving a separation distance of 100 µm. The regional seeding area on the substrate is significantly smaller than current platforms using comparable placement methods. The non-permanent barrier between cell populations allows the cells to remain localized and attach to the substrate while the insert is in place and interact with neighboring regions after removal. The insert was used to simultaneously seed primary rodent hippocampal and cortical neurons onto MEAs. These cells retained their morphology, viability, and function after seeding through the cell insert through 28 days in vitro (DIV). Co-cultures of the two neuron types developed processes and formed integrated networks between the different MEA regions. Electrophysiological data demonstrated characteristic bursting features and waveform shapes that were consistent for each neuron type in both mono- and co-culture. Additionally, hippocampal cells co-cultured with cortical neurons showed an increase in within-burst firing rate (p = 0.013) and percent spikes in bursts (p = 0.002), changes that imply communication exists between the two cell types in co-culture. The cell seeding insert described in this work is a simple but effective method of separating distinct neuronal populations on microfabricated devices, and offers a unique approach to developing the types of complex in vitro cellular environments required for anatomically-relevant brain-on-a-chip devices.

Long-term non-invasive interrogation of human dorsal root ganglion neuronal cultures on an integrated microfluidic multielectrode array platform.

Scientific studies in drug development and toxicology rely heavily on animal models, which often inaccurately predict the true response for human exposure. This may lead to unanticipated adverse effects or misidentified risks that result in, for example, drug candidate elimination. The utilization of human cells and tissues for in vitro physiological platforms has become a growing area of interest to bridge this gap and to more accurately predict human responses to drugs and toxins. The effects of new drugs and toxins on the peripheral nervous system are often investigated with neurons isolated from dorsal root ganglia (DRG), typically with one-time measurement techniques such as patch clamping. Here, we report the use of our multi-electrode array (MEA) platform for long-term noninvasive assessment of human DRG cell health and function. In this study, we acquired simultaneous optical and electrophysiological measurements from primary human DRG neurons upon chemical stimulation repeatedly through day in vitro (DIV) 23. Distinct chemical signatures were noted for the cellular responses evoked by each chemical stimulus. Additionally, the cell viability and function of the human DRG neurons were consistent through DIV 23. To the best of our knowledge, this is the first report on long-term measurements of the cell health and function of human DRG neurons on a MEA platform. Future generations will include higher electrode numbers in customized arrangements as well as integration with different tissue types on a single device. This platform will provide a valuable testing tool for both rodent and human cells, enabling a more comprehensive risk assessment for drug candidates and toxicants.

PhIP metabolites in human urine after consumption of well-cooked chicken.

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.

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 method's sensitivity after real-life exposures.

Identification of urine metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine following consumption of a single cooked chicken meal in humans.

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.

In vivo human metabolism of [2-14C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (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.

The identification of [2-(14)C]2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine metabolites in humans.

[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 protein kinase 5 activity increases in rat brain following ischemia.

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 deprivation inhibits cyclin-dependent kinase activity and decreases cyclin D/CDK4 protein levels in asynchronous MDA-MB-453 human breast cancer cells.

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 blocks cell cycle progression by chelating iron in asynchronous human breast cancer cells.

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.

A clinical screening program for topical chemotherapeutic drugs in psoriasis.

In this national, multicenter cooperative study, a standardized drug screening program was designed and evaluated to test the clinical effectiveness of 30 topically applied chemotherapeutic drugs to psoriasis. Appropriate concentrations and vehicles for topical administration were selected with regard to clinical testing consisted of a double-blind application of test agents to psoriatic plaques under occlusion daily for up to nine days. Drugs known to be topically active in psoriasis, eg, thiotepa, fluorouracil, and betamethasone valerate, were easily detected in the clinical protocol, confirming the validity of this topical drug screening program. Seven drugs produced substantial clinical improvement with evidence of clearing; nine drugs produced slight improvement; 14 drugs had no effect. No systemic toxid reactions occurred. This screen should be useful to test other potential antipsoriatic drugs and to evaluate potential animal model screens for their predictive values with the same drugs.

Staling white pan bread: fundamental causes.

Staling, as it is applied to bakery foods, is a generic term covering a number of changes that occur in the products during normal storage. Consumers judge staleness by direct perception, which provides a subjective estimate that represents an unconscious integration of many factors. This review will discuss the main components of staling: (1) physicochemical changes of bread and related products (firming and texture deterioration of crumb and loss of crispness of crust) and (2) flavor changes. Section I will cover current theories of changes of firming and textural changes. The starch component of flour is generally considered to be responsible for these staling reactions. Consequently, the physicochemical involvement of amylose, amylopectin in these reactions will be fully discussed and current evidence supporting these theories (rheological, chemical, X-rays) will be given. Interactions of starch and surface active agents and other complexing compounds will be presented in Section II. In Section III, contribution of minor flour components and bakery food ingredients will be evaluated. Section IV will focus on organoleptic deterioration of products, presenting flavor changes that were observed during staling bread. Section V will discuss structural changes of breads caused by enzymolysis during bread production and storage as related to staling. Following the theoretical section (Sections I to V), Section VI will focus on practical control of staling. This discussion will cover the following factors: formulation, surfactants, enzymes, storage, freezing, and packaging.