Cytotoxic and genotoxic responses of the RTgill-W1 fish cells in combination with the yeast oestrogen screen to determine the sediment quality of Lagos lagoon, Nigeria.

Economic advancements in developing countries have seen an increase in urbanisation and industrialisation with a rise in the levels of discharge of effluents and municipal waste into aquatic ecosystems. Unfortunately, aquatic environmental regulations in these countries are often rudimentary and the development of environmental monitoring programmes will help identify ecological risks. As an example, the current study assesses the pollution status of 11 sampling sites in Lagos lagoon, Nigeria. The organic solvent sediment extracts were assessed for cytotoxicity and genotoxicity in rainbow trout gill-W1 cells. The induction of oestrogenic activities using the yeast oestrogen screen was also determined. The sediments were analysed for polycyclic aromatic hydrocarbons (PAHs) and other contaminants (polychlorinated biphenyls, organochlorine and organophosphate pesticides). Only sediments from three sites were cytotoxic at both 25 and 12.5mg eQsed/ml using the Alamar Blue cell viability assay. The alkaline Comet assay showed that all sites caused significant DNA damage at 7 mg eQsed/ml; the extent of the damage was site specific. The measure of oxidative damage to DNA via the formamidopyrimidine DNA-glycosylase-modified Comet assay revealed similar results. Toxicity to yeast cells was observed in extracts from six sites; of the remaining sites, only two exhibited oestrogenic activity. There was no strong consistent relationship between sediment PAH concentrations and the cell toxicity endpoints. The dynamic nature of Lagos lagoon with its tides and freshwater inputs are suggested as factors that make it difficult to link the sources of pollution observed at each site with PAH levels and toxic endpoints. The study has demonstrated that the Comet assay is a sensitive endpoint to identify sediments that possess genotoxic contaminants, and this in vitro bioassay has the potential to be incorporated into an environmental monitoring framework for Lagos lagoon.

Environmental monitoring of urban streams using a primary fish gill cell culture system (FIGCS).

The primary fish gill cell culture system (FIGCS) is an in vitro technique which has the potential to replace animals in whole effluent toxicity tests. In the current study FIGCS were transported into the field and exposed to filtered (0.2µm) river water for 24h from 4 sites, on 2 different sampling dates. Sites 1 and 2 are situated in an urban catchment (River Wandle, London, UK) with site 1 downstream of a sewage treatment work; site 3 is located in a suburban park (River Cray, Kent, UK), and site 4 is more rural (River Darent, Kent, UK). The change in transepithelial electrical resistance (TER), the expression of the metal responsive genes metallothionein A (mta) and B (mtb), cytochrome P450 1A1 (cyp1a1) and 3A27 (cyp3a27), involved in phase 1 metabolism, were assessed following exposure to sample water for 24h. TER was comparable between FIGCS exposed to 0.2µm filtered river water and those exposed to synthetic moderately soft water for 24h. During the first sampling time, there was an increase in mta, cyp1a1 and cyp3a27 gene expression in epithelium exposed to water from sites 1 and 2, and during the second sampling period an increase in cyp3a27 gene expression at sites 1 and 4. Urban river water is a complex mixture of contaminants (e.g., metals, pesticides, pharmaceuticals and polyaromatic hydrocarbons) and the increase in the expression of genes encoding mta, cyp1a1 and cyp3a27 in FIGCS is indicative of the presence of biologically active pollutants.

Gill cell culture systems as models for aquatic environmental monitoring.

A vast number of chemicals require environmental safety assessments for market authorisation. To ensure acceptable water quality, effluents and natural waters are monitored for their potential harmful effects. Tests for market authorisation and environmental monitoring usually involve the use of large numbers of organisms and, for ethical, cost and logistic reasons, there is a drive to develop alternative methods that can predict toxicity to fish without the need to expose any animals. There is therefore a great interest in the potential to use cultured fish cells in chemical toxicity testing. This review summarises the advances made in the area and focuses in particular on a system of cultured fish gill cells grown into an epithelium that permits direct treatment with water samples.

Assessment of metabolic capabilities of PLHC-1 and RTL-W1 fish liver cell lines.

Metabolic capabilities of PLHC-1 and RTL-W1 cell lines were investigated since to date, cytochrome P450 (CYP) 1A and glutathione-S-transferase have been almost the unique biotransformation enzymes reported in these cells. Functionality of CYP3A-, CYP2M- and CYP2K-like enzymes was assessed by studying the hydroxylation of testosterone (T) and lauric acid (LA), and glucuronidation and sulfation capacity was assessed by looking at 1-naphthol (1-N) and T conjugation. Only PLHC-1 cells showed the ability to hydroxylate T at 6beta-position (a CYP3A-like catalysed pathway) and LA at (omega-1)-position (a CYP2K-like catalysed pathway). Hydroxysteroid dehydrogenase and steroid reductase enzymes showed comparatively higher activities than CYPs: 5alpha-dihydrotestosterone, androstenedione and 3beta-androstanediol were the major metabolites of T detected in both cell lines. Regarding phase II activities, both cell lines metabolised 1-N to glucuronide and sulfate conjugates. In contrast, when using T as substrate, RTL-W1 formed the glucuronide, whilst PLHC-1 formed the corresponding sulfate. Overall, the observed enzymatic activities are much lower (up to 17.5 x 10(3) times) than those reported in primary cultures of fish hepatocytes. The present study highlights the need of developing new fish cell lines that could be used as alternative in vitro tools for studying xenobiotic metabolism and toxicity in fish.

Effects of ibuprofen on the viability and proliferation of rainbow trout liver cell lines and potential problems and interactions in effects assessment.

Under some conditions ibuprofen was either cytotoxic or cytostatic to rainbow trout cell lines: RTL-W1 (liver) and RTH-149 (hepatoma). Ibuprofen at up to 15 microg/mL was not cytotoxic, regardless of dosing protocols, exposure conditions, viability endpoints, or cell lines. Responses to higher ibuprofen concentrations depended on the test methodology. No cytotoxicity was seen when stock ibuprofen solutions had been prepared in ethanol. For stock solutions in dimethylsulfoxide (DMSO), ibuprofen from 50 to 1500 microg/mL elicited little cytotoxicity in cultures in which the final DMSO concentration was 0.05% (v/v), but was consistently cytotoxic after 24 h for cultures with 0.5% DMSO (v/v). Cytotoxicity was evaluated with alamar Blue (AB) and carboxyfluoroscein diacetate acetoxymethyl ester (CFDA-AM) as measures respectively of metabolic activity and membrane integrity. Effective concentrations (EC50s) for ibuprofen with AB and CFDA-AM depended on whether the stock solution was dosed directly into a culture well or mixed in medium prior to being added to a well. For indirect dosing, ibuprofen was more cytotoxic in medium without fetal bovine serum (FBS), whereas for direct dosing ibuprofen was equally cytotoxic in medium with or without FBS. As judged by AB and CFDA-AM EC50s, dosing ibuprofen was directly 10 to 30 times more cytotoxic. In FBS-containing cultures, which was dosed with increasing ibuprofen and DMSO at 0.05% (v/v), cell proliferation was impaired at 50 and 150 microg/mL ibuprofen. Lipopolysaccharide (LPS) at 50 microg/mL had little influence on these cytotoxic and cytostatic effects of ibuprofen in medium with FBS.

Co-exposure to polystyrene plastic beads and polycyclic aromatic hydrocarbon contaminants in fish gill (RTgill-W1) and intestinal (RTgutGC) epithelial cells derived from rainbow trout (Oncorhynchus mykiss).

Microscopic plastic (MP) particles are a ubiquitous contaminant in aquatic environments, which may bind hydrophobic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), altering their environmental fate and interactions with biota. Using rainbow trout gill (RTgill-W1) and intestinal (RTgutGC) epithelial cells we investigated the effects of polystyrene microbeads (PS-MBs; 220 nm) on the cyto- and genotoxicity of the environmental pollutants benzo[a]pyrene (BaP) and 3-nitrobenzanthrone (3-NBA) over 48 h (0, 0.1, 1 and 10 µM). The Alamar Blue bioassay, used to assess cytotoxicity, showed that both pollutants significantly decreased cell viability by 10-20% at 10 µM in both cell lines after 48 h whereas PS-MBs (5 or 50 µg mL-1) were non-toxic. Cytotoxicity in cells treated with PS-MBs together with BaP or 3-NBA were similar to those observed after exposure to BaP or 3-NBA alone. Using the formamidopyrimidine-DNA glycosylase (FPG)-modified comet assay 3-NBA, but not BaP, induced DNA damage in RTgutGC cells at 10 µM (∼10% tail DNA in the absence and ∼15% tail DNA in the presence of FPG versus ∼1% in controls), whereas PS-MBs alone showed no detrimental effects. Interestingly, comet formation was substantially increased (∼4-fold) when RTgutGC cells were exposed to PS-MBs (50 µg mL-1) and 10 µM 3-NBA compared to cells treated with 3-NBA alone. Further, using 32P-postlabelling we observed strong DNA adduct formation in 3-NBA-exposed RTgutGC cells (∼900 adducts/108 nucleotides). 3-NBA-derived DNA adduct formation was significantly decreased (∼20%) when RTgutGC cells were exposed to MB and 3-NBA compared to cells treated with 3-NBA alone. Our results show that PS-MBs impact on the genotoxicity of 3-NBA, causing a significant increase in DNA damage as measured by the comet assay in the intestinal cell line, providing proof of principle that MPs may alter the genotoxic potential of PAHs in fish cells.

Procedures for the reconstruction, primary culture and experimental use of rainbow trout gill epithelia.

This protocol describes how to reconstruct and culture the freshwater rainbow trout gill epithelium on flat permeable membrane supports within cell culture inserts. The protocol describes gill cell isolation, cultured gill epithelium formation, maintenance, monitoring and preparation for use in experimental procedures. To produce a heterogeneous gill epithelium, as seen in vivo, seeding of isolated gill cells twice over a 2-d period is required. As a consequence, this is termed the double-seeded insert technique. Approximately 5-12 d after cell isolation and seeding, preparations develop electrically tight gill epithelia that can withstand freshwater on the apical cell surface. The system can be used to study freshwater gill physiology, and it is a humane alternative for toxicity testing, bioaccumulation studies and environmental water quality monitoring.

A primary fish gill cell culture model to assess pharmaceutical uptake and efflux: evidence for passive and facilitated transport.

The gill is the principle site of xenobiotic transfer to and from the aqueous environment. To replace, refine or reduce (3Rs) the large numbers of fish used in in vivo uptake studies an effective in vitro screen is required that mimics the function of the teleost gill. This study uses a rainbow trout (Oncorhynchus mykiss) primary gill cell culture system grown on permeable inserts, which tolerates apical freshwater thus mimicking the intact organ, to assess the uptake and efflux of pharmaceuticals across the gill. Bidirectional transport studies in media of seven pharmaceuticals (propranolol, metoprolol, atenolol, formoterol, terbutaline, ranitidine and imipramine) showed they were transported transcellularly across the epithelium. However, studies conducted in water showed enhanced uptake of propranolol, ranitidine and imipramine. Concentration-equilibrated conditions without a concentration gradient suggested that a proportion of the uptake of propranolol and imipramine is via a carrier-mediated process. Further study using propranolol showed that its transport is pH-dependent and at very low environmentally relevant concentrations (ng L(-1)), transport deviated from linearity. At higher concentrations, passive uptake dominated. Known inhibitors of drug transport proteins; cimetidine, MK571, cyclosporine A and quinidine inhibited propranolol uptake, whilst amantadine and verapamil were without effect. Together this suggests the involvement of specific members of SLC and ABC drug transporter families in pharmaceutical transport.

A primary FIsh Gill Cell System (FIGCS) for environmental monitoring of river waters.

Studies were conducted to assess the feasibility of a primary FIsh Gill Cell culture system (FIGCS) for both laboratory and field based environmental monitoring of rivers known to be affected by metal contamination. FIGCS were exposed in the laboratory and in the field to water from the River Hayle, a metal-contaminated system in Cornwall, United Kingdom. Water chemistry, including transition metal concentrations, changes in transepithelial electrical resistance (TEER), cell viability and the expression of metal responsive genes, metallothionein A and B were measured. FIGCS tolerated river water in the laboratory showing no loss in TEER or cell viability following 24h exposure. The cells also tolerated transport to the field (∼1000 km and 30 h) and exposure to unfiltered and filtered river water. Metallothionein A and B, a measure of intracellular biologically active metals, expression was induced in the laboratory and field on exposure to water from sites with elevated metal concentrations compared to those sites where metal levels were below water metal Environmental Quality Standards. This demonstrates that FIGCS detects bioreactive metals in river waters on exposure in the laboratory or field and can be used for on-site environmental monitoring as well as investigations into bioavailability and toxicity of contaminant mixtures in natural waters.

The combined use of the PLHC-1 cell line and the recombinant yeast assay to assess the environmental quality of estuarine and coastal sediments.

Sediment contamination poses a potential risk for both ecosystems and human health. Risk assessment is troublesome as sediments contain complex mixtures of toxicants, and traditional chemical analyses can neither provide information about potential hazards to organisms nor identify and measure all present contaminants. This work combines the use of the PLHC-1 cell line and the recombinant yeast assay (RYA) to assess the environmental quality of estuarine and coastal sediments. The application of multiple endpoints (cytotoxicity, generation of oxidative stress, presence of CYP1A inducing agents, micronucleus formation and estrogenicity) revealed that the organic extracts of those sediments affected by industrial activities or collected near harbours and untreated urban discharges showed significant cytotoxicity, micronuclei and CYP1A induction. The study highlights the usefulness of the applied bioassays to identify those sediments that could pose risk to aquatic organisms and that require further action to improve their environmental quality.

Can pharmaceuticals interfere with the synthesis of active androgens in male fish? An in vitro study.

The in vitro interference of fibrate (gemfibrozil, clofibrate, clofibric acid), anti-inflammatory (ibuprofen, diclofenac), and anti-depressive (fluoxetine, fluvoxamine) drugs with key enzymatic activities-C17,20-lyase and CYP11ß-involved in the synthesis of active androgens in gonads of male carp have been investigated. Among the tested compounds, fluvoxamine and fluoxetine were the strongest inhibitors of C17,20-lyase and CYP11ß enzymes, with IC50s in the range of 321-335 µM and 244-550 µM, respectively. To our knowledge this is the first report on the interaction of pharmaceutical compounds with enzymatic systems involved in the synthesis of oxy-androgens. As oxy-androgens are known to influence spermatogenesis and stimulate reproductive behavior and secondary sexual characteristics in male fish, this work highlights the need for further investigating these endpoints when designing specific in vivo studies to assess the endocrine disruptive effect of pharmaceuticals in fish.

Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11 activities, in accordance with low abcg2 and abcb11 transcript levels. Our data indicate that transporter expression and activity patterns in the different trout cell lines are irrespective of the tissue of origin, but are determined by factors of cell cultivation.

Single and combined toxicity of pharmaceuticals and personal care products (PPCPs) on the rainbow trout liver cell line RTL-W1.

The toxicological implications of the presence of pharmaceuticals and personal care products (PPCPs) in the aquatic environment remain largely unknown. Acute toxicity tests have generally failed to detect the subtle action elicited by those compounds at environmentally relevant concentrations and they have often overlooked the fact that toxicity can be influenced by additive and synergistic effects. The aim of this study was to further assess the cytotoxicity of different pharmaceuticals and synthetic musks as well as their mixtures on the rainbow trout liver cell line RTL-W1. Eleven pharmaceuticals from different therapeutic classes (anti-inflammatory drugs, serotonin re-uptake inhibitors and lipid regulators) and five synthetic musks from the two major groups (nitro- and polycyclic musks) were selected for the study. Two fluorescent dyes were used to monitor cell viability. Among the tested compounds, estimated EC50s (effective concentration causing 50% decline of cell viability) denoted that polycyclic musks (7-25 microM) followed by anti-depressives (7-50 microM) showed the highest potential to induce cytotoxicity, whereas lipid regulators (20-380 microM), anti-inflammatory drugs (160-260 microM) and nitromusks (100-240 microM) had the lowest toxicity. Within a given therapeutic class, combined toxicity of mixtures was additive, following in most cases the concentration addition concept. However, the combined toxicity was higher than additive for those mixtures that included one compound from each class (i.e. dissimilar mixtures). Overall, this study shows that in the aquatic environment, toxicity of PPCPs on non-target organisms may occur at concentrations lower than expected due to synergistic effects between the different toxicants.

The interference of nitro- and polycyclic musks with endogenous and xenobiotic metabolizing enzymes in carp: an in vitro study.

Synthetic musks are widely used as perfuming agents in products, such as cosmetics, detergents, and soaps. The increased detection of these substances in the aquatic environment and their high bioconcentration potential raises concerns about potential effects on aquatic species. This work aimed at assessing the interactions of the most widely used musks: nitromusks (musk xylene, musk ketone) and polycyclic musks (celestolide, galaxolide, and tonalide) with fish enzymatic systems involved in both xenobiotic and endogenous metabolism. Therefore, CYP catalyzed pathways were investigated in carp liver microsomes (CYP1A, CYP3A), ovarian microsomes (CYP19) and testicular mitochondria (CYP17 and CYP11beta) using standard substrates. Phase II activities (UDP-glucuronosyltransferases and sulfotransferases) were determined in carp liver microsomes and cytosol, respectively. Polycyclic musks (galaxolide and tonalide) were stronger inhibitors of CYP3A- (IC(50): 68-74 microM), CYP17- (IC(50): 213-225 microM), CYP11beta- and CYP19-catalyzed activities than nitromusks, while the latter showed higher ability to interfere with CYP1A (IC(50): 35-37 microM). The sulfation of estradiol was also significantly inhibited by tonalide and galoxolide (IC(50): 140-294 microM). Overall, polycyclic musks showed the highest potential to interfere with those activities involved in the synthesis and metabolism of steroids while nitromusks mainly interfered with xenobiotic metabolism (CYP1A-catalyzed reactions). The obtained data suggest that CYP isoforms are potentially sensitive targets of synthetic musk substances in fish.

Gametogenesis correlated with steroid levels during the gonadal cycle of the sea urchin Paracentrotus lividus (Echinodermata: Echinoidea).

The specific mechanism regulating reproduction in invertebrates is a field of topical interest which needs to be explored in detail considering also the intriguing possible comparison with vertebrates. In this paper levels of Testosterone (T) and Estradiol (E2) and their reciprocal ratios were determined in ovaries and testis of the echinoid model species Paracentrotus lividus during the year 2004 by taking into account a putative relationship between steroid levels and reproductive cycle. T levels appeared to significantly vary during male reproductive cycle, thus suggesting a possible role of this hormone in regulation of spermatogenesis as demonstrated for other echinoderms. E2 levels were lower in males with respect to females; consequently E2 involvement in oogenesis is hypothesized. In parallel with steroid levels evaluation, variations in P450-aromatase activity and its possible role on regulation of gametogenesis were also considered. Clear correlations between steroid levels and gonad index (GI), as well as between GI and reproductive cycle were not detected, suggesting that GI alone is not a reliable parameter in describing the reproductive status of the gonads. Altogether the results obtained so far confirm the presence of a relationship between steroid levels and reproductive cycle as suggested by previous results on different echinoderm species.

The interference of pharmaceuticals with endogenous and xenobiotic metabolizing enzymes in carp liver: an in-vitro study.

The interactions of fibrate (clofibrate, fenofibrate, bezafibrate, gemfibrozil), antiinflammatory (ibuprofen, diclofenac, naproxen, ketoprofen), and anti-depressive (fluoxetine,fluvoxamine, paroxetine) drugs with CYP catalyzed pathways (CYP1A, CYP3A-, CYP2K-, and CYP2M-like) and Phase II activities (UDP-glucuronosyltransferases and sulfotransferases), involved in both xenobiotic and endogenous metabolism in fish, were investigated in-vitro by incubating carp liver subcellular fractions in the presence of the substrate and the selected drug. Anti-depressive drugs were strong inhibitors of CYP1A (92-94% inhibition), CYP3A-like (69-80% inhibition), and CYP2K-like (36-69% inhibition) catalyzed activities, while antiinflammatory drugs were potent CYP2M-like inhibitors (32-74% inhibition). Among the lipid regulators, gemfibrozil strongly inhibited CYP2M-catalyzed activity (91% inhibition) and other CYP isoforms (CYP1A and CYP3A-like). Additionally, glucuronidation of naphthol and testosterone were targeted by antiinflammatory drugs, and to a lesser extent, by fibrate drugs (48-78% inhibition). No significant alteration on sulfotransferase activities was observed, apart from a minor inhibitory effect of clofibrate, gemfibrozil, and fluoxetine on the sulfation of estradiol. Overall, gemfibrozil, diclofenac, and the three anti-depressive drugs appear to be the pharmaceuticals with the highest potential to interfere with fish metabolic systems.