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TRANSGENIC hsp 16-lacZ STRAINS OF THE SOIL NEMATODE CAENORHABDITIS ELEGANS AS BIOLOGICAL MONITORS OF ENVIRONMENTAL STRESS
... Few species, however, have been used as test organisms in ecotoxicological studies [15][16][17][18][19][20]. Caenorhabditis elegans has been used in ecotoxicological tests for contaminants in liquid phase [21][22][23][24][25][26][27][28], on agar [29][30][31], and in soil [32,33]. ...
... Various endpoints have been used in ecotoxicological tests with C. elegans. The endpoint most often used has been mortality of the test organism, expressed as the LC50 (lethal concentration, 50% population) [23][24][25]27,28,[30][31][32][33]. Furthermore, fecundity [21,22,28,29], development [21,28,30], morphology [21,29,34], growth [21,22], population growth rate [21], and behavior [35] have been used to assess toxic effects. ...
... Furthermore, fecundity [21,22,28,29], development [21,28,30], morphology [21,29,34], growth [21,22], population growth rate [21], and behavior [35] have been used to assess toxic effects. Recently, assays for C. elegans that measure the induction of stress reporter genes have been developed [25,26]. These tests can be more sensitive than current LC50 tests but are more difficult to perform and technically demanding. ...
We present a method using the free-living nematode Caenorhabditis elegans (Maupas, 1899) to assess toxicity in liquid medium and whole-sediment setups. Test duration is 72 h; endpoints are body length, number of eggs inside worms, percentage of gravid worms, and number of offspring per worm. The effect of CdCl2 on C. elegans in liquid-phase exposures is described as an example. Results from a field study with polluted sediments from the River Elbe (Germany) suggest that nematodes may be useful organisms in assessing toxicity of sediments in the whole phase.
... C. elegans a ainsi été utilisé dans un grand nombre d'études sur les effets de stresseurs variés tels que les polluants organiques (e.g. hydrocarbures aromatiques polycycliques, pesticides) (Anderson & Wild, 1994;Dhawan et al., 1999;Donkin & Williams, 1995;Gomez-Eyles et al., 2009;Höss et al., 2009;Stringham & Candido, 1994), les rayonnements (e.g. UV, radiations ionisantes) (Anderson & Wild, 1994; (Dengg & van Meel, 2004;Williams et al., 2000), les relargages de substances industrielles (Hitchcock et al., 1997) ou les métaux lourds (Cioci et al., 2000;Dutilleul, 2013;Mutwakil et al., 1997;Stringham & Candido, 1994;Swain et al., 2010;Tvermoes & Freedman, 2008). ...
... hydrocarbures aromatiques polycycliques, pesticides) (Anderson & Wild, 1994;Dhawan et al., 1999;Donkin & Williams, 1995;Gomez-Eyles et al., 2009;Höss et al., 2009;Stringham & Candido, 1994), les rayonnements (e.g. UV, radiations ionisantes) (Anderson & Wild, 1994; (Dengg & van Meel, 2004;Williams et al., 2000), les relargages de substances industrielles (Hitchcock et al., 1997) ou les métaux lourds (Cioci et al., 2000;Dutilleul, 2013;Mutwakil et al., 1997;Stringham & Candido, 1994;Swain et al., 2010;Tvermoes & Freedman, 2008). C. elegans est également utilisé dans le cadre d'un programme américain, le programme WormTox, d'analyse des effets toxiques de milliers de composés chimiques à l'aide de méthodes de criblage à haute fréquence (Boyd et al., 2007(Boyd et al., , 2010a(Boyd et al., ,b, 2009Smith et al., 2009 ...
The evolution of toxic effects at a relevant scale is an important challenge for the ecosystem protection. Indeed, pollutants may impact populations over long-term and represent a new evolutionary force which can be adding itself to the natural selection forces. Thereby, it is necessary to acquire knowledge on the phenotypics and genetics changes that may appear in populations submitted to stress over several generations. Usually statistical analyses are performed to analyse such multigenerational studies. The use of a mechanistic mathematical model may provide a way to fully understand the impact of pollutants on the populations' dynamics. Such kind of model allows the integration of biological and toxic processes into the analysis of ecotoxicological data and the assessment of interactions between these processes. The aim of this Ph.D. project was to assess the contributions of the mechanistical modelling to the analysis of evolutionary experiment assessing long-term exposure. To do so, a three step strategy has been developed. Foremost, a multi-generational study was performed to assess the evolution of two populations of the ubiquitous nematode Caenorhabditis elegans in control conditions or exposed to 1.1 mM of uranium. Several generations were selected to assess growth, reproduction, and dose-responses relationships, through exposure to a range of concentrations (from 0 to 1.2 mM U) with all endpoints measured daily. A first statistical analysis was then performed. In a second step, a bioenergetic model adapted to the assessment of ecotoxicological data (DEBtox) was developed on C. elegans. Its numerical behaviour was analysed. Finally, this model was applied to all the selected generations in order to infer parameters values for the two populations and to assess their evolutions. Results highlighted an impact of the uranium starting from 0.4 mM U on both C. elegans' growth and reproduction. Results from the mechanistical analysis indicate this effect is due to an impact on the assimilation of energy from food. Both the mechanistic and the classic approaches highlighted individuals' adaptation to environmental conditions. Despite this, differential evolutions of the individuals from the uranium-selected population were also highlighted. All these results were more in-depth described by the mechanistical analysis. Overall, this work contributes to our knowledge on the effects of pollutants on population dynamics, and demonstrates the contributions of mechanistical modelling which can be applied in other contexts to achieve in fine a better assessment of environmental risks of pollutants.
... C. elegans a ainsi été utilisé dans un grand nombre d'études sur les effets de stresseurs variés tels que les polluants organiques (e.g. hydrocarbures aromatiques polycycliques, pesticides) (Anderson & Wild, 1994;Dhawan et al., 1999;Donkin & Williams, 1995;Gomez-Eyles et al., 2009;Höss et al., 2009;Stringham & Candido, 1994), les rayonnements (e.g. UV, radiations ionisantes) (Anderson & Wild, 1994; (Dengg & van Meel, 2004;Williams et al., 2000), les relargages de substances industrielles (Hitchcock et al., 1997) ou les métaux lourds (Cioci et al., 2000;Dutilleul, 2013;Mutwakil et al., 1997;Stringham & Candido, 1994;Swain et al., 2010;Tvermoes & Freedman, 2008). ...
... hydrocarbures aromatiques polycycliques, pesticides) (Anderson & Wild, 1994;Dhawan et al., 1999;Donkin & Williams, 1995;Gomez-Eyles et al., 2009;Höss et al., 2009;Stringham & Candido, 1994), les rayonnements (e.g. UV, radiations ionisantes) (Anderson & Wild, 1994; (Dengg & van Meel, 2004;Williams et al., 2000), les relargages de substances industrielles (Hitchcock et al., 1997) ou les métaux lourds (Cioci et al., 2000;Dutilleul, 2013;Mutwakil et al., 1997;Stringham & Candido, 1994;Swain et al., 2010;Tvermoes & Freedman, 2008). C. elegans est également utilisé dans le cadre d'un programme américain, le programme WormTox, d'analyse des effets toxiques de milliers de composés chimiques à l'aide de méthodes de criblage à haute fréquence (Boyd et al., 2007(Boyd et al., , 2010a(Boyd et al., ,b, 2009Smith et al., 2009 ...
L'évaluation des effets toxiques à des échelles pertinentes est un challenge important pour la protection des écosystèmes. En effet, les polluants peuvent impacter les populations sur le long terme et représenter une nouvelle force évolutive qui peut s'ajouter aux autres forces de sélection. Il est par conséquent nécessaire d'acquérir des connaissances sur les changements phénotypiques et génétiques apparaissant dans une population exposée à un stress durant plusieurs générations. En général les études multi-générations sont analysées à partir d'approches purement statistiques. La modélisation mécanistique a le potentiel de comprendre pleinement les effets des polluants sur la dynamique des populations. Ce type de modèle permet d'intégrer des processus biologiques et toxiques à l'analyse de données d'écotoxicologie et d'étudier les interactions entre ces processus. L'objectif de ce doctorat était d'étudier les apports de la modélisation mécanistique, par rapport à une analyse statistique classique, dans l'analyse de données d'évolution expérimentale suite à l'exposition sur le long terme à un contaminant. Pour ce faire, une stratégie en trois temps a été menée. Tout d'abord, une expérience multigénérationnelle a été réalisée sur deux populations de C. elegans (contrôle et exposée à 1,1 mM U) dérivées d'une population ancestrale présentant une forte diversité génétique. Toutes les trois générations, des individus ont été extraits des populations et soumis à une gamme de concentrations en uranium (de 0 à 1,2 mM U). Une première analyse statistique classique a alors été menée. Dans un second temps, un modèle bioénergétique adapté à l'analyse de données d'écotoxicologie (DEBtox) a été mis au point pour C. elegans et son comportement numérique a été analysé. Enfin, ce modèle a été appliqué à l'ensemble des générations étudiées afin d'inférer les valeurs des paramètres pour les deux populations et d'étudier leur évolution. Les résultats obtenus ont mis en évidence un impact de l'uranium à la fois sur la croissance et la reproduction de C. elegans à partir de 0,4 mM U. Les résultats de l'analyse mécanistique indiquent que cet effet est la résultante d'un impact sur l'assimilation d'énergie depuis la nourriture. Les deux approches, tant mécanistique que classique, ont mis en évidence une adaptation des individus des deux populations aux conditions expérimentales. Malgré cela, les analyses ont également mis en évidence une évolution différentielle des individus de la population soumise à l'uranium par rapport à ceux de la population témoin. Ces résultats ont été plus finement décrits par l'analyse mécanistique. Globalement, ce travail contribue à accroître nos connaissances sur les effets des polluants sur la dynamique des populations, et démontre les apports de la modélisation mécanistisque qui pourra être appliquée dans d'autres contextes afin de réaliser in fine une meilleure évaluation des risques écologiques des polluants.
The evolution of toxic effects at a relevant scale is an important challenge for the ecosystem protection. Indeed, pollutants may impact populations over long-term and represent a new evolutionary force which can be adding itself to the natural selection forces. Thereby, it is necessary to acquire knowledge on the phenotypics and genetics changes that may appear in populations submitted to stress over several generations. Usually statistical analyses are performed to analyse such multigenerational studies. The use of a mechanistic mathematical model may provide a way to fully understand the impact of pollutants on the populations' dynamics. Such kind of model allows the integration of biological and toxic processes into the analysis of ecotoxicological data and the assessment of interactions between these processes. The aim of this Ph.D. project was to assess the contributions of the mechanistical modelling to the analysis of evolutionary experiment assessing long-term exposure. To do so, a three step strategy has been developed. Foremost, a multi-generational study was performed to assess the evolution of two populations of the ubiquitous nematode Caenorhabditis elegans in control conditions or exposed to 1.1 mM of uranium. Several generations were selected to assess growth, reproduction, and dose-responses relationships, through exposure to a range of concentrations (from 0 to 1.2 mM U) with all endpoints measured daily. A first statistical analysis was then performed. In a second step, a bioenergetic model adapted to the assessment of ecotoxicological data (DEBtox) was developed on C. elegans. Its numerical behaviour was analysed. Finally, this model was applied to all the selected generations in order to infer parameters values for the two populations and to assess their evolutions. Results highlighted an impact of the uranium starting from 0.4 mM U on both C. elegans' growth and reproduction. Results from the mechanistical analysis indicate this effect is due to an impact on the assimilation of energy from food. Both the mechanistic and the classic approaches highlighted individuals' adaptation to environmental conditions. Despite this, differential evolutions of the individuals from the uranium-selected population were also highlighted. All these results were more in-depth described by the mechanistical analysis. Overall, this work contributes to our knowledge on the effects of pollutants on population dynamics, and demonstrates the contributions of mechanistical modelling which can be applied in other contexts to achieve in fine a better assessment of environmental risks of pollutants.
... There are contradictory reports on whether an accompanying reduction of pharyngeal pumping rates might act as a first-line defense to prevent the toxicants from being ingested (Boyd et al., 2003). While there are studies showing that C. elegans decreased feeding in a concentration-dependent manner after exposure to e.g., captan (Jones et al., 1996), Stringham and Candido (1994) found no effects of low toxicant exposure on pumping rates, suggesting that C. elegans were not able to avoid toxicant exposure by reducing pumping rates. Applying this, however, we found for the exposure of synthetic beads that nematodes increased the average pumping rate when PS and silica beads were added to pure K-medium but the stimulation could not be enhanced by raising the bead concentration. ...
Toxicity tests using the model organism Caenorhabditis elegans have shown that exposure to small microplastics such as polystyrene (PS) beads lead to high body burdens and dietary restrictions that in turn inhibit reproduction. Pharyngeal pumping is the key mechanisms of C. elegans for governing the uptake of food and other particles and can be easily monitored by determining the pumping rates. In this study, pharyngeal pumping of C. elegans was examined in response to increasing quantities of food bacteria (E. coli: 10⁶–10¹⁰ cells ml–1) and synthetic particles (10⁷–10⁹ beads ml–1) of similar size (1 µm). While the average pumping rate of C. elegans exposed to E. coli depended on the density of the bacterial cells, this was not the case for the synthetic beads. At 10⁷ items ml⁻¹, bacterial cells and synthetic beads triggered a basic stimulation of the pumping rate, independent of the nutritional value of the particle. At quantities >10⁷ items ml–1, however, the nutritional value was essential to maximize the pumping rate, as it was upregulated only by E. coli cells, which can be chemosensorially recognized by C. elegans. Given the unselective uptake of all particles in the size range of bacteria, restricting the pumping rates for particles with low nutritional value to a basic rate, prevents the nematodes from wasting energy by high-frequency pumping, but still allows a food-quality screening at low food levels.
... This strain contains a reporter transgene that expresses green fluorescence protein (GFP) upregulated by a promoter of small heat shock protein (HSPs), expressed in a stress situation (Feder & Hofmann, 1999). In C. elegans, HSPs are a group of 16 kDa proteins and their expression are induced in response to heavy metals (Stringham & Candido, 1994) or quinones (Link, Cypser, Johnson, & Johnson, 1999). The strain TJ375 contain hsp-16.2::GFP ...
Betalains are nitrogenous plant pigments known for their high antioxidant capacity. For the first time, this antioxidant nature has been studied in an in vivo system using the model organism Caenorhabditis elegans. The oxidative stress caused in the fluorescent strain TJ375 (hsp-16.2::GFP) was reversed by the presence of both natural and semi-synthetic betalains, with an ED50 value around 25 μM for betacyanins and up to 10 μM for betaxanthins, with indicaxanthin, the major pigments in prickly pear fruits, as the most effective betalain. The effect of model betalains on the lifespan of the wild-type N2 strain was carefully studied using the automatic platform “Lifespan Machine”. In a search for different approaches to suppress progeny, pop-1 RNAi was used to avoid FUdR use. The presence of betalains in the medium, both as pure compounds and as enriched Opuntia extracts significantly increased the lifespan of C. elegans.
... The same strategy of studying hsp70 expression was used in transgenic soil nematodes C. elegans for studying the toxic effects of heavy metals. 37 The exposure of arecoline results in the reduction of GSH content and increased GST activity. GSH is a tripeptide and is involved in many biological phenomena, including enzymatic reactions, molecular transport, protein and nucleic acid biosynthesis, microtubule formation, signal transduction, gene expression and protection of cells against oxidative damage. ...
Arecoline is the key component of areca nut and has been suggested as a carcinogenic agent. In the present study, the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg
9
were allowed to feed on a diet having 5, 10, 20, 40 and 80 μM arecoline for 24 h. After the completion of 24 h, the larvae were subjected to ONPG assay, X-gal staining, trypan blue exclusion test, oxidative stress markers, and apoptotic and comet assays. A dose-dependent increase in the β-galactosidase activity, tissue damage, glutathione-S-transferase (GST) activity, lipid peroxidation assay, monoamine oxidase (MAO), caspase-9 and 3, protein carbonyl content (PCC), apoptotic index, and DNA damage and decrease in glutathione (GSH) content, delta aminolevulinic acid dehydrogenase (δ-ALA-D), and acetylcholinesterase (AChE) activity were observed in the larvae exposed to 20, 40 and 80 μM arecoline. The results suggest that arecoline is toxic at 20, 40, and 80 μM toward the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg
9
. Arecoline did not show any toxic effects at 5 and 10 μM.
... In this study, we used the Spodoptera frugiperda 21 (SF21) cell line as cellular level target to determine the uptake, location, and cytotoxic dose range. For further photoactive toxicity research, Caenorhabditis elegans was tested by mortality test, morphological analysis as individual-level model (Williams and Dusenbery 1990;Guven et al. 1994;Stringham and Candido 1994). In consideration of major damage status by insects in China, Chilo suppressalis larvae, a crop endangering insect, were finally selected as a practical field animal model to explore the general photoactive effects and action mechanism of MC-PBP as a photoinsecticide. ...
Our objective was to study if the phycobiliproteins of the cyanobacterium Microcystis aeruginosa rich in phycoerythrin can be used as a photosensitive insecticide. Microcystis, the main genus of freshwater algal blooms, is a rich source of phycobiliproteins (MC-PBP), which are the light-harvesting pigment proteins of cyanobacteria. In this study, we tested the photoactive toxicity of MC-PBP with mortality assays and morphological analysis in a Spodoptera frugiperda 21 (SF21) cell line, Caenorhabditis elegans, and Chilo suppressalis larvae. We found that MC-PBP was mainly localized in the lysosome in the SF21 cells. The LC50 on SF21 was 80 μg mL−1 with sunlight treatment of 880 μmol photons m−2 s−1 for 15 min and 1.5 mg mL−1 on Chilo suppressalis larva in fourth instar with the sunlight irradiation of 650 μmol photons m−2 s−1 for 5 h. Morphological observation demonstrated that autophagic apoptosis is the main death way of C. elegans, and midgut and fat body are the target tissues of C. suppressalis larvae induced by photoactive toxicity of MC-PBP. Interestingly, we also found that MC-PBP can degrade a certain amount of microcystin-LR (MC-LR), one variant of cyanobacterial microcystins. This is the first report showing that MC-PBP has potential for development into natural photosensitive insecticides which are environmental friendly but toxic for nematode and insect cells with degradation capacity for MC-LR.
... If we compare our observations with literature data related to C. elegans, it can be established that Gao et al. also found higher copper concentration in the head of nematodes treated with copper nanoparticles, [27] whereas Jackson et al. observed a homogenous copper distribution in the whole body. [28] Applying a biochemical method, Stringham et al. detected a specific response in the case of CuCl 2 treatment at the anterior end of the pharynx, [36] where our measurements showed also an increased copper content. The distribution of sulfur and copper obtained during our investigations was similar in both investigated areas, and this finding is confirmed by correlation analysis [Fig. ...
Free-living nematodes provide a good tool for environmental monitoring, because the nematode community structure is an appropriate bio-indicator. Soil-inhabiting species can be used for the characterization of soil functioning and heavy-metal pollution. To investigate the uptake and distribution of copper in the soil-inhabiting nematodes Xiphinema vuittenezi, starved animals were kept in CuSO4 and Cu(NO3)2 solutions with a concentration of 1 mmol/dm3 for 24 h. The treatment with CuSO4 was also carried out at higher concentration (0.5 mol/dm3) for 1 h. The nematodes were mounted onto polyimide tape, and after a 2-min-long freezing in liquid nitrogen, they were lyophilized for 72 h. The suitability of the sample preparation method was studied by the scanning electron microscopy and focused ion beam techniques. Elemental maps were recorded with two different lateral resolutions (8 µm ∗ 8 µm and 2 µm ∗ 2 µm). Elevated copper and sulfur content was found along the stylet. The oxidation state of copper was studied by X-ray absorption near edge structure spectroscopy. The spectra of solid standards and copper–amino acid complexes in aqueous solution were obtained as well. The oxidation state of copper within the nematodes was found to be +2 in all cases. The X-ray absorption near edge structure spectra of nematodes treated with different copper compounds showed high similarity, however, the spectrum of untreated animals indicated a different chemical environment of copper.
Environmental quality guideline values and remediation targets, specific to Antarctic ecosystems, are required for the risk assessment and remediation of contaminated sites in Antarctica. Ecotoxicological testing with Antarctic soil organisms is fundamental in determining reliable contaminant effect threshold concentrations. The present study describes the development of optimal culturing techniques, and aqueous toxicity test procedures for an endemic Antarctic soil nematode Plectus murrayi, which lives within interstitial waters between soil particles. Toxicity tests were of extended duration to account for the species’ physiology and life history characteristics. Plectus murrayi was sensitive to aqueous copper with a 50% effective concentration (EC50) for egg hatching success of 139 µg/L. Hatched juveniles that were first exposed to copper as eggs appeared to be less sensitive than those first exposed at the hatched J2 stage, indicating a potential protective effect of the egg. Sensitivity of juveniles to copper increased with exposure duration, with 50% lethal concentrations (LC50s) of 478 and 117 µg/L at 21 and 28 d, respectively. The present study describes new methods for the application of an environmentally relevant test species to the risk assessment of contaminants in Antarctic soil, and provides the first estimates of sensitivity to a toxicant for an Antarctic terrestrial microinvertebrate. This article is protected by copyright. All rights reserved.
Toxins are believed to play a crucial role in Burkholderia pseudomallei pathogenicity, however to date, only a few have been identified. The discovery of additional toxic molecules is limited by the lack of a sensitive indicator of B. pseudomallei toxicity. Previously, from a whole genome transcriptome analysis of B. pseudomallei-infected Caenorhabditis elegans, we noted significant overexpression of a number of worm genes encoding detoxification enzymes, indicating the host’s attempt to clear bacterial toxic molecules. One of these genes, ugt–29, a family member of UDP-glucuronosyltransferases, was the most robustly induced phase II detoxification gene. In this study, we show that strong induction of ugt–29 is restricted to infections by the most virulent species among the pathogens tested. We also noted that ugt–29 is activated upon disruption of host protein synthesis. Hence, we propose that UGT–29 could be a promising biosensor to detect B. pseudomallei toxins that compromise host protein synthesis. The identification of bactobolin, a polyketide-peptide hybrid molecule, as a toxic molecule of B. pseudomallei further verifies the utilization of this surveillance system to search for bacterial toxins. Hence, a ugt–29 based reporter should be useful in screening for other molecules that inhibit host protein synthesis.
The exposure to and toxic effects of heavy metals and other pollutants can be measured in terms of the biochemical responses of organisms, the so-called "molecular biomarkers". This work reviews the main molecular biomarkers induced aquatic organisms in response to heavy metal pollution. The best known are the metallothioneins (MT), described as a group of protein of low molecular weight (6 -12 KDa), rich in cysteine, able to link metallic ions in their molecular structure, and widely distributed in animals and plants. Also, the presence of peptides with higher molecular weights than those of the MT, known as "metal-binding proteins", has been frequently reported. Another interesting group of proteins induced by environmental disturbances, are the "stress proteins" (SP) which seem to be related to the presence of heavy metals in aquatic ecosystems.
We demonstrate here that the nematode Caenorhabditis elegans displays broad hormetie abilities, Hormesis, is the induction of beneficial effects by exposure to lock doses of otherwise harmful chemical or physical agents. Heat as well as pretreatment with hyperbaric oxygen or juglone (a chemical that generates reactive oxygen species) Significantly increased resistance to the Same challenge, Cross-tolerance between juglone and oxygen was also observed, The same heat or oxygen pretreatment regimens that induced subsequent stress resistance also increased life expectancy and maximum life span of populations undergoing normal aging. Pretreatment with ultraviolet or ionizing radiation did not promote subsequent resistance or increased longevity. In dose-response studies, induced thermotolerance paralleled the induced increase in life expectancy, which is consistent with a common origin.
Flubendiamide is a novel class of insecticides against lepidopteran insects. With a view to explore if this chemical is safe for non-targeted organisms, its effect was studied through assessment of heat shock protein (HSP70) expression in third instar larvae of transgenic Drosophila melanogaster, Bg9 (hsp70-lacZ). Dietary concentrations covering a range (5, 10, 20 and 40 μg/ml) of 20% flubendiamide were used for treatment of larvae for different durations (1, 3, 6 and 24 h). Reporter gene assay confirmed that HSP70 expression varied in tissues depending upon treatment concentration and exposure duration. The 5 μg/ml treatment stimulated higher stress response during the initial hours, which declined later (6 and 24 h). Nearly all tissues (humerus, brain, proventriculus, etc.) responded initially with the 20 μg/ml treatment, which declined with increasing exposure. Hence low concentration and short-term exposure of flubendiamide to non-target organisms seems to be highly effective as stressor and thus demands awareness in decreasing irrational use of the chemical.
In the present study the toxic potential of calcium carbide (CaC2) was studied on the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg(9). The third instar larvae were exposed to 2, 4, 8, 16 and 32×10(-3)g/ml of CaC2 in diet for 24h. The results reveal that the dose 2×10(-3)g/ml was not toxic but the remaining doses showed a dose dependent significant increase in the hsp70 expression, β-galactosidase activity, tissue damage, oxidative stress markers (lipid peroxidation and protein carbonyl content), glutathione-S-transferase activity, expression of Caspase 3 and 9, apoptotic index and DNA damage (midgut cells). A significant reduction as compared to control group in total protein, glutathione content and acetylcholinesterase activity was also observed. The Inductively Coupled Plasma Atomic Emission Spectroscopy analysis (ICPAES) reveals the presence of copper, iron, sodium, aluminium, manganese, calcium, nickel and mercury. The toxic effects of CaC2 in the present study may be attributed to the impurities present in it.
The focus of this paper is to consider the applicability of the hsp70 stress protein response as a biomarker in field studies. Stress proteins (or heat shock proteins, hsp) of the hsp70 family are induced by sublethal concentrations of a variety of environmental pollutants. However, few studies have applied these proteins as biomarkers of environmental stress under field conditions. Our laboratory is investigating hsp70 proteins and other responses of Asian clam (Potamocorbula amurensis) as potential biomarkers in laboratory and field studies. Our efforts include two studies presently being conducted in northern San Francisco Bay: (1) monthly collection of clams from four sites along a cadmium contamination gradient; (2) 7 day in situ exposure of clams at two selected sites at Mare Island Naval Shipyard. Here we present results on hsp70 proteins in P. amurensis in field-collected and outplanted clams. Both field projects are ongoing, therefore the results presented here do not represent completed studies; rather, they illustrate a portion of our experience. For this workshop, we illustrate weaknesses and strengths of these proteins as biomarkers, and we underscore where additional work is needed. In field-collected clams (study no. 1), site-specific differences in levels of two hsp70 proteins, hsp70 and hsp76, were measured in May and June 1997. Although an inverse correlation exists between cadmium tissue concentrations and hsp70 protein levels, differences detected may be reflective of a salinity gradient. Results from recent laboratory exposures to cadmium and a range of salinities are discussed. After in situ exposure for 7 days (study no. 2), both hsp70 and hsp76 levels were significantly reduced in clams from site R. However, given a brief heat-shock in the laboratory, hsp70 protein levels were significantly higher in clams from this site than in controls. Results indicate that downregulation as well as upregulation of hsp70 proteins may be indicators of stress in P. amurensis.
Gentamicin is an aminoglycoside antibiotic and is used to treat various Gram negative bacteria. Due to its thermal stability it is useful for the preparation of culture media for various cell lines and microorganisms. It has been reported as ototoxic and nephrotoxic in adult humans. In the present study the toxic effects of gentamicin were studied in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. The third instar larvae were exposed to a wide range of doses, i.e. 60, 70, 80, 90 and 100 μg ml−1 of gentamicin for 6, 12, 24 and 48 hours. The exposure of larvae to the above doses for 6 and 12 hours did not show any toxic effects. The exposure of larvae to 80, 90 and 100 μg ml−1 of gentamicin for 24 hours and 70, 80, 90 and 100 μg ml−1 of gentamicin for 48 hours showed a dose-dependent toxic effect. The dose of 60 μg ml−1 did not show any toxic effects and hence can be suggested as the No Observed Adverse Effect Level (NOAEL). The results suggest that gentamicin is toxic at higher doses and longer duration of exposure.
The present study was carried out to evaluate the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg(9). Cefotaxime at final concentration of 10, 20, 40, 60 and 80μg/ml was mixed in the diet and the larvae were exposed to the selected doses for 6, 12, 24, 48 hrs. The hsp70 expression, trypan blue exclusion test, in situ histochemical β-galactosidase activity, lipid peroxidation, total protein content, glutathione (GSH) content, glutathione-S-transferase (GST) activity, protein carbonyl content, caspase 3 and 9 activity, apoptotic index and comet assay were taken as parameters for the study. The larvae exposed to 40, 60 and 80μg/ml for 12, 24 and 48 hrs showed a dose and duration dependent significant increase in the activity of β-galactosidase and lipid peroxidation but decrease in the total GSH content as compared to unexposed larvae. The decrease in protein content was observed in the larvae exposed to 40, 60 and 80μg/ml of cefotaxime for 24 and 48 hrs. The larvae exposed to 40, 60 and 80μg/ml of cefotaxime for 24 and 48 hrs showed a dose and duration dependent increase in the tissue damage, GST, caspase 3 and 9 activity, PC content, apoptosis and the DNA tail length (Comet assay). The result suggests that the cefotaxime is toxic at 40, 60 and 80μg/ml of doses for the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg(9). Cefotaxime at 10 and 20 μg/ml was not toxic for any duration of exposure.
Copyright © 2015. Published by Elsevier Ireland Ltd.
While in some instances, loss of diversity results from acute toxicity (e.g. major pollution incidents), in most cases it results from long-term sub-lethal effects that alter the relative competitive ability and fitness of certain organisms. In such cases the sub-lethal effects will cause a physiological response in the organism that ultimately leads to community level changes. Very sensitive tools are now available to study sub-lethal responses at the molecular level. However, relating such laboratory measurements to ecological effects represents a substantial challenge that can only be met by investigation at all scales (molecular, individual organism and community level) with an appropriate group of organisms. Among the various in vertebrates which can be used as model organisms in such a way, the soil nematode, Caenorhabditis elegans appear to be a promising biological model to diagnose environmental quality. This paper reviews the current status of multilevel biomarkers in environmental toxicology, and C. elegans as promising organisms for this approach.
Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24 h median lethal concentration (LC50) of different zinc concentrations at different temperatures (15 °C, 20 °C, 25 °C, and 30 °C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1 mM zinc at 37 °C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24 h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity.
The interaction of heavy metals such as hexavalent chromium, Cr (VI) with the environment drastically influences living organisms leading to an ecological imbalance. Caenorhabditis elegans, a saprophytic nematode having 60-80% homology with human genes offers a distinct advantage to be used as a biosensor for the appraisal of heavy metal-induced environmental toxicity and risk monitoring. The present study examines the toxicity effects of K2Cr2O7 as Cr (VI) on stress-related gene expression and morphometric parameters of C. elegans under in vitro conditions to identify genetic markers for environmental pollution. Alterations in growth and modified gene expression were observed in Cr (VI)-exposed N2 worms. The 24-h median lethal concentration for Cr (VI) was observed as 158.5 mgl(-1). Use of the responses of stress-related gene expression suggests that C. elegans can be used as an efficient biosensor for figuring out the precise route of Cr (VI)-induced environmental toxicity in a quick, simple, and inexpensive manner.Journal of Exposure Science and Environmental Epidemiology advance online publication, 23 October 2013; doi:10.1038/jes.2013.66.
Exposure to metal ions induces a stress response by activating β-galactosidase expression in a strain of transgenic nematode (Caenorhabditis elegans strain PC72) carrying an E. coli lacZ gene under the control of an hsp16 heat-shock promoter sequence. This system can also be activated by several organic toxicants, and low β-galactosidase activities are induced in worms exposed to non-ionic Pluronic surfactants. These surfactants have been shown to stimulate worm growth, possibly through enhanced nutrient uptake via membrane pores created by surfactant action. This paper demonstrates that, in the presence of one such surfactant (Pluronic F-127 at 10 ppm throughout), the stress response of transgenic worms to several metal ions (Cd2+, Hg2+, Cu2+, Mn2+ and Zn2+) is markedly enhanced (by 1.5- to four-fold). This enhancement diminishes at high concentrations of Cd2+, possibly due to increased mortality. A three-way ANOVA confirms that both metal concentration and the presence of surfactant have extremely significant effects on β-galactosidase induction, and that there are significant interactions between these factors (generally, the surfactant effect is more pronounced at higher metal concentrations). However, the ANOVA also reveals highly significant variations between repeat runs under the same test conditions, although the trends attributable to metal dose or to surfactant are present consistently in all runs. In situ histochemical staining shows that β-galactosidase is expressed throughout worms treated with metal plus surfactant, in contrast to the localised pharyngeal staining characteristic of worms treated with metal alone. This suggests that Pluronic F-127 may facilitate metal entry into tissues which do not normally display a strong stress response. Tentative support for this is provided by the observation that worms treated with Pluronic F-127 (10 ppm) accumulate slightly (ca. 10%) more Cu2+or Zn2+during the standard exposure period than do control worms exposed to metal only. Thus metal ions are significantly more toxic to C. elegans when combined with a non-ionic surfactant, itself present at sub-toxic (indeed, growth promoting) concentrations.
Toxicity bioassays rely largely on lethality measurements. Such assays are generally lengthy and expensive, and provide little information on mechanisms of toxicity. A desire to understand the mechanisms by which cells respond to physical and chemical stresses has led to interest in measuring stress proteins as toxicological endpoints. Transgenic strains of the nematode Caenorhabditis elegans that carry a reporter enzyme under control of a stress-inducible promoter have been created. The reporter is easily quantified in intact nematodes, and it responds to a wide range of chemical Stressors. Therefore, transgenic C. elegans can provide the basis for a wide range of quick, simple and informative bioassays.
The present study aims to observe the effects of the fungicide Thiovit®Jet on hsp70 expression in third instar larvae of transgenic Bg9 (hsp70-lacZ) strain of Drosophila melanogaster. Various concentrations (40, 50, 200 and 1600 ppm) of Thiovit®Jet in distilled water was added to the food medium. A similar set of control experiments were maintained without any chemical
treatment (normal food). Third instar larvae were assayed for stress gene expression and found to express hsp70 under treated
conditions. Lower doses of treatment elicited positive response in larval skin, parts of midgut and entire hindgut. Higher
doses of the treatment and longer hours of exposure gave somewhat lower positive results excepting with 1600 ppm where an
increase in expression with increased hour of exposure is found initially followed by a subsequent decline in expression to
almost undetectable level. Low dose and short-term exposure of Thiovit®Jet is effective to the flies, which are an innocent target. Negative results may be due to lack of viable cells after a certain
time of exposure causing unavailability of cells expressing hsp70. Bg9 strain of Drosophila melanogaster used for this study
is transgenic with lacZ reporter gene fused with wild type hsp70 gene. The lacZ products confirm the hsp70 expression. Longer hours of exposure may
also make reporter gene products unstable, which do not elicit any positive response.
Expression of hsp70 in the tissues of third-instar larvae of theBg9(hsp70-lacZ) strain ofDrosophila melanogasterafter exposure to hexachlorocyclohexane (HCH) (0.5, 1.0, 1.5, 2.0, and 5.0 ng/ml) in the diet was studied by histochemical staining for β-galactosidase activity and puffing activity at 9E, 87A, and 87C of the salivary gland polytene chromosome was measured. At concentrations of 2.0 and 5.0 ng;shml, the proventriculus of the foregut, gastric caeca (part or whole), brain ganglia, imaginal discs, salivary glands, midgut, and hindgut of the larvae showed β-galactosidase activity. In controls and at dietary concentrations of 0.5 and 1.0 ng/ml, only the proventriculus and part of the salivary gland showed activity, while at 1.5 ng/ml, imaginal discs, the proximal part of brain ganglia, and the whole salivary gland showed positive staining. Among the four isomers of HCH tested, the γ isomer stimulated the most widespread expression of hsp70 in larval tissues (proventriculus, gastric caeca, part of brain ganglia, imaginal discs, salivary gland, midgut, and hindgut) followed by α (proventriculus and salivary gland) and then by the β and δ isomers which produced weak responses in the proventriculus only. Of the metabolites examined in the present study, 2,4,6-trichlorophenol (TCP) evoked the greatest expression of hsp70 compared to the other isomer 2,3,5-TCP and 1,2,3,4-tetrachlorobenzene (TTCB). The puffing pattern in the salivary gland polytene chromosome was similar to the histochemical staining pattern. The present study suggests that there are sites in the larvae vulnerable to HCH, its isomers, and metabolites as evidenced by hsp70 expression.
The nematode, Caenorhabditis elegans, can be grown in liquid culture media supplemented with bacteria as a food source, but growth is limited primarily by lack of oxygen. A novel two-phase liquid culture system has been developed in which the nematodes were grown at an interface between a lower layer of perfluorocarbon and an upper layer of aqueous S medium containing bacteria. By using degassed perfluorodecalin, nematode growth over 3 days was slightly less than in S medium controls above a plastic substrate; however, this difference in growth rate was barely significant over five replica runs. By using oxygen-saturated perfluorodecalin, growth over 3 days was significantly enhanced, as compared both to S-medium controls and to cultures over degassed perfluorodecalin. This much larger effect is attributable to improved oxygenation at the interface on which the worms move. Measurements with an oxygen electrode suggest that dissolved oxygen concentrations were greatly depleted in both the perfluorocarbon and aqueous layers after 24 h. However, during standard 7-h toxicity tests in aqueous media, an underlying layer of oxygenated perfluorocarbon significantly enhanced the sensitivity of PC72 transgenic (hsp16/lacZ) worms to cadmium, increasing expression of the reporter product, β-galactosidase. The utility of this culture system for controlling oxygen availability during nematode growth and toxicity assays is discussed.
Size fractionation has been used to isolate L1L2 larvae from mixed cultures of the nematode, Caenorhabditis elegans. Worm lengths have been compared during growth in synchronized liquid and agar cultures. Supplementation of liquid S medium with 10 ppm surfactant (Pluronics F-68, F-127, F-38, L-35; Tween-20 or Triton X-100) promoted a significant stimulation of growth over three days in all cases. Because of possible poor nutrition and/or aeration in liquid culture, experiments were repeated using the same surfactants in standard NGM agar. Four surfactants again stimulated worm growth significantly whereas two (Tween-20 and Pluronic F-68) did not. Differences between these surfactants were also demonstrated with respect to: (i) toxicity; (ii) induction of stress responses in a transgenic hsp/reporter strain; and (iii) stimulation of amino acid incorporation into soluble protein both initially and after 41 h of surfactant treatment. These surfactants, and in particular, Pluronic F-127, are potentially valuable as culture supplements for enhancing nematode larval growth. Possible mechanisms for growth promotion by surfactants are discussed in light of the fixed somatic cell lineage and the fact that Pluronic F-127 did not speed up maturation from L4 larvae into egg-bearing adults.
As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. Ecotoxicology 21:439-455, 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present.
Background:
Amorphous silica nanoparticles (aSNPs) are used for various applications including food industry. However, limited in vivo studies are available on absorption/internalization of ingested aSNPs in the midgut cells of an organism. The study aims to examine cellular uptake of aSNPs (<30nm) in the midgut of Drosophila melanogaster (Oregon R(+)) owing to similarities between the midgut tissue of this organism and human and subsequently cellular stress response generated by these nanoparticles.
Methods:
Third instar larvae of D. melanogaster were exposed orally to 1-100μg/mL of aSNPs for 12-36h and oxidative stress (OS), heat shock genes (hsgs), membrane destabilization (Acridine orange/Ethidium Bromide staining), cellular internalization (TEM) and apoptosis endpoints.
Results:
A significant increase was observed in OS endpoints in the midgut cells of exposed Drosophila in a concentration- and time-dependent manner. Significantly increased expression of hsp70 and hsp22 along with caspases activation, membrane destabilization and mitochondrial membrane potential loss was also observed. TEM analysis showed aSNPs-uptake in the midgut cells of exposed Drosophila via endocytic vesicles and by direct membrane penetration.
Conclusion:
aSNPs after their internalization in the midgut cells of exposed Drosophila larvae show membrane destabilization along with increased cellular stress and cell death.
General significance:
Ingested aSNPs show adverse effects on the cells of GI tract of the exposed organism thus their industrial use as a food-additive may raise concern to human health.
The effect of extremely low frequency electromagnetic fields (ELF-EMF) on gene expression and early development has been investigated in two different transgenic animals, the nematode Caenorhabditis elegans and Drosophila melanogaster, respectively. ELF-EMF enhanced biological reactions considerably in the presence of a second stressor (mild heat shock). In C. elegans, this effect could be demonstrated at the level of heat shock protein (hsp) gene expression by means of a lacZ reporter gene controlled by an hsp 16 or hsp 70 promoter. In Drosophila, the same experimental strategy led to ELF-EMF induced developmental defects, as well as to a considerable retardation of development. The findings are discussed with respect to possi- ble molecular mechanisms that might explain the observed synergistic ELF-EMF induced enhancement of the stress response. An experimental approach is suggested which may help to unravel the involved signal transduction pathways.
Expression of hsrω stress gene in the third-instar larvae of 951-lacZ2 (hsrω-lacZ having–844pb sequence) and 498-lacZ1 (hrsω-lacZ having –498bp sequence) strains of Drosophila melanogaster at LC50 and lower dietary concentrations of hexachlorocyclohexane (HCH) pentachlorophenol (PCP), and endosulfan was examined in relation to larval mortality by β galactosidase activity, vital dye staining, and salivary gland polytene chromosome puffing. Our results showed that both HCH and PCP at lower concentrations evoked strong hsrω stress gene expression in the larval tissues while endosulfan did not. On the other hand, puffing data revealed that endosulfan at lower doses, induced well-developed puff at the resident site (93D) of the hsrω gene but the transgenic sites (30B in 951-lacZ2 and 44B in 498-lacZ1 strain) did not show any well-developed puff. Regression in hsrω stress gene expression in 951-lacZ2 strain at LC50 concentrations of HCH and PCP after 48 h was concurrent with extensive tissue damage as evident by trypan blue staining. Similarly, strong hsrω expression was accompanied by insignificant trypan blue staining in the larval tissues of this strain after shorter duration of exposure (2–12 h) to these toxicants. Although endosulfan under similar experimental condition did not induce hsrω, strong trypan blue staining indicated extensive tissue damage after 48 h of exposure. The present study suggests that all the three toxicants pose cytotoxic potential to Drosophila. While protective role of this stress gene was evident at the initial stages of exposure, extensive tissue damage in the later stages of intoxication accompanied by autorepression of hsrω led to larval mortality. The study further suggests that –844bp upstream sequence of the gene is adequate for hsrω inducibility against HCH and PCP but not for endosulfan for which responsive elements may be searched further upstream. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:173–186, 2001
We have studied the effect of extremely low frequency electromagnetic fields (ELF-EMF) in the presence of a second stressor (mild heat shock) on the expression of a lacZ reporter gene under the control of hsp16 or hsp70 promoters in two transgenic strains of C. elegans. The expression of the reporter gene was studied by scoring animals with induced β-galactosidase activity after staining in toto or by biochemical quantitation of the enzyme activity, respectively. In our experimental setup we were able to expose the animals to 50 Hz magnetic flux density of 0–150 μT and at the same time control temperature with high precision (±0.1°C). Experimental conditions were defined for which EMF strongly enhances the expression of the reporter gene. Bioelectromagnetics 21:100–106, 2000. © 2000 Wiley-Liss, Inc.
This paper briefly reviews the nomenclature and application of stress proteins as biomarkers, then gives a step by step description of the popular methodologies used to measure stress protein in plant and animal tissues together with a critic of each approach and trouble shooting tips. Stress proteins (previously termed heat shock proteins) are induced by many environmental stresses including exposure to trace metals or organic pollutants, changes in temperature or osmolarity, hypoxia/anoxia, and exposure to ultraviolet radiation. Stress proteins, especially hsp70 and hsp60, have been used as biomarkers in a range of algae, invertebrates, fish, and higher vertebrates. Several suggestions are made to improve the utility of stress proteins as a biomarker of exposure e.g., consideration of the kinetics of stress protein induction relative to the pharmacokinetics of pollutant accumulation in the organism of concern, and selection of the type of stress protein for biomonitoring. The methodologies described include sample preparation, western blotting, slot blotting, and antibody detection methods, especially for hsp70. Western blotting has been the most widely applied technique for assaying stress proteins but dot/slot blotting with the inclusion of an internal standard in each gel improves the approach by simplifying the technique—saving time and materials and producing a more quantitative result.
This paper reviews the current status of nematodes with stress-inducible transgenes as biosensors responsive to a range of
external stressors, e.g., soil or water pollution, microwave radiation or immunological attack. TransgenicCaenorhabditis elegans carrying reporter genes under heat shock promoter control express reporter products only under stressful conditions. Although
relatively insensitive to single metal ions, these worms respond to complex mixtures present in metal-contaminated watercourses
and to laboratory mixtures containing similar constituents, but not to any of their components singly at comparable concentrations.
Responses to metal mixtures are enhanced by a non-ionic surfactant, Pluronic F-127. Metals taken up by food bacteria and insoluble
metal carbonates can also evoke stress responses, both in soil and aqueous media. However, high concentrations of added metals
are needed to induce clear-cut responses in soil, owing to metal sorption onto clays and organic matter. Transgenic worms
are also stressed by exposure to microwave radiation; pulsed signals generate responses that diminish markedly with distance
from the source. Finally, stress responses are inducible by anti-epicuticle antisera and complement, suggesting that immune
attack can also activite the heat shock system. The development of rapid microplate toxicity assays based on transgenic nematodes
is discussed.
The induction of heat shock proteins (HSPs) in the nematode Plectus acuminatus (Nematoda; Plectidae) was studied following exposure to heat, copper, and cadmium. Mini two-dimensional polyacrylamide gel
electrophoresis was used for protein separation and poly- and monoclonal antibodies raised against specific HSPs in various
organisms were used to detect specific HSPs. Both HSP60 and HSP70 could be identified after exposure of nematodes to heat
stress, indicating the broad crossreactivity among species to the antibodies used. The monoclonal antibody LK-2 was selected
for further investigation with the HSP60 response to heavy metals. The induction of HSP60 was related to increased concentrations
of cadmium and copper. For copper, the induction of HSP60 was three orders of magnitude more sensitive than was the EC20 for reproduction. For cadmium, HSP60 induction was one order of magnitude more sensitive. The results point out that HSP60
induction occurred at concentration levels that are realistic for the field situation. It is therefore suggested that HSP60
may be suitable as a potential biomarker to toxicant stress in P. acuminatus.
Zur Beurteilung kontaminierter und sanierter Böden und Substrate sowie zur Abschätzung der chronischen Toxizität von Bodenkontaminationen
wurde ein ökotoxikologischer Test mit der terrestrischen NematodenartPanagrellus redivivus entwickelt. Als Bewertungsgrundlage diente die Populationsdynamik der Nematoden, ausgedrückt als Vermehrungsfaktor (VF),
der nach sieben Tagen Aussagen zur Bodenqualität erlaubt. Aufgrund der kurzen Generationszeit von < 5 Tagen fürPanagrellus wird eine Testdauer von sieben Tagen als ausreichend erachtet. Überprüfungen zur Eignung des längerfristigen Nematodentests
erfolgten an gezielt mit Lindan und einem polychlorierten Biphenyl (PCB 52) belasteten Böden.
For the assessment of contaminated and remediated soils and substrates, and the estimation of chronic toxicity of soil contaminations,
an ecotoxicological test was developed using the terrestrial nematodePanagrellus redivivus. The population dynamics expressed by the reproduction factor (VF) allows an estimation of soil quality within seven days.
Due to the rapid generation time ofPanagrellus (< 5 d), the short test duration is considered to be sufficient. The suitability of this test duration is considered to be
sufficient. The suitability of this test was evaluated by investigating soils which had been contaminated using applications
of specific pollutants (lindane and PCB 52).
The effect of endosulfan (0.02-2.0μgmL(-1)) to Drosophila melanogaster (Oregon R(+)) at the cellular and organismal levels was examined. Third instar larvae of D. melanogaster and the strains transgenic for hsp70, hsp83 and hsp26 were exposed to endosulfan through food for 12-48h to examine the heat shock proteins (hsps), reactive oxygen species (ROS) generation, anti-oxidant stress markers and xenobiotic metabolism enzymes. We observed a concentration- and time-dependent significant induction of only small hsps (hsp23>hsp22) in the exposed organism in concurrence with a significant induction of ROS generation, oxidative stress and xenobiotic metabolism markers. Sub-organismal response was to be propagated towards organismal response, i.e., delay in the emergence of flies and decreased locomotor behaviour. Organisms with diminished locomotion also exhibited significantly lowered acetylcholinesterase activity. A significant positive correlation observed among ROS generation and different cellular endpoints (small hsps, oxidative stress markers, cytochrome P450 activities) in the exposed organism indicate a modulatory role of ROS in endosulfan-mediated cellular toxicity. The study thus suggests that the adverse effects of endosulfan in exposed Drosophila are manifested both at cellular and organismal levels and recommends Drosophila as an alternative animal model for screening the risk caused by environmental chemicals.
In most sediments and soils, nematodes are the dominant organism group among the metazoans and play an important role for the food web. In biomonitoring studies, these endobenthic organisms provide several advantages over most macrobenthic organisms, including their omnipresence, high abundance and diversity of species and trophic groups. Single species and community level bioassays, as well as assessments of in situ nematode communities proved to be suitable tools for evaluting the quality of sediments and soils.
Adaptation to sublethal exposure to crude oil by phytoplankton is poorly understood. Use of chemical dispersants for oil spill remediation increases petroleum hydrocarbon concentrations in water, while exposing marine organisms to potentially toxic concentrations of dispersant. Heat shock proteins (hsps) have been found to serve as an adaptive and protective mechanism against environmental stresses. The objective of this project was to examine the induction of hsps in Isochrysis galbana, a golden-brown algae, following exposure to the water-accommodated fraction (WAF) of Prudhoe Bay crude oil (PBCO) and PBCO chemically dispersed with Corexit 9527® (dispersed oil: DO). Initial experiments using 35S-labeled amino acids and 2-dimensional electrophoresis with subsequent western blotting identified and confirmed hsp60, a member of the chaperonin family of stress proteins, as being efficiently induced by heat shock in this species. One-dimensional SDS PAGE and western blotting, with hsp60 antibodies and chemiluminesence detection, were used to quantitate hsp60 following exposure to a range of environmental temperatures and concentrations of WAF and DO preparations. I. galbana cultured in 22 parts per thousand (‰) salinity showed a statistically significant increase (p<0.05) in hsp60 after exposure to 25, 30, and 35°C for 1 h compared to controls (20°C), while 34‰ cultures showed no significant increase in hsp60 with increasing temperature. Cultures (22 and 34‰) exposed to varying doses of WAF and DO exhibited statistically significant (p<0.05) increases in levels of hsp60 in all cases. A dose-related response was observed in all exposures (r2≥0.90) with the exception of 34‰ WAF. In independent exposures, a dose-related hsp60 response to naphthalene, a relatively abundant water-soluble aromatic hydrocarbon, was observed in both 22 and 34‰ cultures. Results of this study are consistent with previous studies in other species documenting increases in hsp60 levels with exposure to xenobiotics. Further studies are investigating the protective function of hsp60 against the toxic effects of exposure to WAF and DO preparations.
1.1. The patterns of HSP70 expression induced in Caenorhabditis elegans by mild (31°C) or severe (34°C) heat shock, and by cadmium ions at 31°C, have been compared with those expressed constitutively in 20°C controls by 1- and 2-dimensional immunoblotting.2.2. The 2D spot patterns become more complex with increasing severity of stress (34°C > 31°C + Cd > 31°C > 20°C).3.3. A stress-inducible transgene construct is minimally active at 31°C, but is abundantly expressed in the presence of cadmium or at 34°C.4.4. Differing degrees of types of stress may differentially induce available hsp70 promoters.
Agricultural run off that is contaminated with pesticides enters water bodies, thereby polluting the aquatic environment. The sensitivity of the freshwater prawn, Macrobrachium malcolmsonii, to such pesticides is well-documented. However, the stress response to sublethal concentrations of pesticides has scarcely been investigated. In the present study, the effect of two different sublethal concentrations (1/5th and 1/10th of LC50 value) of endosulfan and carbaryl on the synthesis of hsp70 in various tissues of the prawn M. malcolmsonii was evaluated by Western blotting analysis. Among the tissues analyzed (gill, skeletal muscle, and hepatopancreas), induction of hsp70 synthesis was recorded only in the gill tissue of the prawns that had been exposed to either the high or low sublethal concentration of endosulfan. Induction of synthesis of hsp70 was not observed in any of the tissues sampled from the prawns that had been exposed to either the low or the high sublethal concentration of carbaryl. Pesticides interfere with the hydrophobic core of protein, thus leading to denaturation of the protein by exposing the lipophilic moieties to the aqueous environment. Specific induction of hsp70 in the gill possibly occurred since the gill is the route of exposure for pesticides in the prawns. The absence of induction of hsp70 synthesis in prawns that had been exposed to carbaryl suggests that induction of hsp70 synthesis is stressor-specific.
To investigate the dose–response relationship of the expression of heat shock proteins hsp 60 and hsp 70 following exposure to environmentally-relevant contaminants, zebra mussels (Dreissena polymorpha) were exposed to sublethal concentrations of copper and tributyltin (TBT). Mussels were exposed to Cu (0–500 μg/l) or TBT (0–75 μg/l) for 24 h. Hsp 60 and hsp 70 expression relative to the controls was analyzed by western blotting and densitometry. Contaminant concentrations in the exposure medium and mussel tissues were measured by atomic absorption spectroscopy (AAS) or high resolution gas chromatography with flame photometric detection (HRGC-FPD) for Cu and TBT, respectively. Following copper exposure, hsp 60 showed a biphasic expression pattern, with a maximal expression of three times control levels at 22 μg Cu/g dry wt., while hsp 70 concentrations reached a plateau of approximately 2.5 times control levels after crossing an induction threshold at tissue concentrations of less than 29 μg Cu/g dry wt. In contrast, concentrations of both hsp 60 and hsp 70 were increased to approximately 2.5–3 times control levels in TBT-exposed mussels at all tested doses. The results of this study demonstrate that the nature of the dose–response curves depend on both the form of stress protein investigated (hsp 60 or hsp 70) and on the contaminant. The implications for the use of hsp 60 and hsp 70 as biomarkers in ecotoxicological research are discussed.
AChE inhibition is widely regarded as a good biomarker of exposure to organophosphorus insecticides (OPIs), suggesting increase in the cholinergic transmission and consequent accumulation of acetylcholine in the organism. This might lead to behavioral changes and create widespread disturbances in the normal physiology of an organism. In the present study we have employed the model organism Caenorhabditis elegans to evaluate the biochemical and behavioral alterations induced by dichlorvos, a well known OPI. Exposure of worms to dichlorvos (at sublethal concentrations: 5, 50, 100, 150 and 200 μM) induced a concentration and time dependent AChE inhibition, and accumulation of acetylcholine. Further, we also observed cessation in feeding (by 72%), shutting of pharyngeal pumping, inhibition of egg laying (34–55%), contraction of nose (45%) and significant paralysis (50%) after 4 h of exposure. Significant correlation was observed between biochemical effects and behavioral parameters clearly suggesting the implications of sublethal concentrations of dichlorvos on non-target invertebrate organism such as C. elegans. These data further suggest that assessment of subtle neurophysiological parameters may serve as useful indicators of OPI exposure.
A transgenic strain of the nematode Caenorhabditis elegans which carries a stress-inducible lacZ reporter gene has been evaluated in terms of its response to several environmental toxicants. Optimal sensitivity is obtained by exposing these worms to toxicants at a temperature just below that required for heat induction of the transgene. Under these circumstances, several heavy metals (Cd2+, Zn2+, Hg2+, Mn2+, Sn2+, Ag+) cause dose-dependent transgene expression, which can be monitored as β-galactosidase enzyme activity or by in situ histochemical staining. A simple assessment procedure has been developed so that staining patterns can be compared between runs. The induced enzyme activity is localised in a single band (of apparent size 170 kD) on Western blots, as shown both by histochemical staining and immunoprobing. Endogenous heat-shock proteins (hsp70) are optimally induced under the same assay conditions, but modest induction is also apparent under control conditions (sub-heat-shock temperatures alone). Our system requires relatively high concentrations (ppm) of metallic ions for clear-cut induction, but is apparently more sensitive to certain organic and organo-metallic compounds (lindane and tributyltin are effective at ppb levels). This system works well within strictly defined assay conditions, but some toxicants are more effective inducers than others (e.g. Cd2+ versus Mn2+), while some give paradoxical dose-response curves possibly due to enzyme poisoning at high toxicant concentrations (e.g. Ag+). However, similar problems are likely to be encountered with any transgenic assay system based on the heat-shock response when used to monitor environmental pollution.
Representing toxicant‐induced changes in biological systems, biomarkers can serve as linkers between contamination (cause) and biological effects, and therefore provide unique information on ecosystem health. Hence, they are increasingly used for assessing the exposure of organisms to environmental contamination. Here, application of the induction of cytochrome P4501A (CYP1A), certainly the best studied biomarker, in field trials with freshwater and marine fish is reviewed. Moreover, the use of a number of other biomarkers, alone or in concert with CYP1A, is evaluated. Determination of CYP1A in field studies with fish has to consider several influencing factors, such as species, age, reproductive stage, temperature, and possible inhibitors. The overview shows that CYP1A induction is significantly related to contaminant levels in the environment in 93% of the investigated field studies. It is apparent that only certain classes of xenobiotica act as inducers of CYP1A (e.g., PAHs, coplanar PCBs, polychlorinated dibenzofurans, and dibenzodioxins). Induction of CYP1A was found to be more sensitive than other biomarkers at the biochemical, cellular, organ, and population level. Thus, induction of CYP1A can serve as a powerful tool for assessing environmental contamination and health, and its measurement, preferably together with other biomarkers and chemical analysis, is strongly recommended in monitoring programs.
Caenorhabditis elegans strains carrying stress-reporter green fluorescent protein transgenes were used to explore patterns of response to metals. Multiple stress pathways were induced at high doses by most metals tested, including members of the heat shock, oxidative stress, metallothionein (mtl) and xenobiotic response gene families. A mathematical model (to be published separately) of the gene regulatory circuit controlling mtl production predicted that chemically similar divalent metals (classic inducers) should show additive effects on mtl gene induction, whereas chemically dissimilar metals should show interference. These predictions were verified experimentally; thus cadmium and mercury showed additive effects, whereas ferric iron (a weak inducer) significantly reduced the effect of mercury. We applied a similar battery of tests to diluted samples of soil pore water extracted centrifugally after mixing 20% w/w ultrapure water with air-dried soil from an abandoned lead/zinc mine in the Murcia region of Spain. In addition, metal contents of both soil and soil pore water were determined by ICP-MS, and simplified mixtures of soluble metal salts were tested at equivalent final concentrations. The effects of extracted soil pore water (after tenfold dilution) were closely mimicked by mixtures of its principal component ions, and even by the single most prevalent contaminant (zinc) alone, though other metals modulated its effects both positively and negatively. In general, mixtures containing similar (divalent) metal ions exhibited mainly additive effects, whereas admixture of dissimilar (e.g. trivalent) ions often resulted in interference, reducing overall levels of stress-gene induction. These findings were also consistent with model predictions.
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In this study, specific developmental stage for adults from day 1 to day 10 was selected to evaluate the chronic metal toxicity, because the population of dead nematodes and the accumulation of intestinal autofluorescence increased sharply after day 10. Chronic exposure to Cr, Pb, Cu, and Hg caused a significant elevation in fractions of dead animals after day 4, and resulted in a significant induction of hsp-16.2::gfp expression at all assayed metal concentrations. Moreover, chronic exposure to Ag, Cr, Pb, Cu, Hg, and Cd would induce a more severe stress response than exposure to Zn and Mn in intestine, and chronic exposure to Pb, Hg, Cr, Zn, and Mn would induce a more severe stress response than exposure to Ag, Cu and Cd in head neurons. Therefore, in determining the usefulness of animals in metal toxicity assessment, this study established a method using nematodes in testing the chronic metal toxicity.
Indiscriminate use of agrochemicals worldwide, particularly, persistent organic pollutants (POPs), is of concern. Endosulfan, a POP, is used by various developing/developed nations and is known to adversely affect the development and the hormonal profiles of humans and animals. However, little is known about the molecular players/pathways underlying the adverse effects of endosulfan. We therefore analyzed the global gene expression changes and subsequent adverse effects of endosulfan using Drosophila. We used Drosophila melanogaster keeping in view of its well annotated genome and the wealth of genetic/molecular reagents available for this model organism. We exposed third instar larvae of D. melanogaster to endosulfan (2.0 μg mL(-1)) for 24 h and using microarray, we identified differential expression of 256 genes in exposed organisms compared to controls. These genes are associated with cellular processes such as development, stress and immune response and metabolism. Microarray results were validated through quantitative PCR and biochemical assay on a subset of genes/proteins. Taking cues from microarray data, we analyzed the effect of endosulfan on development, emergence and survival of the organism. In exposed organisms, we observed deformities in hind-legs, reminiscent of those observed in higher organisms exposed to endosulfan. In addition, we observed delayed and/or reduced emergence in exposed organisms when compared to their respective controls. Together, our studies not only highlight the adverse effects of endosulfan on the organism but also provide an insight into the possible genetic perturbations underlying these effects, which might have potential implications to higher organisms.
Heat shock proteins (Hsp) are a family of stress proteins, which are elicited in response to a variety of stressors in organisms. The objective of this study was to explore the potential of cypermethrin, a synthetic pyrethroid insecticide, on induction of small heat shock proteins (Hsp16) and feeding response in transgenic Caenorhabditis elegans PC72 (Hsp16-LacZ). A concentration-dependent inhibition in feeding was evident in worms exposed to cypermethrin (31%, 46% and 56% at 5, 10, 15 mM, respectively) beyond 4h while marked induction of heat shock protein-16 expression was evident after 12h exposure (as evident from quantitative analysis). Maximum expression of Hsp16 was observed throughout the body of the worms 24h after exposure similar to that evident in the worms exposed to heat shock at 30 degrees C. These data suggest that cypermethrin possesses the potential to induce Hsp16 as well as inhibit feeding in C. elegans at non-lethal concentrations. C. elegans (PC72) thus could serve as a convenient model to study the early toxic effects of xenobiotics.
The response to stress triggers activation of the genes involved in cell survival and/or cell death. Stress response is a ubiquitous feature of cells that is induced under stress conditions. As a part of this response a set of genes called stress genes are induced to synthesize a group of proteins called heat shock proteins (Hsps). The Hsps play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, and by preventing their aggregation. Because of their sensitivity to even minor assaults, Hsps are suitable as an early warning bio-indicator of cellular hazard. Despite having enormous use in toxicology, the current state of knowledge in defining a mechanism of action or accurately predicting toxicity based on stress gene expression warrants further investigation. The goal of this review is to summarize current developments in the application of stress genes and their products 'Hsps' in toxicology with a brief discussion of the caveats. While focusing on hsp70 because of its higher conservation across the taxa and since it is one of the first to be induced under stress conditions, we will also discuss other members of the stress gene family.
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