Toxicological assessment of dust from sanding micronized copper-treated lumber in vivo.

Micronized copper azole (MCA) is a lumber treatment improve longevity. In this study, the in vivo response to PM2.5 sanding dust generated from MCA-treated lumber was compared to that of untreated yellow pine (UYP) or soluble copper azole-treated (CA-C) lumber to determine if the MCA was more bioactive than CA-C. Mice were exposed to doses (28, 140, or 280 µg/mouse) of UYP, MCA, or CA-C sanding dust using oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) lactate dehydrogenase activity was increased at 1 day post-exposure to 280 µg/mouse of MCA and CA-C compared to UYP. BALF polymorphonuclear cells were increased by MCA and CA-C. There were increases in BALF cytokines in MCA and CA-C-exposed groups at 1 day post-exposure. Lung histopathology indicated inflammation with infiltration of neutrophils and macrophages. Pulmonary responses were more severe in MCA and CA-C-exposed groups at 1 day post-exposure. MCA caused more severe inflammatory responses than CA-C at 1 day post-exposure. These findings suggest that the MCA and CA-C sanding dusts are more bioactive than the UYP sanding dust, and, moreover, the MCA sanding dust is more bioactive in comparison to the CA-C sanding dust. No chronic toxic effects were observed among all observed sanding dusts.

Assessment of false negative rates of lactate dehydrogenase-based malaria rapid diagnostic tests for Plasmodium ovale detection.

Currently, malaria rapid diagnostic tests (RDTs) are widely used for malaria diagnosis, but test performance and the factors that lead to failure of Plasmodium ovale detection are not well understood. In this study, three pLDH-based RDTs were evaluated using cases in China that originated in Africa. The sensitivity of Wondfo Pf/Pan, CareStart pLDH PAN and SD BIOLINE Pf/Pan in P. ovale detection was 70, 55 and 18%, respectively. CareStart was worse at detecting P. o. curtisi (36.5%) than at detecting P. o. wallikeri (75.0%), and SD could not detect P. o. curtisi. The overall detection ratio of all three RDTs decreased with parasite density and pLDH concentration. Wondfo, CareStart and SD detected only 75.0, 78.1 and 46.9% of the P. ovale cases, respectively, even when the parasitemia were higher than 5000 parasites/µL. Subspecies of P. ovale should be considered while to improve RDT quality for P. ovale diagnosis to achieve the goal of malaria elimination.

The use of Fionet technology for external quality control of malaria rapid diagnostic tests and monitoring health workers' performance in rural military health facilities in Tanzania.

INTRODUCTION: Internal and external quality control (QC) of rapid diagnostic tests (RDTs) is important to increase reliability of RDTs currently used to diagnose malaria. However, cross-checking of used RDTs as part of quality assurance can rarely be done by off-site personnel because there is no guarantee of retaining visible test lines after manufacturers' recommended reading time. Therefore, this study examined the potential of using Fionet™ technology for remote RDT quality monitoring at seven clinics, identifying reasons for making RDT processing and interpretation errors, and taking corrective actions for improvement of diagnosis and consequently improved management of febrile patients. METHODS: The study was conducted at seven military health facilities in Mainland Tanzania and utilized RDTs capable of detecting Plasmodium falciparum specific Histidine-rich protein 2 (Pf-HRP2) and the genus specific Plasmodium lactate dehydrogenase (pLDH) for other species of plasmodium (P. vivax, P. malariae or P. ovale; pan-pLDH). Patients' data and images of processed RDTs from seven clinics were uploaded on a Fionet web portal and reviewed regularly to monitor preparation procedures and visual interpretation of test results compared to automated analysis using the Deki reader of RDT. Problems detected were rapidly communicated to remote laboratory personnel at the clinic for corrective action and follow-up of patients who were falsely diagnosed as negative and missed treatment. Factors contributing to making errors in visual interpretation of RDT results were analyzed during visits to the health facilities. RESULTS: A total of 1,367 (1.6%) out of 83,294 RDT test images uploaded to the Fionet portal had discordant test results of which 822 (60.1%) and 545 (39.9%) were falsely reported as negative and positive, respectively. False negative and false positive test results were common for a single test line in 515 (62.7%) and 741 (54.2%) tests, respectively. Out of 1,367 RDT images assessed, 98 (7.2%) had quality problems related to preparation procedures of which 95(96.9%) errors were due to putting too much blood on the sample well or insufficient buffer in the respective wells. The reasons for discrepant results included, false reporting of none existent lines in 526 (38.5%) tests, missing a faint positive line in 493 (36.1%), missing a strong positive line in 248(18.1%) and errors caused by poorly processed RDTs in 96 (7.2%) tests. Among the false negative tests (n = 822), 669 (48.9%) patients were eligible for follow-up and only 339 (48.5%) were reached and 291 (85.8%) received appropriate anti-malaria therapy. CONCLUSION: Fionet technology enabled remote monitoring of RDT quality issues, identifying reasons contributing to laboratory personnel making errors and provided a rapid method to implement corrective actions at remote sites to improve malaria diagnosis and consequently improved health care management of febrile patients infected with malaria.

Immunomagnetic capture and colorimetric detection of malarial biomarker Plasmodium falciparum lactate dehydrogenase.

We report a sensitive, magnetic bead-based colorimetric assay for Plasmodium falciparum lactate dehydrogenase (PfLDH) in which the biomarker is extracted from parasitized whole blood and purified based on antigen binding to antibody-functionalized magnetic particles. Antigen-bound particles are washed, and PfLDH activity is measured on-bead using an optimized colorimetric enzyme reaction (limit of detection [LOD] = 21.1 ± 0.4 parasites/µl). Enhanced analytical sensitivity is achieved by removal of PfLDH from the sample matrix before detection and elimination of nonspecific reductases and species that interfere with the optimal detection wavelength for measuring assay development. The optimized assay represents a simple and effective diagnostic strategy for P. falciparum malaria with time-to-result of 45 min and detection limits similar to those of commercial enzyme-linked immunosorbent assay (ELISA) kits, which can take 4-6 h. This method could be expanded to detect all species of malaria by switching the capture antibody on the magnetic particles to a pan-specific Plasmodium LDH antibody.

PET/CT Assessment of Follicular Lymphoma and High Grade B Cell Lymphoma - Good Correlation with Clinical and Histological Features at Diagnosis.

BACKGROUND: Follicular lymphoma is a common type of non-Hodgkin's lymphoma observed in Western countries. The diagnosis of this disease is based primarily on morphological and immunohistochemical assessment. The proliferative index Ki67 correlates with histological grading and clinical aggressiveness. Currently, positron emission tomography/computed tomography scanning are not applied for standard staging at diagnosis of follicular lymphoma and its use is limited to those patients for whom the identification of residual disease is crucial for therapeutic decisions and only when transformation to a high-grade lymphoma is suspected. OBJECTIVES: Our aim was to assess whether a correlation exists between the maximal standardized uptake value (SUVmax) at the biopsy site as detected via positron emission tomography/computed tomography and pathological (Ki67 and FL histological grade) and clinico-biological features (e.g. LDH, beta-2-microglobulin, Ann Arbor stage and FL International Prognostic Index--FLIPI) at diagnosis. MATERIAL AND METHODS: We retrospectively identified 16 patients during the previous 3.5 years in whom node biopsies were guided, taking into account the SUVmax as detected upon PET/CT scan at diagnosis. The results of these biopsies were diagnostic of follicular lymphoma. We also included 6 patients with high grade B cell lymphoma: 5 diffuse large B cell lymphoma (DLBCL) and 1 FL 3b histological grade. A 2-tailed non-parametric Spearman's correlation analysis of the SUVmax with Ki67, histological grade, LDH and b-2-microglobuline was performed. RESULTS: The Ki67 (r=0.73) and follicular lymphoma histological grade (r=0.75) at the biopsy displayed a significant correlation with the SUVmax at diagnosis (p<0.01). CONCLUSIONS: Our results suggest that SUV detected by positron emission tomography/computed tomography correlates with histological grade in follicular lymphoma/high grade B cell lymphoma, Ki67 and LDH. Positron emission tomography/computed tomography should be considered for guiding lymph node biopsy when transformation to a high-grade B cell lymphoma is suspected.

Technical note: comparison of the PrestoBlue and LDH release assays with the MTT assay for skin viability assessment.

MTT assay is the gold standard for assessing skin sample viability but it is time-consuming. Here we compared the MTT test with two other assays for the assessment of skin viability. The MTT, PrestoBlue (colorimetric method) and LDH release assays were applied to fresh and cryopreserved skin. Skin viability was considered proportional to the optical density values of the relevant analytes. PrestoBlue did not reliably distinguish between fresh and cryopreserved skin. The LDH release assay did not allow us to establish a viability index. We recommend the MTT assay for assessing skin viability.

Correlation between health-care costs and salivary tests.

The burden of health-care costs relative to gross domestic product in Japan is increasing. A large percentage (7.6% in 2009) of the Japanese gross domestic product has been spent on health care, and this percentage has been increasing annually. Soaring health-care costs have been recognised as a serious social problem. In this study, we attempted to estimate the relationship between periodontal disease and health-care costs. Subjects consisted of teachers and staff members (35 men, 26 women; mean age, 45 ± 9 years) from two high schools. The salivary levels of lactate dehydrogenase and haemoglobin were adopted as biomarkers to assess periodontal disease. After salivary tests, data for the health-care costs over the subsequent 6 months were provided by the mutual association of the public schools on an individual basis. Curve-fit estimations were then performed where health-care costs were used as a dependent variable and age or salivary levels of haemoglobin or lactate dehydrogenase were used as independent variables. However, no good fitness was obtained. Subsequently, multilayer perceptron neural networks were applied. With the neural networks, good fitness was obtained by using lactate dehydrogenase as an independent variable. The results of this study show that oral health, particularly periodontal disease, is correlated with total health-care costs. The data presented in this study suggests that, from the perspective of both oral and systemic health, oral health can be a signpost in well-being and health promotion.

[Use of oral fluid metabolic rates for the assessment of reparative osteogenesis after bone augmentation procedures].

The use of osteoplastic materials allows extending indications for dental implants placement by considerable alveolar bone atrophy. The aim of the study was to reveal bone tissue metabolites which can be used as early bone destruction markers after bone augmentation procedure before any radiological signs occur. For this purpose the content of osteocalcinum, C-telopeptides, alkaline phosphatase and lactate dehydrogenase was examined in oral fluid. The increase of osteocalcinum and C-telopeptides and decrease activity of alkaline phosphatase and lactate dehydrogenase in comparison with control represented the pattern specific for bone destruction.

The value of machine perfusion biomarker concentration in DCD kidney transplantations.

BACKGROUND: Donation after cardiac death (DCD) increases the number of donor kidneys but is associated with more primary nonfunction (PNF) and delayed graft function (DGF). It has been suggested that biomarkers in the preservation solution of machine perfused kidneys may predict PNF, although evidence is lacking. METHODS: We analyzed the diagnostic accuracy of the perfusate biomarkers glutathione S-transferase, lactate dehydrogenase (LDH), heart-type fatty acid binding protein, redox-active iron, interleukin (IL)-18, and neutrophil gelatinase-associated lipocalin to predict PNF and DGF in 335 DCD kidneys preserved by hypothermic machine perfusion at our center between 1 January 1997 and 1 January 2008. The diagnostic accuracy of these biomarkers to predict PNF was evaluated with the area under the receiver operating characteristics curves. Additionally, the risk of DGF and graft failure was assessed. RESULTS: LDH and IL-18 concentrations were associated with PNF (odds ratio [95% confidence interval], 1.001 [1.000-1.002]; P=0.005 and 1.001 [1.000-1.002]; P=0.003, respectively) in a multivariate analysis; the diagnostic accuracy for PNF was "poor" for all biomarkers but increased to "fair" for redox-active iron and IL-18 in a multivariate analysis (area under the receiver operating characteristics curves, 0.701 and 0.700, respectively). LDH and IL-18 concentrations were associated with DGF; biomarker concentration was not associated with 1-year graft survival. CONCLUSIONS: The diagnostic accuracy of the perfusate biomarkers glutathione S-transferase, LDH, heart-type fatty acid binding protein, redox-active iron, IL-18, and neutrophil gelatinase-associated lipocalin to predict viability of DCD kidneys varies from "poor" to "fair". Therefore, DCD kidneys should not be discarded because of high biomarker perfusate concentration.

Assessment of neutrophil gelatinase-associated lipocalin and lactate dehydrogenase in peritoneal fluids for the screening of bacterial peritonitis.

BACKGROUND: Manual microscopy remains the gold standard for enumeration and classification of nucleated cells in peritoneal fluids, especially for diagnosing bacterial peritonitis. However, this approach carries several drawbacks, so that the use of simple and automated tests may be a viable option for initial screening of peritoneal fluids. MATERIALS AND METHODS: Neutrophil gelatinase-associated lipocalin (NGAL), lactate dehydrogenase (LDH), proteins and glucose were assessed in peritoneal fluids from patients with new onset nonmalignant ascites, along with nucleated cell count and differentiation. RESULTS: One hundred and eleven specimens were analyzed, 26 of which (23%) with polymorphonuclear leukocyte (PMN) count≥250/µL, thus compatible with bacterial peritonitis. The median concentration of LDH and NGAL was 3.4 and 3.7-fold higher in samples with ≥250 PMN/µL. The concentration of proteins was also higher in samples with ≥250 PMN/µL, whereas that of glucose was lower. The PMN count significantly correlated with peritoneal fluid values of LDH (r=0.859), NGAL (r=0.774) and proteins (r=0.268), but not with glucose (r=-0.069). The area under ROC curve was 0.88 for LDH, 0.89 for NGAL and 0.94 for their combination (both tests positive), whereas that of proteins and glucose was 0.80 and 0.71, respectively. Sensitivities and specificities were 0.81 and 0.87 for LDH≥227 U/L, 0.96 and 0.75 for NGAL≥120 ng/mL, 0.77 and 0.95 for their combination. The agreement with PMN count was 0.86 for LDH, 0.80 for NGAL, and 0.91 for their combination. CONCLUSIONS: These results suggest that assessment of NGAL in peritoneal fluids, especially in combination with LDH, may be a reliable approach for screening of bacterial peritonitis in patients with new onset nonmalignant ascites.

Health status of Pomatoschistus microps populations in relation to pollution and natural stressors: implications for ecological risk assessment.

Effects induced on wild populations by recurrent environmental contamination may difficult the ecological risk assessment of punctual pollution events such as oil spills. Here, the issue was addressed by comparing the health status of Pomatoschistus microps populations from four NW Iberian estuaries, using an integrated chemical-biological monitoring. Despite high seasonal variability, the parameters measured discriminated estuaries with different contamination levels and associated biological effects with chemical and abiotic stress. The decreased health status of fish from polluted sites strengthens the need of considering pollution-induced background effects and seasonal variability when assessing impacts and risks of oil and other chemical spills.

Proliferation assessment of primary human mesenchymal stem cells on collagen membranes for guided bone regeneration.

PURPOSE: Human mesenchymal stem cells (hMSCs) hold the potential for bone regeneration because of their self-renewing and multipotent character. The goal of this study was to evaluate the influence of collagen membranes on the proliferation of hMSCs derived from bone marrow. A special focus was set on short-term eluates derived from collagen membranes, as volatile toxic materials washed out from these membranes may influence cell behavior during the short time course of oral surgery. MATERIALS AND METHODS: The proliferation of hMSCs seeded directly on a collagen membrane (BioGide) was evaluated quantitatively using the cell proliferation reagent WST-1 (4-3-[4-iodophenyl]-2-[4-nitrophenyl]-2H-[5-tetrazolio]-1, 3--benzol-disulfonate) and qualitatively by scanning electron microscopy. Two standard biocompatibility tests, namely the lactate dehydrogenase and MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazoliumbromide) tests, were performed using hMSCs cultivated in eluates from membranes incubated for 10 minutes, 1 hour, or 24 hours in serum-free cell culture medium. The data were analyzed statistically. RESULTS: Scanning electron microscopy showed large numbers of hMSCs with well-spread morphology on the collagen membranes after 7 days of culture. The WST test revealed significantly better proliferation of hMSCs on collagen membranes after 4 days of culture compared to cells cultured on a cover glass. Cytotoxicity levels were low, peaking in short-term eluates and decreasing with longer incubation times. CONCLUSION: Porcine collagen membranes showed good biocompatibility in vitro for hMSCs. If maximum cell proliferation rates are required, a prewash of membranes prior to application may be useful.

A decision tree for differentiating tuberculous from malignant pleural effusions.

OBJECTIVE: To improve physicians' ability to discriminate tuberculous from malignant pleural effusions through a simple clinical algorithm that avoids pleural biopsy. DESIGN: We retrospectively compared the clinical and pleural fluid features of 238 adults with pleural effusion who satisfied diagnostic criteria for tuberculosis (n=64) or malignancy (n=174) at one academic center (derivation cohort). Then, we built a decision tree model to predict tuberculosis using the C4.5 algorithm. The model was validated with an independent sample set from another center that included 74 tuberculous and 293 malignant effusions (validation cohort). RESULTS: Among 12 potential predictor variables, the classification tree analysis selected four discriminant parameters (age>35 years, pleural fluid adenosine deaminase>38U/L, temperature>or=37.8 degrees C, and pleural fluid LDH>320U/L) from the derivation cohort. The generated flowchart had 92.2% sensitivity, 98.3% specificity, and an area under the ROC curve of 0.976 for diagnosing tuberculosis. The corresponding operating characteristics for the validation cohort were 85.1%, 96.9% and 0.958. CONCLUSIONS: Applying a decision tree analysis that contains simple clinical and laboratory data can help in the differential diagnosis of tuberculous and malignant pleural effusions.

Cerebrospinal fluid biomarkers showing neurodegeneration in dogs with GM1 gangliosidosis: possible use for assessment of a therapeutic regimen.

The present study investigated cerebrospinal fluid (CSF) biomarkers for estimating degeneration of the central nervous system (CNS) in experimental dogs with GM1 gangliosidosis and preliminarily evaluated the efficacy of long-term glucocorticoid therapy for GM1 gangliosidosis using the biomarkers identified here. GM1 gangliosidosis, a lysosomal storage disease that affects the brain and multiple systemic organs, is due to an autosomal recessively inherited deficiency of acid beta-galactosidase activity. Pathogenesis of GM1 gangliosidosis may include neuronal apoptosis and abnormal axoplasmic transport and inflammatory response, which are perhaps consequent to massive neuronal storage of GM1 ganglioside. In the present study, we assessed some possible CSF biomarkers, such as GM1 ganglioside, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), neuron-specific enolase (NSE) and myelin basic protein (MBP). Periodic studies demonstrated that GM1 ganglioside concentration, activities of AST and LDH, and concentrations of NSE and MBP in CSF were significantly higher in dogs with GM1 gangliosidosis than those in control dogs, and their changes were well related with the months of age and clinical course. In conclusion, GM1 ganglioside, AST, LDH, NSE and MBP could be utilized as CSF biomarkers showing CNS degeneration in dogs with GM1 gangliosidosis to evaluate the efficacy of novel therapies proposed for this disease. In addition, we preliminarily treated an affected dog with long-term oral administration of prednisolone and evaluated the efficacy of this therapeutic trial using CSF biomarkers determined in the present study. However, this treatment did not change either the clinical course or the CSF biomarkers of the affected dog, suggesting that glucocorticoid therapy would not be effective for treating GM1 gangliosidosis.

Assessment of GaN chips for culturing cerebellar granule neurons.

In this work, the behaviors of cerebellar granule neurons prepared from 7-day-old Wistar rats on gallium nitride (GaN) were investigated. We believe that this is the first time that the GaN has been used as a substrate for neuron cultures to examine its effect on cell response in vitro. The GaN surface structure and its relationship with cells were examined by atomic force microscopy (AFM), metallography microscopy, scanning electron microscopy (SEM), lactate dehydrogenase (LDH) release and Western blot analysis. GaN is a so-called III-V compound semiconductor material with a wide bandgap and a relatively high bandgap voltage. Compared with silicon used for most neural chips, neurons seeded on GaN were able to form an extensive neuritic network and expressed very high levels of GAP-43 coincident with the neurite outgrowth. Therefore, the GaN structure may spatially mediate cellular response that can promote neuronal cell attachment, differentiation and neuritic growth. The favorable biocompatibility characteristics of GaN can be used to measure electric signals from networks of neuronal cells in culture to make it a possible candidate for use in a microelectrode array.

Ecological impact assessment of metallurgic effluents using in situ biomarker assays.

An ecological impact study was performed based on in situ biomarker assays with the waterflea Daphnia magna. The effects of metallurgic effluents on the energy metabolism, anti-oxidative metabolism and DNA damage were assessed in caged daphnids during a 4-week study. In situ survival and reproduction studies demonstrated a clear impact on these parameters in organisms exposed in the most polluted areas. At the downstream--sublethal--zone the organisms were disturbed within their tolerance limits, resulting in alterations of their energy metabolism. These data suggest an acclimation hypothesis, which was tested through the analysis of the energy metabolism of resident species: isopods and amphipods. These organisms had shifted to a decrease in their overall energy metabolism compared to the upstream region. This change in some biochemical processes suggests a selective advantage to cope with the prevailing environmental conditions. In addition, we found clear genotoxic effects caused by the industrial discharges that might correlate with a reduction in (long-term) survival.

Viability assessment in sandwich-cultured rat hepatocytes after xenobiotic exposure.

Troglitazone, bosentan and glibenclamide inhibit the bile salt export pump (Bsep) which transports taurocholate into bile. Sandwich-cultured rat hepatocytes maintain functional sodium taurocholate co-transporting polypeptide and Bsep transport proteins, and may be useful to study inhibition of transport by xenobiotics at concentrations below the lowest observable adverse effect level (LOAEL). The purpose of this study was to compare viability assessments determined with the neutral red, lactate dehydrogenase (LDH), alamar blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide assays in sandwich-cultured rat hepatocytes following exposure to xenobiotics known to inhibit Bsep, and to define the LOAEL for these xenobiotics in this system. The neutral red assay was not amenable to use in this model due to crystal formation on the collagen. Troglitazone decreased viability in every assay examined, with a LOAEL approximately 100 microM. Bosentan also decreased viability as measured by the LDH, MTT and propidium iodide assays, with a LOAEL approximately 200 microM; however, a significant decrease in viability was not observed with the alamar blue assay. Glibenclamide did not decrease viability with any assay at the xenobiotic concentrations examined in this study. Based on the results of this study, the LDH or propidium iodide assays would be the methods of choice to assess viability in sandwich-cultured rat hepatocytes after xenobiotic exposure.

In vitro genotoxicity assessment of commercial quartz flours in comparison to standard DQ12 quartz.

Crystalline silica has been classified as a human carcinogen, but there is still considerable debate on its variable fibrogenic and carcinogenic potential. We investigated genotoxicity of a panel of four quartz flours in comparison to DQ12 standard quartz with similar size and surface area, using single cell gel electrophoresis (SCGE) or comet assay. A549 human lung epithelial cells were incubated for 4 hours with different concentrations of quartz ranging from 1.6 to 200 micrograms/cm2 and cytotoxicity was assessed using leakage of lactate dehydrogenase (LDH), trypan blue exclusion and conversion of a metabolic substrate (MTT). DNA strand breakages were seen with all quartzes at an in vitro concentration of 200 micrograms/cm2. At this concentration all tests and quartz samples showed significant cytotoxicity. The most toxic quartz flour (Qz 2/1-C) but not DQ12, showed an increase in strand breaks at 40 micrograms/cm2 in cell culture. At this concentration no cytotoxicity was seen with LDH and MTT, but a significant increase in cells with trypan blue uptake was noted. No differences in tail moment percentage were observed at equal concentrations of different quartz flours. Also no correlation between DNA damage and OH-radical generation or surface radicals as measured by electron spin resonance was observed. We conclude that quartzes do not cause strand breaks without concomitant cell toxicity and a sufficient in vitro concentration of > 40 micrograms/cm2 can only be reached in vivo with instillation of massive doses (> 100 mg). Therefore, in vitro genotoxicity found here is unlikely to explain the genotoxicity observed in in vivo studies with the same and other quartzes.

Comparative pulmonary toxicity assessment of single-wall carbon nanotubes in rats.

The aim of this study was to evaluate the acute lung toxicity of intratracheally instilled single-wall carbon nanotubes (SWCNT) in rats. The lungs of rats were instilled either with 1 or 5 mg/kg of the following control or particle types: (1) SWCNT, (2) quartz particles (positive control), (3) carbonyl iron particles (negative control), (4) phosphate-buffered saline (PBS) + 1% Tween 80, or (5) graphite particles (lung tissue studies only). Following exposures, the lungs of PBS and particle-exposed rats were assessed using bronchoalveolar lavage (BAL) fluid biomarkers and cell proliferation methods, and by histopathological evaluation of lung tissue at 24 h, 1 week, 1 month, and 3 months postinstillation. Exposures to high-dose (5 mg/kg) SWCNT produced mortality in ~15% of the SWCNT-instilled rats within 24 h postinstillation. This mortality resulted from mechanical blockage of the upper airways by the instillate and was not due to inherent pulmonary toxicity of the instilled SWCNT particulate. Exposures to quartz particles produced significant increases versus controls in pulmonary inflammation, cytotoxicity, and lung cell parenchymal cell proliferation indices. Exposures to SWCNT produced transient inflammatory and cell injury effects. Results from the lung histopathology component of the study indicated that pulmonary exposures to quartz particles (5 mg/kg) produced dose-dependent inflammatory responses, concomitant with foamy alveolar macrophage accumulation and lung tissue thickening at the sites of normal particle deposition. Pulmonary exposures to carbonyl iron or graphite particles produced no significant adverse effects. Pulmonary exposures to SWCNT in rats produced a non-dose-dependent series of multifocal granulomas, which were evidence of a foreign tissue body reaction and were nonuniform in distribution and not progressive beyond 1 month postexposure (pe). The observation of SWCNT-induced multifocal granulomas is inconsistent with the following: (1) lack of lung toxicity by assessing lavage parameters, (2) lack of lung toxicity by measuring cell proliferation parameters, (3) an apparent lack of a dose response relationship, (4) nonuniform distribution of lesions, (5) the paradigm of dust-related lung toxicity effects, (6) possible regression of effects over time. In addition, the results of two recent exposure assessment studies indicate very low aerosol SWCNT exposures at the workplace. Thus, the physiological relevance of these findings should ultimately be determined by conducting an inhalation toxicity study.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry.

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome p450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.