Quantitation of metallothionein mRNA from the New Zealand common bully (Gobiomorphus cotidianus) and its implications for biomonitoring

Metallothionein (MT) is a protein found in most eukaryotes where its primary role is the regulation of homeostasis of the essential metals copper and zinc. MTs have an additional protective role through their binding of toxic metals such as cadmium and mercury. Increased expression of MT in response to harmful levels of these metals has been demonstrated for several aquatic species. In this study, the expression of MT in the liver tissue of the New Zealand common bully Gobiomorphus cotidianus was measured by quantifying MT mRNA. A statistically significant increase in MT expression was observed in fish exposed to copper for 48 h, but not those exposed to zinc. Field sampling of common bullies showed a wide variation in individual hepatic MT mRNA levels which did not correlate with fish age, sex, or sampling location. A comparison of two populations of common bullies from a polluted and a control site showed a two‐fold higher mean MT mRNA level in fish from the polluted site. Implications for using MT expression in the common bully as a bio‐monitoring tool are discussed.     

Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.     

The effect of estrogen on cadmium distribution in rainbow trout (Oncorhynchus mykiss)

The effects of injections of 17β-estradiol (E2) and Cd, on the distribution of Cd and the induction of metallothionein (MT) mRNA and vitellogenin mRNA was investigated. Bone and liver were the main organs accumulating Cd. However, E2 redirected the metal accumulation from the bone and liver to the gill, gut and muscle upon exposure to E2. Cd did not induce the hepatic MT mRNA levels in animals treated with E2. The VTG mRNA levels were also reduced following co-injection of E2 and Cd. However, the kidney responded to Cd exposure by upregulating MT mRNA even in the presence of E2 treatment. In the liver the reduced MT mRNA induction led to a redistribution of CD from MT to non-MT proteins. The toxicological significance of these alterations in Cd handling remains to be determined.     

Induction of vitellogenin in vivo and in vitro in the model teleost medaka (Oryzias latipes): comparison of gene expression and protein levels

Quantification of the egg yolk precursor vitellogenin (VTG) in fish has become a standard technique to detect estrogenic effects of known chemicals and environmental samples. In the present study, we have analysed VTG induction by estradiol, ethynylestradiol and genistein exposure in the model teleost medaka (Oryzias latipes) and demonstrate that the medaka is a suitable model system to analyse estrogenic effects. By comparing VTG gene expression and protein levels we show that in principal both techniques can be used to study VTG induction in vivo (juvenile and adult males) and in vitro (primary cultures of male liver cells). If a short term in vivo or in vitro exposure is performed, detection of mRNA might be sufficient. For long term studies with the need to detect weak estrogenic chemicals and a precise quantification, immuno-chemical detection may be favoured.     

Kinetics of vitellogenin protein and mRNA induction and depuration in fish following laboratory and environmental exposure to oestrogens

Plaice, flounder and sand goby were exposed to ethynylestradiol (EE2) for 21 days and then followed for up to 31 days after removal of the oestrogen. Plasma vitellogenin (VTG) and hepatic VTG mRNA were determined in groups of fish sampled during the induction and post-exposure phases. VTG mRNA increased slightly earlier than plasma protein, but both reached maxima by 21 days. In contrast, VTG mRNA decayed much more rapidly than protein after EE2 exposure was terminated (typical values t(1/2) mRNA 3 days, protein 15-30 days). Vitellogenin and VTG mRNA thus measure different temporal events and this is illustrated by field data of male flounder in which both parameters have been determined. Few fish show co-ordinate increased VTG mRNA and vitellogenin but rather more fish have increased vitellogenin. Low level increases of VTG mRNA (5 X) is observed in some fish without increased vitellogenin and this may represent polymorphic differences between individual fish.     

Cellular responses and disease expression in oysters (Crassostrea virginica) exposed to suspended field contaminated sediments.

Exposure of oysters to water soluble fractions derived from field-contaminated sediments (FCS) containing predominantly lower molecular weight organic aromatic compounds, has been previously demonstrated to enhance pre-existing infections caused by the protozoan parasite, Perkinsus marinus (Dermo), and the prevalence of experimentally induced infections. To further explore the role of pollution on the onset and progression of disease, effects of suspended FCS from an estuarine creek in Virginia, USA, dominated by higher molecular weight polycyclic aromatic hydrocarbons (PAHs) on cellular responses and Dermo disease expression in oysters (Crassostrea virginica) were examined. Sediments were collected from a PAH polluted estuarine creek in Virginia, USA. To test effects on cellular response, oysters from Maine were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 70.2, 105, or 140 microg PAHs, respectively) for 5, 10, 20, and 40 days. Hemocyte activities and plasma lipid, protein and lactate dehydrogenase (LDH) levels were then measured. Exposure stimulated neutral red uptake, MTT reduction, and 3H-leucine incorporation in oyster hemocytes at various exposure times, but did not affect the plasma protein, lipid and LDH levels. To test effects on Dermo expression, oysters from a Dermo enzootic area, with an initial estimated infection prevalence of 39%, were exposed daily to 0, 1.0, 1.5, or 2.0 g suspended FCS (corresponding to 0, 75.0, 113, or 150 microg PAHs, respectively) for 30 days. Exposure enhanced disease expression in oysters. However, no significant change was noted in any measured cellular or humoral parameters.     

Evidence for resistance to benzo[a]pyrene and 3,4,3'4'-tetrachlorobiphenyl in a chronically polluted Fundulus heteroclitus population

Halogenated aromatic hydrocarbons (HAHs) and polynuclear aromatic hydrocarbons (PAHs) are major environmental contaminants. Fish species that are chronically exposed to these compounds can develop resistance to their toxic effects. In all fish species studied to date, toxicant resistance has been accompanied by decreased inducibility of the xenobiotic metabolizing enzyme, cytochrome P450 1A (CYPIA). CYP1A induction is mediated through the Aryl Hydrocarbon Receptor (AHR). Although these compounds mediate their effects through this pathway, there have been resistant populations in which one chemical class cannot induce CYPIA expression (HAHs) while the other (PAHs) can. Resistance to PAHs was examined in a HAH-resistant population of Fundulus heteroclitus collected from a site contaminated with both compound classes (Newark Bay, NJ). Fish were injected intraperitoneally with the HAH 3,4,3',4'-tetrachlorobiphenyl (PCB77), benzo[a]pyrene (B[a]P, a PAH) or vehicle and sacrificed after 2 (B[a]P) or 5 days (PCB77, vehicle). We found no significant increase in CYP1A mRNA levels in resistant Newark Bay F. heteroclitus treated with either B[a]P or PCB77, while there was a 3.9 fold (PCB77) and 4.2 fold (B[a]P) increase in CYP1A mRNA in Flax fish relative to controls. AHR labeling studies revealed significantly (P < 0.05) lower levels of hepatic AHR in Newark fish (1,770 +/- 1,693.2 DPM) relative to Flax fish (6,082.5 +/- 1,709.9 DPM). Overall, these data suggest Newark F. heteroclitus are resistant to both PAHs and HAHs at the level of CYP1A mRNA, which might be mediated, in part, though lower expression of AHR. We are currently studying the promoter sequence to determine its role in chemical resistance.     

Identification of transcriptional effects of ethynyl oestradiol in male plaice (Pleuronectes platessa) by suppression subtractive hybridisation and a nylon macroarray

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.     

Estrogenic responses of larval sunshine bass (Morone saxatilis x M. Chrysops) exposed to New York City sewage effluent

To determine the estrogenicity of effluents from sewage treatment plants (STPs) to larval fish, 2-day-old sunshine bass were exposed to effluents from three STPs serving New York City (NYC), varying in size and treatment level. Estrogenic response was evaluated by measuring vitellogenin (VTG) and estrogen receptor (ER) expression in cytosolic fractions of whole body homogenates. Concentrations of the presumptive endocrine disruptors in the effluents were also measured. VTG and ER levels in sewage-exposed fish were 3-5 times that observed in controls. Combined concentrations of estradiol and estrone ranged from 5 to 13 ng/l and nonylphenol-ethoxylate metabolites (NPEOs: 4-nonylphenol, and 1-, 2-, and 3-nonylphenol-ethoxylates) ranged from 180 to 470 microg/l in chlorinated effluent. Results indicate that both ER and VTG can be used as biomarkers for endocrine disruption in larval fish, and that 4-day exposure to sewage effluent is sufficient to elicit significant expression of these markers in sunshine bass larvae. The extremely higher concentrations of NPEOs found in effluent relative to hormones (approximately 40,000-fold) indicates that surfactant metabolites may be contributing significantly to the estrogenic effects observed.     

Oestrogenic markers and reduced population fertile egg production in a sand goby partial life-cycle test

Understanding the ecological significance of oestrogenic biomarker responses, seen in field surveys of some UK estuarine fish, requires a suitable model to link biochemical and reproductive effects. The sand goby (Pomatoschistus minutus) is a common inshore fish that has been successfully bred in laboratory aquaria. Juvenile sand goby were continuously exposed to 17beta-oestradiol (E2) during gonadal maturation, and their subsequent reproductive success investigated. Exposure endpoints included hepatic vitellogenin (VTG) mRNA expression and maturation (male nuptial colouration, sperm duct gland- and gonado-somatic indices (SDGSI, GSI)). Reproductive endpoints included spawning behaviour, brood size, fertility and population-level fertile egg production. Measured E2 exposure concentrations were <5 (controls), 17, 71 and 530 ngl(-1). Male maturation decreased with increasing E2 concentration. VTG mRNA expression increased with E2 concentration, but not with duration of exposure. Reproductive endpoints showed progressive impairment with increasing E2 concentration. For most endpoints, the lowest observed effect concentration (LOEC) was 71 ngl(-1), and EC50s were in the range 87-165 ngl(-1). Pre-breeding male colouration, VTG expression and SDGSI were good predictors of subsequent population-level fertile egg production, but GSI was not. The sensitivity of oestrogenic markers in sand goby are comparable to those used in other species, and feral fish populations exhibiting such oestrogenic responses at molecular and organ levels are likely to have reduced fertile egg production.     

Evidence for estrogenic endocrine disruption in an offshore flatfish, the dab (Limanda limanda L.)

Dab (Limanda limanda) caught in UK offshore waters show evidence of being exposed to estrogenic endocrine disrupters at a relatively low level. Two of 449 males caught between June and July 2005 had markedly elevated levels of vitellogenin (VTG; 21 and 750 microg/ml) and the remainder ranged from <0.01 to 8.6 microg/ml. Omitting the two outliers, there was a very significant positive relationship with the mass of individual males (a feature noted in previous studies on cod). Mean VTG concentrations in males differed significantly between sites. The site with the highest mean (1.6 microg/ml) was North East of the Dogger Bank and the site with the lowest (0.04 microg/ml) was in Cardigan Bay. Mean VTG concentrations in all North Sea fish were significantly higher than English Channel and Irish Sea fish, but this difference disappeared when fish mass was taken into account. VTG concentrations showed no relationship to water depth, stage of sexual maturity or age of the males. Sixty selected male plasmas were assayed for 17beta-estradiol but only two had measurable amounts (assay limit 0.04 ng/ml). Despite being the start of summer, the gonads of many of the males and females (especially those caught in the North Sea) showed signs of sexual maturity (presence of sperm in males and vitellogenic eggs in females). Many females had high VTG concentrations (up to 14 mg/ml) and 78 out of 80 had measurable concentrations of 17beta-estradiol. The cause of elevated VTG levels in male dab is unknown. As seen in cod, the presence of affected males does not appear to be linked to proximity to land or to known point sources of endocrine disrupters. However, our data, showing that larger fish are more likely to have elevated VTG concentrations, suggests a gradual accumulation by marine fish, probably through feeding, of persistent (probably relatively weak) estrogenic compounds.     

Identification of transcriptional effects of ethynyl oestradiol in male plaice (Pleuronectes platessa) by suppression subtractive hybridisation and a nylon macroarray

Suppression subtractive hybridisation (SSH) was used to generate cDNA libraries representing genes differentially expressed in response to ethynyl oestradiol (EE2) exposure in liver from male plaice (Pleuronectes platessa) previously analysed for vitellogenin (VTG) induction. Characterisation of the cDNA clones identified many as VTG (2 genes) and zona radiata proteins (ZRP) (3 genes), but 40 encoded other proteins, with more than half cryptic. Further analysis identified 85 non-redundant clones suitable for array on nylon membrane. Radiolabelled cDNAs were prepared from hepatic mRNA from EE2 treated plaice (0 and 21 days) and hybridised with the arrayed clones. Analysis of the data showed that 11/17 novel, 21/22 VTG, 13/14 ZRP, 2/2 liver aspartic proteinase (LAP) and 8/10 other mRNAs were up-regulated by EE2 exposure.     

Polycyclic aromatic hydrocarbons in recent sediments from a subtropical estuary in Brazil

The scope of this work was to evaluate the levels of polycyclic aromatic hydrocarbons (PAHs) and their possible sources in two shallow sediment cores from an estuarine area located in the south region of Brazil, the Guaratuba Bay. The estuary is encompassed by an Environmental Protection Area (EPA) of 1306 km², which is still considered to be pristine, despite recent urban growth. To assess levels of 14 selected PAHs in the sediments, among those listed as “priority pollutants” by the US Environmental Protection Agency, the cores were divided in several sections, followed by extraction, clean-up, fractionation and subsequent analysis using HPLC coupled to a fluorescence detector. Among the PAHs detected, phenanthrene and fluoranthene showed the highest concentrations. PAHs with more than four rings were usually present in low concentrations or were undetectable. To assist in the identification of possible sources, the ratios An/(An + Phe) and Flt/(Flt + Py) (anthracene to anthracene plus phenanthrene and fluoranthene to fluoranthene plus pyrene) and a PCA model were also used. Total concentrations of PAHs ranged between 1.5 and 3130 ng/g (mean 495) dry weight for the core collected in the inner part of the estuary, and between 78.5 and 3270 ng/g (mean 899) dry weight for the other core, collected in the outer part. Compared to values found in other studies in coastal zones along South American eastern margin, this area can be considered to range from low polluted to moderately polluted. From the data, it was also possible to conclude that there is predominance of petroleum sources, and essentially close to the more urbanised areas. Combustion sources have only minor contribution and are episodic, when compared to the latter.     

Effect of temperature and size on metallothionein synthesis in the gill of Mytilus galloprovincialis exposed to cadmium

Although the degree of metal contamination is expected to be a primary determinant of metallothionein (MT) induction in marine mussels, at least at polluted sites, variability caused by temperature, and biotic factors such as size, may need to be considered when interpreting field data. To test the effects of these variables, mussels, Mytilus galloprovincialis, of different sizes (small: 3.5 +/- 0.5 cm and large: 5.2 +/- 0.7 cm) were exposed to Cd (100 microg l(-1)) at different water temperatures (5, 18 and 25 degrees C) for 34 days. Resultant Cd and MT concentrations in gills were shown to be size dependent and increased significantly with temperature. At the highest temperature tested (25 degrees C) there appears to be a disproportionate effect on Cd accumulation, which raises MT synthesis to exceptionally high levels. The effect of size on MT concentrations was also temperature-dependent: at 18 and 25 degrees C, large mussels exhibited higher MT levels than smaller individuals, whilst at 5 degrees C there were no significant differences between contaminated and control mussels, in either size-group, as a result of the reduced level of MT production at this temperature. When considering the use of MT levels in mussels as a biochemical indicator of metal contamination, the potential influence of factors such as size and temperature on MT-metal relationships needs to be considered. Samples should be of uniform size as far as possible, and collection should be limited to a fixed season (avoiding climatic extremes) to ensure that the effects of these factors on baseline levels of MT is minimised.     

Metallothionein in liver of eels Anguilla anguilla from the Thames Estuary: an indicator of environmental quality?

The purpose of this study was to examine the role of the cysteine-rich protein metallothionein (MT) in the detoxification and metabolism of metals in livers of eels Anguilla anguilla from the Thames Estuary, and to assess the value of MT measurements in environmental monitoring. Hepatic MT levels and associated metal concentrations were determined in A. anguilla collected on three occasions during 1998 at sites along the tidal Thames (from freshwater to the sea) and also from a reference site (Tamar Estuary) in southwest England. MT was present at basal levels of 2 mg g(-1) dry weight (dw), predominantly in association with the essential metals Cu and Zn. MT concentrations were variable within each eel 'population' but were generally highest (up to 11 mg g(-1) in individual eels) at the more contaminated upper- and mid-estuarine sites in the Thames (e.g. Brentford and Kew). Lowest MT levels were those in eels from Blythe Sands at the mouth of the estuary. Season, sex, reproductive status and salinity may have influenced MT levels to some extent, although metals were the most significant factor. Hepatic MT concentrations were highly correlated with associated metal burdens (Zn, Cu, Ag and Cd),--notably reflecting Cu and Ag enrichment in the upper- and mid-estuary. With the exception of Zn, metals in the supernatant fraction of eel livers were primarily associated with MT. Proportions of Cu, Ag and Cd bound to MT increased as a function of accumulated metal burdens, with no indication of saturation. Thus, despite causing induction of MT, excess bioavailable Cu, Ag and Cd appear to be successfully detoxified in eels over the range of environmental contamination encountered along the Thames Estuary. Paradoxically, it may be argued from conventional doctrine that the presence of raised levels of MT, at upstream sites, constitutes a response to contamination and, by definition, signals that the fish are affected by metals; however, obvious signs of deleterious effects were not detected. Superficially, from liver MT data, it would seem that eel populations have become biochemically adapted to metal contamination. The approach tested here has proved viable in helping to interpret trends in bioavailability and the toxicological significance of accumulated metal burdens in Thames eels. A. anguilla is a common inhabitant of estuarine and fresh waters throughout Europe, and related species occur world wide. Therefore, determination of hepatic MT (and associated metals) in eels could be useful for monitoring responses to metal exposure and environmental quality on a much broader basis. Ideally these determinations should be performed in tandem with other sublethal-effects measurements to maximise information.     

Natural modulation of hepatic metallothionein and cytochrome P4501A in flounder, Platichthys flesus L.

The use of biomarkers such as cytochrome P4501A (CYP1A) or metallothionein (MT) for pollution monitoring is based on the assumption that an increased activity or concentration is primarily caused by exposure to contaminants. Previous studies have shown that the response of biomarkers may be affected by factors such as season, temperature, gender, nutritional status or size. The objective of the present study was to identify natural factors that affect the hepatic activity of CYP1A and hepatic concentration of MT in flounder (Platichthys flesus L.). Thirty flounder were sampled at each of two sites in the Hvaler archipelago, southern Norway, monthly through one year. A set of variables were recorded for each sampling and individual including water temperature, salinity, external and internal lesions, intestinal content, sex, size and organ weights. Hepatic CYP1A activity (EROD), MT and metal concentrations were determined for each individual flounder. The influence of environmental and endogenous factors on the response of these two biomarkers was assessed in multiple regression models. For both biomarkers, 50–60% of the total variability could be explained from factors directly related to season, gender and maturation. Season contributed significantly in the model for EROD, as did external lesions. Size, condition and diet did not contribute greatly when the above factors were included. The results confirm previous findings that season, gender and maturation must be taken into account in biomarker monitoring, but also indicate that other factors such as external lesions should be considered.     

Differential sensitivity of flounder (Platichthys flesus) in response to oestrogenic chemical exposure: an issue for design and interpretation of monitoring and research programmes

This study was conducted as an initial investigation of 'differential response' in one of the main sentinel organisms used for monitoring programmes in United Kingdom estuaries, the flounder Platichthys flesus. It has been hypothesised that monitoring using species with a wide geographical spread and limited migration, such as flounder, might result in the comparison of different genetic stocks and certainly of populations with differing early life stage contaminant exposure histories. Furthermore, it is probable that these pre-exposure and genetic differences could manifest themselves in an ability to respond differently to contaminant exposure, so-called 'differential response'. It is important that the extent and nature of this response is understood, if we want to be able to fully interpret the monitoring data from such programmes. During this study, flounder were collected from four separate sources; wild caught fish from the estuaries of the Rivers Alde, Mersey and Tyne, and farmed flounder from Port Erin Farm, Isle of Man. Under controlled laboratory conditions, groups of fish from each source were exposed to water-borne concentrations of the synthetic oestrogen ethynylestradiol (EE2) at a nominal concentration of 50 ng/l. Plasma was taken from each male fish after 6 and 10 days exposure and analysed for the presence of vitellogenin (VTG) using an ELISA technique. Significant levels of VTG induction were evident in fish from all sources after both 6 and 10 days exposure. Flounder from the Mersey were the only fish with significantly elevated initial background levels of VTG (day 0) and this appeared to be reflected in that these specimens showed the highest induction response after day 6. However, after day 10, fish from all other sites had a slightly higher mean VTG than those from the Mersey which showed significantly (p < 0.05) lower mean plasma VTG. It is suggested that other differential responses may have been masked by the use of a high dose of EE2 which produced maximum induction in nearly all fish. The findings of the study are discussed in terms of implications for further research into the differential response issue and how the initial plasma VTG figures contribute to a time-series from the Mersey, Tyne and Alde estuaries.