Heavy metal-induced gene expression in fish and fish cell lines.

Abstract

Two isoforms of metallothionein (MT) have been isolated from rainbow trout livers following CdCl2 injections. These MTs have been identified by standard procedures and appear to be similar to mammalian MTs. Total RNA from such induced livers was shown to contain high levels of MT-mRNA activity when translated in cell free systems. This activity was demonstrated to be in the 8 to 10S region of a sucrose gradient. The RNA fractions also showed homology to a mouse MT-I cDNA probe. The exposure of rainbow trout hepatoma (RTH) cells to various concentrations of CdCl2 and ZnCl2 induced the expression of MT and MT-mRNA. Exposure of Chinook salmon embryonic (CHSE) cells to these metals, however, did not result in MT synthesis, suggesting that the MT genes have not become committed to transcription. Instead, an unknown low molecular weight (MW 14 kDa) protein was induced. This metal-inducible protein (MIP) was capable of binding 1"Cd and was stable to heating, while the binding of the metal to this protein was not. These characteristics have been reported for a protein induced in rainbow trout liver following environmental exposure to cadmium. We suggest that both MT and MIP may function in detoxification of heavy metals. IntroductionThe prevalence of toxic heavy metals in the environment is of increasing concern to both the environmental and medical communities. Since aquatic animals are often the first life forms to come into contact with these poisons, the detrimental effects, as well as the mechanisms by which these animals cope with such poisons is ofgreat interest. By such studies it may be possible to establish a biological index for heavy metal toxicity in the aquatic environment.One apparently universal method by which eukaryotes cope with heavy metals is by synthesizing a family of proteins known as metallothioneins (MTs) (1). These low molecular weight, thiol-rich proteins are induced at the transcriptional level in response to heavy metals as well as a variety of other agents and environmental stresses (2-5).It is widely believed that MTs function by binding to toxic metals such as Cd and Hg thus detoxifying the system in question (1). In addition, MTs function in the homeostasis of essential trace metals such as Cu and Zn (6,7). MTs have been quite well characterized in a number of fish such as rainbow trout (8-10), salmon (11), plaice (12), carp (13), and eels (14), among others.We report here the induction of MT and MT mRNA ResultsWe have previously shown (8) that when rainbow trout are subjected to a series of intraperitoneal injections of CdCl2, they respond by producing two isoforms of the low molecular weight metal binding protein metallothionein. These two isoforms, MT-I and MT-II, were purified by standard techniques involving heat denaturation, G-75 column chromatography and ion-exchange chromatography. Figure 1 shows an SDS-polyacrylamide gel demonstrating the MT purification scheme, and Table 1 shows the amino acid analysis of purified MT-I and MT-II. We have also shown (8) that RNA isolated from...

Structure of the rainbow trout metallothionein B gene and characterization of its metal-responsive region.

“…Rainbow trout hepatoma (RTH-149) (10) cells were propagated at 18°C in Eagle minimum essential medium with Earle salts supplemented with 5% fetal bovine serum-penicillin (100 U/ml)-streptomycin (100 ,ug/ml)-sodium bicarbonate (15%)-amphotericin B (2.8 ,ug/ml; Fungizone; GIBCO Diagnostics, Madison, Wis.) in an atmosphere of 5% CO2. The conditions for metal treatment of these cells have been described previously (28,29 by banding it twice by cesium chloride equilibrium gradient centrifugation, as described by Gorman et al (11). Transfections were carried out by the calcium phosphate precipitation method (11).…”

“…(24), Drosophila melanogaster (22), mice (35), and sea urchins (39) to complex multigene families in primates such as humans (19,38). Although MT-like proteins have been identified in several fish tissues and cell lines (3,13,28,29), no information about the structure of MT genes in a major taxonomic group like fish has been available until now. We have recently reported results of cloning of two distinct MT cDNA isoforms from rainbow trout (Salmo gairdnerii) whose overall amino acid structure resembled that of mammalian MTs (4), although a number of significant differences were found on closer examination.…”

The midgut epithelium of aquatic arthropods: a critical target organ in environmental toxicology.

“…Because midgut epithelial cells secrete the components of the PM, we used a molecular biological approach to identify MRGs and other genes in the arthropod midguts. Although the metallothionein genes were considered to be likely candidates as biomarkers for heavy-metal pollution (15,22,23), other metal-responsive genes were also candidates to be induced (8,9,13,14,(24)(25)(26).…”

“…In Colorado it is estimated that heavy metals from over 5,000 abandoned waste dumps or tailing pipes impact over 2,600 km of streams (1). These hazardous waste sites expose aquatic organisms and other wildlife to multiple heavy metals as well as to other pollutants, which can exert impacts on aquatic ecosystems at different levels of organization (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Theoretically, organisms such as aquatic arthropods that experience long-term exposure to the mixtures would be most impacted by such heavy-metal pollution.…”

Difference in hepatic metallothionein content in Antarctic red-blooded and haemoglobinless fish: undetectable metallothionein levels in haemoglobinless fish is accompanied by accumulation of untranslated metallothionein mRNA

“…MTs have been detected and characterized in a large number of fish, including trout [6][7][8], goldfish [9,10], flounder [11] and carp [5]. As with many other vertebrate species, resistance to heavy-metal toxicity in fish is related to their ability to overexpress MT genes after exposure to metal ions [12][13][14][15][16][17][18].…”