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.     

Kinetics of hepatic phase I and II biotransformation reactions in eight finfish species

Hepatic microsomes and cytosols of channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), red tilapia (Oreochromis sp.), largemouth bass (Micropterus salmoides), striped bass (Morone saxatilis), hybrid striped bass (M. saxatilis × M. crysops), and bluegill (Lepomis macrochuris) (n = 8) were used to study the kinetics of phase I (ECOD, EROD, PROD, BROD) and phase II (UDP-glucuronosyltransferase (UDPGT)-, sulfotransferase (ST)- and glutathione-s-transferase (GST)-mediated) reactions. The best catalytic efficiency for ECOD and GST activities was performed by channel catfish, Atlantic salmon, rainbow trout and tilapia. The highest EROD catalytic efficiency was for Atlantic salmon. None of the species had either PROD or BROD activities. Rainbow trout had very similar UDPGT catalytic efficiency to tilapia, channel catfish, Atlantic salmon, largemouth bass and bluegill. Sulfotransferase conjugation had no significant differences among the species. In summary, tilapia, channel catfish, Atlantic salmon and rainbow trout had the best biotransforming capabilities; striped bass, hybrid striped bass and bluegill were low metabolizers and largemouth bass shared some capabilities with both groups.     

A fish hepatocyte model for the investigation of the effects of environmental contaminants

An isolated fish hepatocyte culture system was developed as a models ystem to investigate the mechanisms of action of environmental contaminants. Hepatocytes were isolated from striped bass (Morone saxatilis) by an adaptation of the two stage perfusion technique of Seglen.¹ The system was used to evaluate metal binding protein (MBP) induction in response to cadmium, a primary inducer of metallothionein (MT) in rat hepatocytes. Striped bass hepatocytes appeared to be refractory to the induction of MBP by cadmium, since there was no significant increase in the synthesis of MBP for any of the doses at any of the time points investigated. However, when a similar experiment was performed using rat hepatocytes there was induction of MBP that was related to both dose and time. These comparative experiments indicate that although there are similarities between the hepatocytes of the two species in regard to ³⁵S incorporation into low molecular weight metal-binding proteins, there appear to be significant quantitative differences as well in regard to MBP kinetics. This in vitro model system could potentially be utilized to investigate the toxicological properties of other environmental contaminants.     

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 (Cu(2+), Cd(2+), Hg(2+), Ni(2+), Pb(2+) and Zn(2+)) were able to induce hepatic MT mRNA levels. Renal MT mRNA levels of Cd(2+) and Zn(2+) 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.     

Bioaccumulation and trophic transfer of mercury in striped bass (Morone saxatilis) and tautog (Tautoga onitis) from the Narragansett Bay (Rhode Island,USA)

We examined the bioaccumulation and trophic transfer of mercury in two marine finfish species, striped bass (Morone saxatilis) and tautog (Tautoga onitis), collected from the Narragansett Bay (Rhode Island, USA). For each of these target fish, white muscle tissue was analyzed for total mercury (Hg) and results were evaluated relative to fish age, body size, and Hg content of preferred prey. Dietary and stable isotope analysis was also used to elucidate the effect of trophic processes on Hg concentrations in fish. The Hg content of muscle tissue was positively correlated with fish age and length for both species, although striped bass accumulated Hg faster than tautog. Accelerated Hg bioaccumulation in striped bass is consistent with its high trophic level (trophic level = 4.07) and Hg-enriched prey (forage fish and macrocrustaceans; mean Hg content = 0.03 mg Hg kg wet wt^?1). In contrast, tautog maintain a lower trophic status (trophic level = 3.51) and consume prey with lower Hg levels (mussels and crabs; mean Hg content = 0.02 mg Hg kg wet wt^?1). Despite differences in Hg bioaccumulation between target fish, the mean Hg concentration of tautog exceeded levels in striped bass (0.24 and 0.16 mg Hg kg wet wt^?1, respectively) due to a disparity in age-at-catch between sampled groups (mean age of tautog and bass = 11.3 and 4.3 yr, respectively). Taking into account legal minimum catch lengths further revealed that 75.0% of legal-size striped bass (>70.2 cm TL; n = 4) and 44.8% of tautog (>40.6 cm TL; n = 29) had Hg levels beyond the US EPA regulatory threshold of 0.3 mg Hg kg wet wt^?1. Moreover, Hg-length relationships suggest that each target fish meets this threshold near their minimum legal catch length. Our findings reiterate the value of species ecology to improve predictions of fish Hg and permit better management of human contamination by this important dietary source.     

Comparative vitellogenic responses in three teleost species: extrapolation to in situ field studies.

Induction of vitellogenin (VTG) was compared among three teleostean species to determine their relative sensitivity of exposure to 17 beta-estradiol (E2). Japanese medaka (Oryzias latipes), sunshine bass (Morone saxatalis x Morone chrysops) and channel catfish (Ictalurus punctatus) were exposed to aqueous concentrations of E2 ranging from 10 to 100,000 ng/l for 21 days. Respective EC50 values for plasma VTG detected by western blot in medaka, catfish and bass were 200, 170 and 1560 ng E2/l. Since these EC50 values are based on VTG induction curves calculated relative to control values, they indicate differences in species' sensitivity to E2 exposure. Catfish and bass VTG responses obtained in laboratory exposures were compared to VTG responses previously observed with 21-day wastewater treatment plant effluent exposures. Plasma VTG induction in effluent-exposed fish ranged from 14 to 82% above reference values depending on species. Extrapolation of field responses with laboratory-exposed fish indicate catfish and bass were exposed to the equivalent of 27-240 ng E2/l in sewage effluent.     

Influence of capture stress, salinity and reproductive status on zinc associated with metallothionein-like proteins in the livers of three marine teleost species

The influence of capture stress, salinity and reproductive status on zinc associated with the metallothionein-like (MT-like) proteins in the livers of three marine teleost species was investigated. In striped mullet, Mugil cephalus L., the zinc associated with the MT-like protein, as determined by gel-permeation HPLC, increased significantly from approximately 102 to 1162 nmol zinc bound/g wet wt 7 days following capture in the field, handling, transportation to the laboratory and transfer to holding tanks. Significant increases were also observed following exposure to reduced salinity for 7 days. Smaller increases were observed in cultured red drum, Sciaenops ocellatus, livers following exposure to reduced salinity and physical trauma for 7 days. Finally, there was approximately a 2·5-fold increase in the zinc associated with the MT-like protein in livers of female spotted seatrout, Cynoscion nebulosus, collected during the reproductive season compared to regressed animals. These findings emphasize the need for establishing the natural fluctuations in MT levels as well as the response to common physical environmental variables before measurement of MT can be used reliably as a biochemical indicator of environmental contamination.     

Role of biotransformation in determining aldicarb toxicity in fish

The carbamate pesticide, aldicarb, demonstrates significant acute toxicity in fish, and is readily biotransformed by most organisms studied. In fish, both the cytochrome P450 (CYP) and the flavin monooxygenase systems (FMO) are involved in bioactivating aldicarb to aldicarb sulfoxide, which is a more potent inhibitor of acetylcholinesterase (AChE). Channel catfish (Ictalurus punctatus), along with many other fresh water species, do not express FMO and are relatively resistant to the effects of aldicarb. This project examined the toxicity, AChE inhibition, metabolism, and toxicokinetic of aldicarb in channel catfish, and compared these values with an aldicarb-sensitive species, rainbow trout, which expresses FMO. Studies of in vitro and in vivo aldicarb biotransformation in catfish suggest that a low rate of bioactivation (10 times slower V_max), resulting in less initial conversion to the activated metabolite, aldicarb sulfoxide, may be a contributing factor to resistance of channel catfish to aldicarb toxicity. These data are supported by toxicokinetic and enzyme inhibition studies. This work demonstrates that differences in FMO expression among fish species may have significant influence on toxicity resulting from exposure to some xenobiotics.     

Modelling Kepone in the striped bass food chain of the James River estuary

A mathematical model that computes the accumulation of Kepone in the striped bass food chain of the James River estuary was developed. The purpose of the model was to help understand the relationship of Kepone levels in important fish species to sediment and water column Kepone concentrations and then to address the question of why these levels still exceed Food and Drug Administration limits eight years after discharge ceased. The model considers exposure through diet and respiration at rates based on species bioenergetics. It was successfully calibrated to the Kepone concentrations observed in the period 1976 through 1982 in striped bass, white perch, and Atlantic croaker. The model indicates that for the upper levels of the food chain, diet is the major route of contamination, accounting for 87–88% of the observed concentration in croaker and white perch and 91% of the observed concentration in striped bass. The two Kepone sources; sediment and water column, contribute approximately equally to the croaker and white perch. The water column is more significant for striped bass, being the original source for approximately 60% of the observed body burdens. It was estimated that a criterion requiring Kepone concentrations in fish to be at or below 0·3 μg g^?1 would require dissolved water column and sediment Kepone concentrations to be reduced to somewhere between 3 and 9 ng l^?1 and 13–39 ng g^?1, respectively, depending on the species. Striped bass require the greatest reductions in dissolved water column and sediment Kepone concentrations to somewhere between 3 and 5 ng l^?1 and 13 and 24 ng g^?1, respectively.     

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.     

Marine mammals from northeast Atlantic: relationship between their trophic status as determined by delta13C and delta15N measurements and their trace metal concentrations

The relationship between trophic position through delta13C and delta15N and trace metal concentrations (Zn, Cd, Cu and Hg) was investigated in the tissues of six marine mammal species from the Northeast Atlantic: striped dolphin Stenella coeruleoalba, common dolphin, Delphinus delphis, Atlantic white-sided dolphin Lagenorhynchus acutus, harbour porpoise Phocoena phocoena, white beaked-dolphin Lagenorhynchus albirostris, grey seal Halichoerus grypus stranded on French Channel and Irish coasts. White-beaked dolphins, harbour porpoises, white-sided dolphins, common and striped dolphins display the same relative and decreasing trophic position, as measured by delta15N values, along both the Irish and French channel coasts, reflecting conservative trophic habits between these two places. Hepatic and renal Cd concentrations were significantly correlated to muscle delta13C and delta15N values while Hg, Zn and Cu did not. These results suggest that Cd accumulation is partly linked to the diet while other factors such as age or body condition might explain Hg, Zn or Cu variability in marine mammals. Combined stable isotope and trace metal analyses appear to be useful tools for the study of marine mammal ecology.     

The precautionary approach to non-native fisheries—The case of striped bass in Texas

Fisheries managers have long relied on non-native fish to improve, sustain, or create recreational fishing opportunities, often without adequate consideration of potentially negative ecological consequences. There is growing advocacy for using the precautionary approach to avoid potentially serious or irreversible harm to the environment in the face of scientific uncertainty. This paper uses striped bass (Morone saxitilis) in Texas to examine possible effects that might have resulted had the precautionary approach been applied before stocking began in the 1960s when the species was considered non-native to Texas. The current range of reproducing striped bass populations extends from the North Atlantic Ocean to the U.S.–Mexico border. However, the western extent of the species current range beyond Louisiana is probably the result of stocking begun in 1965 that continues today. The historic range is yet unresolved. The best available information suggests that striped bass are not native to Texas, fish stocking has resulted in beneficial economic impacts, and any negative effects (i.e., harm) are currently unknown. It seems unlikely that application of the precautionary approach would have led to different outcomes in Texas than currently exist. However, the complete striped bass story has yet to be written.     

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.     

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.     

Estrogenic responses to xenobiotics in channel catfish (Ictalwus punctatus)

Several environmentally relevant contaminants are characterized as xenoestrogens by virtue of their ability to induce responses similar to 17β-estradiol (E2). There is concern that exposure to these xenoestrogens may result in endocrine and, thus, reproductive disruption. The objective of the present research was to determine whether xenobiotics known to elicit estrogenic responses in mammals were estrogenic in channel catfish, and if these compounds were capable of altering normal physiological response to E2. Sexually immature catfish were exposed by intraperitoneal injection to E2, suspected xenoestrogens or combination doses of E2 and xenoestrogen. Appearance of vitellogenin (Vg) in serum was used as the bioindicator of estrogenicity; Vg was measured by ELISA 7 days after injection. The ED₅₀ of E2 for the appearance of Vg in blood, 0.6 mg/kg, was used as the positive control. Synthetic estrogens, ethinylestradiol, mestranol and DES were effective in inducing Vg; the antiestrogen tamoxifen inhibited response to E2 when given in a combination dose. Treatment by both methoxychlor and p-nonylphenol resulted in Vg appearance in serum; the doses required were 300 times that of E2, and the vitellogenic response was less when compared to the E2-induced response. In addition, a combination dose of methoxychlor with E2 decreased the magnitude of the response to E2. Other mammalian xenoestrogens, o,p′-DDT, chlordecone, lindane and β-HCH, were not able to produce a vitellogenic response in this study.     

Metallothionein and mercury tolerance in the killifish, fundulus heteroclitus

Synthesis of metallothionein (MT) as a possible mechanism by which Fundulus heteroclitus adapts to heavy metal pollution has been investigated in relation to responses to methylmercuric chloride (meHg) and HgCl₂(Hg²⁺) at different life stages.Clutches of embryos from unpolluted sites exhibit teratogenic responses to a standard dose of meHg or of Hg²⁺ which range from little or no effect (tolerant) to severely affected or almost complete inhibition of development (susceptible). At the time of hatching, untreated embryos from tolerant clutches had twice as much of a protein which co-migrates in SDS-PAGE gels with Fundulus MT as do those of susceptible clutches. This difference could be a mechanism for conveying tolerance to Hg²⁺ and also to meHg if demethylation occurs. However, eggs at the time of deposition had virtually no MT, suggesting that the protein is synthesized too late to provide tolerance in the early stages of development which are critical in the etiology of malformations. Treatment of embryos with non-teratogenic doses of either meHg or Hg²⁺ did not increase the amount of MT-like protein. This may indicate that the thionein gene is already fully active, and would also explain observations that Hg²⁺ pretreatment of embryos does not enhance tolerance to Hg²⁺ in the larval stage.MT, isolated from livers of Hg²⁺- or Cu²⁺-dosed or control adults, is associated with Cu and Zn but not with Hg, although it can bind Hg²⁺in vitro. Fundulus, like other fish, methylates much (ca. 75 %) of its Hg burden, and meHg is considered by some not to react with MT. Fundulus liver has its Hg burden distributed between homogenate sediment (25 %), the cytosol (60 %) and the remainder in lipid. The liver responds to Hg²⁺ treatment by increasing lipid content. This may allow sequestration of some lipid-soluble meHg. Of the cytosolic Hg, most is associated with larger proteins and some (ca. 25 %) is dialyzable.These data suggest that metallothionein is not a significant factor in Hg tolerance in Fundulus adults, and has a questionable role in embryonic and larval tolerance to Hg²⁺ and none to the more toxic meHg.     

Metallothionein and stress protein levels in shore crabs (Carcinus maenas) along a trace metal gradient in the Fal estuary (UK)

A field study was conducted to assess the potential use of metallothionein (MT) and stress proteins as biomarkers of trace metal exposure. Shore crabs (Carcinus maenas) were collected from five sites: three located in the metal-contaminated Fal estuary (Southwest England) and two control sites located on the south coast of England. Two methods for determining MT levels in crab midgut gland were compared. A direct method in which MT was quantified by spectrophotometric measurement of SH content, and an indirect method in which the metal content (Cu and Zn) of partially purified MT was measured. Stress protein levels (stress-70) were quantified in crab gills using an improved western blotting technique. Elevated Cu and Cd concentrations compared to values in control crabs were found in the midgut glands of crabs from the Fal estuary. No significant variation was seen in Zn concentrations among the five sites. The two techniques used for MT analysis yielded similar results. Metallothionein concentrations were slightly higher in crabs from Restronguet Creek (the most metal-contaminated site) than in crabs from the other four sites, however this difference was not significant (p > 0.05). Stress protein levels did not reflect the metal gradient.     

Hatchery programs,stock enhancement,and cost effectiveness: A case study of the Albemarle Sound/Roanoke River stocking program 1981–1996

Fish hatchery programs commonly are used to enhance depleted fish populations. While these programs are highly valued by the public, most likely due to their nonrestrictive approach to restoring a fishery, the effectiveness of these programs has been often questioned. This study investigates economic and ecological effectiveness of the Albemarle Sound/Roanoke River (AR) striped bass stocking program from 1981 to1996 as a case study.