Metallothioneins failed to reflect mercury external levels of exposure and bioaccumulation in marine fish--considerations on tissue and species specific responses

The suitability of metallothioneins (MT) in fish as biomarker of exposure to mercury has been questioned. Therefore, this study aimed at investigating the relationship between external levels of exposure, mercury accumulation and MT content, assessing species and tissue specificities. Two ecologically different fish species - Dicentrarchus labrax and Liza aurata - were surveyed in an estuary historically affected by mercury discharges. Total mercury (T-Hg) and MT content were determined in gills, blood, liver, kidney, muscle and brain. All tissues reflected differences in T-Hg accumulation in both species, although D. labrax accumulated higher levels. Regarding MT, D. labrax revealed a depletion in brain MT content and an incapacity to induce MT synthesis in all the other tissues, whereas L. aurata showed the ability to increase MT in liver and muscle. Tissue-specificities were exhibited in the MT inducing potential and in the susceptibility to MT decrease. L. aurata results presented muscle as the most responsive tissue. None of the investigated tissues displayed significant correlations between T-Hg and MT levels. Overall, the applicability of MT content in fish tissues as biomarker of exposure to mercury was uncertain, reporting limitations in reflecting the metal exposure levels and the subsequent accumulation extent.     

Molecular cloning and expression of novel metallothionein (MT) gene in the polychaete Perinereis nuntia exposed to metals

To report a novel metallothionein (MT) gene and evaluate its potency as a biomarker, we clone this MT gene and measured the expression levels in the metal-exposed polychaete Perinereis nuntia. Accumulated metal contents and metallothionein-like proteins (MTLPs), which have been recognized as potential biomarkers, were compared with the relative mRNA expressions of the MT gene of P. nuntia (Pn-MT). In addition, the metal-binding affinity was estimated by recombinant Pn-MT protein. Pn-MT having high cysteine residues with three metal response elements in the promoter region closely clusters with those of other invertebrates. The accumulation patterns of metals were dependent on the exposure times in lead (Pb), cadmium (Cd), and copper (Cu) exposure. Particularly, both MTLP levels and relative mRNA expressions of MT were increased with accumulated metal contents and exposure time in P. nuntia exposed to Pb and Cd. There was no significant modulation of the Pn-MT gene in polychaetes exposed to Zn and As. However, the metal-binding ability of the recombinant Pn-MT protein provides a clear evidence for a high affinity of MT to several metal elements. These results suggest that Pn-MT would play an important role in the detoxification and/or sequestration of specific metals (e.g., Pb and Cd) in P. nuntia and have potential as a molecular biomarker in the monitoring of the marine environment using a polychaete.     

Vitellogenin gene expression in males of the Antarctic fish Trematomus bernacchii from Terra Nova Bay (Ross Sea): a role for environmental cadmium?

Synthesis of vitellogenin (VTG) in male fish is a widely recognized effect for estrogenic pollutants in temperate environments, while similar investigations are still lacking for Antarctic organisms. In this study, a preliminary characterization of vitellogenin gene expression was performed by RT-PCR in the key species Trematomus bernacchii sampled in different phases of reproductive cycle and food availability. Females exhibited the highest gene expression during the spawning period, but VTG mRNA was always detected also in males; a significant increase of gene expression was observed both in males and females at the end of the feeding season. These results were not fully supported by a differential exposure to phyto- or anthropogenic estrogens during the planctonic cycle; on the other side, the endocrine properties of cadmium, naturally elevated in Terra Nova Bay and increasing during algal bloom, would explain both the presence of VTG mRNA in males and the seasonal changes of gene induction. Laboratory exposures did not reveal short-term estrogenic effects of cadmium while an elevated responsiveness of T. bernacchii was observed toward a classical estrogenic receptor agonist (17beta-estradiol). Different hypotheses were considered to suggest delayed endocrine effects of cadmium, including the early interaction with other cellular detoxification systems or alterations at multiple levels of the hypothalamus-pituitary-gonad-liver axis. Although molecular mechanisms of VTG gene expression in males of T. bernacchii remain unclear, obtained results provide interesting insights on this species which should stimulate future research activities.     

Environmental monitoring by gene expression biomarkers in Barbus graellsii: laboratory and field studies

Barbel (Barbus graellsii) is a freshwater fish used as a sentinel species in environmental monitoring programmes and ecotoxicological studies in northern Spain, particularly in the Ebro River basin, the largest freshwater resource in Spain. We developed specific primers for the quantification of CYP1A and metallothionein (MT)-1 and -2 gene expression by QRT-PCR in barbel in order to assess their suitability in biological effect monitoring of dioxin-like compounds and metals. All three genes responded as expected in laboratory tests, using model inducers. Hepatic CYP1A mRNA levels showed a twofold induction in fish injected intraperitoneally with beta-naphthoflavone, whereas MT-1 and MT-2 gene expression was strongly induced by cadmium (15- and 13-fold, respectively) and mercury (five- and eightfold). Barbel populations from different sites on the Ebro River basin showed a good correlation between the historical records of organochlorine pollution, CYP1A expression levels and EROD activity. Nevertheless, although metallothionein protein levels in the liver of wild fish correlated with hepatic levels of mercury, MT-1 and MT-2 gene expression did not correlate with the mercury content or with the concentration of metals in sediments from the sites inhabited by the fish. These results demonstrate the utility of barbel CYP1A-mRNA expression, but not that of MT-1 or MT-2, as a biomarker in field studies. The tools and protocols developed here are likely to apply to other species of the genus Barbus, with some 700 species distributed throughout most of the Old World.     

Cadmium accumulation and Cd-binding proteins in marine invertebrates--a radiotracer study

Tissue and subcellular accumulation of cadmium were studied in different tissues of three marine invertebrates (blue mussel Mytilus edulis, the tunicate Ciona intestinalis and the sea star Asterias rubens) using radioactive ¹⁰⁹Cd as a tracer. The organisms were exposed to 0.05, 2 and 50 μg Cd l⁻¹ for 21 days. Quantitative data were obtained by dissecting, weighing and subsequently measuring radioactivity in organs and tissues. Differences between each exposure and each tissue with regard to the amount of radioactivity and metallothionein (MT) content were evaluated. Obvious interspecies differences in Cd accumulation were observed, as well as differences between tissues of the three species. The highest concentrations of Cd in all exposure treatments were found in the hepatopancreas of M. edulis and body wall of A. rubens. Taking all treatments into account, Cd accumulation in the tunic of C. intestinalis was high compared to other tissues from this species. Over 60% of Cd was present in the S50 fraction in all treatments in all three species. Metallothionein levels were increased at the highest Cd-exposure in all species and tissues, except in branchial pharynx of C. intestinalis where the highest MT level was reached following exposure to 2 μg Cd l⁻¹. The most surprising finding was that even the lowest Cd exposure concentration (0.05 μg Cd l⁻¹) caused MT induction in pyloric caeca of A. rubens, but there was no dose-dependent increase in MT at higher exposure levels.     

mRNA expression of a cadmium-responsive gene is a sensitive biomarker of cadmium exposure in the soil collembolan Folsomia candida

The gene expression of environmental organisms is useful as a biomarker of environmental pollution. One of its advantages is high sensitivity. We identified the cDNA of a novel cadmium-responsive gene in the soil collembolan Folsomia candida. The deduced protein, designated “metallothionein-like motif containing protein” (MTC), was cysteine-rich and contained a metallothionein-like motif with similarity to metallothionein, but had a much longer sequence than metallothionein and contained repeated sequences of amino acids. Expression of MTC mRNA was sensitively induced by cadmium exposure at 0.3 mg/kg of dry food, a concentration at which toxic effects are not observed, but expression was not affected by γ-ray exposure (an inducer of oxidative stress). These findings suggest that MTC is involved in cadmium-binding processes rather than in oxidative-stress responses. In conclusion, we suggest that gene expression of MTC may be a candidate biomarker for detecting low levels of cadmium contamination in soil.     

Long‐term mercury accumulation in the presence of cadmium and lead in oseochromis aureus (Steindachner)

Abstract

The effects of cadmium and lead on chronic mercury accumulation were investigated in O. aureus. After 140 days’ exposure the accumulation of mercury in the liver, brain, gill filaments, intestine, caudal muscle, spleen, trunk kidney and eye was analysed. The exposure concentrations were 0.05, 0.10 and 0.20 mg/L for mercury alone. O. aureus was also exposed to mixtures of 0.05 mg/L mercury with lead (0.05 mg/L and 0.50 mg/L or cadmium (0.05 mg/L) and 0.10 mg/L mercury with 0.10 mg/L cadmium. In food fish, a knowledge of toxic metal accumulation patterns is of great importance because of their contribution to the human diet and, as fishmeal, to the diet of agricultural animals. The trunk kidney consistently accumulated higher concentrations of mercury than any of the other tissues investigated.     

Effect of cadmium on ATPase activities in rats fed on iron-deficient and sufficient diets

Male Sprague-Dawley rats were fed on different levels of cadmium mixed with purified diets containing iron or no iron for 8 weeks. The body weight gain, tissue weights, hemoglobin, hematocrit, liver and brain ATPase were measured at 2, 4 and 8 weeks after feeding. The hemoglobin and hematocrit values were the same in all rats fed on cadmium. The rats fed on iron-deficient diets mixed with cadmium showed a significant decrease in body weight gain. However, the rats receiving only the 100 ppm of cadmium in iron-sufficient diet showed a significant decrease in body weight gain. There were no significant changes in the weights of thymus, spleen, kidney, heart, brain and testes. However, the liver weights were decreased in the highest treatment of cadmium but the liver weight/body ratios were uneffected. Na+-K+ activated ATPase activity in brains of rats fed on cadmium were significantly decreased at 2, 4 and 8 weeks of treatment. The decrease was more pronounced in rats fed on iron-deficient diets. Oligomycin-sensitive (Mitochondrial) Mg2+ ATPase activity was also significantly decreased in liver and brain tissues of rats fed on cadmium. Oligomycin-insensitive Mg2+ ATPase activity, however, was not altered in any tissues tested. It appears that cadmium may be interfering with energy (ATP) production and utilization processes in rat brain and liver tissues.     

低浓度Pb2+、Cd2+对鲫鱼肝脏组织中 HSP70诱导的影响

以鲫鱼(Carassius auratus)作为实验对象,经过40 d Pb2+、Cd2+不同浓度的暴露后,运用SDS-PAGE和Western Blotting方法检测鱼肝脏组织内应激蛋白HSP70的诱导表达情况.结果表明,在实验浓度下,与对照组相比,Pb2+、Cd2+对鱼肝脏内HSP70有显著的诱导(P＜0.05),但在Cd2+浓度为0.2 mg/L有停止表达HSP70的现象,可能是由于浓度过高造成了组织病理损伤而破坏了诱导表达机制.实验还发现,在实验浓度低于国家渔业用水标准时,HSP70仍然表现为明显诱导(P＜0.05),充分说明运用分子生物学指标要比传统的环境检测指标敏感,具有对污染物早期预警的作用.     

Long-term effect of temperature on bioaccumulation of dietary metals and metallothionein induction in Sparus aurata

Previous studies have demonstrated that the commercial feed of aquacultured fish contains trace amounts of toxic and essential metals which can accumulate in tissues and finally be ingested by consumers. Recently rising temperatures, associated to the global warming phenomenon, have been reported as a factor to be taken into consideration in ecotoxicology, since temperature-dependent alterations in bioavailability, toxicokinetics and biotransformation rates can be expected. Sparus aurata were kept at 22 °C, 27 °C and 30 °C for 3 months in order to determine the temperature effect on metallothionein induction and metal bioaccumulation from a non-experimentally contaminated commercial feed. A significant temperature-dependent accumulation of cadmium (Cd), copper (Cu), mercury (Hg), zinc (Zn), lead (Pb) and iron (Fe) was found in liver, together with that of manganese (Mn), Fe and Zn in muscle. Hg presented the highest bioaccumulation factor, and essential metal homeostasis was disturbed in both tissues at warm temperatures. An enhancement of hepatic metallothionein induction was found in fish exposed to the highest temperature.     

Biochemical effects of clomazone herbicide on piava (Leporinus obtusidens)

This study aims to verify the effects of the clomazone concentration used in rice fields on acetylcholinesterase (AChE), thiobarbituric acid reactive substances (TBARS), protein carbonyl and catalase activity in tissues of piava (Leporinus obtusidens). LC(50)-96h was 5.0 mg L(-1) and the fish were exposed to 1/10 of LC(50)-96 h: 0.5 mg L(-1) of clomazone for 96 and 192h. The same parameters were also assayed after a recovery period of 192 h in clean water. AChE activity was reduced only in the brain and heart of fish exposed for 96 h. AChE activity was decreased in the brain, muscle and heart tissues after 192 h of exposure. After 192 h of recovery period, AChE activity remained diminished in brain and muscle and showed a decrease in eye. However, after 192 h of recovery, AChE activity in heart was recovered. Fish showed increased TBARS levels in brain at all experimental periods. TBARS levels decreased in liver and muscle tissues after 192 h of exposure. The increase in muscle TBARS persisted in fish transferred to clean water. Protein carbonyl in the liver was increased in all periods studied including the recovery period. Catalase activity was reduced during all periods. The present study demonstrates the occurrence of disorders in AChE, TBARS, protein carbonyl and catalase activity in piava. The results also show changes in fish after exposure to an environmentally relevant concentration of clomazone. Most effects observed persisted after the recovery period. Thus, these parameters may be used to monitor clomazone toxicity in fish.     

Environmental cadium exposure and metabolic responses of the Nile tilapia, Oreochromis niloticus

The contamination of water by metal compounds is a worldwide environmental problem. This study was undertaken to evaluate the impact of short-term cadmium exposure on metabolic patterns of the freshwater fish Oreochromis niloticus. The fish were exposed to 320, 640, 1,280 and 2,560 microg/l sublethal concentrations of Cd++ (CdCl2) in water for 7 days. The specific activities of the enzymes phosphofructo kinase (PFK-E.C.2.7.1.11.), lactate dehydrogenase (LDH-E.C.1.1.1.27.) and creatine kinase (CKE.C.2.7.3.2.) were decreased in white muscle after cadmium treatments, indicating decreases in the capacity of glycolysis in this tissue. Cadmium exposure induced increased glucose concentration in white muscle of fish. On the other hand, cadmium exposure at sublethal concentrations increased phosphofructo kinase and LDH in red muscle of fish. Cadmium significantly decreased total protein concentrations in liver and white muscle regardless of tissue glycogen levels. The data suggest that cadmium acts as a stressor, leading to metabolic alterations similar to those observed in starvation.     

Uptake and accumulation of mercury from dental amalgam in the common goldfish,Carassius auratus

In this study, the bioavailability and accumulation of mercury from external environmental exposure to mixed, cured, milled, sieved and proportioned dental amalgam was examined in the common goldfish, Carassius auratus. Fish were exposed to dental amalgam (particle size range from < 0.10 to 3.15 mm) in order to represent the particle size and distribution of that found within the typical dental office wastewater discharge stream. Experimental amalgam water loadings were 0 g/l, 0.5 g/l and 1 g/l in glass aquaria at 15 degrees C for 28 days. Fish tissues were sampled at 5 min and 28 days of exposure, and the liver, brain, muscle and whole body analyzed for total mercury using cold vapor atomic fluorescence spectroscopy. Mercury was found in several tissues examined and generally increased with exposure to higher amounts of dental amalgam. The highest levels were found in the whole body (17.68 +/- 5.73 microg/g) followed by the liver (0.80 +/- 0.16 microg/g) and muscle (0.47 +/- 0.16 microg/g). The lowest concentrations were seen in the brain (0.28 +/- 0.19 microg/g). Compared to controls, concentrations in the whole body, muscle and liver in fish exposed for 28 days to the highest concentration of amalgam were 200-, 233-, and 40-fold higher, respectively. This study shows that mercury from an environmental exposure to representative samples of dental amalgam typically found within the dental wastewater discharge stream is bioavailable to fish and may accumulate in internal tissues.     

Biological effect monitoring in dab (<Emphasis Type="Italic">Limanda limanda</Emphasis>) using gene transcript of CYP1A1 or EROD—a comparison

BACKGROUND, AIM, AND SCOPE: Gene expression analyses with real-time (RT)-polymerase chain reaction (PCR) gains importance in marine monitoring. This new technique has to be compared to the classical approaches like the well known biomarker ethoxyresorufin-O-deethylase (EROD) to test their suitability for monitoring programmes. The goal of the present study is to compare EROD activity and CYP1A1 mRNA expression in the important monitoring fish species dab (Limanda limanda) and to answer the question of whether these parameters reflect the polycyclic aromatic hydrocarbon (PAH) contamination of the fish. Further on, glyceraldehyd-3-phosphate dehydrogenase (GAPDH) was investigated as a potential housekeeping gene. MATERIALS AND METHODS: Female dab were caught in the summer of 2004 in the North Sea and in the Baltic. EROD activity was determined in liver samples by a kinetic fluorimetric assay according to a standard protocol. The gene expression of CYP1A (cytochrome P450 1A) and GAPDH were determined by means of RT-PCR. Results were compared to gonado somatic index and to the concentration of PAH metabolite 1OHPyr (1-hydroxypyrene) analysed in the bile fluids of the fish, respectively. RESULTS: Dab from all stations showed a considerable individual variation in the levels of both CYP1A mRNA and EROD. Highest mean values for CYP1A mRNA and EROD were detected in the northern part of the sampling area. In contrast, the PAH metabolite 1OHPyr was found at the highest concentration in fish caught near the German coast. CYP1A mRNA and EROD showed only a minor but significant correlation (r = 0.32, p < 0.05, n = 123). 1OHPyr in bile correlated significantly (p < 0.05) with the amount of GAPDH mRNA content in the liver. DISCUSSION: The significant but low correlation of CYP1A mRNA and EROD activity on an individual basis illustrates that these two parameters are apparently not closely linked. However, maximum EROD values correspond with maximum CYP1A mRNA concentrations when station means are regarded. Because EROD and CYP1A mRNA in dab follow different physiological principles, their application will lead to related but not identical monitoring results. This should be taken into account when future marine monitoring programmes are designed. The results also indicate that PAH are not the crucial factor for CYP1A and EROD levels in dab from the off-shore areas in the North Sea. This is remarkable because the PAH metabolism is known to be CYP1A-dependent and the widely used biomarker EROD has been recommended for monitoring PAH-related effects in fish from the North Sea. Due to a correlation between GAPDH and 1OHPyr, GAPDH was not suitable as housekeeping gene for dab. CONCLUSIONS: Neither the results from EROD nor from CYP1A1 mRNA measurements in dab reflected their exposure to PAH as measured by the PAH metabolite 1OHPyr. Thus, the question arises of whether EROD or CYP1A mRNA is a suitable biomarker at all to indicate PAH exposure in dab from the open North Sea. RECOMMENDATIONS AND PERSPECTIVES: For future biological effect monitoring, it is advisable to measure more and predominately independent parameters by RT-PCR and to incorporate more components of the detoxification system.