Conventional Bioassays Research Articles

A new light on Alkaptonuria: A Fourier-transform infrared microscopy (FTIRM) and low energy X-ray fluorescence (LEXRF) microscopy correlative study on a rare disease

BackgroundAlkaptonuria (AKU) is an ultra-rare disease associated to the lack of an enzyme involved in tyrosine catabolism. This deficiency results in the accumulation of homogentisic acid (HGA) in the form of ochronotic pigment in joint cartilage, leading to a severe arthropathy. Secondary amyloidosis has been also unequivocally assessed as a comorbidity of AKU arthropathy. Composition of ochronotic pigment and how it is structurally related to amyloid is still unknown. MethodsWe exploited Synchrotron Radiation Infrared and X-Ray Fluorescence microscopies in combination with conventional bio-assays and analytical tools to characterize chemical composition and morphology of AKU cartilage. ResultsWe evinced that AKU cartilage is characterized by proteoglycans depletion, increased Sodium levels, accumulation of lipids in the peri-lacunar regions and amyloid formation. We also highlighted an increase of aromatic compounds and oxygen-containing species, depletion in overall Magnesium content (although localized in the peri-lacunar region) and the presence of calcium carbonate fragments in proximity of cartilage lacunae. ConclusionsWe highlighted common features between AKU and arthropathy, but also specific signatures of the disease, like presence of amyloids and peculiar calcifications. Our analyses provide a unified picture of AKU cartilage, shedding a new light on the disease and opening new perspectives. General significanceOchronotic pigment is a hallmark of AKU and responsible of tissue degeneration. Conventional bio-assays have not yet clarified its composition and its structural relationship with amyloids. The present work proposes new strategies for filling the aforementioned gap that encompass the integration of new analytical approaches with standardized analyses.

The chemical exposure toxicity space (CETS) model: Displaying exposure time, aqueous and organic concentration, activity, and onset of toxicity.

A 1‐compartment toxicokinetic model is used to characterize the chemical exposure toxicity space (CETS), providing a novel graphic tool that can aid in the design of aquatic toxicity tests for fish and for interpreting their results. The graph depicts the solution to the differential equation describing the uptake kinetics of a chemical by a modeled fish under conventional bioassay conditions. The model relates the exposure concentration in the water to a dimensionless time and the onset of toxicity as determined by an estimated or assumed critical body residue or incipient lethal aqueous concentration. These concentration graphs are specific to each chemical and exposure and organism parameters and clearly demonstrate differences in toxicity between chemicals and how factors such as hydrophobicity influence the toxic endpoint. The CETS plots can also be used to assess bioconcentration test conditions to ensure that concentrations are well below toxic levels. Illustrative applications are presented using a recent set of high‐quality toxicity data. Conversion of concentrations to chemical activities in the plots enables results for different baseline toxicants to be superimposed. For chemicals that have different modes of toxic action, the increased toxicity then becomes apparent. Implications for design and interpretation of aquatic toxicity tests are discussed. The model, and pictorial visualization of the time‐course of aquatic toxicity tests, may contribute to improvements in test design, implementation, and interpretation, and to reduced animal usage. Environ Toxicol Chem 2017;36:1389–1396. © 2016 The Authors. Environmental Toxicology and Chemistry Published by Wiley Periodicals, Inc. on behalf of SETAC.

Aneugenicity and clastogenicity in freshwater fish Oreochromis niloticus exposed to incipient safe concentration of tannery effluent

Conventional effluent bioassays mostly rely on overt responses or endpoints such as apical and Darwinian fitness. Beyond the empirical observation, laboratory toxicity testing needs to rely on effective detection of prognostic biomarkers such as genotoxicity. Indeed, characterization of tannery effluent requires slotting in of genotoxic responses in whole effluent toxicity testing procedures. Hence, the prime objective of the present experimental investigation is to apply the technique of biological assay as a tool of toxicity testing to evaluate the induction of micronuclei (MN) in peripheral erythrocytes, and exfoliated cells of gill and kidney of O.niloticus exposed to Maximum tolerable concentrations (MTCs) of composite Modjo tannery effluent (CMTE) and to compare the sensitivity of each cells origin to the induction of MN. After 72h of exposure, cellular aberrations were detected using MN and nuclear abnormality (NA) tests. The induction of MN was significantly higher in exposed groups (P<0.05) when compared to the control group; moreover the tissue specific MN response was in the order, gill cells>peripheral erythrocyte>kidney. Total NA was found to increase significantly (P<0.05), when compared to the non-exposed group. NA was also further ramified as blebbed (BL), bi-nucleated (BN), lobbed (LB) and notched (NT) abnormalities. The result of each endpoint measured has demonstrated that at a concentration of total chromium (0.1, 0.73 and 1.27mg/L), a perceptible amount cellular aberration was measured, further implicating somber treat of genotoxicity to fishes, if exposed to water contaminated with tannery effluent. This further highlight that conventional effluent monitoring alone cannot reveal the effects expressed at cellular and genetic levels further demanding the incorporation of effluent bioassays in risk assessment and risk management/abatement programs.

Alternative Multiorgan Initiation–Promotion Assay for Chemical Carcinogenesis in the Wistar Rat

The medium-term multiorgan initiation-promotion chemical bioassay (diethylnitrosamine, methyl-nitrosourea, butyl-hydroxybutylnitrosamine, dihydroxypropylnitrosamine, dimethylhydrazine [DMBDD]) with the Fischer 344 rat was proposed as an alternative to the conventional 2-year carcinogenesis bioassay for regulatory purposes. The acronym DMBDD stands for the names of five genotoxic agents used for initiation of multiorgan carcinogenesis. The Brazilian Agency for the Environment officially recognized a variation of this assay (DMBDDb) as a valid method to assess the carcinogenic potential of agrochemicals. Different from the original protocol, this DMBDDb is 30-week long, uses Wistar rats and two positive control groups exposed to carcinogenesis promoters sodium phenobarbital (PB) or 2-acetylaminofluorene (2-AAF). This report presents the experience of an academic laboratory with the DMBDDb assay and contributes to the establishment of this alternative DMBDD bioassay in a different rat strain. Frequent lesions observed in positive groups to evaluate the promoting potential of pesticides and the immunohistochemical expressions of liver cytochrome P450 (CYP) 2B1/2B2 and CYP1A2 enzymes were assessed. Commonly affected organs were liver, kidney, intestines, urinary bladder, and thyroid. PB promoting activity was less evident than that of 2-AAF, especially in males. This study provides a repository of characteristic lesions occurring in positive control animals submitted to a modified alternative 2-stage multiorgan protocol for carcinogenesis in Wistar rat.

Bioassay of prion-infected blood plasma in PrP transgenic Drosophila.

In pursuit of a tractable bioassay to assess blood prion infectivity, we have generated prion protein (PrP) transgenic Drosophila, which show a neurotoxic phenotype in adulthood after exposure to exogenous prions at the larval stage. Here, we determined the sensitivity of ovine PrP transgenic Drosophila to ovine prion infectivity by exposure of these flies to a dilution series of scrapie-infected sheep brain homogenate. Ovine PrP transgenic Drosophila showed a significant neurotoxic response to dilutions of 10-2 to 10-10 of the original scrapie-infected sheep brain homogenate. Significantly, we determined that this prion-induced neurotoxic response in ovine PrP transgenic Drosophila was transmissible to ovine PrP transgenic mice, which is indicative of authentic mammalian prion detection by these flies. As a consequence, we considered that PrP transgenic Drosophila were sufficiently sensitive to exogenous mammalian prions to be capable of detecting prion infectivity in the blood of scrapie-infected sheep. To test this hypothesis, we exposed ovine PrP transgenic Drosophila to scrapie-infected plasma, a blood fraction notoriously difficult to assess by conventional prion bioassays. Notably, pre-clinical plasma from scrapie-infected sheep induced neurotoxicity in PrP transgenic Drosophila and this effect was more pronounced after exposure to samples collected at the clinical phase of disease. The neurotoxic phenotype in ovine PrP transgenic Drosophila induced by plasma from scrapie-infected sheep was transmissible since head homogenate from these flies caused neurotoxicity in recipient flies during fly-to-fly transmission. Our data show that PrP transgenic Drosophila can be used successfully to bioassay prion infectivity in blood from a prion-diseased mammalian host.

Silk-Encapsulated Plasmonic Biochips with Enhanced Thermal Stability.

Because of their high sensitivity, cost-efficiency, and great potential as point-of-care biodiagnostic devices, plasmonic biosensors based on localized surface plasmon resonance have gained immense attention. However, most plasmonic biosensors and conventional bioassays rely on natural antibodies, which are susceptible to elevated temperatures and nonaqueous media. Hence, an expensive and cumbersome "cold chain" system is necessary to preserve the labile antibodies by maintaining optimal cold temperatures during transport, storage, and handling. Herein, we introduce a facile approach to preserve the antibody activity on a biosensor surface even at elevated temperatures. We show that silk fibroin film could be used as a protective layer to preserve the activity of a model antibody (Rabbit IgG) and cardiac troponin antibody at both room temperature and 40 °C over several days. Furthermore, a simple aqueous rinsing process restores the biofunctionality of the biosensor. This energy-efficient and environmentally friendly method represents a novel approach to eliminate the cold chain and temperature-controlled packing of diagnostic reagents and materials, thereby extending the capability of antibody-based biosensors to different resource-limited circumstances such as developing countries, an ambulance, an intensive care unit emergency room, and battlefield.

An improved method for determining microbially available phosphorus in drinking water

Microbially available phosphorus (MAP) is the labile phosphorus that is readily assimilated by microorganisms, which is linearly correlated to bacterial re-growth in drinking water in some regions. The conventional MAP bioassay for drinking water was originally developed by Markku based on the growth potential of Pseudomonas fluorescens P17 (P17). However, the bioassay bears some demerits, such as time-consuming and labor-intensive enumeration. For convenience, an alternative method based on a similar principle was developed to assess the content of MAP in drinking water, in which natural microbial consortium was used as inoculum instead of pure culture P17, cell number was counted using flow cytometry (FCM), and cultivation at 30 °C was adopted. Natural microbial consortium is able to efficiently utilize organic phosphorus and exhibit high sensitivity since more cells are produced per μg P utilized. FCM is a rapid method to count all bacteria growing in drinking water. With incubation temperature increasing up to 30 °C, there is a shorter test period (64 h), excellent sensitivity and better utilization efficiency for organic phosphorus. The results show that the developed bioassay is sensitive, time-saving and easily operated.

Impedimetric quantification of cells encapsulated in hydrogel cultured in a paper-based microchamber

Recently, 3D cell culture technique was proposed to provide a more physiologically-meaningful environment for cell-based assays. With the development of microfluidics technology, cellular response can be quantified by impedance measurement technique in a real-time and non-invasive manner. However, handling of these microfluidic systems requires a trained engineering personnel and the operation is not compatible to traditional biological research laboratories. In this work, we incorporated the impedance measurement technique to paper-based 3D cell culture model and demonstrated non-invasive quantification of cells encapsulated in hydrogel during the culture course. A cellulose filter paper was patterned with an array of circular microchambers. Cells were encapsulated in hydrogel and loaded to the microchambers for culturing cells in 3D environment. At the preset schedule during the culture course, the paper was placed on a glass substrate with measurement electrodes for the impedance measurement. Cells in each microchamber was represented by impedance magnitude and cell proliferation could be studied over time. Also, conventional bio-assay was performed to further confirm the feasibility of the impedimetric quantification of cells encapsulated in hydrogel cultured in the paper-based microchamber. This technique provides a convenient, fast, and non-invasive approach to monitor cells cultured in 3D environment. It has potential to be developed for routine 3D cell culture protocol in biological research laboratories.

Lipid raft biomaterial as a mass screening affinity tool for rapid identification of potential antitumor Chinese herbal medicine

IntroductionThe quest for novel cancer drugs is one of the most significant studies in natural products, and Chinese herbs with its promising antitumor properties cannot be exempted from this chemotherapeutic bracket. This study was therefore aimed at piloting the use of receptor-rich lipid raft biomaterial as a mass screening tool for rapid isolation of effective antitumor components from some Chinese herbs used in the treatment of diseases consistent with cancer symptoms. MethodAqueous crude extracts from 10 different Chinese herbs (Paeonia suffruticosa, Vaccaria segetalis, Terminalia chebula, Trichosanthes kirilowii, Arisaema erubescens, Morus australis, Centella asiatica, Rhematoxylon campechianum, Trachelospermum jasminoides and Taraxacum officinale) were screened using lipid raft silica beads. The identified bioactive products were separated into various fractions (petroleum ether, ethyl acetate, n-butanol and raffinate extracts) with another affinity screening analysis. Conventional MTT bioassay was employed to authenticate the susceptibility of liver cancer cells to the final bioactive isolates. ResultsThe results showed that only Vaccaria segetalis and Arisaema erubescens significantly interacted with the system. The MTT bioassay further confirmed the cytotoxicity of these plants and the data correlated very well with its reported cancer-related activities. ConclusionThese findings support the reliable application of the lipid raft biomaterial as a prospective mass screening technique for antitumor natural products.

Electrochemical Nanobiosensors for Cancer Diagnosis

Cancer is one of the most leading cause of death worldwide and can take over 200 diverse forms, including lung, prostate, breast, cervical, ovarian, hematologic, colon cancer, and leukemia. It has been found that environmental factors (eg, alcohol, radiation, smoke, and carcinogenic chemical compounds) as well as genetic factors (eg, autoimmune dysfunction and hereditary mutations) are linked with an increased threat in the development and progression of cancer [1]. In addition microorganisms are also reported to be associated with some types of cancer (eg, stomach cancer and cervical cancer etc) [2]. In view of such an important medical condition, several methods have already been discovered to diagnose cancer and many more methods are in the process of development [3]. Conventional clinical approaches to detect cancers are based on biopsy followed by histopathology [4], biomarkers using protein levels or nucleic acid content and its expression in the cancer suspects [5]. Biopsy is the most widely used technique, however, it is an invasive technique and cannot always be used. Furthermore, it cannot be applied when cancer biomarkers are present in an extremely low concentrations in the body fluids and in malignant tissues. Thus, the development of highly sensitive and new techniques of cancer diagnosis is extremely interesting and significant in medical science. Due to high interest in interdisciplinary research in the last decade several nanobiosensors based on spectrophotometric or optical methods, fluorescence immunoassay, chemiluminescence analysis, electrochemistry, radioimmunoassay, capillary electrophoresis and chromatographic analysis have been developed to detect cancer biomarkers (proteomic and genetic markers) and cells [6,7]. The major issues in cancer diagnosis are sensitivity and to develop a miniaturized platforms that can be used as point-of-care medical device and can be applied in the remote areas. The development of electrochemical nanobiosensors composed of nanomaterials and biological receptors (antibody, aptamer, peptide etc) are likely the most encouraging approach to solve the problems related to sensitivity, rapidity, selectivity, and low cost [8-12]. This approach is expected to be very effective for cancer diagnosis due to the combination of conventional bioassay (antibody, aptamer, peptide etc) coupled with nanomaterials and electrochemical measurement. Another advantage of the electrochemical biosensor include its ability to be miniaturized as an onsite medical device, low cost, small, and handy size [13-17]. These features of electrochemical nanobiosensors may serve as a smart alternative to support fast cancer diagnosis, thereby designing better therapeutic strategies which will be extremely helpful in decreasing patient stress.

Inorganic lanthanide nanoprobes for background-free luminescent bioassays

Luminescent bioassay techniques have been widely adopted in a variety of research and medical institutions. However, conventional luminescent bioassays utilizing traditional bioprobes like organic dyes and quantum dots often suffer from the interference of background noise from scattered lights and autofluorescence from biological matrices. To eliminate this disadvantage, the use of inorganic lanthanide (Ln3+)-doped nanoparticles (NPs) is an excellent option in view of their superior optical properties, such as the long-lived downshifting luminescence, near-infrared triggered anti-Stokes upconverting luminescence and excitation-free persistent luminescence. In this review, we summarize the latest advances in the development of inorganic Ln3+-doped NPs as sensitive luminescent bioprobes from their fundamental physicochemical properties to biodetection, including the chemical synthesis, surface functionalization, optical properties and their promising applications for background-free luminescent bioassays. Future efforts and prospects towards this rapidly growing field are also proposed.

A comprehensive comparison of assays for detection and identification of Ralstonia solanacearum race 3 biovar 2.

To determine the reliable combination of protocols for specific detection and identification of R. solanacearum race 3 biovar 2 (R3bv2) through a comprehensive comparison among currently available techniques. Sensitivity and specificity of the conventional isolation, bioassay, serological assays, conventional and real-time PCR and multiplex PCR were assessed for the detection of 25 strains of R. solanacearum biovars 1, 2 and 3 (Phylotypes I, II, III and IV) in spiked potato saps. Results indicated that all assays evaluated varied in complexity and sensitivity and should be applied strategically in indexing schemes to maximize efficiency of testing without compromising accuracy of the results. The TaqMan PCR assay, with an internal reaction control and confirmation by melting curve and electrophoretic analysis, achieved best sensitivity at 10(2) -10(3 ) CFU ml(-1) for all eighteen strains of R. solanacearum R3bv2. Selective enrichment on mSMSA medium plates enhanced the detection sensitivity up to 10-100 CFU ml(-1) for the conventional PCR-based assays. This is the first time nine different assays were compared side by side for their sensitivity and specificity in detection and identification of R. solanacearum R3bv2. The data accumulated here will provide basis for regulatory applications for low level detection and rapid identification of latently infections caused by R. solanacearum R3bv2.

Evaluation of Whole Wastewater Effluent Impacts on HepG2 using DNA Microarray-based Transcriptome Analysis

DNA microarray-based transcriptome analysis with human hepatoma HepG2 cells was applied to evaluate the impacts of whole wastewater effluents from the membrane bioreactors (MBRs) and the activated sludge process (AS). In addition, the conventional bioassays (i.e., cytotoxicity tests and bioluminescence inhibition test), which were well-established for the evaluation of the overall effluent toxicity, were also performed for the same samples. Transcriptome analysis revealed that 2 to 926 genes, which were categorized to 0 to 225 biological processes, were differentially expressed after exposure to the effluents and the raw wastewater. Among the tested effluents, the effluent from a MBR operated at a relatively long solid retention time (i.e., 40 days) and small membrane pore size (i.e., 0.03 μm) showed the least impacts on the HepG2 even at the level comparable to tap water. The observed gene expression responses were in good agreement with the results of cytotoxicity tests, and provided additional molecular mechanistic information on adverse effects occurred in the sublethal region. Furthermore, the genes related to "lipid metabolism", "response to endogenous stimulus", and "response to inorganic substance" were selected as potential genetic markers, and their expression levels were quantified to evaluate the cellular impacts and treatability of wastewater effluents. Although the harmful impacts and innocuous impacts could not be distinguished at present, the results demonstrated that the DNA microarray-based transcriptome analysis with human HepG2 cells was a powerful tool to rapidly and comprehensively evaluate impacts of whole wastewater effluents.

Host‐specific salivary elicitor(s) of European corn borer induce defenses in tomato and maize

Plants turn on induced defenses upon insect herbivory. In the current study, we evaluated the role of European corn borer (ECB) elicitors (molecules secreted by herbivores) that either induce/suppress defenses in Solanum lycopersicum (tomato) and Zea mays (maize), two very important crop plants that are grown for food and/or fuel throughout the world. We used a combination of molecular, biochemical, confocal and scanning electron microscopy, caterpillar spinneret ablation/cauterization, and conventional insect bioassay methods to determine the role of ECB elicitors in modulating defenses in both tomato and maize crop plants. Our results clearly demonstrate that the components present in the ECB saliva induce defense-related proteinase inhibitors in both tomato (PIN2) and maize (MPI). Presence of glucose oxidase in the ECB saliva induced defenses in tomato, but not in maize. However, ECB saliva induced genes present in the jasmonic acid biosynthesis pathway in both tomato and maize. Although ECB saliva can induce defenses in both tomato and maize, our results suggest that host-specific salivary components are responsible for inducing host plant defenses. Proteomic analysis of ECB salivary elicitors and plant receptors/signaling mechanisms involved in recognizing different ECB elicitors remains to be determined.

Abstract 1561: An evaluation of the use of genetically modified mouse models in human cancer risk assessment.

Abstract The development of short-term or accelerated cancer bioassays using genetically modified (GM) animals that exhibit high sensitivity to chemically induced cancers and that act through mode(s) of action relevant to humans continues to be an important focus of research. However, to date only a few GM mouse models such as the Trp53+/−, the Tg.AC and the rasH2 models have effectively been evaluated to substitute for the two-year rodent cancer bioassays. Whether these models provide an acceptable replacement for the conventional rodent cancer bioassay for assessing human cancer risks is the subject of ongoing debate. The objective of this study was to evaluate the current status of the use of GM mice for accelerated cancer bioassays in assessing the potential human health risks associated with exposure to carcinogenic agents. We compared the published data from the GM bioassays with results obtained using the National Toxicology Program's conventional 2-year mouse cancer bioassay presently used in human cancer risk assessment. The results demonstrate a moderate success for these GM models to distinguish carcinogens from non-carcinogens. Although the GM models are less efficient in detecting rodent carcinogens, they are more consistent in identifying non-carcinogens. Our analysis also shows that there continue to be concerns about the assay design and protocols used for the GM models, including issues related to sample size, study duration, genetic instability, and reproducibility. Additional issues of concern include specific toxicity pathway-dependent effects, and differences in the potential carcinogenic mechanisms operating in GM and non-GM animals, topics that still need to be investigated. In addition, the dose-responses of the GM models can vary substantially from those seen in conventional cancer bioassays. Overall, we conclude that the existing GM mouse models are of value for hazard identification, but are of limited use for dose-response analysis with currently used study designs. Hence, until these models are thoroughly validated using more robust study designs and the dose-response issues adequately addressed, caution should be exercised when using the current GM models to assess the carcinogenic risks of chemicals to humans. Disclaimer: The views expressed in this abstract are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency or the National Institute of Environmental Health Sciences. Citation Format: David A. Eastmond, Suryanarayana V. Vulimiri, John E. French, Babasaheb Sonawane. An evaluation of the use of genetically modified mouse models in human cancer risk assessment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1561. doi:10.1158/1538-7445.AM2013-1561

WISH cell line: From the antiviral system to a novel reporter gene assay to test the potency of human IFN-α and IFN-β

Interferons (IFNs) are potent biologically active proteins that are widely used as biopharmaceuticals, so their potency must be correctly identified. Usually, the biological activity is quantified by a bioassay based on its capacity to induce an antiviral state in target cells, but this type of assays is subject to virus manipulation-related issues and they show considerable intra- and inter-assay variability.In this work, we generated a reporter gene assay (RGA) supported on the WISH-Mx/eGFP reporter cell line to determine human type I IFN activity. WISH cells were stably transfected with the enhanced green fluorescent protein (eGFP) gene under the control of type I IFN-inducible Mx2 promoter.This system implies the use of a standardized cell line for human IFN-potency analysis such as WISH cells and the simultaneous use of the sensitive reporter gene eGFP, having also several advantages when compared to antiviral activity assays and other RGAs: it can determine the potency of hIFN-α and hIFN-β using only one cell line showing the highest expression of eGFP after 28h and being only observed in cells treated with type I IFNs due to the specificity of the Mx promoter. It is a sensitive assay and it represents a safe alternative when compared with the conventional antiviral tests. The cell line showed the same sensitivity along 57 generations, allowing its use during two months of successive culture. The inter- and intra-assay coefficients of variation were lower than 20%, demonstrating its reproducibility. In addition, this reporter cell line can be used for the conventional antiviral assay, either for hIFN-α or hIFN-β.In conclusion, we have developed an alternative reporter system for the analysis of type I IFNs, in which its performance make it a suitable candidate to replace or complement conventional bioassays that are currently employed to measure IFN potency.

Effect of novel phosphoramidate on growth and respiratory metabolism of Paramecium aurelia

The continuous increase in the number of new chemicals as well as the discharges of solid and liquid wastes triggered the need for simple and inexpensive bioassays for routine testing. In recent years, there has been increasing development of methods (particularly rapid tests) for testing environmental samples. This paper describes the quick toxic evaluation of a novel synthetic compound: Phosphoramidate derivative B at different concentrations (2, 4 and 8 μM) for 72 h on Paramecium aurelia. We showed that B concentrations affect the growth of Paramecium in concentration- dependent manner; also it decreases the growth rate and increases response percentage in concentration- dependent manner. The value of LC50 obtained for these protozoa was estimated at 4.9693 μM after 24 hours of exposure. The respiratory metabolism of protozoan is perturbed at three concentrations, noting that the oxygen consumption was significantly increased at high concentrations after 18 hours of exposure. The results indicate that the Paramecium toxicity assay could be used as a complementary system to rapidly elucidate the cytotoxic potential of insecticides. The major advantages associated with these tests are: inexpensive, simple, rapid and seem to be attractive alternatives to conventional bioassays

Toxic Marine Puffer Fish in Thailand Seas and Tetrodotoxin They Contained

A total of 155 puffers caught from two of Thailand’s seas, the Gulf of Siam and the Andaman seas, during April to July 2010 were included in this study. Among 125 puffers from the Gulf of Siam, 18 were Lagocephalus lunaris and 107 were L. spadiceus which were the same two species found previously in 2000-2001. Thirty puffers were collected from the Andaman seas, 28 Tetraodon nigroviridis and two juvenile Arothron reticularis; the two new species totally replaced the nine species found previously in 1992-1993. Conventional mouse bioassay was used to determine the toxicity in all fish tissue extracts, i.e., liver, reproductive tissue, digestive tissue and muscle. One of each of the species L. lunaris and L. spadiceus (5.56 and 0.93%, respectively) were toxic. All 28 T. nigroviridis and 2 A. reticularis (100%) from the Andaman seas were toxic. The toxicity scores in T. nigroviridis tissues were much higher than in the respective tissues of the other three fish species. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) revealed that the main toxic principle was tetrodotoxin (TTX). This study is the first to report TTX in L. spadiceus. Our findings raised a concern for people, not only Thais but also inhabitants of other countries situated on the Andaman coast; consuming puffers of the Andaman seas is risky due to potential TTX intoxication.

Vascular Hydrophytes for Bioassay of Phosphate Enrichment in Fresh Waters: A Pilot Study

Vascular hydrophytes were shown to have the potential to be useful for straightforward, low-technology, bioassay of fresh water quality, specifically in relation to phosphate enrichment by effluent from sewage treatment works. Field-collected shoots of Elodea canadensis and Callitriche sp. made greater extension growth when incubated in canal water from downstream of discharges, indicating enrichment. This was supported by phosphate analysis and by conventional Selenastrum bioassay. Growth of shoots incubated in phosphate-augmented water from upstream of discharges equalled that in downstream water, confirming that bioassay, using vascular plants, is effective in detecting enrichment by phosphate.

Development of a Real-Time PCR Assay for Detection and Quantification of Rhizobium leguminosarum Bacteria and Discrimination between Different Biovars in Zinc-Contaminated Soil

Primers were designed to target 16S rRNA and nodD genes of Rhizobium leguminosarum from DNA extracted from two different soil types contaminated with Zn applied in sewage sludge. Numbers of rhizobia estimated using 16S rRNA gene copy number showed higher abundance than those estimated by both nodD and the most-probable-number (MPN) enumeration method using a plant trap host. Both 16S rRNA gene copies and the MPN rhizobia declined with increased levels of Zn contamination, as did the abundance of the functional gene nodD, providing compelling evidence of a toxic effect of Zn on R. leguminosarum populations in the soil. Regression analysis suggested the total Zn concentration in soil as a better predictor of rhizobial numbers than both NH4NO3-extractable and soil solution Zn. R. leguminosarum bv. viciae nodD gene copies were generally less sensitive to Zn than R. leguminosarum bv. trifolii nodD. The latter were generally below detection limits at Zn levels of >250 mg kg(-1). Although there were differences in the actual numbers estimated by each approach, the response to Zn was broadly similar across all methods. These differences were likely to result from the fact that the molecular approaches assess the potential for nodulation while the MPN approach assesses actual nodulation. The results demonstrate that the use of targeted gene probes for assessing environmental perturbations of indigenous soil rhizobial populations may be more sensitive than the conventional plant bioassay and MPN methods.