In vitro investigation of oxide nanoparticle and carbon nanotube toxicity and intracellular accumulation in A549 human pneumocytes.

If released in the environment, nanomaterials might be inhaled by populations and cause damage to the deepest regions of the respiratory tract, i.e., the alveolar compartment. To model this situation, we studied the response of A549 human pneumocytes after exposure to aluminium oxide or titanium oxide nanoparticles, and to multi-walled carbon nanotubes. The influence of size, crystalline structure and chemical composition was investigated. After a detailed identification of nanomaterial physico-chemical characteristics, cells were exposed in vitro and viability and intracellular accumulation were assessed. In our conditions, carbon nanotubes were more toxic than metal oxide nanoparticles. Our results confirmed that both nanotubes and nanoparticles are able to rapidly enter into cells, and distribute in the cytoplasm and intracellular vesicles. Among nanoparticles, we demonstrate significant difference in biological response as a function of size, crystalline phase and chemical composition. Their toxicity was globally lower than nanotubes toxicity. Among nanotubes, the length did not influence cytotoxicity, neither the presence of metal catalyst impurities.

Cellular accumulation and distribution of uranium and lead in osteoblastic cells as a function of their speciation.

Uranium (U) and lead (Pb) are accumulated and fixed for long periods in bone, impairing remodeling processes. Their toxicity to osteoblasts, the cells responsible for bone formation, is poorly documented. It has been previously shown that cytotoxicity and phenotypic effects of both metals on osteoblasts are highly influenced by metal speciation. Differences in sensitivity between cell types have been underlined as well. In this paper, cellular accumulation of U and Pb in cultured and primary osteoblastic cells was assessed by trace element analysis. Distribution of different species at the cell scale was investigated by electron microscopy. Internalization of both metals was shown to be correlated to cytotoxicity and population growth recovery after exposure. For each metal, the amount of metal uptake leading to 50% cell death was shown to be speciation-dependent. Scanning and transmission electron microscopy showed the formation of precipitates with phosphate in lysosomes for both metals, whose role in toxicity or cell defence remains to be clarified. Although a clear link was established between cytotoxicity and accumulation, differences in sensitivity observed in terms of speciation could not be fully explained and other studies seem necessary.

Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation.

Bone is one of the main retention organs for uranium (U) and lead (Pb). The clinical effects of U or Pb poisoning are well known: acute and chronic intoxications impair bone formation. However, only few studies dealt with the cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate acute cytotoxicity of U and Pb and their phenotypic effects on rat and human osteoblasts, the cells responsible for bone formation. The most likely species of the toxicants in contact with cells after blood contamination were selected for cell exposure. Results showed that the cytotoxic effect of U and Pb is highly dependent on their speciation. Thus, Pb was cytotoxic when left free in the exposure medium or when complexed with carbonate, cysteine or citrate, but not when complexed with albumin or phosphate, under an insoluble form. U was cytotoxic whatever its speciation, but differences in sensitivity were observed as a function of speciation. Population growth recovery could be obtained after exposure to low doses of U or Pb, except for some U-carbonate complexes which had irreversible effects whatever the dose. The activation of two markers of bone formation and mineralization, osteocalcin and bone sialoprotein (BSP), was observed after exposure to non-toxic doses or non-toxic species of U or Pb while their inhibition was observed after toxic exposure to both metals. This work provides new elements to better understand the complex mechanisms of U and Pb toxicity to osteoblasts. Our results also illustrate the importance of a strictly controlled speciation of the metals in toxicological studies.

Development of a single ion hit facility at the Pierre Sue Laboratory: a collimated microbeam to study radiological effects on targeted living cells.

A single ion hit facility is being developed at the Pierre Süe Laboratory (LPS) since 2004. This set-up will be dedicated to the study of ionising radiation effects on living cells, which will complete current research conducted on uranium chemical toxicity on renal and osteoblastic cells. The study of the response to an exposure to alpha particles will allow us to distinguish radiological and chemical toxicities of uranium, with a special emphasis on the bystander effect at low doses. Designed and installed on the LPS Nuclear microprobe, up to now dedicated to ion beam microanalysis, this set-up will enable us to deliver an exact number of light ions accelerated by a 3.75 MV electrostatic accelerator. An 'in air' vertical beam permits the irradiation of cells in conditions compatible with cell culture techniques. Furthermore, cellular monolayer will be kept in controlled conditions of temperature and atmosphere in order to diminish stress. The beam is collimated with a fused silica capillary tubing to target pre-selected cells. Motorisation of the collimator with piezo-electric actuators should enable fast irradiation without moving the sample, thus avoiding mechanical stress. An automated epifluorescence microscope, mounted on an antivibration table, allows pre- and post-irradiation cell observation. An ultra thin silicon surface barrier detector has been developed and tested to be able to shoot a cell with a single alpha particle.

Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity.

Uranium is a naturally occurring heavy metal. Its extensive use in the nuclear cycle and for military applications has focused attention on its potential health effects. Acute exposures to uranium are toxic to the kidneys where they mainly cause damage to proximal tubular epithelium. The purpose of this study was to investigate the biological consequences of acute in vitro uranyl exposure and the influence of uranyl speciation on its cytotoxicity. NRK-52E cells, representative of rat kidney proximal epithelium, were exposed to uranyl-carbonate and -citrate complexes, which are the major complexes transiting through renal tubules after acute in vivo contamination. Before NRK-52E cell exposure, these complexes were diluted in classical or modified cell culture media, which can possibly modify uranyl speciation. In these conditions, uranium cytotoxicity appears after 16 h of exposure. The CI50 cytotoxicity index, the uranium concentration leading to 50% dead cells after 24 h of exposure, is 500 microM (+/-100 microM) and strongly depends on uranyl counterion and cell culture medium composition. Computer modeling of uranyl speciation is reported, enabling one to draw a parallel between uranyl speciation and its cytotoxicity.

Impact of emerging technologies and regulations on the role of POCT.

Point-of-care testing (POCT) can be introduced by laboratory directors or clinicians in response to the need for rapid results to guide treatment. The professional responsibility for ensuring reliable and accurate results is well-defined in some countries such as France, but the role and responsibility of the laboratory is less clear in many other places. When point-of-care instrument technology and the intrinsic design of the device leads to device-specific parameters or analytical differences from laboratory-based equipment, there is a risk of misinterpretation and erroneous treatment decisions. Laboratory staffs are more often aware of the interaction of analytical technology and result interpretation than clinicians, making it more rational to involve the laboratory in the selection of point-of-care equipment, procedures, and therapeutic decisions based on the results. The design control and risk-analysis provisions of emerging regulations such as the EU in vitro diagnostic medical device (IVD) can be interpreted as engaging the manufacturer's responsibility, even when the equipment is functioning according to specification. This is especially true of device-specific parameters in which cross-calibration or traceability to a reference material or method is not possible. This is a further argument for involvement of the laboratory in the selection and implementation of point-of-care testing devices.

Red cell effects on lithium measurements by ion-selective electrode.

The possible interference of haematocrit was studied in an evaluation of the analytical performance of the NOVA 11 analyzer. For lithium concentrations between 0.56 and 0.68 mmol/L, the within-run imprecision was less than 1.06%, and less than 2.8% in day-to-day imprecision, measured in quality control solutions. Comparison of whole blood with plasma measurement showed satisfactory correlation with deviations up to 0.06 mmol/L for haematocrits from 35-53% (y = 0.73 x + 0.12). Measurement on a blood specimen not containing lithium, whose haematocrit was varied, allowed us to quantify a specific red cell interference. With samples from treated patients, and a sample spiked to various lithium concentrations, we obtained differences dependent on haematocrit and on lithium concentrations. In subtracting the specific red cell effect, we could systematically observe that apparent lithium levels diminish as haematocrit increases. This observation should allow development of an appropriate algorithm for obtaining exact values on whole blood samples.

[Pertinence of simultaneous measurements of pO2 and sO2 on ABL 510]. / Pertinence des mesures simultanées de pO2 et sO2 sur l'ABL 510.

Blood tonometry is the only method to assess the accuracy of the pO2 determination on blood gas analyzers. ABL instruments by Radiometer were tested by two types of tonometry (film and bubble tonometry) and the validity of the algorithm for pO2 correction was analysed with these results. The role of the presentation of the specimen is also discussed. For the precision study on the ABL 510 analyzer, coefficients of variation for pO2 were < 0.37% and < 1.7% for within-run and day-to-day series respectively. pO2 accuracy was excellent. Linearity was verified between 0-620 mmHg (82.5 kPa), and interinstrument comparisons demonstrated a strict correlation with the Ciba-Corning 178 instrument. ABL 510 measures simultaneously oxygen saturation by spectrophotometry. Analytical results showed an acceptable level of imprecision, but the definition and the clinical significance of this parameter are ambiguous.

Assessing the ABL 500 blood gas analyser.

The ABL 500 blood gas analyser from Radiometer has cordless electrodes and does not use a humidifier for calibrating gases. During the evaluation of the analytical performance of this instrument, the problem of p0(2) accuracy was approached by comparing the values obtained with two kinds of tonometry (film and bubble). An acceptable level of imprecision was demonstrated for all measured parameters. For within-run precision, with tonometry, coefficients of variation (CV) were similar, not equals0.37% for pO(2) and similar, not equals0.52% for pCO(2). A CV of 1.76% was found for day-to-day precision for both p0(2) and pCO(2). In the linearity study, with both tonometry methods, and in the inter-instrument comparisons (the ABL was compared with the Ciba Corning 178), pO(2) values obtained on the ABL 500 exhibited a slight overestimation above 150 mmHg (2.2-3.4% at 600 mmHg). This minor discrepancy is discussed with reference to the new design of the pO(2) electrode, the algorithm for pO(2) correction and the tonometry procedure. The results reported in this paper stress the importance of pO(2) accuracy assessment for the evaluation of blood gas analysers.

[Determination of pH in the body: methodologic review]. / La détermination du pH dans l'organisme: rappels méthodologiques.

The maintenance of a defined free H+ concentration within narrow limits is a prerequisite and feature of living organisms. In recent years the different disciplines of biological science have made considerable progress in the elucidation of the mechanisms involved in pH homeostasis. Recent advances have occurred also in the field of pH measurement. This review focuses mainly on the modern instruments for pH and blood gas analysis. The techniques of intracellular pH currently in general use are described together with some of the techniques for determination of pH in subcellular compartments.

Electrode measurement of glucose and urea in undiluted samples.

The development of ion-selective electrodes (ISEs) for electrolyte measurements necessitates a re-evaluation of the biological and clinical interpretation of a result. In pathological situations (e.g., hyperlipidemia and hyperproteinemia) direct potentiometry is the method of choice for ion measurements in blood. However, the "plasma water effect" exists also in normal samples, requiring new reference values for physiological ranges. A compromise between medical and instrumentation workers retained the old reference values (flame photometry for Na+ and K+) by introducing correction factors into the ISE instruments, so that the results for direct ISE and flame photometry are the same for "normal" samples. Analyses of "abnormal" samples will reveal biases between the two methods. Now, a new generation of electrodes for assaying additional metabolites reopens the issue. Although classical methods measure a quantity of substance in a predetermined volume of sample, the majority of the substance is usually in the aqueous phase, and the volumes occupied by lipid and protein are not taken into consideration. In evaluating the NOVA 12 instrument (NOVA Biomedical), using electrodes for direct measurement in serum or plasma of Na, K, Cl, total CO2, urea, and glucose, we have demonstrated the inadequacy of classical measurements of urea and glucose, especially in pathological situations characterized by a large variation in the plasma water fraction.

[Ionized calcium. An essential parameter for the study of calcium metabolism]. / Le calcium ionisé. Un paramètre essentiel pour l'étude du métabolisme calcique.

Calcium ions in plasma are either free (ionized calcium) or bound to protein and small anions. Ionized calcium is the physiologically active fraction. Several factors have recently been combined to change the measurement of ionized calcium from a specialized to a routine test. Reliable instrumentation, easy to maintain, is now available with simultaneous pH measurement. This paper focuses on both the analytical consideration required to perform reliable laboratory measurements and the clinical situations where ionized calcium has proved to provide a substantially different clinical interpretation than the measurement of total calcium.

A reliable potentiometric measurement of ionized calcium and pH on the ICA2 Radiometer in clinical practice.

A second generation analyser for potentiometric measurement of ionized calcium and pH (ICA2 Radiometer, Copenhagen) was tested with respect to analytical performance in two different laboratories. Newly-developed aqueous buffered solutions were used at different Ca2+ concentrations as well as biological specimens prepared from human subjects. The overall precision of ICA2 was very good: CV = 0.0-0.4% for within-run and 0.4-1.7% for day-to-day precision with protein-free solutions and CV = 0.3-0.9% for within-run precision on biological specimens. The adjusted value of Ca2+ to pH 7.40 is an analytical advantage for which the calculation is correct but the assumption made for this algorithm may limit its clinical relevance. Measurements were comparable on ICA2 when performed on either whole blood, serum or plasma. Pre-analytical conditions should be observed such as the choice of anticoagulant and anaerobic conditions. Such a reliable and accurate instrument may promote the use of ionized calcium measurement in daily clinical practice.

Blood chemistry by disposable ISE with the Chempro 500 system.

Potentiometric measurements are usually done with multiple determination electrodes. Disposable electrodes were first introduced on the Ektachem systems by Kodak with the concept of differential potentiometric measurement between the specimen and a reference fluid delivered on two parts of a single slide. In the Chempro 500 system a calibrating solution and the specimen are sequentially measured by the same potentiometric system with a classic reference electrode and selective electrodes present on a disposable device in various combinations. This device is connected to a compact analyzer with a microprocessor, a display and a printer. The authors evaluated the analytical performance of the Chempro 500 on whole blood, plasma and serum specimens over a two months period. Results and practicability are discussed and compared to those of reference instruments, according to requirements of decentralized analysis.

Assessment of recently developed blood gas analysers: a multicentre evaluation.

Providing guidelines for testing expected inaccuracy and imprecision is still a matter under debate. The Expert Panel of the French Society of Clinical Chemistry has developed a protocol, which was based on a comparative multi-centre evaluation of four instruments: the Ciba-Corning 278, the Instrumentation Laboratory 1306, the Nova SP 5 and the ABL 330. The purpose was to evaluate the analytical performance and efficiency of the analysers. Another aim was to design a valid approach for evaluating any new system. As buffered aqueous solutions and fluorocarbon emulsions give only partial information, tonometered blood was used at different levels of gas mixture, even though it is both difficult and time-consuming. Comparisons have been established on patients' blood samples with the analysers currently used in the evaluation sites. The tests showed that the four analysers have the same degree of precision, and interinstrument comparisons demonstrated a very high degree of reliability.This analysis emphasizes that the evaluation of instruments for pH and blood gas analysis is neither easy nor is it often done, mainly due to the choice of a quality-control material and the lability of the measured parameters.

Evaluation of Roche fructosamine test: use for diabetic patient monitoring.

A fructosamine assay using commercially available reagents on an IL Multistat III centrifugal analyser was evaluated. Within- and between-batch precision were less than 4% (CV). Results were similar using serum or oxalated plasma, but were 5% lower than those using sodium heparinate. We compared serum fructosamine and Hb A1c results in insulin-dependent and non-insulin-dependent diabetics: serum fructosamine correlated with Hb A1c (r = 0.80) and gave at least as good a separation of patients from normal controls as did Hb A1c.