Active Pharmaceutical Ingredient Uptake by Zebrafish (Danio rerio) Oct2 (slc22a2) Transporter Expressed in Xenopus laevis Oocytes.

Uptake of active pharmaceutical ingredients (APIs) across the gill epithelium of fish is via either a passive or facilitated transport process, with the latter being more important at the lower concentrations more readily observed in the environment. The solute carrier (SLC) 22A family, which includes the organic cation transporter OCT2 (SLC22A2), has been shown in mammals to transport several endogenous chemicals and APIs. Zebrafish oct2 was expressed in Xenopus oocytes and the uptake of ranitidine, propranolol, and tetraethylammonium characterized. Uptake of ranitidine and propranolol was time- and concentration-dependent with a km and Vmax for ranitidine of 246 µM and 45 pmol/(oocyte × min) and for propranolol of 409 µM and 190 pmol/(oocyte × min), respectively. Uptake of tetraethylammonium (TEA) was inhibited by propranolol, amantadine, and cimetidine, known to be human OCT2 substrates, but not quinidine or ranitidine. At external media pH 7 and 8 propranolol uptake was 100-fold greater than at pH 6; pH did not affect ranitidine or TEA uptake. It is likely that cation uptake is driven by the electrochemical gradient across the oocyte. Uptake kinetics parameters, such as those derived in the present study, coupled with knowledge of transporter localization and abundance and API metabolism, can help derive pharmacokinetic models. Environ Toxicol Chem 2022;41:2993-2998. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

An X-ray micro-computer tomography study of the Malpighian tubules of the Blue Bottle Blow Fly (Calliphora vomitoria) Diptera: Calliphoridae.

Malpighian tubules are the insect equivalent of mammalian kidneys and normally drain into the gut at the junction between the mid and hind gut. The Malpighian tubules of the fruit fly Drosophila melanogaster are increasingly being used as a model for studying human renal tract development, histology, nephrolithiasis and urolithiasis. In the present study we report when using X-ray micro-computer tomography techniques, the larval, intrapuparial and adult stages of the larger Calliphora vomitoria can contain large amounts of calcium-rich concretions which are tightly packed in the lumen of both anterior Malpighian tubules. We show that it is feasible to utilise these calcium-rich concretions as a form of marking agent to delineate the various developmental stages of the Malpighian tubules including the crucial phase when the Malpighian tubules reconnect with the hind gut. In the majority of cases during the intrapuparial period the ureters of the Malpighian tubules did not start to re-canalise and thus reconnect with the developing hind gut until the 7th day of the 10-11 day. Just prior to ecdysis, virtually all the radio-opaque concretions in the Malpighian tubules had emptied into the hind gut and had then been completely excreted by the time the imago emerged from its puparium. In contrast, we show that in flies developing from larvae previously stained by ingesting Rhodamine B, a known substrate for both the Multi Xenobiotic Resistance and Multi Drug Resistant membrane transport systems, the efficiency with which these calcium-rich concretions are excreted by the imago as it emerges from its intrapuparial period can be significantly impaired. Therefore, it might be useful to include C. vomitoria as a model when studying renal tract development and urolithiasis using X-ray micro-computer tomography.

Effect of Water pH on the Uptake of Acidic (Ibuprofen) and Basic (Propranolol) Drugs in a Fish Gill Cell Culture Model.

Water pH is predicted to affect the uptake of ionizable pharmaceuticals in fish. The current study used an in vitro primary fish gill cell culture system to assess the effect of pH values in the range of 4.5-8.75 on the uptake rates of the base propranolol (pKa 9.42) and the acid ibuprofen (pKa 4.59). The rate-limiting step in the uptake was the diffusive supply flux of the unionized form from the water to the apical membrane, with subsequent rapid transfer across the epithelium. Computed uptake rate based on the unionized fraction best described the uptake of propranolol and ibuprofen over the range of pH values 5-8 and 6-8.75, respectively. For ibuprofen, the computed uptake rate overestimated the uptake below pH 6 where the unionized fraction increased from 4% at pH 6 to 55% at pH 4.5. As the unionized fraction increased, the uptake rate plateaued suggesting a saturation of the transport process. For both drugs, large variations in the uptake occur with only small fluctuations in pH values. This occurs between pH values 6 and 8, which is the pH range acceptable in regulatory test guidelines and seen in most of our freshwaters.

Developing in vitro models to assess fish gill excretion of emerging contaminants.

Advances in analytical methods have enabled the detection of emerging contaminants at ever lower concentrations in freshwaters. However, such measurements need to be linked to effect-based assays to identify risks. The bioconcentration factor (BCF) forms part of a chemical's environmental risk assessment (ERA), and current regulatory testing guidelines to calculate fish BCFs use hundreds of fish per chemical. Due to ethical concerns a reduction in the numbers of animals used is desired, and there is a need to identify in vitro or in silico alternatives which meet regulatory acceptance. This study describes the successful demonstration of a FIsh Gill Cell culture System (FIGCS) to assess an often overlooked parameter in pharmacokinetics: the excretion of drugs across the gill. The FIGCS tolerates the application of natural waters on its apical surface, mimicking the situation of the live fish, and thus in combination with advanced analytical methods, offers an opportunity to take lab-based testing used for ERA, such as compound uptake, biotransformation or excretion directly into field for validation with natural waters. Here we used the basic drug propranolol and the acidic ibuprofen as a demonstration of the FIGCS utility in three separate experiments. Excretion across the apical membrane showed saturation kinetics, suggesting the involvement of carrier-mediated processes. Both propranolol and ibuprofen were excreted across the epithelium from the media (internal blood equivalent) to the water, with ibuprofen excretion being considerably slower than propranolol excretion. Further studies indicate that ibuprofen may be complexing with fetal bovine serum (FBS) reducing bioavailability; in contrast propranolol efflux rate was unaffected, indicating that drugs behave differently in the presence of FBS and other plasma proteins. A key issue in future ERA is to better understand the effects of mixtures of different pollutant classes found in environmental samples, and this model offers an ethical path to do this.

Use of X-ray micro-computed tomography to study the moult cycle of the freshwater amphipod Gammarus pulex.

Stages of the moult cycle of the amphipod Gammarus pulex have been previously characterised based on the examination of either apolysis of the 3rd dactyl, or the whole body and eye appearance. In the current study the aim was to compare these two established moult staging techniques with a novel X-ray micro-computed tomography (micro-CT) scan method. The micro-CT provides information on the degree of calcification of the external integument and of the internal structures, such as the gastric mill. The degree of calcification is predicted to change during the moult cycle. Successful micro-CT scans were obtained from 80 G. pulex specimens and the radiological appearance of the 28 specimens immediately immersed in 4 % PFA were not different to the 52 specimens stored in 4 % PFA for at least 28 days prior to scanning. These specimens could be classified into moult stages A, B, C, early D or late D based on the degree of calcification. Good agreement was obtained between all three methods of moult stage classification if fresh specimens were used, but if specimens had been preserved in 4% Paraformaldehyde (PFA) for more than 24 hours the loss of colour from the whole body and eye meant these methods were not suitable. This is the first time that a micro-CT method has been used to study G. pulex and shows that this method of moult staging is accurate and reliable.

Hatching gland development and hatching in zebrafish embryos: A role for zinc and its transporters Zip10 and Znt1a.

Zinc transporters of the ZIP (Slc39, importers) and ZnT (Slc30, exporters) protein families have evolutionary conserved roles in biology. The aim of the present study was to explore the role of zinc, and zinc transporters Zip10 and Znt1a in zebrafish hatching gland development and larval hatching. In the study, knockdown of genes for Zip10 and Znt1a in zebrafish embryos was achieved using morpholino-modified oligonucleotides. A partial loss-of-function Znt1a mutant (Znt1asa17) allowed comparison with the Znt1a morphant. Free Zn2+ in embryos and apoptosis were investigated using fluorescent dyes whereas gene expression was investigated by whole-mount in situ hybridization (WISH). The results showed high levels of free Zn2+ in the hatching gland cells (HGC) along with abundant expression of zip10 and znt1a in normal embryo. Knockdown of zip10 reduced free Zn2+ in HGC, ceased their normal developmental apoptosis, and resulted in displacement and later disappearance of hatching glands and hatching enzymes he1a and catL1b, and inability to hatch. Conversely, knockdown of znt1a or the Znt1asa17 mutation accelerated hatching and coincided with high expression of hatching enzymes and free Zn2+ in the HGC. Thus, Zip10 and free Zn2+ in the HGC are required both for their development and function. This study also demonstrated the opposite functions of the two zinc transporters, ZIP10 and ZnT1 as well as shedding light on the role of Zn2+ in regulation of the human hatching enzyme homologue, ovastacin, which is activated by zinc and cleaves the zona pellucida protein, ZP2, to prevent polyspermy.

The zebrafish Znt1asa17 mutant reveals roles of zinc transporter-1a in embryonic development.

BACKGROUND: Zinc is one of the vital micronutrients required through various developmental stages in animals. Zinc transporter-1 (ZnT1; Slc30a1) is essential in vertebrates for nutritional zinc uptake and cellular zinc extrusion. Knockout of ZnT1 is lethal in vertebrates and there are therefore few functional studies of this protein in vivo. METHODS: In the present study we characterised the embryonic development in a zebrafish Znt1a mutant (Znt1asa17) which is lacking the last 40 amino acids of Znt1a as generated by TILLING. In parallel experiments, we compared the development of a zebrafish embryo Znt1a morphant (Znt1aMO) which was generated by knockdown of Znt1a using morpholino-modified oligonucliotides. RESULTS: The homozygous Znt1asa17 embryo is viable, but displays a subtle phenotype informing on the biological roles of Znt1a. The Znt1asa17 fish have delayed development, including attenuated epiboly. They further show a decrease in phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2), retarded yolk resorption, and impaired clearance of free Zn2+ from the vitelline fluid and its storage in hatching gland cells. All these aberrations are milder versions of those observed upon knockdown of Znt1a by morpholinos. Interestingly, the phenotype could be rescued by addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) to the incubation medium and was aggravated by addition of zinc(II). Thus, the Znt1asa17 mutant has a reduced ability to handle zinc and can be characterised as a hypomorph. CONCLUSION: This study is the first to show that the last 40 amino acids of Znt1a are of importance for its role in zinc homeostasis and ability to activate the MAPK/ERK pathway contrary to what was previously thought.

Sublethal exposure to copper supresses the ability to acclimate to hypoxia in a model fish species.

Hypoxia is one of the major threats to biodiversity in aquatic systems. The association of hypoxia with nutrient-rich effluent input into aquatic systems results in scenarios where hypoxic waters could be contaminated with a wide range of chemicals, including metals. Despite this, little is known about the ability of fish to respond to hypoxia when exposures occur in the presence of environmental toxicants. We address this knowledge gap by investigating the effects of exposures to different levels of oxygen in the presence or absence of copper using the three-spined sticklebacks (Gasterosteus aculeatus) model. Fish were exposed to different air saturations (AS; 100%, 75% and 50%) in combination with copper (20 µg/L) over a 4 day period. The critical oxygen level (Pcrit), an indicator of acute hypoxia tolerance, was 54.64 ± 2.51% AS under control conditions, and 36.21 ± 2.14% when fish were chronically exposed to hypoxia (50% AS) for 4 days, revealing the ability of fish to acclimate to low oxygen conditions. Importantly, the additional exposure to copper (20 µg/L) prevented this improvement in Pcrit, impairing hypoxia acclimation. In addition, an increase in ventilation rate was observed for combined copper and hypoxia exposure, compared to the single stressors or the controls. Interestingly, in the groups exposed to copper, a large increase in variation in the measured Pcrit was observed between individuals, both under normoxic and hypoxic conditions. This variation, if observed in wild populations, may lead to selection for a tolerant phenotype and alterations in the gene pool of the populations, with consequences for their sustainability. Our findings provide strong evidence that copper reduces the capacity of fish to respond to hypoxia by preventing acclimation and will inform predictions of the consequences of global increases of hypoxia in water systems affected by other pollutants worldwide.

Influence of urban river restoration on nitrogen dynamics at the sediment-water interface.

River restoration projects focused on altering flow regimes through use of in-channel structures can facilitate ecosystem services, such as promoting nitrogen (N) storage to reduce eutrophication. In this study we use small flux chambers to examine ammonium (NH4+) and nitrate (NO3-) cycling across the sediment-water interface. Paired restored and unrestored study sites in 5 urban tributaries of the River Thames in Greater London were used to examine N dynamics following physical disturbances (0-3 min exposures) and subsequent biogeochemical activity (3-10 min exposures). Average ambient NH4+ concentrations were significantly different amongst all sites and ranged from 28.0 to 731.7 µg L-1, with the highest concentrations measured at restored sites. Average NO3- concentrations ranged from 9.6 to 26.4 mg L-1, but did not significantly differ between restored and unrestored sites. Average NH4+ fluxes at restored sites ranged from -8.9 to 5.0 µg N m-2 sec-1, however restoration did not significantly influence NH4+ uptake or regeneration (i.e., a measure of release to surface water) between 0-3 minutes and 3-10 minutes. Further, average NO3- fluxes amongst sites responded significantly between 0-3 minutes ranging from -33.6 to 97.7 µg N m-2 sec-1. Neither NH4+ nor NO3- fluxes correlated to sediment chlorophyll-a, total organic matter, or grain size. We attributed variations in overall N fluxes to N-specific sediment storage capacity, biogeochemical transformations, potential legacy effects associated with urban pollution, and variations in river-specific restoration actions.

The Use of Molecular Descriptors To Model Pharmaceutical Uptake by a Fish Primary Gill Cell Culture Epithelium.

Modeling approaches such as quantitative structure-activity relationships (QSARs) use molecular descriptors to predict the bioavailable properties of a compound in biota. However, these models have mainly been derived based on empirical data for lipophilic neutral compounds and may not predict the uptake of ionizable compounds. The majority of pharmaceuticals are ionizable, and freshwaters can have a range of pH values that affect speciation. In this study, we assessed the uptake of 10 pharmaceuticals (acetazolamide, beclomethasone, carbamazepine, diclofenac, gemfibrozil, ibuprofen, ketoprofen, norethindrone, propranolol, and warfarin) with differing modes of action and physicochemical properties (p Ka, log S, log D, log Kow, molecular weight (MW), and polar surface area (PSA)) by an in vitro primary fish gill cell culture system (FIGCS) for 24 h in artificial freshwater. Principal component analysis (PCA) and partial least-squares (PLS) regression was used to determine the molecular descriptors that influence the uptake rates. Ionizable drugs were taken up by FIGCS; a strong positive correlation was observed between log S and the uptake rate, and a negative correlation was observed between p Ka, log D, and MW and the uptake rate. This approach shows that models can be derived on the basis of the physicochemical properties of pharmaceuticals and the use of an in vitro gill system to predict the uptake of other compounds. There is a need for a robust and validated model for gill uptake that could be used in a tiered risk assessment to prioritize compounds for experimental testing.

Considering aspects of the 3Rs principles within experimental animal biology.

The 3Rs - Replacement, Reduction and Refinement - are embedded into the legislation and guidelines governing the ethics of animal use in experiments. Here, we consider the advantages of adopting key aspects of the 3Rs into experimental biology, represented mainly by the fields of animal behaviour, neurobiology, physiology, toxicology and biomechanics. Replacing protected animals with less sentient forms or species, cells, tissues or computer modelling approaches has been broadly successful. However, many studies investigate specific models that exhibit a particular adaptation, or a species that is a target for conservation, such that their replacement is inappropriate. Regardless of the species used, refining procedures to ensure the health and well-being of animals prior to and during experiments is crucial for the integrity of the results and legitimacy of the science. Although the concepts of health and welfare are developed for model organisms, relatively little is known regarding non-traditional species that may be more ecologically relevant. Studies should reduce the number of experimental animals by employing the minimum suitable sample size. This is often calculated using power analyses, which is associated with making statistical inferences based on the P-value, yet P-values often leave scientists on shaky ground. We endorse focusing on effect sizes accompanied by confidence intervals as a more appropriate means of interpreting data; in turn, sample size could be calculated based on effect size precision. Ultimately, the appropriate employment of the 3Rs principles in experimental biology empowers scientists in justifying their research, and results in higher-quality science.

The evolution, structure and function of the ray finned fish (Actinopterygii) glucocorticoid receptors.

Basal ray-finned fish (Actinopterygii) possess a single glucocorticoid receptor (GR) and when compared to the lobe-finned vertebrate (Sarcopterygii) GR possess nine additional amino acids between the zinc-finger of the DNA binding domain. A whole genome duplication event which occurred between 320 and 350MYA in the teleost lineage following the split from the basal ray-finned fish resulted in 2 GRs: one GR group, GR1, has retained the 9 amino acids insert whereas the other group, GR2, has not. The exception to this is the zebrafish, that have lost one of the GRs, but they do possess 2 GRs with a splice variant that lacks the C-terminal portion of the GR to form GRß which acts as a dominant-repressor of the wildtype GR. Another splice variant sees the basal ray-finned GR and teleost GR1 without the 9 amino acids insert. The molecular basis for GRs retention is beginning to be unravelled. In Pantadon buchholzi, rainbow trout, carp, marine and Japanese medaka GR2 is more sensitive to glucocorticoids (GC), thus potentially playing a more significant role in regulating gene expression at basal circulatory GC concentrations. However, this division in GC sensitivity is not seen in other species. The few studies to evaluate the significance of the 9 amino acid insert have shown that it affect maximal transactivational activity the extent to which is dependent on the number of glucocorticoid response elements (GREs) present in the reporter plasmid. The retention of these GRs would suggest there was an evolutionary advantage, which saw the development of a complex regulatory process to mediate the actions of the glucocorticoids.

Lagos lagoon sediment organic extracts and polycyclic aromatic hydrocarbons induce embryotoxic, teratogenic and genotoxic effects in Danio rerio (zebrafish) embryos.

An expansion of anthropogenic activity around Lagos lagoon, Nigeria, has raised concerns over increasing contaminants entering the lagoon's ecosystem. The embryotoxicity, teratogenicity and genotoxicity of sediment organic extracts from four sampling zones around Lagos lagoon, Ilaje, Iddo, Atlas Cove and Apapa, as well as the dominant polycyclic aromatic hydrocarbons (PAHs) identified in water measured during the wet season (naphthalene, phenanthrene, pyrene, benzo[a]pyrene and a mixture of these), were assessed with Danio rerio embryos. Embryos were exposed to varying concentrations of toxicants from 0-72 h post-fertilization (hpf). Embryotoxicity at 72 hpf showed a dose-dependent increase in mortality upon exposure to extracts from all zones, except Atlas Cove. Similarly, higher levels of teratogenic effects, such as increased oedema, and haemorrhage and developmental abnormalities resulted from exposure to extracts from Ilaje, Iddo and Apapa zones. Treatment with single PAHs revealed that significant levels of detrimental effects were obtained only for phenanthrene. The modified comet assay revealed that the oxidative damage to DNA was generally low (<12 %) overall for all sediment extracts, but was significantly elevated with Ilaje and Iddo sediment extracts when compared with solvent controls. Oxidative damage was observed with the single PAHs, phenanthrene and benzo[a]pyrene, as well as with the PAH mixture. This study highlights that Lagos lagoon sediment extracts have teratogenic, embryotoxic and genotoxic properties, which are likely due to the high molecular weight PAHs present in the extracts, some of which are known or are suspected human carcinogens.

Hypoxia Suppressed Copper Toxicity during Early Development in Zebrafish Embryos in a Process Mediated by the Activation of the HIF Signaling Pathway.

Hypoxia is a global and increasingly important stressor in aquatic ecosystems, with major impacts on biodiversity worldwide. Hypoxic waters are often contaminated with a wide range of chemicals but little is known about the interactions between these stressors. We investigated the effects of hypoxia on the responses of zebrafish (Danio rerio) embryos to copper, a widespread aquatic contaminant. We showed that during continuous exposures copper toxicity was reduced by over 2-fold under hypoxia compared to normoxia. When exposures were conducted during 24 h windows, hypoxia reduced copper toxicity during early development and increased its toxicity in hatched larvae. To investigate the role of the hypoxia signaling pathway on the suppression of copper toxicity during early development, we stabilized the hypoxia inducible factor (HIF) pathway under normoxia using a prolyl-4-hydroxylase inhibitor, dimethyloxalylglycine (DMOG) and demonstrated that HIF activation results in a strong reduction in copper toxicity. We also established that the reduction in copper toxicity during early development was independent of copper uptake, while after hatching, copper uptake was increased under hypoxia, corresponding to an increase in copper toxicity. These findings change our understanding of the current and future impacts of worldwide oxygen depletion on fish communities challenged by anthropogenic toxicants.

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.

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.

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.

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.

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.

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.