Toxic and transcriptional responses of PC12 cells to soluble tungsten alloy surrogates.

There is increasing evidence that metals have a role in the etiology of diverse neurological diseases. This study used PC12 cells as an in vitro model to examine the toxicity of tungsten alloys that have important military applications. Initially, the relative concentrations of tungsten (W), nickel (Ni), and cobalt (Co) mobilized from pellets of a weapons-grade tungsten alloy incubated in physiologically relevant solutions were determined. Dosing solutions of soluble metal salts that were equivalent in ratio to those mobilized from these alloy pellets were used to treat nerve growth factor (NGF) differentiated PC12 cells. Treatments consisted of single (W, Ni or Co), paired (W/Ni, W/Co or Ni/Co) or complete (W/Ni/Co) metal exposures for 24 h followed by measurement of cytotoxicity, viability, and microarray analysis to examine their impact on survival and viability, global gene expression, and biological processes. Gene expression changed dramatically with addition of NGF. Addition of Ni or Co either singly or in combination further impacted gene expression. An observed additive effect of Ni and Co on gene expression was unaffected by the addition of W. The work showed that tungsten, as found in this tungsten alloy, had minimal relative toxicity as compared to the other alloy components when used either alone or in combination.

Assessment of 3-nitro-1,2,4-triazol-5-one as a potential endocrine disrupting chemical in rats using the Hershberger and uterotrophic bioassays.

The explosive 3-nitro-1,2,4-triazol-5-one (NTO) is an insensitive formulation developed to replace high energetics that are susceptible to accidental detonation from heat, shock, and impact. Although studies have shown NTO to be nontoxic at acute exposures, recent subacute and subchronic tests have demonstrated effects on testes and subsequent sperm production in rats. This study assessed endocrine disruption as a potential mechanism for these reproductive effects via the Hershberger and uterotrophic bioassays. These assays are 2 of the US Environmental Protection Agency's tier 1 in vivo screens for the Endocrine Disruptor Screening Program that measure differences in androgen- and estrogen-sensitive tissue weights in castrated and ovariectomized rats. The gonadectomized rats were orally exposed to NTO in a corn oil vehicle at doses of 250, 500, or 1000 mg/kg body weight (bw)/d for 10 and 3 days for the Hershberger and uterotrophic assays, respectively, according to standard protocols. Male rats also received testosterone (0.2 mg/kg/d, subcutaneous) and antiandrogenic flutamide (3mg/kg/d, oral) as negative and positive controls, and females received 17 α-ethynyl estradiol (0.3 µg/d, subcutaneous) as positive controls. 3-Nitro-1,2,4-triazol-5-one caused neither a decrease in androgen-sensitive male reproductive selected tissue (seminal vesicles with fluid/without fluid, glans penis, Cowper gland, ventral prostrate, and levator ani-bulbocavernosus) weights nor a change in uterine weights. The results of this study provide no evidence to suggest that NTO acts like an estrogenic or antiandrogenic endocrine disruptor in rats at these doses.

In vivo corrosion, tumor outcome, and microarray gene expression for two types of muscle-implanted tungsten alloys.

Tungsten alloys are composed of tungsten microparticles embedded in a solid matrix of transition metals such as nickel, cobalt, or iron. To understand the toxicology of these alloys, male F344 rats were intramuscularly implanted with pellets of tungsten/nickel/cobalt, tungsten/nickel/iron, or pure tungsten, with tantalum pellets as a negative control. Between 6 and 12 months, aggressive rhabdomyosarcomas formed around tungsten/nickel/cobalt pellets, while those of tungsten/nickel/iron or pure tungsten did not cause cancers. Electron microscopy showed a progressive corrosion of the matrix phase of tungsten/nickel/cobalt pellets over 6 months, accompanied by high urinary concentrations of nickel and cobalt. In contrast, non-carcinogenic tungsten/nickel/iron pellets were minimally corroded and urinary metals were low; these pellets having developed a surface oxide layer in vivo that may have restricted the mobilization of carcinogenic nickel. Microarray analysis of tumors revealed large changes in gene expression compared with normal muscle, with biological processes involving the cell cycle significantly up-regulated and those involved with muscle development and differentiation significantly down-regulated. Top KEGG pathways disrupted were adherens junction, p53 signaling, and the cell cycle. Chromosomal enrichment analysis of genes showed a highly significant impact at cytoband 7q22 (chromosome 7) which included mouse double minute (MDM2) and cyclin-dependant kinase (CDK4) as well as other genes associated with human sarcomas. In conclusion, the tumorigenic potential of implanted tungsten alloys is related to mobilization of carcinogenic metals nickel and cobalt from corroding pellets, while gene expression changes in the consequent tumors are similar to radiation induced animal sarcomas as well as sporadic human sarcomas.

Preclinical characterization of 2-[3-[3-[(5-ethyl-4'-fluoro-2-hydroxy[1,1'-biphenyl]-4-yl)oxy]propoxy]-2-propylphenoxy]benzoic acid metabolism: in vitro species comparison and in vivo disposition in rats.

Assessment of the pharmacokinetics of [14C]2-[3-[3-[(5-ethyl-4'-fluoro-2-hydroxy[1,1'-biphenyl]-4-yl)oxy]propoxy]-2-propylphenoxy-]benzoic acid ([14C]LY293111), an experimental anti-cancer agent, suggested long-lived circulating metabolites in rats. In vivo metabolites of LY293111 were examined in plasma, bile, urine, and feces of Fischer 344 (F344) rats after oral administration of [14C]LY293111. Metabolites were profiled by high-performance liquid chromatography-radiochromatography, and identified by liquid chromatography (LC)/mass spectrometry and LC/NMR. The major in vivo metabolites of LY293111 identified in rats were phenolic (ether), acyl, and bisglucuronides of LY293111. Measurement of radioactivity in rat plasma confirmed that a fraction of LY293111-derived material was irreversibly bound to plasma protein and that this bound fraction increased over time. This was consistent with the observed disparity in half-lives between LY293111 and total radioactivity in rats and monkeys, and is likely due to covalent modification of proteins by the acyl glucuronide. In vitro metabolism of [14C]LY293111 in liver slices from CD-1 mice, F344 rats, rhesus and cynomolgus monkeys, and humans indicates that glucuronidation was the primary metabolic pathway in all species. The acyl glucuronide was the most prevalent radioactive peak (16% of total 14C) produced by F344 rat slices, whereas the ether glucuronide was the major metabolite in all other species (26-36% of total 14C). Several minor hydroxylated metabolites were detected in F344 rat slice extracts but were not observed in other species. The data presented suggest that covalent modification of proteins by LY293111 acyl glucuronide is possible in multiple species, although the relative reactivity of this metabolite appears to be low compared with those known to cause adverse drug reactions.

Disposition of LY333531, a selective protein kinase C beta inhibitor, in the Fischer 344 rat and beagle dog.

1. Studies were conducted in the Fischer 344 rat and beagle dog to determine the disposition of LY333531 and its equipotent active des-methyl metabolite, LY338522, both potent and selective inhibitors of the beta-isozyme of protein kinase C. 2. Male Fischer 344 rats and female beagle dogs received a single 5-mg kg(-1) oral dose of (14)C-LY333531. Urine, faeces, bile and plasma were collected and analysed for (14)C, LY333531 and LY338522. 3. LY333531 was eliminated primarily in the faeces (91% by 120 h in rat, 90% by 96 h in dog). Bile contributed the majority of the radioactivity excreted in the faeces in rat (66% in the cannulated bile duct study) and a variable but significant proportion in dog. 4. Pharmacokinetics following a single 5 mg kg(-1) oral dose of (14)C-LY333531 to the male rat produced C(max) and AUC(0-infinity ) for LY333531 of 14.7 ng ml(-1) and 60.8 ng h ml(-1), respectively, with a half-life of 2.5 h. LY338522 and total radioactivity showed similar profiles. 5. In the female dog at the same dose, C(max) and AUC(0-infinity ) of LY333531 were higher, producing 245 +/- 94 ng ml(-1) and 1419 +/- 463 ng h ml(-1), respectively, with a half-life of 5.7 h. 6. The data indicate that the disposition of LY333531 is similar in rat and dog.

The role of anion exchange in the uptake of Pb by human erythrocytes and Madin-Darby canine kidney cells.

Anion exchange (AE) plays a critical role in regulating intracellular pH in erythrocytes and epithelial cells and has been suggested to facilitate the transport of lead (Pb) across the erythrocyte cell membrane. In this study we examined the role of AE in the uptake of Pb by human erythrocytes and by Madin-Darby canine kidney (MDCK) cells, the kidney epithelial cell line. Functional AE in MDCK cells was evidenced by: increased uptake of SO(4)(2-) at pH 6.0 over pH 7.0, and inhibition of SO(4)(2-) uptake by the AE inhibitor 4, 4'-diisothiocyanostilbene-2, 2'- disulfonic acid (DIDS) as well as by non-halide anions. Accumulation of Pb into MDCK cells was time and temperature dependent. DIDS inhibited uptake of Pb into human erythrocytes but not MDCK cells. In conclusion, uptake of Pb into erythrocytes but not kidney epithelial cells occurs through AE.

An exploration of community psychiatric nursing: a Northern Ireland perspective.

The Audit Commission's (1994) report Finding a Place states that mental health problems are a major cause of disruption and difficulty in people's lives and that in any one year more than a quarter of all people suffer to some degree. The report asserts that the majority visit their general practitioner (GP) and less than half of the people concerned are recognized as having a mental health problem. Many recover over a period of weeks or months and are best served by community services. Only those with the most serious conditions need specialized care or admission to hospital. On a local level in Northern Ireland, the policy of reducing the number of long-stay patients began in the 1960s with a call for people with mental illness to be integrated into the community. This has had major implications for community mental health services and for community psychiatric nursing. The aim of this study into one community psychiatric nursing service (CPNS) in Northern Ireland is to inform managers and professionals about the nature and shape of this service and to facilitate wider discussion on how to plan and deliver it in the future.

The influence of zinc supplementation on morbidity due to Plasmodium falciparum: a randomized trial in preschool children in Papua New Guinea.

Zinc is crucial for normal immune function and can reduce morbidity from multiple infectious diseases. To determine the influence of zinc on malaria morbidity we conducted a randomized placebo-controlled trial of daily zinc supplementation in children residing in a malaria endemic region of Papua New Guinea. A total of 274 preschool children aged 6 to 60 months were given 10 mg elemental zinc (n = 136) or placebo (n = 138) for 6 days a week for 46 weeks. Slide-confirmed malaria episodes were detected by surveillance of cases self-reporting to a local health center. Cross-sectional surveys were conducted at the beginning, middle, and end of the study to assess infection rates, parasite density, spleen enlargement, and hemoglobin levels. Zinc supplementation resulted in a 38% (95% CI 3-60, P = 0.037) reduction in Plasmodium falciparum health center-based episodes, defined as parasitemia > or = 9200 parasites/microl with axial temperature > or = 37.5 degreesC or reported fever. Episodes accompanied by any parasitemia were also reduced by 38% (95% CI 5-60, P = 0.028), and episodes with parasitemia > or = 100,000/microl were reduced by 69% (95% CI 25-87, P = 0.009). There was no evidence of the effects of zinc on Plasmodium vivax morbidity or on health center attendance for causes other than P. falciparum. Zinc had no consistent effect on cross-sectional malariometric indices. Although P. falciparum prevalence tended to be lower at the end of the study in children given the placebo, such changes were absent at the mid-study survey. These results suggest that improved dietary zinc intake may reduce morbidity due to P. falciparum.

Kinetic analysis of the activation of Zymomonas mobilis glucokinase by phosphate.

A detailed kinetic analysis of glucokinase EC 2.7.1.2 from Zymomonas mobilis has been carried out. This enzyme has an absolute requirement for inorganic phosphate as activator, and the kinetic behaviour can be interpreted as a steady-state ordered mechanism in which glucose is the first substrate. Values for each of the kinetic constants have been obtained for the conditions I = 0.12, 30 degrees C, and pH 7.0. Direct binding studies have confirmed that ATP does not bind to the enzyme without glucose present. Phosphate does not affect ATP binding to the enzyme-glucose complex; when saturated with both ATP and glucose, the dissociation constant for phosphate (determined kinetically) is 0.045 mM. When saturated with the other substrate and phosphate, the Km values for glucose and MgATP are 0.095 mM and 0.19 mM, respectively. The ionic form of phosphate is not important, as the apparent Km for phosphate did not change significantly over the pH range 6.4 to 7.5. Raising the temperature increased Vmax at the high rate of 10% per degree, which correlates well with the fermentation rates between 20 and 30 degrees C, giving further support to the concept that glucokinase is the rate-controlling enzyme in Z. mobilis glucose fermentation.

Pilot study of sources of lead exposure in Moscow, Russia.

This preliminary investigation of sources of lead exposure in Moscow, Russia, by Russian and US collaborators measured lead in paint, interior dust, and drinking water in seven day-care centres, and in petrol, soil and canned food. Some paint samples exceeded US regulatory standards for lead in paint on surfaces (0.5%). Dust lead loadings were < 1.7 µg cm(-2) and below the guidance levels of the US EPA. Drinking water lead concentrations were at or below the US drinking water standard of 15 µg L(-1). Lead concentrations in petrol from Moscow vehicles and petrol stations were consistent with a regulation banning the sale of leaded petrol within the Moscow City limits. Except for baby food, lead levels were higher in the Russian canned foods (range 6 to 1240 µg kg(-1), dry weight) compared to corresponding US canned foods, with ratios of Russian to US levels of up to 120:1 for evaporated milk. Lead concentrations in soil generally ranged from 500 to 2000 µg g(-1), levels that would trigger hazard reduction measures according to US EPA guidance. These findings, together with the use of lead in petrol outside Moscow, indicate multiple sources of lead exposure in Russia. Priorities for future research are discussed including the establishment of interlaboratory quality control programmes.

Graphite furnace atomic absorption spectroscopic measurement of blood lead in matrix-matched standards.

Now that the level of concern for a toxic blood lead concentration is 0.482 mumol/L (10 micrograms/dL), laboratories must meet new requirements to shorten analysis times and increase accuracy and precision of blood lead determinations. We used a matrix-matching method to estimate the lead concentration in blood by graphite furnace atomic absorption spectroscopy (GFAAS). For CDC proficiency samples and the NIST-Certified Blood Reference standard, the performance of this method compared favorably with that of previously published GFAAS methods and of the anodic stripping voltammetric method routinely used in our laboratory. At lead concentrations of 0.242 mumol/L (5.01 micrograms/dL) and 1.478 mumol/L (30.63 micrograms/dL), within-run CVs were 2.78% and 0.68%, respectively; between-run CVs were 4.9% and 1.35%. In 52 study samples with lead content ranging from 0.097 to 3.812 mumol/L (2 to 79 micrograms/dL), 87% of results by the matrix-modified method were within 0.048 mumol/L (1 microgram/dL) of consensus values.

Comparison of a wipe and a vacuum collection method for the determination of lead in residential dusts.

Lead-containing house dust is an important source of childhood lead exposure. Standard methods for collection of settled dust for evaluation of lead content have not been established. Little is known about the relationships between the various wipe and vacuum-based methods employed in past studies, preventing meaningful comparisons of results. This study characterized the relationship between a frequently used wipe dust collection method and a vacuum-based in-line filter method used to collect dust in a national survey of lead in paint and dust in U.S. housing. The correlation coefficient was 0.82 for estimates of lead loadings (PbD, mg/m2) from 71 pairs of side-by-side wipe and vacuum dust samples collected from uncarpeted floors, window sills, and exterior window wells in six dwellings. Geometric mean (GM) wipe PbD estimates exceeded those for vacuum samples by a factor of 3.9 and 5.7 for floors and window sills, respectively, findings consistent with the multiple sources of sample loss associated with the vacuum sampler. For window wells, the GM vacuum PbD estimate exceeded the GM wipe PbD estimate by a factor of 3.4, possibly due to the use of an alternative vacuum nozzle. The resulting increase in the estimated prevalence of U.S. homes with elevated dust lead loadings had wipe-sampling instead of vacuum-sampling methods been used in the national survey is discussed.

Synthesis of [11C]dapoxetine.HCl, a serotonin re-uptake inhibitor: biodistribution in rat and preliminary PET imaging in the monkey.

[11C]Dapoxetine.HCl, S-(+)-N,N-dimethyl-a-[2-(naphthalenyloxy)ethyl] benzenemethanamine hydrochloride, a potent serotonin re-uptake inhibitor was prepared from its mono-methyl precursor, S-(+)-N-methyl-a-[2-(naphthalenyloxy)ethyl]benzene methanamine hydrochloride. Biodistribution was determined in rats at 5, 30 and 60 min after injection and preliminary PET studies were performed in a Rhesus monkey. 11CH3I was bubbled into a solution of S-(+)-N-methyl-alpha-[2-(naphthalenyloxy)ethyl]benzene methanamine hydrochloride (3.0 mg in DMSO) and the mixture was heated at 110 degrees C for 8 min. [11C]Dapoxetine.HCl was purified by HPLC on a C18 cartridge eluted with MeOH:phosphate buffer, pH 7,2 (75:25) with a 10% yield (end of synthesis). The time required for the synthesis was 40 min, from the end of bombardment. Radiochemical purity of the final product was > 99% and specific activity was routinely > 400 mCi/mumol [EOS]. In the biodistribution studies the highest concentration (%ID/g +/- SEM) of dapoxetine.HCl was detected in lung: 4.56 +/- 0.27 (5 min), 1.28 +/- 0.18 (30 min) and 0.67 +/- 0.04 (60 min). Brain accumulation was 0.76 +/- 0.02 (5 min), 0.46 +/- 0.04 (30 min) and 0.27 +/- 0.01 (60 min). Preliminary PET studies demonstrated significant displaceable binding in the cerebral cortex and subcortical grey matter. These results demonstrate that [11C]dapoxetine.HCl can be prepared in high purity and may be useful for the in vivo evaluation of serotonin re-uptake mechanisms.

Retrograde continuous warm blood cardioplegia: maintenance of myocardial homeostasis in humans.

Recent clinical reports have suggested that continuous delivery of oxygenated warm blood cardioplegia through the coronary veins (retrograde cardioplegia) produces good myocardial preservation during aortic cross-clamping. No data exist, however, about actual myocardial metabolism/homeostasis during retrograde warm blood cardioplegia. We studied 100 consecutive patients undergoing coronary artery bypass grafting, aortic valve replacement, or both who received retrograde continuous warm blood cardioplegia (4:1 dilution) during aortic cross-clamping for 54 to 174 minutes. We measured pH, oxygen tension, carbon dioxide tension, HCO3, base excess, and oxygen content of the inflow cardioplegia and the blood egressing from coronary arteries during each arteriotomy for bypass grafting (arteries act as postcapillary veins with retrograde cardioplegia) or the left and right coronary orifices during aortic valve replacement. We also measured these variables from the coronary sinus effluent 1 minute after release of the aortic cross-clamp. Retrograde cardioplegia flow ranged from 50 to 250 mL/min (mean flow, 150 mL/min). All patients were maintained at normothermia during bypass. A total of 460 samples were analyzed (4.6 per patient). Neither the duration of aortic cross-clamping nor the artery sampled affected myocardial blood gases. The pH dropped from 7.41 +/- 0.05 for the inflow cardioplegia to 7.32 +/- 0.1 when sampled from coronary arteries, and the oxygen tension fell from 181 +/- 25 to 28 +/- 5 mm Hg, respectively. Carbon dioxide tension rose from 31.0 +/- 4.1 to 41.4 +/- 9.8 mm Hg. Coronary sinus blood gases 1 minute after cross-clamp removal showed no acidosis or oxygen debt.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiwell chamber chemotaxis assays: improved experimental design and data analysis.

The chemotaxis assay using the Boyden transfilter technique has become widely used in recent years for assessing migratory responses of a wide variety of cell types. In the study reported here we examined the migratory responses of mouse peritoneal macrophages using a multiwell chamber. The experiments were designed to analyze the components of variance in the assay method, to optimize the experimental design, and to develop objective statistical criteria for choosing among experiments with disparate results. Cell counts were obtained with the aid of an image analyzer coupled to a light microscope. Microcomputer software was developed to drive the image analyzer, collect data and conduct statistical analyses. Nested analysis of variance (ANOVA, either 2- or 3-level) was employed to partition the components of variance and F-tests were used to determine their significance. Significant sources of experimental error were identified both within and among wells and were attributed mostly to variability in the chamber/filter assembly and counting procedure. Statistical analyses demonstrated that there was significant variation among assays conducted in different multiwell chambers on the same day, among assays where the same agent was tested on different days in the same chamber, and among replicate counts of the same assay. The following recommendations were made: use ANOVA to distinguish differences due to biological effects from those due to experimental error, design experiments so that all relevant comparisons are included in the same chamber and the same assay, avoid pooling data from different assays unless ANOVA treatment variances are comparable, and when replicate assays yield disparate results choose the assay with the lowest percentage of variation due to experimental error.

Bone-derived macrophage chemotactic factors: methods of extraction and further characterization.

Mononuclear phagocytes have been implicated as important cellular elements in the process of bone resorption. We have postulated that the recruitment and migration of mononuclear phagocytes to bone occurs via a mechanism(s) in which bone-derived chemotactic factors (BDCF) are released from foci undergoing resorption. In the experiments presented here we have used newborn mouse calvaria and examined a variety of extraction protocols, both dissociative and non-dissociative, as means of obtaining stable and reproducible chemotactic activity for mouse peritoneal macrophages. Chemotaxis and chemokinesis were assessed using a multi-well chamber modification of the Boyden transfilter method. Further, we have attempted to purify the BDCF by both molecular sieve and anion exchange chromatography. Our results indicated that non-dissociative extraction with 0.5 M EDTA in the presence of 1% DMSO yielded the most potent and reproducible chemotactic activity. The results of molecular sieve and anion exchange chromatography suggested that there were several BDCF activities in these preparations and that their molecular weights were probably in the range of from 14,000-67,000 daltons. Anion exchange chromatography also demonstrated the presence of a fraction, eluted with 2 M NaCl, with high chemotactic activity and minimal protein concentration. These observations confirmed the suggestion that there are several macrophage chemotactic factors in bone which have as yet to be identified, and suggest methods for pursuing their isolation.