Cytosolic and Metallothionein-Bound Hepatic Metals and Detoxification in a Sentinel Teleost, Dules auriga, from Southern Rio de Janeiro, Brazil.

Dules auriga, a native Brazilian teleost, was applied as a sentinel species regarding metal contamination at Ilha Grande Bay, previously considered a reference site in Southeastern Brazil. Cytosolic (S50) and metallothionein-bound (HTS50) hepatic iron (Fe), zinc (Zn), copper (Cu), manganese (Mn), cadmium (Cd), and silver (Ag) were determined by inductively coupled plasma optical emission spectrometry (ICP-OES), while metallothionein (MT) concentrations were determined by polarography. Ag concentrations in both cytosolic fractions were below the limit of detection. All other HTS50 metal contents were significantly lower than S50 contents. No significant associations were found for MT. Fe and Mn S50 were positively and moderately correlated to total length, as well as HTS50 Mn, while total weight was correlated to both Mn fractions, suggesting that environmental Mn and Fe concentrations may influence fish growth. A moderate correlation between the condition factor and the S50 Cu fraction was observed, also indicating that Cu may affect fish growth. Inter-element correlations were observed, including between Cd, a toxic element, and Mn and Zn, both essential elements. Calculated molar ratios indicate that both Mn and Zn are in molar excesses compared with Cd, corroborating literature assessments regarding protective Mn and Zn effects against Cd. Lack of MT correlations suggests that metal concentrations may not be high enough to reach an MT induction threshold and that MT variability is probably linked to environmental metal concentrations. Therefore, the increased environmental contaminant levels observed in the study area indicate the need for biomonitoring efforts aiming at the application of efficient mitigation measures.

Hepatic Metal and Metallothionein Levels in a Potential Sentinel Teleost, Dulesauriga, from a Southeastern Brazilian Estuary.

The identification of estuarine sentinel species is of paramount importance. The potential of the species Dules auriga sampled from Guanabara Bay, Brazil regarding metal contamination was assessed. Hepatic metallothionein (MT) and Fe, Zn, Cu, Mn, Cd and Ag concentrations were determined in cytosolic fractions (S50 and HT S50) by polarography and ICP-OES, respectively. HT S50 Fe, Mn and Zn were lower than in the S50 fraction, indicating MT-detoxification. MT was correlated to HT S50 Zn, indicating Zn homeostasis. Zn was negatively correlated to weight (TW) and length (TL), suggesting environmental Zn influence. A moderate negative correlation between HT S50 Cu and the condition factor (CF) was observed, indicating that increasing environmental Cu concentrations may decrease D. auriga CF. Several inter-elemental correlations were observed. No MT, TW and TL correlations were found. Thus, MT variability is probably linked to environmental metal concentrations. D. auriga fulfills most sentinel requisites, and MT may be an adequate metal contamination biomarker in this species.

Metal speciation of metallothionein in white sea catfish, Netuma barba, and pearl cichlid, Geophagus brasiliensis, by orthogonal liquid chromatography coupled to ICP-MS detection.

Metal speciation analysis in MTs was carried out in two tropical fish species of Brazil, the freshwater fish pearl cichlid (Geophagus brasiliensis) and the marine fish white sea catfish (Netuma barba), that are presently used to monitor the effects of heavy metal pollution in aquatic ecosystems in Brazil. In order to obtain the MT fraction, liver cytosols from both fish species where subjected to size exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. That MT fraction was then separated by anion-exchange (AE)-FPLC, whose optimal chromatographic conditions were optimized for the separation of the different hepatic MT isoforms existing in both fish species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with the ICP-MS instrument. The analytical results showed that MTs of these fish species, unknown so far, exhibited unique characteristics in comparison with standard MTs and other fish liver MTs. In fact, MT isoforms of N. barba turned out to be very anionic, as indicated by their high retention in the Mono Q column and the strong ionic strength required to separate them. As for G. brasiliensis, cadmium was exclusively present in only one of the peaks of the MT isoforms showing a unique metal-binding behavior for MT in this fish species. The differences between the MTs among these species and the different association of metals in particular MT isoforms display the importance of the metal speciation analysis in these proteins prior to its use as bioindicators.

Development of a stable isotope approach for the inductively coupled plasma-mass spectrometry determination of oxidized metallothionein in biological materials.

The use of isotope dilution analysis (IDA) with inductively coupled plasma-mass spectrometry (ICP-MS) for the determination of oxidized metallothionein (MT) by a Cd-saturation method is investigated. The method developed here is a modification of an earlier methodology which used a radioactive Cd isotope ((109)Cd). While retaining the many advantages of this previous approach, the procedure presented here uses stable isotope ratio measurements ((114)Cd/(111)Cd) for the determination of MT. Experimental parameters governing the instrumental precision and accuracy for isotope ratio measurements of Cd by ICP-MS were characterized. Systematic errors, including mass bias, detector dead time, and spectroscopic interferences, could be easily corrected. The isotope dilution ICP-MS method was validated by the determination of very low levels of cadmium in biological certified reference materials (NIST SRM 2670 freeze-dried urine, IAEA H-8 horse kidney, and BCR TP-25 lichens). Finally, the IDA procedure was evaluated for the determination of oxidized MT by a Cd-saturation method previously developed using radioactive (109)Cd. The final procedure was applied to the quantification of MT in Long-Evans Cinnamon rat liver cytosol samples and the results were compared with data obtained for the same samples using the reference (109)Cd methodology. A good agreement between the analytical values obtained by both methods was observed.

Significance of alpha-fragment of metallothionein in cadmium binding.

In order to evaluate the significance of alpha- and beta-fragments of metallothionein with regard to Cd binding in biosynthetic processes, the Cd-binding ability of four mutant metallothioneins was examined using the Escherichia coli expression system. The features of the mutant metallothioneins were proteins in which cysteine residues in the alpha- or beta-fragment were replaced with alanine residues, or that the sequential order of the fragments was altered. The expressed mutant metallothioneins having an intact alpha-fragment showed the constructive abilities of the Cd-thiolate cluster. On the other hand, mutant metallothionein having an alpha-fragment lacking metal-binding sites exhibited no Cd-binding ability. The condition for maintaining the Cd-binding capability of metallothionein was that the alpha-fragment remains intact irrespective of the sequential order of the two fragments. The alpha-fragment is an indispensable component in metal-binding processes of Cd-metallothionein.

Expression of human metallothionein-2 in Escherichia coli: cadmium tolerance of transformed cells.

A genetic approach was undertaken to investigate the physiological roles of human metallothionein-2. A constructed expression plasmid, pEXPMTII, in which human metallothionein-IIA cDNA was inserted downstream of a tryptophan-lactose promoter, was used to transform Escherichia coli JM105 strain. Cadmium-binding metallothionein was successfully expressed in E. coli in the medium containing cadmium, while copper and zinc-metallothioneins were scarcely observed in copper- or zinc-containing medium. The amino acid composition and sequence of the biosynthesized cadmium-metallothionein were analyzed. The selectivity of metals bound to metallothionein and the stability of metal-binding forms of metallothionein in E. coli were discussed. In addition, cadmium, zinc, or copper resistance of the cells expressing metallothionein was examined. Cells transformed with the plasmid pEXPMTII and cultured in a medium containing cadmium exhibited tolerance only to cadmium. It was demonstrated that human metallothionein-2 functioned for cadmium detoxification in E. coli.