Effects of mercury on the feeding behavior of the mummichog, Fundulus heteroclitus from a polluted habitat

Fish living in contaminated environments may exhibit reduced growth which may be due, in part, to the impact of toxicants on prey capture ability. We studied the effects of methylmercury (meHg) and mercuric chloride (Hg) on feeding of Fundulus heteroclitus from Piles Creek, a polluted habitat in Linden, New Jersey. We found that exposure to 0·01 mg/liter of meHg for one week had less impact on the prey capture rate than exposure to a similar concentration of Hg. Exposure to 0·01 mg/liter for two weeks or to 0·02 mg/liter for one week, of either meHg or Hg, produced significant reductions in feeding rate, compared to controls. In general, the fish were affected to a greater degree by exposure to Hg than to meHg. This is the reverse of the usual order of toxicity, but is in keeping with previous observations on this population, in which we have noted that gametes and embryos had developed tolerance to meHg, but were more susceptible to Hg.     

Regulation of Tilapia metallothionein gene expression by heavy metal ions

Tilapia is a common fish species inhabiting inland waters and estuarine regions in Hong Kong and Southeast Asia, and useful for bio-monitoring of metal pollution. Metallothionein (MT) gene expression in fish tissues has been useful to sub-lethal risk assessment as biomarker of exposure to metal ions in fishes inhabiting metal contaminated area. To investigate metal inductions of Tilapia MT gene expression in vivo, Tilapias were injected with different concentrations of heavy metals and tissues were then removed for quantitative PCR assay using mimic PCR methods. All of the metal ions tested (Cu²⁺, Cd²⁺, Hg²⁺, Ni²⁺, Pb²⁺ and Zn²⁺) were able to induce hepatic MT mRNA levels. Renal MT mRNA levels of Cd²⁺ and Zn²⁺ treated fish was not induced with significant fold induction, however MT mRNA levels in gills were sensitive to the administrations of these metal ions. These data indicated that Tilapia MT mRNA levels in gills and liver are sensitive biomarker of exposure to various metal ions.     

A rapid change in methylmercury tolerance in a population of killifish,Fundulus heteroclitus,from a golf course pond

Embryos of killifish (Fundulus heteroclitus) from Bullhead Bay, Southampton, New York, showed considerable variation in tolerance to methylmercury (meHg) in 1980, 1981 and 1983, in that some females produced eggs that were tolerant to the teratological effects of 0·05 ppm, while other females produced susceptible eggs. However, in 1982, over 40% of the females produced clutches of non-viable eggs (which could not be tested for meHg tolerance) and those fish which produced viable eggs generally produced tolerant ones. After hatching, the larvae were also more tolerant. Accompanying the shift in meHg tolerance was a trend of increasing fin ray count of the females, a parameter which has previously been correlated with the production of more tolerant embryos. In 1982, a nearby population exhibited a distribution of embryonic and larval tolerance and fin ray count comparable with that of the first population in the other years.We hypothesize that the unusually high rainfall in June, 1982 caused an inflow of pesticides and heavy metals from a golf course adjacent to the first site, and that the contaminants were responsible for the striking changes in reproductive success and meHg tolerance. Residues of chlorinated hydrocarbon insecticides were found in fish of the first, but not of the second, population. The variability in the original population may have permitted a segment of it to withstand the inflow of pollutants, while the susceptible individuals produced non-viable eggs. This may have been the cause of the rapid shift of overall tolerance in the population.     

Inhibition of essential amino acid absorption in marine fishes by mercury

In polluted coastal waters marine fish can concentrate heavy metals in the intestine two or more orders of magnitude above those found in the water column and several-fold higher than the sediment level. These accumulations are primarily due to ingestion of contaminated food and drink. Relatively long residence time of gut content, low permeability coefficient of divalent cations through the lipid bilayers of membranes and profuse binding to the negative charge sites on the mucosal side of the gut, all combine to increase the concentration of heavy metals in the lumen. Do these high levels of heavy metals interfere with the normal processes of nutrient absorption? We have measured the effect of HgCl₂ and CH₃HgCl on the absorption of the essential amino acids, l-leucine, l-isoleucine, l-lysine and l-methionine, by the intestine of Fundulus and toadfish in vitro and in vivo. At concentrations expected in the gut, HgCl₂ can inhibit 20–80% of the leucine absorption; CH₃HgCl is less potent in this respect. While the action of Hg²⁺ appears to be limited to the luminal side, the more lipophilic CH₃Hg⁺ rapidly penetrates the epithelial cell layer and can inhibit the intracellular energy production needed for transport. The implications of these findings for growth and survival of marine fishes are discussed.     

青蛤(Cyclina sinensis)金属硫蛋白及硫氧还蛋白基因的克隆与表达分析

利用构建的cDNA文库及高通量测序方法,获得金属硫蛋白及硫氧还蛋白基因全长。结果表明,青蛤金属硫蛋白和硫氧还蛋白的cDNA全长分别为776bp和804bp,金属硫蛋白基因编码74个氨基酸,包含7个CXC特征结构;硫氧还蛋白基因编码165氨基酸,包含5个氨基酸构成的活性中心。采用实时定量PCR方法分析了两种基因在Cd2+胁迫下的表达变化,结果显示,金属硫蛋白基因在Cd2+胁迫下6—12hmRNA表达量急剧上升,与对照组有显著性差异(P<0.01)。硫氧还蛋白基因在Cd2+胁迫下mRNA在6h明显升高,在6—24h与对照组出现显著性差异(P<0.05)。说明Cd2+能够诱导青蛤金属硫蛋白和硫氧还蛋白基因表达的时序性升高,二者的转录过程反映了贝类抵御重金属胁迫的分子调控过程。     

Mycobacteria, but not mercury, induces metallothionein (MT) protein in striped bass, Morone saxitilis, phagocytes, while both stimuli induce MT in channel catfish, Ictalurus punctatus, phagocytes

Recent advances in molecular immunology indicate that the expression of inducible pro-inflammatory proteins is increased in vertebrates in response to both infectious disease agents and various xenobiotics. For example, iNOS, COX-2, and CYP1A are induced by both inflammation and AhR ligands. Moreover, the expression of these proteins in response to stimuli varies among individuals within populations. Little is known of the differences among fish in the inducibility of proinflammatory proteins in response to both infectious agents and xenobiotics. Through random screening of a striped bass, Morone saxitilis, peritoneal macrophage cDNA library, a full length metallothionein (MT) gene was cloned and sequenced. MT is a low-molecular weight (6–8 kDa), cysteine-rich metal binding protein. Metals are required by pathogenic bacteria for growth, and by the host defense system by serving as a catalyst for the generation of reactive oxygen intermediates (ROIs) by phagocytes. A recombinant striped bass MT (rMT) was expressed and purified, then used to generate a specific mAb (MT-16). MT protein expression was followed in freshly isolated striped bass and channel catfish, Ictalurus punctatus, phagocytes after in vitro exposure to the naturally occurring intracellular pathogen Mycobacteria fortuitum or to 0.1 and 1 μM mercury (Hg), as HgCl₂. MT expression was increased by 24 h in both channel catfish and striped bass phagocytes as a result of exposure to M. fortuitum cells. On the other hand, MT was induced by Hg in channel catfish cells, but not those of striped bass. These results indicate that metal homeostasis in phagocytes is different between catfish and striped bass. In addition, these data suggest that care should be taken to distinguish between inflammation-induced vs. metal-induced MT when using MT expression as a biomarker of metal exposure.     

汞胁迫对拟穴青蟹(Scylla paramamosain) 主要消化和免疫因子的影响

采用实验生态学方法,开展了汞胁迫下拟穴青蟹(Scylla paramamosain)的消化和免疫因子变动的研究。在实验室条件下,测定了0.00、0.005、0.01、0.02、0.04、0.08mg/L浓度Hg2+在1d、3d、5d、7d、9d胁迫时间下的消化和免疫因子变化。结果表明,汞胁迫下拟穴青蟹AMS活性快速升高,但激活效应随胁迫时间延长而逐渐下降,后期表现为抑制效应。汞胁迫能激发Pepsin活性,短期内Pepsin活性和胁迫浓度、胁迫时间呈正相关;长期高浓度胁迫(0.04、0.08mg/L),则激发效应减弱。汞胁迫对LPS活性的影响主要表现为抑制作用。免疫因子的研究结果表明,汞胁迫对AKP有激发作用,可迅速刺激机体AKP活力上升。低浓度汞胁迫对AKP活性提升最显著(0.005、0.01、0.02mg/L),高浓度组AKP活性先升后降。汞胁迫对ACP活性有激发作用,且存在显著的时间效应和浓度效应。汞胁迫下SOD活性短期内即显著升高,且SOD活力随胁迫时间延长而持续升高。汞胁迫对LZM活性有激活作用,且激活效应存在时间效应和浓度效应。汞胁迫对PO活力快速产生抑制作用,抑制作用存在时间效应而无显著的浓度效应。汞胁迫对拟穴青蟹消化和免疫因子能产生胁迫效应,对Pepsin、AMS、AKP、ACP、SOD、LZM表现为激发,而对LPS、PO表现为抑制。     

菲律宾蛤仔(Ruditapes philippinarum)金属硫蛋白基因克隆及Cd~(2+)胁迫下的表达分析

金属硫蛋白(Metallothionein,MT)是一类富含半胱氨酸的小分子蛋白质,参与机体重金属解毒和金属元素代谢等生理过程。本研究采用RACE技术,克隆获得了菲律宾蛤仔(Ruditapes philippinarum)金属硫蛋白(RpMT)的全长cDNA序列。RpMT的cDNA全长为570bp,编码75个氨基酸,包含15个MT所特有的Cys-Xn-Cys结构。采用实时荧光定量PCR技术,分析了两种壳色菲律宾蛤仔(白蛤和斑马蛤)RpMT基因在Cd2+暴露后的表达变化。结果发现:Cd2+急性和亚慢性暴露均可导致两种壳色蛤仔消化腺和鳃组织RpMT基因表达量的显著上调;暴露后两种壳色蛤仔鳃组织RpMT基因表达量的增加幅度均高于消化腺组织,且以白蛤鳃组织基因表达水平的上调幅度较高。上述结果表明,RpMT可能在菲律宾蛤仔抵御Cd2+胁迫过程中发挥了重要作用。     

Effects of heavy metals on phagocytosis by Molluscan hemocytes

The effects of ten heavy metal cations on phagocytosis of polystyrene latex spheres by hemocytes of the American oyster, Crassostreavirginica, were investigated. Exposure to 1 and 5 ppm Cd²⁺, 5 ppm Co²⁺, 1 ppm Cr³⁺, 1 ppm Cu²⁺, 0·5 ppm Fe³⁺, 0·05 ppm Hg²⁺, 1 and 5 ppm Mn²⁺, 1 and 5 ppm Pb²⁺, 1 ppm Sn²⁺, and 1 and 5 ppm Zn²⁺ resulted in no alteration of phagocytic ability. However, exposure to 1 ppm Co²⁺, 5 ppm Cu³⁺, 5 ppm Cu²⁺, 1 and 5 ppm Fe³⁺, 0·1 ppm Hg²⁺ and 5 ppm Sn²⁺ resulted in significantly enhanced uptake of polystyrene spheres. Finally, exposure to 0·5, 1 and 5 ppm Hg²⁺ resulted in inhibition of phagocytosis and increased cell death.     

Effects of the duration of exposure to mercuric chloride on the embryogenesis of the estuarine teleost, Fundulus heteroclitus

Embryos of Fundulus heteroclitus in the 4–8 cell stage were exposed nine Hg⁺⁺ (as mercuric chloride) concentrations from 0 to 100 μg/litre (ppb) for 32 days, 5 days, 2 days and 1 day. All but the chronically (32-day) exposed embryos were allowed to continue development in mercury-free water (20% S) after the exposure period to determine the effect of duration of exposure on three parameters of development. Ninety-six hour survival, total hatching success and per cent incidence of lateral spinal curvature were measured. Only embryos exposed to mercury for a single day showed a significant increase in survival in comparison with embryos exposed for a longer period of time after four days of development. Under all conditions of exposure, survival was reduced at concentrations greater than 40 ppb Hg⁺⁺. Hatching success of chronically exposed embryos was significantly reduced at concentrations greater than 10 ppb Hg⁺⁺. Significant increases in total hatchability were effected by reducing the duration of exposure to five days and one day. Of eleutheroembryos emerging following chronic exposure to mercury, increases in the incidence of lateral spinal curvature were noted at all concentrations above 20 ppb Hg⁺⁺. Significant reductions of this disorder were observed among eleutheroembryos exposed to mercury for 5 days, 2 days or 1 day. Of the three parameters measured, the incidence of lateral spinal curvature and the resulting VH₅₀ value gave a more sensitive evaluation of the embryotoxic effects of Hg⁺⁺ as mercuric chloride on the estuarine teleost, Fundulus heteroclitus.     

Complexation of dimethylsulfide with mercuric ion in aqueous solutions

The tendency of dimethylsulfide (DMS) to form complexes with heavy metal ions in aqueous solutions and the factors that influence it have been investigated. Among five heavy metal ions examined (Cu²⁺, Cd²⁺, Zn²⁺, Pb²⁺ and Hg²⁺), only Hg²⁺ bound significantly with DMS in aqueous solutions in which Hg²⁺ concentration was increased to much higher levels than that of natural seawater. The complexation capacity of Hg²⁺ for DMS was influenced by pH and media. The affinity of Hg²⁺ for DMS was generally lower at high than at low pH, presumably due to the competition of hydroxide ion to form hydroxomercury species. In different solutions, the affinity of Hg²⁺ for DMS followed the following sequence: ultra-purified water > 35‰ NaCl solution > seawater. It seems apparent that chloride had a negative impact on the complexation of DMS by Hg²⁺, owing to the competition of chloride with DMS for complexing Hg²⁺. In addition, the affinity of Hg²⁺ for DMS in the bulk seawater appeared to be higher than that in the surface microlayer seawater. The tendency of Hg²⁺ to form complexes with DMS in aqueous solution can be reduced by the presence of 2 mM amino-acid such as glycine, alanine, serine and cysteine, as these ligands give stable mercury complexes. However, the presence of 2 mM acetate in experimental solutions had no significant effect on the complexation of Hg²⁺ with DMS, even though this ligand has a relatively strong complexing capacity for Hg²⁺. Although mercury ions appeared to have a strong affinity for DMS, the concentration of mercury in seawater is too low to produce a great effect on the distribution of DMS in oceans.     

镉和汞两种重金属离子对四角蛤蜊的急性毒性

采用毒理学实验方法,比较研究了Cd~(2+)和Hg~(2+)对四角蛤蜊(Mactra veneriformis Reeve)急性毒性效应的差异,以期为进一步评价重金属离子对四角蛤蜊的毒性效应和作用机理等提供理论依据.死亡概率单位和实验液浓度对数的线性回归分析表明,Cd~(2+)对四角蛤蜊在24、48和96 h的半致死浓度(LC_(50))分别为15.961、5.149和2.383 mg/L,Hg~(2+)的LC_(50)分别为3.714、0.607和0.207 mg/L;Cd~(2+) 和Hg~(2+) 对四角蛤蜊的安全质量浓度分别为0.023 8 mg/L和0.002 1 mg/L,分别是我国渔业水质标准(GB11607-89)限定量的4.76倍和4.02倍.     

Behavioral effects in mummichog larvae (Fundulus heteroclitus) following embryonic exposure to methylmercury

Embryos of the mummichog, Fundulus heteroclitus, were exposed to concentrations of methylmercury (meHg) below those that can cause morphological abnormalities. After hatching, larvae were kept in clean water and tested periodically for ability to capture prey (Artemia salina) or for their swimming performance and predator avoidance. Embryonic exposure to 10 μg/liter meHg resulted in reduced prey capture ability by early larvae, measured as total Artemia caught in 1 and 5 min. However, older larvae recovered from the behavioral deficits, perhaps owing to a compensation for early CNS insult. Fish from a chronically polluted environment (Piles Creek (PC), a tributary of the Arthur Kill), previously shown to have high tolerance to the teratogenic effects of higher concentrations of meHg, proved to be less susceptible to these behavioral effects than fish from a relatively uncontaminated site (EH). Also, the PC control larvae captured prey far more effectively than the EH larvae, although they made more miscues. When tested for swimming performance after embryonic exposure to 10 μg/liter, EH larvae had increased performance when compared to controls. This effect was also transitory. The improved swimming performance did not correspond to improved ability to avoid predation by grass shrimp (Palaemonetes pugio). The increased swimming performance may correspond to greater total activity, making them more likely to attract the predator's attention and resulting in increased capture. Thus, negative, though temporary, effects were observed in both prey capture and predator avoidance after embryonic exposure to meHg.     

Effects of water-borne copper on metallothionein and lipid peroxidation in the marine amphipod gammarus locusta

The aim of the current work was to determine over 10 days the effects of water-borne exposure of the marine amphipod Gammarus locusta to 4 μgCu l⁻¹ on the metallothionein (MT; measured by differential pulse polarography) protection system and lipid peroxidation (LP; thiobarbituric acid-reactive malondialdehyde equivalents) as a measure of oxidative damage. MT levels in exposed animals increased significantly at day 2 (36% > control; P<0.001) and remained high at days 6 and 10 (55 and 38%, respectively, P<0.001). The maximum level of MT at day 6 coincided with the highest Cu body-burden. LP increased within 1 day of exposure, indicative of Cu as an oxidative stressor. However, in contrast to MT, the highest LP level was seen at day 4 (68% > control, P<0.001) before returning to control values by day 6, indicating a protective role of MT against the pro-oxidant effects of Cu.     

Alterations in hepatic metallothionein content in perch, Perca fluviatilis, environmentally exposed to cadmium

During recent years laboratory studies have demonstrated the presence of metallothionein in several species of teleosts.^1–3 The role of metallothionein (MT) in protection against cadmium toxicity has been investigated by exposing fish to high levels of cadmium in the water.^4–6 So far, however, few field studies have focused on the existence of MT and its role in subcellular distribution of cadmium.⁷ In the present study the synthesis of MT as a possible mechanism by which perch adapt to cadmium contamination was investigated in a wild population of perch. An increased MT content of perch liver was found to correlate to an increase in cadmium present in the liver.     

Factors influencing the oxidation,reduction, methylation and demethylation of mercury species in coastal waters

The objective of this study was to examine the redox reactions and other transformations of mercury (Hg) species in surface waters, and the factors determining the rates of these reactions. For the redox studies completed at the Chesapeake Biological Laboratory (CBL), two isotopes (¹⁹⁹Hg^II and ²⁰²Hg⁰) were added into different types of filtered water (fresh to seawater) to examine the oxidation and reduction reactions. Further studies of both the redox reactions and methylation/demethylation reactions of Hg were conducted with unfiltered water on board research vessels during cruises in May and July 2005 on the Chesapeake Bay and shelf. While CH₃¹⁹⁹Hg^II was added to allow the examination of demethylation, ²⁰¹Hg^II was used to examine both reduction and methylation, and ²⁰²Hg⁰ was used to examine oxidation. Overall, the results showed that both Hg oxidation and reduction were simultaneously occurring and were photochemically mediated in the waters investigated. In contrast to the previously assumed “unreactive” nature of Hg⁰, the studies found that the magnitude of the rate constant for Hg⁰ oxidation was greater than that for reduction, indicating its importance in estuarine and coastal waters. In addition, both experiments at CBL and on board ship showed that Hg^II reduction was similar in magnitude, suggesting that biotic processes were relatively unimportant. While no measurable methylation occurred during the incubation period during the on board studies, concentration of CH₃¹⁹⁹Hg^II decreased over the time during the experiments. It appeared that the demethylation processes were not dominantly photochemically driven, but could be microbially mediated. Further studies are needed in order to help better understand Hg redox and transformations in natural water systems.     

Inhibitory effects of divalent metal ions on liver microsomal 7-ethoxyresorufin O-deethylase (EROD) activity of leaping mullet

The purpose of the present study was to elucidate in vitro effects of Hg²⁺, Zn²⁺, Ni²⁺ and Cd²⁺ on cytochrome P4501A1 (CYP1A1) dependent EROD activities in leaping mullet liver microsomes. Fish captured from the most polluted part of Izmir Bay, had highly elevated EROD activities, and induced CYP1A1 protein levels as determined by Western blotting. Although all of the metal ions caused inhibition of the initial velocity of the reaction, Hg²⁺ and Cd²⁺ exhibited much higher inhibitory effect at lower concentrations and they were evidently more potent inhibitors than others. The inhibitor concentration giving 50% inhibition (IC₅₀ values) of Zn²⁺, Ni²⁺, Cd²⁺ and Hg²⁺ of initial EROD activity were 107, 16, 1.3 and 0.15 micromolar, respectively. Glutathione (GSH) at 0.5 mM final concentration, completely reversed Ni²⁺ and Cd²⁺ inhibition of EROD activity indicating the protective action of GSH.     

4种重金属离子对彩虹明樱蛤(Moerella iridescens)的急性致毒效应

以彩虹明樱蛤养殖壳高优势组(平均壳高为17.35±1.01mm)为实验动物,进行4种重金属离子Hg2 、Cr6 、Cu2 、Zn2 对彩虹明樱蛤的急性毒性和加和等毒性强度联合毒性试验。结果表明,彩虹明樱蛤对重金属的耐药性均随实验时间延长而缓慢降低,4种重金属离子毒性大小依次为Cu2 、Hg2 、Zn2 、Cr6 ,其对彩虹明樱蛤96h的半致死质量浓度分别为0.0554mg/L、0.1099mg/L、2.2605mg/L、19.6277mg/L;Hg2 、Cr6 、Cu2 、Zn2 离子两两组合在加和等毒性强度下对彩虹明樱蛤96h联合急性毒性所呈现的致毒特征与离子间毒性强度匹配情形密切相关。     

Behavior and brain chemistry correlates in mummichogs (Fundulus heteroclitus) from polluted and unpolluted environments

The ability of Fundulus from a polluted environment to capture a prey organism, the grass shrimp (Palaemonetes pugio), was compared to that of Fundulus from an unpolluted reference site. Fish from the unpolluted site took significantly more prey (p < 0.1) during the first 5–10 min of each trial than fish from the polluted site.Analysis of the neurotransmitters dopamine (DA) and serotonin (5-HT) and their metabolites, DOPAC, homovanillic acid (HVA), and 5-hydroxy-indolacetic acid (5-HIAA) in cerebellums and medullas was performed by HPLC. Mummichogs from the polluted site had significantly lower concentrations of 5-HT and its metabolite 5-HIAA in their medullas, but not cerebellums, than fish from the reference site (p < 0.05). Concentrations of DA, DOPAC and HVA were not different between populations in either part of the brain. Neurotransmitter concentrations were shown to be independent of size and therefore, presumably, age.Analysis of Hg in livers and brains of these mummichogs by atomic absorption spectrophotometry showed significant differences between the two populations for both organs. Earlier work showed that the behavioral deficit could be elicited by Hg; thus, this was the putative neurotoxic agent. However, there are other candidates which may contribute to neurotoxicity in the study site.     

The effects of silver nanoparticles on oyster embryos

Nanoparticles may be introduced into aquatic environments during production processes and also as a result of release following their use in various commercial formulations and biologic applications. Filter-feeding bivalve mollusks such as oysters are valuable model species for characterizing nanoparticle bioavailability and interactions with basic cellular processes. The adults release their gametes into the environment, so their embryos and larvae are also likely targets of nanoparticles. The purpose of these studies was to characterize the toxicity of metal nanoparticles on embryonic development of oysters, Crassostrea virginica and to compare the relative sensitivity of embryos to adults. Newly-fertilized oyster embryos were exposed to silver nanoparticles (AgNP) and then the percent normal development after 48 h was assessed. Studies were conducted with adult oysters in which they were also exposed to AgNP for 48 h, and the effects on lysosomal destabilization were determined. The expression of metallothionein (MT) gene expression was also assessed in both embryos and adults. Adverse effects on embryonic development were observed at concentrations similar to those that caused both statistically and biologically significant effects on lysosomal destabilization of adults. Significant increases in MT mRNA levels were observed in both embryos and adult oysters, and MT levels were highly induced in embryos. While we do not know whether the toxicity and gene expression responses observed in this study were due to the nanoparticles themselves or the Ag ions that dissociated from the nanoparticles, these kinds of basic studies are essential for addressing the potential impacts of nanoengineered particles on fundamental cellular processes as well as aquatic organisms.