Response characteristics of arsenic-sensitive bioreporters expressing the gfp reporter gene

This paper describes the development of an analytical technique for arsenic analyses that is based on genetically-modified bioreporter bacteria bearing a gene encoding for the production of a green fluorescent protein (gfp). Upon exposure to arsenic (in the aqueous form of arsenite), the bioreporter production of the fluorescent reporter molecule is monitored spectroscopically. We compared the response measured as a function of time and concentration by steady-state fluorimetry (SSF) to that measured by epi-fluorescent microscopy (EFM). SSF is a bulk technique; as such it inherently yields less information, whereas EFM monitors the response of many individual cells simultaneously and data can be processed in terms of population averages or subpopulations. For the bioreporter strain used here, as well as for the literature we cite, the two techniques exhibit similar performance characteristics. The results presented here show that the EFM technique can compete with SSF and shows substantially more promise for future improvement; it is a matter of research interest to develop optimized methods of EFM image analysis and statistical data treatment. EFM is a conduit for understanding the dynamics of individual cell response vs. population response, which is not only a matter of research interest, but is also promising in the practical terms of developing micro-scale analysis.

Effect of groundwater composition on arsenic detection by bacterial biosensors.

A luminescent bacterial biosensor was used to quantify bioavailable arsenic in artificial groundwater. Its light production above the background emission was proportional to the arsenite concentration in the toxicologically relevant range of 0 to 0.5 mu M. Effects of the inorganic solutes phosphate, Fe(II) and silicate on the biosensor signal were studied. Phosphate at a concentration of 0.25 g L-1 phosphate slightly stimulated the light emission, but much less than toxicologically relevant concentrations of the much stronger inducer arsenite. No effect of phosphate was oberved in the presence of arsenite. Freshly prepared sodium silicate solution at a concentration of 10 g L-1 Si reduced the arsenite-induced light production by roughly 37%, which can be explained by transient polymerization leading to sequestration of some arsenic. After three days of incubation, silicate did not have this effect anymore, probably because depolymerization occurred. In the presence of 0.4 g L-1 Fe(II), the arsenite-induced light emission was reduced by up to 90%, probably due to iron oxidation followed by arsenite adsorption on the less soluble Fe(III) possibly along with some oxidation to the stronger adsorbing As(V). Addition of 100 mu M EDTA was capable of releasing all arsenic from the precipitate and to transform it into the biologically measurable, dissolved state. The biosensor also proved valuable for monitoring the effectiveness of an arsenic removal procedure based on water filtration through a mixture of sand and iron granules.

Field testing of arsenic in groundwater samples of Bangladesh using a test kit based on lyophilized bioreporter bacteria.

A test kit based on living, lyophilized bacterial bioreporters emitting bioluminescence as a response to arsenite and arsenate was applied during a field campaign in six villages across Bangladesh. Bioreporter field measurements of arsenic in groundwater from tube wells were in satisfying agreement with the results of spectroscopic analyses of the same samples conducted in the lab. The practicability of the bioreporter test in terms of logistics and material requirements, suitability for high sample throughput, and waste disposal was much better than that of two commercial chemical test kits that were included as references. The campaigns furthermore demonstrated large local heterogeneity of arsenic in groundwater, underscoring the use of well switching as an effective remedy to avoid high arsenic exposure.

Development of bacteria-based bioassays for arsenic detection in natural waters.

Arsenic contamination of natural waters is a worldwide concern, as the drinking water supplies for large populations can have high concentrations of arsenic. Traditional techniques to detect arsenic in natural water samples can be costly and time-consuming; therefore, robust and inexpensive methods to detect arsenic in water are highly desirable. Additionally, methods for detecting arsenic in the field have been greatly sought after. This article focuses on the use of bacteria-based assays as an emerging method that is both robust and inexpensive for the detection of arsenic in groundwater both in the field and in the laboratory. The arsenic detection elements in bacteria-based bioassays are biosensor-reporter strains; genetically modified strains of, e.g., Escherichia coli, Bacillus subtilis, Staphylococcus aureus, and Rhodopseudomonas palustris. In response to the presence of arsenic, such bacteria produce a reporter protein, the amount or activity of which is measured in the bioassay. Some of these bacterial biosensor-reporters have been successfully utilized for comparative in-field analyses through the use of simple solution-based assays, but future methods may concentrate on miniaturization using fiberoptics or microfluidics platforms. Additionally, there are other potential emerging bioassays for the detection of arsenic in natural waters including nematodes and clams.

Compact portable biosensor for arsenic detection in aqueous samples with Escherichia coli bioreporter cells.

We present a compact portable biosensor to measure arsenic As(III) concentrations in water using Escherichia coli bioreporter cells. Escherichia coli expresses green fluorescent protein in a linearly dependent manner as a function of the arsenic concentration (between 0 and 100 μg/L). The device accommodates a small polydimethylsiloxane microfluidic chip that holds the agarose-encapsulated bacteria, and a complete optical illumination/collection/detection system for automated quantitative fluorescence measurements. The device is capable of sampling water autonomously, controlling the whole measurement, storing and transmitting data over GSM networks. We demonstrate highly reproducible measurements of arsenic in drinking water at 10 and 50 μg/L within 100 and 80 min, respectively.

Tunable reporter signal production in feedback-uncoupled arsenic bioreporters.

Escherichia coli-based bioreporters for arsenic detection are typically based on the natural feedback loop that controls ars operon transcription. Feedback loops are known to show a wide range linear response to the detriment of the overall amplification of the incoming signal. While being a favourable feature in controlling arsenic detoxification for the cell, a feedback loop is not necessarily the most optimal for obtaining highest sensitivity and response in a designed cellular reporter for arsenic detection. Here we systematically explore the effects of uncoupling the topology of arsenic sensing circuitry on the developed reporter signal as a function of arsenite concentration input. A model was developed to describe relative ArsR and GFP levels in feedback and uncoupled circuitry, which was used to explore new ArsR-based synthetic circuits. The expression of arsR was then placed under the control of a series of constitutive promoters, which differed in promoter strength, and which could be further modulated by TetR repression. Expression of the reporter gene was maintained under the ArsR-controlled Pars promoter. ArsR expression in the systems was measured by using ArsR-mCherry fusion proteins. We find that stronger constitutive ArsR production decreases arsenite-dependent EGFP output from Pars and vice versa. This leads to a tunable series of arsenite-dependent EGFP outputs in a variety of systematically characterized circuitries. The higher expression levels and sensitivities of the response curves in the uncoupled circuits may be useful for improving field-test assays using arsenic bioreporters.

La toxicologie forensique, une discipline scientifique en plein essor [Forensic toxicology, a growing scientific discipline]

La recherche de substances ayant pu jouer un rôle direct ou indirect dans la cause de la mort ou ayant pu modifier le comportement d'un individu constitue la mission première de la toxicologie forensique. L'augmentation de la consommation de substances illégales et de médicaments dans les sociétés modernes a fait connaître à la toxicologie forensique un essor très important ces dernières décennies. D'autre part, les développements technologiques analytiques ont permis d'obtenir des outils très sensibles et spécifiques pour la recherche et le dosage d'une multitude de substances dans une grande variété d'échantillons biologiques, parfois présentes à des concentrations très faibles, conséquence d'une dose équivalente à la prise d'une seule unité galénique. Forensic toxicology has to bring evidence of substances that could have been involved directly or indirectly in the cause of death or that could influence the behaviour of somebody. The increase of the consumption of illegal and legal drugs in modern societies during last decades gave a boost to forensic toxicology. Moreover, improvement with analytical technology gave tools with high degrees of sensitivity and specificity for the screening and quantification of a large amount of substances in various biological specimens, even with very low concentration resulting of a single dose of medication.

Arsenic(V) adsorption onto α-Al2O3 between 25 and 70°C

The adsorption of As(V) onto alpha -Al2O3 was investigated at 25, 50 and 70 degreesC using batch adsorption experiments. Results indicate that As is strongly adsorbed at low pH and gets progressively released to the fluid with increasing pH above 7. At any pH, increasing temperature favors aqueous species of As over surface species. Surface complexation constants were determined at the experimental temperatures by fitting the adsorption data. Adsorption reactions were then converted to semi-isocolumbic reactions, i.e, reactions with balanced like-charged aqueous species. Intrinsic adsorption constants of semi-isocolumbic reactions change linearly when plotted against inverse temperature, suggesting that the heat capacity of these reactions remains constant over the temperature range considered. This permitted thermodynamic parameters of intrinsic surface complexation constants to be determined. Changes in surface complexation constants result in a change in the surface speciation with increasing temperature. This change is similar to the one observed for aqueous species, i.e. increasing temperature favors less negatively charged species below a pH of 9 and more negatively charged species above a pH of 10. Comparison with the stability of As surface complexes with Fe suggests that surface complexes with Al are more stable. (C) 2001 Elsevier Science Ltd. All rights reserved.

Dispersion of natural arsenic in the Malcantone watershed, Southern Switzerland: field evidence for repeated sorption-desorption and oxidation-reduction processes

In recent years, elevated arsenic concentrations have been found in waters and soils of many, countries, often resulting in a health threat for the local population. Switzerland is not an exception and this paper deals with the release and subsequent fate of arsenic in a 200-km(2) mountainous watershed, characterized by crystalline silicate rocks (gneisses, schists, amphibolites) that contain abundant As-bearing sulfide ore deposits, some of which have been mined for iron and gold in the past. Using analytical methods common for mineralogical, ground water and soil studies (XRD, XRF, XAS-XANES and -EXAFS, electron microprobe, extraction, ICP, AAS with hydride generator, ion chromatography), seven different field situations and related dispersion processes of natural arsenic have been studied: (1) release by rock weathering, (2) transport and deposition by water and ice; (3) release of As to the ground and surface water due to increasing pH; (4) accumulation in humic soil horizons; (5) remobilization by reduction in water-saturated soils and stagnant ground waters; (6) remobilization by using P-rich fertilizers or dung and (7) oxidation, precipitation and dilution in surface waters. Comparison of the results with experimental adsorption studies and speciation diagrams from the literature allows us to reconstruct and identify the typical behavior of arsenic in a natural environment under temperate climatic conditions. The main parameters identified are: (a) once liberated from the primary minerals, sorption processes on Fe-oxy-hydroxides dominate over Al-phases, such as Al-hydroxides or clay minerals and limit the As concentrations in the spring and well waters between 20 and 300 mug/l. (b) Precipitation as secondary minerals is limited to the weathering domain, where the As concentrations are still high and not yet too diluted by rain and soils waters. (c) Although neutral and alkaline pH conditions clearly increase the mobility of As, the main factor to mobilize As is a low redox potential (Eh close or below 0 mV), which favors the dissolution of the Fe-oxy-hydroxides on which the As is sorbed. (d) X-ray absorption spectroscopy (XAS) of As in water-logged humic forest soils indicates that the reduction to As III only occurs at the solid-water interface and that the solid contains As as As V (e) A and Bh horizons of humic cambisols can effectively capture As when As-rich waters flow through them. Complex spatial and temporal variation of the various parameters in a watershed results in repeated mobilization and immobilization of As, which continuously transports As from the upper to the lower part of a watershed and ultimately to the ocean. (C) 2004 Elsevier B.V. All rights reserved.

Fatal tolperisone poisoning: autopsy and toxicology findings in three suicide cases.

Tolperisone (Mydocalm) is a centrally acting muscle relaxant with few sedative side effects that is used for the treatment of chronic pain conditions. We describe three cases of suicidal tolperisone poisoning in three healthy young subjects in the years 2006, 2008 and 2009. In all cases, macroscopic and microscopic autopsy findings did not reveal the cause of death. Systematic toxicological analysis (STA) including immunological tests, screening for volatile substances and blood, urine and gastric content screening by GC-MS and HPLC-DAD demonstrated the presence of tolperisone in all cases. In addition to tolperisone, only the analgesics paracetamol (acetaminophen), ibuprofen and naproxen could be detected. The blood ethanol concentrations were all lower than 0.10 g/kg. Tolperisone was extracted by liquid-liquid extraction using n-chlorobutane as the extraction solvent. The quantification was performed by GC-NPD analysis of blood, urine and gastric content. Tolperisone concentrations of 7.0 mg/l, 14 mg/l and 19 mg/l were found in the blood of the deceased. In the absence of other autopsy findings, the deaths in these three cases were finally explained as a result of lethal tolperisone ingestion. To the best of our knowledge, these three cases are the first reported cases of suicidal tolperisone poisonings.