Vertebrate Vitellogenin Gene Duplication in Relation to the “3R Hypothesis”: Correlation to the Pelagic Egg and the Oceanic Radiation of Teleosts

The spiny ray-finned teleost fishes (Acanthomorpha) are the most successful group of vertebrates in terms of species diversity. Their meteoric radiation and speciation in the oceans during the late Cretaceous and Eocene epoch is unprecedented in vertebrate history, occurring in one third of the time for similar diversity to appear in the birds and mammals. The success of marine teleosts is even more remarkable considering their long freshwater ancestry, since it implies solving major physiological challenges when freely broadcasting their eggs in the hyper-osmotic conditions of seawater. Most extant marine teleosts spawn highly hydrated pelagic eggs, due to differential proteolysis of vitellogenin (Vtg)-derived yolk proteins. The maturational degradation of Vtg involves depolymerization of mainly the lipovitellin heavy chain (LvH) of one form of Vtg to generate a large pool of free amino acids (FAA 150–200 mM). This organic osmolyte pool drives hydration of the ooctye while still protected within the maternal ovary. In the present contribution, we have used Bayesian analysis to examine the evolution of vertebrate Vtg genes in relation to the “3R hypothesis” of whole genome duplication (WGD) and the functional end points of LvH degradation during oocyte maturation. We find that teleost Vtgs have experienced a post-R3 lineage-specific gene duplication to form paralogous clusters that correlate to the pelagic and benthic character of the eggs. Neo-functionalization allowed one paralogue to be proteolyzed to FAA driving hydration of the maturing oocytes, which pre-adapts them to the marine environment and causes them to float. The timing of these events matches the appearance of the Acanthomorpha in the fossil record. We discuss the significance of these adaptations in relation to ancestral physiological features, and propose that the neo-functionalization of duplicated Vtg genes was a key event in the evolution and success of the teleosts in the oceanic environment

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. © 2004 Elsevier Ltd. All rights reserved

Effects of individual and a mixture of pharmaceuticals and personal-care products on cytotoxicity, EROD activity and ROS production in a rainbow trout gonadal cell line (RTG-2)

The presence of pharmaceuticals and personal-care products (PPCPs) in aquatic environments is of concern. Although measured concentrations of individual substances are low, little consideration has been given to the likely chronic nature of the exposures or to the potential for mixture effects. The purpose of the present study was to use the RTG-2 rainbow trout cell line to analyse sub-lethal and cytotoxic effects of PPCPs present in a wastewater-treatment-plant (WWTP) effluents and their mixtures. Interactions with cytochrome P450 1A enzyme, oxidative stress, cellular senescence and cell viability were assessed using 7-ethoxyresorufin-o-deethylase (EROD), reactive oxygen species (ROS), ß-galactosidase (ß-gal) and neutral red (NR) uptake assays, respectively. Not all of the compounds that were tested exhibited significant effects. The lowest-observed-effect concentrations and half maximal effective concentrations (EC50) were within the range 0.15 to 784.47μg l-1. Clear dose-response curves were found for cells exposed to different mixtures of PPCPs. The lowest-observed-effect concentrations and concentrations causing EC50 were within the range 0.05 to 54.61μg l-1. Four out the seven tested mixtures induced EROD activity. ROS production was detected in two mixtures. The ß-gal inhibition response was observed in six out the seven tested mixtures and occurred at a higher concentration than was observed for EROD induction activity or ROS generation. The present study clearly shows that the stress response through which cells mount a homeostatic response to toxicants can be potentially used for an initial, rapid and cost-effective assessment of the complex mixtures of PPCP that present in WWTP effluents are difficult and expensive to analyse chemically. © 2012 John Wiley & Sons, Ltd

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

This study proposed a battery of endpoints based on in vitro fish cell lines. Two fish cell lines and four toxicity endpoints are considered. The tetracycline (TC), oxytetracycline (OTC), sulfachlorpyridazine (SCP), and septrin® (ST) were selected as model antimicrobials and chlorpyrifos as positive control. EROD was induced by septrin® (formulation containing sulfamethoxazole and trimethoprim) at concentrations higher than 20 mg/L, but inhibited by tetracycline, oxytetracycline and chlorpyrifos. Dose dependent inhibition responses were observed for β-galactosidase (β-Gal) activity in cells exposed to septrin®, tetracycline or oxytetracycline. The EROD EC50s ranged between 2.29 × 10-2 mg/L (chlorpyrifos) and 167.63 mg/L (tetracycline). The β-Gal EC50s ranged between 22.1 (septrin®) and 84.59 mg/L (tetracycline). Data suggest that in vitro testing using a battery of endpoints can be a cost-effective solution for screening the toxicity of antimicrobials on fish. The absence of in vitro effects at concentrations well above those expected/measured in the environment may replace the need for conducting acute lethality tests on fish. © 2004 Elsevier B.V. All rights reserved

In vitro evaluation of cellular responses induced by ZnO nanoparticles, zinc ions and bulk ZnO in fish cells

Zinc oxide nanoparticles (ZnO-NPs) are inevitably released into the environment and are potentially dangerous for aquatic life. However, the potential mechanisms of cytotoxicity of zinc nanoparticles remain unclear. Studying the toxicity of ZnO-NPs with In vitro systems will help to determine their interactions with cellular biomolecules. The aim of this study was to evaluate the cytotoxic potentials of ZnO-NPs in established fish cell lines (RTG-2, RTH-149 and RTL-W1) and compare them with those of bulk ZnO and Zn2+ ions. Membrane function (CFDA-AM assay), mitochondrial function (MTT assay), cell growth (KBP assay), cellular stress (β-galactosidase assay), reductase enzyme activity (AB assay), reactive oxygen species (ROS), total glutathione cellular content (tGSH assay) and glutathione S-transferase (GST) activities were assessed for all cell lines. ZnO-NPs cytotoxicity was greater than those of bulk ZnO and Zn2+. ZnO-NPs induced oxidative stress is dependent on their dose. Low cost tests, such as CFDA-AM, ROS, GST activity and tGSH cell content test that use fish cell lines, may be used to detect oxidative stress and redox status changes. Particle dissolution of the ZnO-NPs did not appear to play an important role in the observed toxicity in this study. © 2013

Application of bioassays for the ecotoxicity assessment of contaminated soils

The use of bioassays for soil characterization is receiving significant attention as a complementary tool to chemical analysis. Bioassays consist of direct toxicity assays of environmental samples that are transferred to the laboratory and analyzed for toxicity against selected organisms. Such soil samples contain the combination of the different pollutants present in situ and enable factors such as the bioavailability of contaminants or the interactions (synergic and antagonic) between them to be simultaneously studied.In this chapter, methods for soil toxicity assessment based in the guidelines developed by OECD for single substances are described. These methods have been adapted for their application to the assessment of complex matrices such as soils. The field sample can be tested undiluted and/or diluted with "uncontaminated" soil to create a pollution gradient. In the diluted samples, concentration/response relationships may be obtained. Toxicity assays to soil organisms include earthworms, plants, and microorganisms tests. In addition, toxicity assays with soil extracts are recommended. Assays of extracts with algae, daphnia, and fish (in vitro test using fish cell lines) are also described

Short communication. An "in vitro" approach for ecotoxicity testing of toxic and hazardous wastes

In waste-characterization, chemicals with a low acute toxicity but with a high long-term hazard such as PBTs (persistent, bioaccumulable, and toxic chemicals) are difficult to identify, leading to an inaccurate assessment of their risk to the environment. This study assesses the sensitivity of in vitro assays with rainbow trout cell line RTG-2 as a tool for the ecotoxicological testing of hazardous wastes using a battery of bioassays, including a neutral red (NR) assay for predicting cytotoxicity, a Beta-galactosidase (Betagal) assay for predicting cellular defence, and two sub-lethal damage assays: ethoxy resorufin-O-deethylase (EROD) and glutathione S-transferase (GST) to check for induction of cytochrome and for alteration of antioxidant defences respectively. Six samples of complex waste, covering a wide range of liquid (A to C) and solid (1 to 3) matrices were tested. The EC50-values (NR) ranged from 0.09 mL/100 mL (B) to 2.01 mL/100 mL (2). A hormesis effect (Betagal) at low concentrations was observed for the A (0.03-0.18 mL/100 mL), 1 (0.03-0.29 mL/100 mL) and 2 (0.03-0.29 mL/100 mL) samples. A dose-dependent stimulation of enzymatic activity was observed for the B ((EROD (0.58-4.68 mL/100 mL), Betagal (0.58-4.68) and GST (0.14-1.17 mL/100 mL)) and C ((EROD (0.29-4.68 mL/100 mL) and Betagal (2.34-4.68)) samples. No effects were observed at the highest concentration tested (74 mL/100 mL) for sample 3. The in vitro approach can be considered to be an efficient, rapid and cost effective screening system to provide basic information on toxic and hazardous waste for further analysis and risk evaluation.La caracterización de residuos se complica cuando contienen PBTs (sustancias tóxicas persistentes y bioacumulables) y sustancias con baja toxicidad aguda y mucha peligrosidad crónica, impidiendo la evaluación del riesgo ambiental. Este trabajo utiliza la línea celular de trucha RTG-2 para estudiar la sensibilidad de los ensayos in vitro, como herramienta en la evaluación ecotoxicológica de residuos peligrosos. La batería de bioensayos incluye la medida de citotoxicidad mediante el test de rojo neutro (RN), de protección celular mediante la actividad Beta-galactosidasa (Betagal) y la medida de la alteración del citocromo y de las defensas antioxidantes mediante las actividades etoxi resorufin-O-dietilasa (EROD) y glutation S-transferasa (GST) respectivamente. Se ensayaron tres residuos líquidos (A a C) y tres sólidos (1 a 3) representando un amplio rango de muestras. Los valores de EC50 (RN) variaron entre 0,09 mL/100 mL (B) y 2,01 mL/100 mL (2). Se detectaron fenómenos de hormesis (Betagal) a bajas concentraciones en las muestras A (0,03-0,18 mL/100 mL), 1 (0,03-0,29 mL/100 mL) y 2 (0,03-0,29 mL/100 mL). Se detectó aumento de las actividades enzimáticas en las muestras B ((EROD (0,58-4,68 mL/100 mL), Betagal (0,58-4,68 mL/100 mL) y GST (0,14-1,17 mL/100 mL)) y C ((EROD (0,29-4,68 mL/100 mL) y Betagal (2,34-4,68 mL/100 mL)), mientras que con la muestra 3 no se observaron efectos a la concentración más alta (74 mL/100 mL). La propuesta in vitro puede ser un método eficaz, rápido y barato para proporcionar información básica que permita orientar análisis posteriores y evaluar el riesgo de los residuos tóxicos y peligrosos

Rapid serotyping of infectious pancreatic necrosis virus by one-step enzyme-linked immunosorbent assay using monoclonal antibodies

A one-step ELISA has been developed for detection and serotyping of infectious pancreatic necrosis virus (IPNV) in infected cell cultures using monoclonal antibodies (mAb) raised against strains representing the Sp, Ab and VR 299 serotypes of IPNV. This assay uses a serotype-specific mAb as capture and a mAb directed against a common epitope as detector antibody. Avidin-peroxidase was employed for amplification. The assay was performed in 90 min by simultaneous incubation of the samples, the biotin labelled mAb and the avidin-peroxidase, and detected 37 ng/ml of purified virus. Serotyping of 34 isolates from different areas of Europe, Asia and America showed a total concordance with the results obtained by the neutralization assay. © 1991

Detection of African horsesickness virus in infected spleens by a sandwich ELISA using two monoclonal antibodies specific for VP7

A sandwich enzyme-linked immunosorbent assay (ELISA) for rapid detection of African horsesickness virus (AHSV) in infected spleens or cell culture supernatant was developed. This method uses two monoclonal antibodies (MAbs) which recognize two non-overlapping epitopes of the major core protein (VP7) to coat the solid phase, and one labeled with biotin as second antibody. This ELISA was evaluated for its ability to detect AHSV in infected spleens resulting in a sensitivity of 97.4% and a specificity of 100% compared with virus isolation in cell culture, and can be used for the detection of the nine different AHSV serotypes. © 1992

Inhibition of IL-2R and SLA class II expression on stimulated lymphocytes by a suppressor activity found in homogenates of African swine fever virus infected cultures

Virus free supernatants (VFS) obtained by ultracentrifugation of homogenates of African swine fever (ASF) virus infected cultures inhibited the proliferative response and the expression in peripheral blood mononuclear cells of two activation molecules, the IL-2 receptor (IL-2R) and the swine MHC class II antigens (SLA II), induced by several stimuli (lectins, PMA plus the calcium ionophore A23187 or specific antigen). This inhibition was time dependent no effect was seen on IL-2R expression when VFS was added after 48 h, when the expression of this molecule reached its maximum. However at this time the proliferative response was still inhibited. The presence of VFS in the cultures was necessary to inhibit both the IL-2R expression and the proliferation of cells. In these conditions the addition of exogenous IL-2 to the cultures failed to restore the IL-2R expression and the proliferation shown by control stimulated cells. Furthermore, the IL-2 activity found in supernatants from cell cultures stimulated with Con A in the presence of VFS was even higher than in cultures stimulated without VFS. The inhibition observed suggests an important impairment of host immunocompetence in ASF infected swine. © 1995 Springer-Verlag