Fish Cell Line RTG-2 Research Articles

Toxic Effects Produced by Microcystins from a Natural Cyanobacterial Bloom and a Microcystis aeruginosa Isolated Strain on the Fish Cell Lines RTG-2 and PLHC-1

Toxic cyanobacterial blooms are a worldwide problem, causing serious water pollution and public health hazard to humans and livestock. The intact cells as well as the toxins released after cellular lysis can be responsible for toxic effects in both animals and humans and are actually associated with fish kills. Two fish cell lines-PLHC-1 derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss were exposed to several concentrations of extracts from a natural cyanobacterial bloom and a Microcystis aeruginosa-isolated strain. After 24 hours, morphologic and biochemical changes (total protein content, lactate dehydrogenase leakage, neutral red uptake, methathiazole tetrazolium salt metabolization, lysosomal function, and succinate dehydrogenase [SDH] activity) were investigated. The most sensitive end point for both cyanobacterial extracts in PLHC-1 cells was SDH activity, with similar EC(50) values (6 microM for the cyanobacterial bloom and 7 microM for the isolated strain). RTG-2 cells were less susceptible according to SDH activity, with their most sensitive end point lysosomal function with an EC(50) of 4 microM for the M. aeruginosa-isolated strain and 72 microM for the cyanobacterial bloom. The lysosomal function was stimulated at low concentrations, although SDH activity increased at high doses, indicating lysosomal and energetic alterations. Increased secretion vesicles, rounding effects, decreased cell numbers and size, hydropic degeneration, esteatosis, and apoptosis were observed in the morphologic study. Similar sensitivity to the M. aeruginosa-isolated strain was observed in both cell lines, whereas the cyanobacterial bloom was more toxic to the PLHC-1 cell line.

The use of the fish cell lines RTG-2 and PLHC-1 to compare the toxic effects produced by microcystins LR and RR

Cyanobacterial toxins, especially microcystins (MC), are found in eutrophied waters through the world. Acute poisonings of animals and humans has been reported following MC exposure. In the present study, two fish cell lines, PLHC-1 and RTG-2, were evaluated after exposure to the cyanobacterial toxins MC-LR and MC-RR. The effects of different concentrations of the toxins were investigated in both cell lines at morphological and biochemical levels (total protein content, lactate dehydrogenase leakage, lysosomal activity and succinate dehydrogenase activity). The results obtained showed a decrease in protein content and no relevant increase in cell disruption, except for MC-LR in PLHC-1 cells. Morphological changes produced by microcystins were cellular swelling, blebbling, rounding, reduction in the cell number and increase in the number and size of lysosomal bodies. In addition, steatosis was produced in hepatoma PLHC-1 cells, particularly with MC-RR. Furthermore, the fish PLHC-1 cell line was more sensitive than RTG-2 cells to the cyanobacterial toxins compared, being the stimulation of the lysosomal function and the induction of steatosis the most specific changes detected.

In vitro toxicity of selected pesticides on RTG-2 and RTL-W1 fish cell lines

The rainbow trout fish cell lines RTG-2 and RTL-W1 were used to determine the cytotoxic effects of the pesticides bifenthrin, cypermethrin, cyhalothrin, λ-cyhalothrin, quinalphos and chlorpyrifos. Cytotoxicity was measured by EROD and β-Gal enzymatic activities, the neutral red (NR) uptake assay, and the FRAME KB protein (KBP) assay. The β-Gal activity was unaffected by the pesticide exposure. The EROD activity was induced by cyhalothrin and λ-cyhalothrin (RTG-2 and RTL-W1) and by bifenthrin (RTL-W1). Dose dependent inhibition responses were observed for EROD activity in cells exposed to quinalphos (RTL-W1) and chlorpyrifos (RTG-2 and RTL-W1). RTL-W1 offered a better response for EROD induction. The EC50 values on EROD endpoint were more sensitive than NR and KBP. The acute fish toxicity of chlorpyrifos and quinalphos depends highly on the species; the species sensitivity distributions cover several orders of magnitude and the values obtained for EROS were within the lowest part of the reported ranges.

In vitro assessment of DNA damage after short- and long-term exposure to benzo(a)pyrene using RAPD and the RTG-2 fish cell line

Genotoxins present in the aquatic environment are often associated with the decline or disappearance of many wild populations. The hazard assessment of chemicals requires sensitive and specific tests to study the genotoxic effects in order to establish the maximum allowable chemical concentrations prior to the release to the environment. We have previously shown that an established fish cell line (RTG-2) together with the random amplified polymorphic DNA (RAPD) technique, can be used to detect alterations in the DNA caused by direct acting genotoxins. The current study takes this a step further and examines in the same system the effect of a pro-mutagen benzo(a)pyrene (B(a)P) at different concentrations (0.05, 0.1, and 0.5 μg/ml) and at different exposure periods (1, 2, 3, 15, and 30 days). After comparing DNA fingerprints from control and exposed cells, both qualitative and quantitative analysis show an increase in the instability in the DNA fingerprint of exposed cells over a time- and concentration-dependent manner. At the higher concentration (0.5 μg/ml) three out the four primers showed altered bands after 1 day of exposure, while after 3 days all used primers showed an altered pattern. At the lower concentration of B(a)P (0.05 μg/ml) the appearance of new bands was observed with a 100% level of reproducibility after 30 days of exposure suggesting an inheritance of the altered DNA. We conclude that this in vitro system is useful to evaluate genotoxic effects, both after acute and chronic exposures and of direct and non-direct acting genotoxins. Cultured cells can be considered as genetically homogenous populations. Therefore, in vitro systems permits us to undertake mechanistic studies avoiding the interference of polymorphisms inherent in the in vivo systems. Furthermore, the RTG-2 fish cell line combined with a RAPD assay could be used in studies of hazard identification in risk assessment protocols of chemicals.

Ecotoxicological evaluation of carbamazepine using six different model systems with eighteen endpoints

The occurrence of pharmaceutically active compounds in the aquatic environment has been recognized as one of the emerging issues in environmental chemistry. However, the ecotoxicological effects of pharmaceuticals have still not been researched adequately. Carbamazepine, an anticonvulsant commonly present in surface and groundwater, was studied, using six ecotoxicological model systems with eighteen endpoints evaluated at different exposure time periods. The battery included the immobilization of Daphnia magna, bioluminescence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction and root growth inhibition in the plant Allium cepa. Cell morphology, neutral red uptake, total protein content, MTS metabolization, lactate dehydrogenase leakage and activity and glucose-6-phosphate dehydrogenase activity were studied in the salmonid fish cell line RTG-2. The total protein content, LDH activity, neutral red uptake and MTT metabolization in Vero monkey kidney cells were also investigated. The most sensitive system to carbamazepine was the Vero cell line, followed by Chlorella vulgaris, Vibrio fischeri, Daphnia magna, Allium cepa, and RTG-2 cells. EC 50 values from 19 μ m in Vero cells at 72 h to more than 1200 μ m in other systems, were obtained. Comparing the concentrations in water and the toxicity quantified in our assay systems, carbamazepine is not expected to produce acute toxic effects in the aquatic biota under these circumstances, but chronic and synergistic effects with other chemicals cannot be excluded.

A test battery for the ecotoxicological evaluation of pentachlorophenol

Experimental bioassays are currently used in ecotoxicology and environmental toxicology to provide information for risk assessment evaluation of new chemicals and to investigate their effects and mechanisms of action; in addition, ecotoxicological models are used for the detection, control and monitoring of the presence of pollutants in the environment. As a single bioassay will never provide a full picture of the quality of the environment, a representative, cost-effective and quantitative test battery should be developed. The effects of pentachlorophenol were studied using a battery of ecotoxicological model systems, including immobilization of Daphnia magna, bioluminiscence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction in the plant Allium cepa. The inhibition of cell proliferation and MTT reduction were investigated in Vero cells. Neutral red uptake, cell growth, MTT reduction, lactate dehydrogenase leakage and activity were studied in the salmonid fish cell line RTG-2, derived from the gonad of rainbow trout. Pentachlorophenol was very toxic for all biota and cells. The system most sensitive to pentachlorophenol, was micronuclei induction in A. cepa, followed by D. magna immobilization, bioluminescence inhibition in V. fischeri bacteria at 60 min and cell proliferation inhibition of RTG-2 cells at 72 h. Inhibition of cell proliferation and MTT reduction on Vero monkey cells showed intermediate sensitivity.

Toxicological characterisation of sludge from sewage treatment plants using toxicity identification evaluation protocols based on in vitro toxicity tests

The ecotoxicological characterisation of complex mixtures, such as sludge from sewage treatment plants, is complex. Toxicity identification evaluation (TIE) protocols, developed by the United States Environmental Protection Agency (US EPA); to identify toxic pollutants in complex effluents, are useful tools in this context; to solve the difficulties in assessing unknown organic pollutants by analytical methods, the usefulness of bioassays to detect the relevant (toxic) organic compounds present in complex samples, and the possibilities of in vitro cytotoxicity tests as screening tools, offers a profitable combination. The sludge obtained from a sewage treatment plant was extracted by acetonitrile using a microwave extractor and fractionated in an HPLC system. The toxicity of every fraction was assayed using a RTG-2 cytotoxicty test, based on the fibroblastic RTG-2 fish cell line (ATCC, CCL N. 55). At exponential growth, three endpoints, β-galactosidase activity, culture viability assayed by the neutral red assay (NR) and inhibition of growth rate using the FRAME KB protein assay (KBP), were used. By plotting the toxicity of each fraction vs elution time, the corresponding “toxicograms” were built. The UV and fluorescence chromatograms are compared to the three toxicograms (one for each toxicity endpoint).

Flow cytometric measurement of micronuclei induced in a permanent fish cell line as a possible screening test for the genotoxicity of industrial waste waters.

An in vitro micronucleus assay using the permanent fish cell line RTG-2 (rainbow trout gonads) was developed to test industrial waste waters for their genotoxic potential. Comparison of flow cytometric measurement and microscopic scoring of micronucleus frequency with the reference chemicals 1,4-butane sultone (0.2-1 mM), ethylmethane sulphonate (2-10 mM), potassium dichromate (20-100 microM) and benzo[a]pyrene (5-25 microM) showed similar dose-effect relationships. Thirty-eight industrial waste waters from 11 different branches of industry obtained from the Bavarian state office for water research were tested using the flow cytometric method (18 from metal processing, 10 from combined waste water, two from synthetic fibre production, one sample each from settlement wastes, non-iron metal manufacturing, leather production, sulphuric acid production, ore processing, graphite film production, cellulose production and flue gas washing). Fourteen of them showed a significant increase in micronucleus frequency.

Light and Scanning Electron Microscopic Cytopathology of 3,5-Dichlorophenol in the Permanent Fish Cell Line RTG-2

The permanent fibrocyte-like fish cell line RTG-2 from rainbow trout (Oncorhynchus mykiss) gonads was investigated by means of light and scanning electron microscopy with respect to alterations as a consequence of sublethal exposure to 0, 1, 10, 16, 25, and 50 mg/liter 3,5-dichlorophenol (3,5-DCP) for 24 h. Control RTG-2 cells were spindle-like in shape and almost three times longer than wide. In subconfluent cultures, they displayed widely spread, flat leading lamellae and formed small groups. Along the edges of the cell protrusions, numerous retraction fibrils could be identified. Except for shallow ridges and small folds, the surface of untreated cells was completely smooth. Following exposure to 3,5-DCP, distinct dose-dependent alterations in cell shape, the appearance of surface blebs and invaginations, as well as progressive retraction of cell extensions could be observed from the lowest test concentration. Morphological changes could be correlated to suppression of lactate dehydrogenase and reduced neutral red retention capacity.

Characterization of RTG-2 Fish Cell Line by Random Amplified Polymorphic DNA

The increasing presence of genotoxic chemicals in the aquatic environment has led to the development of bothin vivoandin vitroassays for target species. The fish population represents an important level of aquatic ecosystems that can be threatened by increased environmental pollution. The authors have studied the DNA pattern of the RTG-2 fish cell line, a fibroblast-like cell line, derived from rainbow trout (Oncorhynchus mikyss), to use this cell line as anin vitrosystem to study genotoxicity by means of random amplified polymorphic DNA primers (RAPDs). A constant pattern in the DNA band is essential when an organism or cell line is used to detect DNA alterations produced by genotoxic environmental chemicals. DNA fingerprints with RAPDs were obtained for RTG-2 by testing 26 single and 70 pairwise combinations of primers. Different methods of DNA extraction (chelating resin, salting out, and phenolization), the influence of spectrometric measures at 320 nm in the 260/280 quotient to quantify DNA extracts, genomic DNA and primer concentrations, annealing temperatures, and cell line passage were studied in the cell line characterization. RAPD products were identified by agarose gel electrophoresis. The good results obtained should allow the use of this system as a possible tool for detection of the genotoxicity of aquatic pollutants.

Application of NR-assay on RTG-2 fish cell line for consideration of toxicity of chemical substances and preparations

Application of NR-assay on RTG-2 fish cell line for consideration of toxicity of chemical substances and preparations

Application of NR-assay on RTG-2 fish cell line for consideration of toxicity of chemical substances and preparations

Anticoagulant rodenticides are highly toxic compounds that are widely used for pest control of rodents, but that also may threaten the wildlife's health. This work aimed to assess the exposure to first- and second-generation anticoagulant rodenticides (ARs) in six birds of prey species from the Canary Islands (Spain). The concentrations of seven widely used ARs were determined by LC–MS/MS in 104 liver samples of six species of birds of prey (Buteo buteo, Accipiter nisus, Falco pelegrinoides, Falco tinnunculus, Asio otus, and Tyto alba). We determined that 61% of the livers had detectable residues of at least one AR. The most frequently detected AR was bromadiolone, which was detected in 60.3% of the positive cases. The detection frequencies of these compounds varied widely, depending on the species. More than 75% of the A. nisus, T. alba, and A. otus individuals had detectable rodenticide residues in the liver. However, F. tinnunculus exhibited the highest concentrations of AR, with median values above 100 ng/g w.w. We did not detect first-generation ARs in any of the samples. When grouped, nocturnal species exhibited higher AR concentrations than diurnal species (P < 0.001). The residue levels were higher among small mammal-eaters than bird-eaters (P < 0.01). While most animals exhibited no macroscopic signs of coagulation disorders, approximately 35% exceeded the threshold levels of toxicity, which suggests that these compounds could weaken these animals in their natural environment. In conclusion, the control of rodent populations by ARs suggests that these compounds will enter the food chain and thus threaten the vulnerable populations of raptors on the Canary Islands. Our findings require authorities to ban or strictly control the use of these rodenticides in the natural environment for the conservation of raptors and other predatory species.

Long Primers for RAPD Mapping and Fingerprinting of Grape and Pear

BioTechniquesVol. 20, No. 3 BenchmarksOpen AccessLong Primers for RAPD Mapping and Fingerprinting of Grape and PearG.-N. Ye, M. Hemmat, M.A. Lodhi, N.F. Weeden & B.I. ReischG.-N. YeNew York State Agricultural Experiment Station, Cornell University, Geneva, NY, USASearch for more papers by this author, M. HemmatNew York State Agricultural Experiment Station, Cornell University, Geneva, NY, USASearch for more papers by this author, M.A. LodhiNew York State Agricultural Experiment Station, Cornell University, Geneva, NY, USASearch for more papers by this author, N.F. WeedenNew York State Agricultural Experiment Station, Cornell University, Geneva, NY, USASearch for more papers by this author & B.I. Reisch*Address correspondence to Bruce I. Reisch, Department of Horticultural Sciences, New York State Agricultural Experiment Station, Cornell University, Geneva, New York 14456, USA. Internet: E-mail Address: bruce_reisch@cornell.eduNew York State Agricultural Experiment Station, Cornell University, Geneva, NY, USASearch for more papers by this authorPublished Online:2 Aug 2018https://doi.org/10.2144/19962003368AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByCAAT box- derived polymorphism (CBDP): a novel promoter -targeted molecular marker for plants19 March 2013 | Journal of Plant Biochemistry and Biotechnology, Vol. 23, No. 2Start Codon Targeted (SCoT) Polymorphism: A Simple, Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants16 September 2008 | Plant Molecular Biology Reporter, Vol. 27, No. 1Study of genetic diversity in Chamomile (Matricaria chamomilla) based on morphological traits and molecular markersScientia Horticulturae, Vol. 117, No. 3Polymorphism of Some Native Sistan Grapes Assessed by Long and Short Primers for RAPD MarkersPakistan Journal of Biological Sciences, Vol. 10, No. 12FluoMEP: A new genotyping method combining the advantages of randomly amplified polymorphic DNA and amplified fragment length polymorphismELECTROPHORESIS, Vol. 28, No. 4A gene controlling sex in grapevines placed on a molecular marker-based genetic mapGenome, Vol. 43, No. 2Sports and seedlings of rose varieties analysed with molecular markersPlant Breeding, Vol. 119, No. 1Plant Breeding: Genetic Mapping in Woody CropsCharacterization of RTG-2 Fish Cell Line by Random Amplified Polymorphic DNAEcotoxicology and Environmental Safety, Vol. 40, No. 1-2Effective pairwise combination of long primers for RAPD analyses in roses28 April 2006 | Plant Breeding, Vol. 117, No. 2 Vol. 20, No. 3 Follow us on social media for the latest updates Metrics Downloaded 121 times History Published online 2 August 2018 Published in print March 1996 Information© 2018 Author(s)PDF download

Assessing organic toxic pollutants in fish-canning factory effluents using cultured fish cells

The fibroblastic RTG-2 fish cell line has been used to assess the cytotoxicity of organic compounds present in the effluents discharged by a canning factory. Organic compounds of the effluents were extracted and concentrated, and their cytotoxicity determined by measuring three end points: the cellular mass, the cell viability and the intracellular ATP content of the cells. Three out of the four organic concentrates were found to be highly cytotoxic, and these three toxic concentrates were fractionated, into 210 organic fractions, using an HPLC system. Up to six toxic fractions, present in all three organic concentrates, could be detected. Thus, when assessing the environmental effect of fish-canning factory effluents, the presence of specific toxic chemicals must be considered, in addition to toxicological problems due to fish-offal sewage.