A comparison of ecotoxicological tests

A simple, inexpensive and rapid method of determining toxicity by using a bacterium as the indicator organism was developed and compared with 23 other tests. The average correlation coefficient when comparing these 23 tests with the present test was 0.800, ranging from 0.580 to 0.950. Eleven of the tests were compared in detail by using 35 of the chemicals on the Multicentre Evaluation of In Vitro Cytotoxicity list of test chemicals. Comparing results from the present test with test results for these 35 chemicals with Microtox, Biotox, Daphnia magna, rat hepatocytes and ascites tumour cell resulted in correlation coefficients ranging from 0.871 to 0.933. Comparisons of the test data with rodent LD50 values, human lethal dose estimates from autopsies and human lethal doses obtained from the literature provided correlation coefficients ranging from 0.580 to 0.770, indicating that the test compares less favourably with these methods. This test provides data comparable to data from other ecotoxicological tests.     

Lymphocyte phenylvaleric acid hydroxylase activity (L-PVH), a new marker of peripheral neurotoxicity]

A reduction in the level of a new enzymatic assay--a phenyl valerate hydrolase (PVH)--has been found during the clinical evolution of toxic neuropathies (as almitrine-bismetilate ones) as well as alcoholic or diabetic neuropathies. The substrate and the enzymatic function are different from those used by M.K. Johnson for NTE. The method follows procedures comparable to NTE (differential determination after inhibition by paraoxon and by paraoxon plus mipafox or DFP). It may be useful to test possible neurotoxicity of drugs and chemicals.     

Transcriptional activation analysis using bioluminescent reporter assays

Analysis of promoter and enhancer DNA sequences provides the researcher with valuable information regarding the expression patterns of genes. Insertion of small DNA fragments containing the regulatory sequence of interest into vectors carrying reporter genes allows for the accurate quantitative analysis of the gene's expression patterns and responses to various stimuli. The use of bioluminescent reporter genes provides a simple, rapid, and inexpensive system that generates virtually no toxic or radioactive waste products. In addition, bioluminescent reporter vectors are more sensitive than previous methods such as the chloramphenicol acetyl transferase (CAT) systems, that require the use of hazardous chemicals and isotopically labeled reagents.     

Use of an established cell line in the evaluation of the cytotoxic effects of various chemicals.

The HTC hepatoma cell line was used as an "in vitro" model to detect the cytotoxicity of eighteen chemicals, chosen on the basis of different biological activities and physicochemical characteristics. Two different cytotoxicity assays measuring cell lethality (CS) or inhibition of cell growth (CF) were applicated to confluent cell monolayers or to colony-forming cells, respectively. Cells were exposed to the chemicals at doses ranging from 10(-6) M to 10(-2) M for 24 h. The results indicated a wide range of IC 50 (the concentration resulting in 50% inhibition of toxicity parameters) from as low as 1 microM (Potassium dichromate) to as high as 407.5 mM (Ethanol), the sensitivity of the CF test being greater than that of the CS test. A battery of cytotoxicity tests could be established in order to offer simple, rapid and economic methods which can be complementary and, in part, alternative to the use of laboratory animals.     

Application of the chemiluminescent assay to cytotoxicity test: detection of menadione-catalyzed H2O2 production by viable cells.

Menadione-catalyzed H2O2 production by viable cells is proportional to viable cell number. The correlations between the viable cell number and the concentration of H2O2 produced are determined with the rapid chemiluminescent assay (S. Yamashoji, T. Ikeda, and K. Yamashoji, 1989, Anal. Biochem. 181, 149-152). This chemiluminescent assay of viable cells requires only 10 min and is much faster than NR (neutral red) inclusion and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assays, which require 3-5 h. When viable cells are incubated with antitumor drugs, detergents, mycotoxins, and glycoalkaloids for 24-48 h, a decrease in menadione-catalyzed H2O2 production in a dose- or incubation time-dependent manner is observed. In general, the 50% inhibition concentration determined by the chemiluminescent assay is lower than that determined by NR inclusion and MTT reduction assays, and the order of relative cytotoxic effects of agents is the same among these assays. Furthermore, clear cytotoxic effects are observed by the chemiluminescent assay after 1 h exposure of trypsinized cells to toxic compounds. Therefore, the chemiluminescent assay is expected to be more useful for the rapid detection of cytotoxic compounds than NR inclusion and MTT reduction assays.     

Simple and rapid F+ coliphage culture, latex agglutination, and typing assay to detect and source track fecal contamination

Simple, rapid, and reliable fecal indicator tests are needed to better monitor and manage ambient waters and treated waters and wastes. Antibody-coated polymeric bead agglutination assays can fulfill these needs and are inexpensive and portable for nonlaboratory settings, and their reagents can be stored at ambient temperatures for months. The goal of this study was to develop, optimize, and validate a rapid microbial water quality monitoring assay using F+ coliphage culture, latex agglutination, and typing (CLAT) to detect F+ coliphage groups with antibody-coated particles. Rapid (180 min) F+ coliphage culture gave comparable results to those with the 16- to 24-h culture time used in EPA method 1601 and was amenable to CLAT assay detection. CLAT was performed on a cardboard card by mixing a drop of coliphage enrichment culture with a drop of antibody-coated polymeric beads as the detection reagent. Visual agglutination or clumping of positive samples occurred in <60 seconds. The CLAT assay had sensitivities of 96.4% (185/192 samples) and 98.2% (161/164 samples) and specificities of 100% (34/34 samples) and 97.7% (129/132 samples) for F+ RNA and DNA coliphages, respectively. CLAT successfully classified F+ RNA coliphages into serogroups typically obtained from human (groups II and III) and animal (groups I and IV) fecal sources, in similar proportions to those obtained with a nucleic acid hybridization assay. This novel group-specific antibody-based particle agglutination technique for rapid and simple detection and grouping of F+ coliphages provides a new and improved tool for monitoring the microbiological quality of drinking, recreational, shellfishing, and other waters.     

Development of ecotoxicity assay based on inhibition of respiring activity in microbial community using XTT reduction

The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC(50)) value of 3,5-dichlorophenol (3,5-DCP). The EC(50) value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.     

The radioreceptor assay: a simple, sensitive and rapid analytical procedure

Radioreceptor assays (RRAs) are analogous in concept to radioimmuno assays. Characteristic to both methods is the saturable, specific, competitive and reversible ligand/receptor interaction. The RRA is simple, sensitive and reproducible and provides a degree of precision comparable to more sophisticated analytical techniques. Since RRAs require little specialized equipment, they can be used routinely by any laboratory engaged in biochemical research or, as an inexpensive exercise to teach the fundamentals of ligand binding and analytical pharmacology.     

Spreeta-based biosensor assays for endocrine disruptors

The construction and performance of an automated low-cost Spreeta-based prototype biosensor system for the detection of endocrine disrupting chemicals (EDCs) is described. The system consists primarily of a Spreeta miniature liquid sensor incorporated into an aluminum flow cell holder, dedicated to support a Biacore chip frame, in combination with a simple pressurized air-driven fluid system. During the optimization, a monoclonal antibody (MAb)-based immunoassay for the estrogenic compound bisphenol A (BPA) was used as a model. After the optimization two thyroxine transport protein inhibition assays for thyroid endocrine disruptors were implemented. The average noise of the system for 1 min of baseline was 1.1 microRIU (refractive index units) and it could be operated in the range of 18-22 degrees C with a minimum baseline drift (5-10 microRIU/100 min). Optimum signal to noise ratio (S/N R) was obtained using a flow cell height of 100 microm and a flow rate of 180 microl/min. The sensitivity of the Spreeta-based biosensor inhibition assays implemented (50% inhibition concentration (IC50) of 30.2 nM for BPA using MAb12 and 12.3 and 11.6 nM for thyroxine (T4) using thyroxine-binding globulin (TBG) and recombinant transthyretin (rTTR), respectively) was comparable to the sensitivity previously obtained using a Biacore 3000 (IC50 of 39.9 nM for BPA and 8.6 and 13.7 nM, respectively, for T4). The results indicate that the alternative prototype system can be used in combination with ready-to-use biosensor chip surfaces and it is potentially a useful tool for the bioeffect-related screening of EDCs.     

Bacterial motility as a potential tool for rapid toxicity assays

Summary Bacterial motility was evaluated as a potential tool for the rapid assay of the toxicity of chemicals to microorganisms. The level of toxicity was evaluated by the chemical concentration which caused a 50% reduction in motile bacteria at the 60 s of exposure to toxicants. Assay results showed a good correlation with those obtained from a conventional growth inhibition test.     

Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation

Hematoxylin, a natural dye commonly used as a histological stain, generates superoxide upon oxidation to its quinonoid product, hematein. The parameters affecting this reaction were assessed in developing a new and versatile assay for superoxide dismutase. The autoxidation of hematoxylin to hematein was accompanied by an increase in absorbance between 400 and 670 nm. The autoxidation rate was proportional to hematoxylin concentration and increased with pH above 6.55. Trace metals accelerated the autoxidation and this effect was eliminated by EDTA. Superoxide dismutase inhibited the autoxidation 90-95% below pH 7.8, but above pH 8.1 the rate was augmented by superoxide dismutase. The rate inhibition at low pH was proportional to the superoxide dismutase concentration up to 70% inhibition. The rate acceleration at high pH was proportional to superoxide dismutase concentration up to approximately 200% acceleration. The autoxidation rate was not significantly affected by ethanol, cyanide, azide, hydrogen peroxide, or catalase. However, the reaction was inhibited by the reducing agents NADH, reduced glutathione, ascorbate, and dithiothreitol, and by undialyzed extracts of Escherichia coli B. When cell extracts were dialyzed prior to assay, the degree of inhibition observed was proportional to the concentration of superoxide dismutase in the extract. These observations form the basis for negative and positive assays of superoxide dismutase which are inexpensive and simple to perform. The negative assay has the added advantage of being applicable at physiological pH.     

The improvement of in vitro cytotoxicity testing for the assessment of acute toxicity in fish

The use of fish cell line cytotoxicity tests as alternatives to acute lethality tests with fish is hampered by the clearly lower sensitivity of the fish cell line tests. Recently, it has been shown that this is not a unique feature of fish cells. In fact, the sensitivity of mammalian and human cell lines toward the cytotoxic actions of chemicals, in general, is comparable to that of fish cell lines. Reviewing some of our recent investigations, the objective of this paper is to show that the sensitivity of in vitro cytotoxicity testing and the correspondence between in vitro cytotoxic and acute fish toxic concentrations (LC50) can be increased, if: a) inhibition of cell growth instead of cell death is used as the endpoint; and b) the bioavailable free cytotoxic concentration (ECu50) of chemicals in vitro, instead of the nominal cytotoxic concentration (EC50), is used as the measure of cytotoxic potency. Based on these results, a pragmatic in vitro testing strategy for estimating the minimal aquatic toxic potency of chemicals is proposed.     

Protein concentration of cerebrospinal fluid by precipitation with Pyrogallol Red prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis

The Pyrogallol Red Molybdate (PRM) and Coomassie Brilliant Blue (CBB) protein dye-binding assays have been applied to samples of cerebrospinal fluid (CSF) to investigate protein concentration by dye precipitation prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration values of the CSF samples (N=62) showed good agreement between the PRM and CBB assays as indicated by linear regression analysis (y(PRM)=1.033x(CBB)+1.004 in units of mg/l, r=0.99) but the PRM assay was optimal for protein concentration as the PRM protein-dye complex was less soluble allowing protein recovery over a wider working range. Dye precipitation using PRM is recommended as a simple, rapid and economic method for protein concentration of samples of CSF prior to SDS-PAGE.     

Development of a rapid colorimetric time-kill assay for determining the in vitro activity of ceftazidime and tobramycin in combination against Pseudomonas aeruginosa

It is standard clinical practice to use a combination of two or more antimicrobial agents to treat an infection caused by Pseudomonas aeruginosa. The antibiotic combinations are usually selected empirically with methods to determine the antimicrobial effect of the combination such as the time-kill assay rarely used as they are time-consuming and labour intensive to perform. Here, we report a modified time-kill assay, based on the reduction of the tetrazolium salt, 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT), that allows simple, inexpensive and more rapid determination of the in vitro activity of antibiotic combinations against P. aeruginosa. The assay was used to determine the in vitro activity of ceftazidime and tobramycin in combination against P. aeruginosa isolates from cystic fibrosis patients and the results obtained compared with those from conventional viable count time-kill assays. There was good agreement in interpretation of results obtained by the XTT and conventional viable count assays, with similar growth curves apparent and the most effective concentration combinations determined by both methods identical for all isolates tested. The XTT assay clearly indicated whether an antibiotic combination had a synergistic, indifferent or antagonistic effect and could, therefore, provide a useful method for rapidly determining the activity of a large number of antibiotic combinations against clinical isolates.     

A simplified method for staining mast cells with astra blue

The copper phthalocyanin dye astra blue has been used to stain differentially mast cells of the intestine; however; the procedure has not been used widely because of the difficulty in preparing and using the dye solution. Described here is a simple, reliable, and consistent method for selectively staining mast cells using a dye solution that may be prepared in any laboratory without the aid of sophisticated pH metering equipment. Astra blue is mixed with an alcoholic solution containing MgCl2-6H2O and the pH indicator pararosaniline hydrochloride. Concentrated hydrochloric acid is added dropwise, changing the dye mixture from purple to violet and then to blue. In this low range the weakly ionizing ethanol provides a more stable hydrogen ion concentration than the corresponding aqueous solutions used previously. Alcoholic acid fuchsin is a convenient counterstain, and this simple procedure then provides good contrast between the blue staining mast cell granules and the red tissue background.     

Isolation and characterization of Acidithiobacillus caldus from a sulfur-oxidizing bacterial biosensor and its role in detection of toxic chemicals

A novel toxicity detection methodology based on sulfur-oxidizing bacteria (SOB) has been developed for the rapid and reliable detection of toxic chemicals in water. The methodology exploits the ability of SOB to oxidize sulfur particles in the presence of oxygen to produce sulfuric acid. The reaction results in an increase in electrical conductivity (EC) and a decrease in pH. The assay is based on the inhibition of SOB in the presence of toxic chemicals by measuring changes in EC and pH. We found that SOB biosensor can detect toxic chemicals, such as heavy metals and CN−, in the 5-2000 ppb range. One bacterium was isolated from an SOB biosensor and the 16S rRNA gene of the bacterial strain has 99% and 96% sequence similarity to Acidithiobacillus sp. ORCS6 and Acidithiobacillus caldus DSM 8584, respectively. The isolate was identified as A. caldus SMK. The SOB biosensor is ideally suited for monitoring toxic chemicals in water having the advantages of high sensitivity and quick detection.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Estradiol attenuation of β-amyloid-induced toxicity: A comparison of MTT and calcein AM assays

17β-estradiol (βE2) has been shown to attenuate the toxicity of β-amyloid peptides (Aβ) in neuronal cultures with the effective concentration of βE2 ranging from low nM to high μM. This study compares the effective neuroprotective concentration of βE2 against both Aβ-mediated toxicity in a human neuroblastoma cell line, SK-N-SH using cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an endpoint to the effective βE2 concentration obtained using a calcein acetoxymethyl ester (calcein AM) viability assay. The minimum βE2 concentration required for protection varied 1000-fold between the two viability assays with 1 nM βE2 conferring significant protection in the calcein AM assay but 1 μM βE2 required for significant protection in the MTT assay. Interestingly, the maximal inhibition of MTT reduction occured at sub-toxic Aβ concentrations and did not correlate with other markers of cellular viability including calcein fluorescence, dye exclusion (propidium iodide or trypan blue), cellular ATP levels, or reduction of another tetrazolium dye, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium (MTS). By contrast, there was no difference between the MTT and calcein AM assays with respect to H₂O₂ toxicity or the neuroprotective effectiveness of 10 nM βE2 against H_{2 2} toxicity. These results indicate that low concentrations of βE2 can attenuate Aβ and H₂O₂ toxicity in a human neuroblastoma cell line. Further, these results suggest that the MTT assay is not an appropriate assay for the determination of βE2-mediated attenuation of Aβ toxicity.     

Quantitative Determination of Gap Junction Intercellular Communication by Scrape Loading and Image Analysis

Gap junction intercellular communication (GJIC) consists of intercellular exchange of low molecular weight molecules. Chemically induced alterations of this communication have been suggested to result in abnormal cell growth and tumour promotion. Several in vitro assays have been developed to determine the effect of chemicals on gap junction communication in cultured cells. The scrape loading dye transfer technique is based on studying the transfer of the fluorescent dye Lucifer Yellow in cells where the dye is loaded through a cut in the cell monolayer. This technique is rapid and relatively uncomplicated, but has only been used to qualitatively demonstrate communication, due to lack of an appropriate method for quantification of the dye spreading. We show here that analysis of digital fluorescence images of cells scrape loaded with Lucifer Yellow can be used for quantitative determination of GJIC. We have analysed the images both by means of distance of diffusion of the dye in the cell monolayer, as well as by area of dye-coupled cells. The results are consistent with that obtained using microinjection of Lucifer Yellow and the method offers a simple way for quantitative determination of GJIC.