Characteristics of the adaptive response in cultured salmon cells exposed to ionizing radiation.

The aim of this study was to investigate the influence of "priming" doses of ionizing irradiation on salmon cell survival in vitro prior to being challenged with subsequent higher doses. A radiation-induced adaptive response (AR) was examined in the Chinook salmon embryo cell line (CHSE-214). Cells were initially irradiated with a range of priming (conditioning) doses of (60)Co gamma (gamma) rays (0.25-0.75 Gy), followed by a challenge dose of 7.50 Gy at intervals of 24, 48, and 72 hr. The AR was assessed using a colony-forming assay. Cell survival was determined by counting the number of colonies (viable clones) after 40 days of culture. This study revealed that cells that received a priming dose of 0.50 Gy before delivering the higher challenge dose became more radiation resistant with an increase in cell survival of 29% over cells receiving the challenge dose alone. The cells showed maximum resistance to ionizing radiation when the priming dose was given 72 hr prior to the higher challenge dose. This study is one of the first to demonstrate an AR using an in vitro piscine system, and is generally consistent with other studies of both in vitro and in vivo systems across the taxa.

Comparison of direct and bystander effects induced by ionizing radiation in eight fish cell lines.

PURPOSE: To determine bystander and direct effects of ionizing radiation on eight fish cell lines. MATERIALS AND METHODS: Fish cell lines were irradiated at a range of doses from 0.5 - 5 Gy. The Irradiated Cell Conditioned Medium (ICCM) was then harvested and placed onto a HPV-G, reporter cell line as well as onto autologous fish cell lines. Cloning efficiency (CE) was the end point used. The HPV-G reporter cell line was chosen because this cell line is capable of transmitting and producing the bystander effect. RESULTS: Four of the eight fish cell lines were clonogenic. These, with the exception of RTG-2 cells, showed increased CE when ICCM was tested on unirradiated autologous cells or on HPV-G cells. ICCM from RTG-2 cells reduced survival. The non-clonogenic cells ICCM tested on HPV-G all showed increased CE. CONCLUSIONS: The results show that both bystander signal production and cellular response varies depending on the cell line and that in general signals from established fish cells do not produce death inducing bystander effects. Thus, the comparison of the effect from fish cell ICCM on autologous cells or HPV-G human cells allowed us to separate signal production from response. In almost all cases, for both non-clonogenic and clonogenic fish cell lines, the HPV-G recipient cell line showed an increase in percent survival compared to controls while the clonogenic fish cell lines do not appear to respond.

Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L.

Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.

A two-species biomarker model for the assessment of sediment toxicity in the marine and estuarine environment using the comet assay.

Sediments frequently cause damage to biota due to the accumulation of toxic compounds and the bioavailability of sediment-associated contaminants. Damage can be assessed using biomarkers, such as the degree of genotoxic impact following in vivo exposure to contaminants. Genotoxic damage, expressed as single-strand DNA breaks, was measured in cells isolated from haemolymph/blood, gill and digestive gland/liver from the clam Tapes semidecussatus and turbot Scophthalmus maximus, using the single cell gel electrophoresis (Comet Assay). Both animals were exposed for three weeks to sediment samples collected from a polluted site and a 'clean' reference site. The level of DNA damage was assessed using an image analysis package and expressed as % tail DNA. Throughout the study, significant differences in DNA damage were recorded for each tissue type, in both species, between animals exposed to the two sediment samples. However, turbot appeared to be a more sensitive indicator species, because, due to lower background levels, they were able to detect a significant difference between reference site and background values. This suggests that turbot, rather than clams, are more suitable as a sentinel species for the assessment of genotoxic impact of low-level contamination in aquatic sediments and highlights the need for a two- or multi-species approach.

Cellular responses in primary epidermal cultures from rainbow trout exposed to zinc chloride.

In this study, we examined the effects of zinc chloride (ZnCl(2)) (0-200mg/L) on primary epidermal cultures from Oncorhynchus mykiss. Increases in the rate and amount of mucus released were detected post-exposure, as was a dose-dependent increase in the synthesis of acidic glycoproteins. The cytotoxicity of ZnCl(2) to the cultures was significantly increased (P< or =0.05) when exposures were conducted in serum-free medium as opposed to medium containing serum. Significant increases in the levels of apoptosis and necrosis were observed with increasing exposure concentration, but these were seen to decrease over time. The in vitro cytological and pathological changes observed in this study were found to be in accordance with previously published in vivo studies on the effects of heavy metals on the integument. This model system may help to further elucidate the effects of ecotoxicants on the external innate immune system of fish.

Identification of a multixenobiotic resistance mechanism in primary cultured epidermal cells from Oncorhynchus mykiss and the effects of environmental complex mixtures on its activity.

Multixenobiotic resistance (MXR) is a mechanism analogous to the mammalian multidrug resistance (MDR) phenotype, whereby, simultaneous resistance is conferred against the intracellular accumulation of structurally and functionally diverse, natural, endogenous and environmental toxicants. Expression of P-glycoproteins (P-gp), ATP-dependent transporters encoded for by the mdr1 gene that have been implicated in this xenobiotic efflux mechanism, have previously been detected in normal teleost tissues involved in a secretory, absorption or a barrier function. The presence of these proteins in the epidermis of fish species has not to our knowledge previously been investigated. In the present study, primary cultures of epidermis from the rainbow trout Oncorhynchus mykiss were employed to investigate whether an MXR mechanism is functional in the epidermis of fish. The efflux of the fluorescent mdr1 substrate rhodamine 123 from the cells was significantly inhibited by verapamil, a compound known to interfere with P-gp mediated transport. The cultured epidermal cells were also observed to accumulate this fluorescent dye in a verapamil sensitive manner, thus indicating the presence of an mdr1-like mechanism. Immunocytochemical analysis, using a monoclonal antibody (JSB1) directed against a conserved cytoplasmic P-gp epitope, also demonstrated the presence of P-gp-like proteins. Sediment elutriate extracts were employed as models of environmental complex mixtures to evaluate the potential of the epidermal cultures to discriminate between samples of varying contaminant burden using MXR activity as an endpoint. The induction of P-gp expression was found to be in accordance with the level of contamination detected in the sediments from which the elutriates were extracted. The findings of the functional study also demonstrated that environmental pollutants, which interfere with P-gp function, could be identified using this model.

Expression of delayed cell death (DCD) in the progeny of fish cells surviving 2,4-dichloroaniline (2,4-DCA) exposure.

Interest in and concern for the quality of the environment has prompted a great deal of research into methods of measuring and assessing changes in it. One problem of major interest is that of increasing amounts of mutagenic/carcinogenic chemicals generated and released into marine and freshwater ecosystems. Numerous techniques involving whole animals and cell culture for these genotoxic changes have been devised to assay specific chemicals. Little has been done to determine the effects of potential genotoxicants on aquatic organisms. The purpose of this study was to investigate if 2,4-Dichloroaniline (2,4-DCA) (CASRN: 554-00-7), induced delayed cell death (DCD) or delayed reproductive cell death a.k.a. as lethal mutations in a teleost cell line, CHSE-214. Delayed expression of cell death in the progeny of cells, which survived a toxic insult, was first shown for ionizing radiation and is one of the signs of induced genomic instability. The survival of cells initially treated with 2,4-DCA and the survival of their progeny were determined. When cells are exposed to a toxic insult, the component cells of a normal appearing survivor colony or clone were commonly thought to have proliferative capacity equivalent to that of the untreated cells. In this study, however, it was found that CHSE-214 cells surviving 2,4-DCA exposure carried heritable lethal defects, which came to light only after numerous apparently successful divisions, in the form of plating efficiencies, which were reduced below those of the untreated, control cells. DCD expression did not appear to be dose-dependent with poor cell survival occurring at the lower end of 2,4-DCA exposure and remained constant until recovering to something like 60% of the controls. A study of the CHSE-214 kinetics post-exposure showed that the apparent reduced growth rate of the cells was due to reduced numbers of reproductively viable cells in the population. Results showed that the expression of DCD occurred persistently post-treatment and was relatively independent of dose once a threshold level of 40 microM (Fig. 1) had been reached.

Rainbow trout primary epidermal cell proliferation as an indicator of aquatic toxicity: an in vitro/in vivo exposure comparison.

Little or no work has been carried out on primary cell cultures in terms of cellular proliferation and toxicity studies. Cell proliferation represents one of the most relevant cellular functions. Anti-PCNA antibodies have aroused considerable interest recently as potential immunocytochemical markers of proliferation for use in toxicity studies. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to rainbow trout (Oncorhynchus mykiss (R.)) primary cultured epidermal cells exposed in vivo i.e. whole animal exposures and in vitro for the study of the ecotoxicological potential of the aromatic amine, 2,4-dichloroaniline (2,4-DCA), a member of a little studied and widespread class of aquatic pollutants. There are many approaches to assess the proliferative activity of cells. Immunocytochemical methods offer a high sensitivity and specificity. The immunohistochemical avidin-biotin complex (ABC) method was used for the detection and quantification of PCNA, one of the best-known endogenous proliferation markers, applying the mammalian monoclonal antibody PC-10 to formalin-fixed primary cultures of rainbow trout skin. Here we describe our experience with the immunocytochemical detection and quantification of this proliferation marker. Results indicate that the antibody cross reacts with the corresponding rainbow trout epitope and that the alterations in PCNA labelling in the in vivo and in vitro exposed cultures followed similar patterns. This paper presents data on the validation of rainbow trout primary epidermal culture as an in vitro ecotoxicity model with epidermal proliferation as an endpoint. It can be concluded that cellular proliferation could be used as an indicator of the aquatic toxicity potential of xenobiotics. Correlations between cellular proliferation responses in primary cultures derived from in vivo exposed rainbow trout and primary cultures exposed in vitro were assessed. A dose-response was evidenced in both approaches, however the in vivo exposures appeared to be approximately two orders of magnitude more sensitive than the in vitro exposures. Responses in vitro occurred between 200 and 1000 micro M while in vivo responses were between 2 and 10 micro M. The good qualitative correspondence between the in vitro and in vivo results indicates that studies using trout epidermal cells allow the identification of xenobiotic effects in fish skin. However, further work is required before quantitative predictions i.e. effective concentrations in vivo, can be made from in vitro studies. This study suggests that the in vitro exposed rainbow trout primary cultured cell model with proliferation as an endpoint can be used as an alternative testing procedure to the whole animal assay.