Quantification of metallothionein isoforms in fish liver and its implications for biomonitoring

Abstract

A rapid, reproducible, and sensitive ion-exchange chromatography (IEC) method combined with graphite-furnace atomic absorption spectrometry (GF-AAS) was developed to separate and quantify basal amounts of both metallothionein (MT) isoforms in dab (Limanda limanda) liver samples. Dab liver homogenates were saturated with Cd, and obtained cytosols were purified by a two-step acetone precipitation prior to chromatographic analysis. Metallothionein isoforms were separated by IEC and were subsequently quantified indirectly by GF-AAS via their Cd contents. The amount of Cd needed for saturation was optimized. The efficiency of Cd saturation and acetone precipitation was proven by gel permeation chromatography (GPC) and metal distribution analysis. Based on the method of standard addition, a recovery for MT was 98% after acetone precipitation and 68% after IEC. The repeated determination of MT isoforms in a dab liver homogenate resulted in coefficients of variation of approximately 12% for both isoforms. Based on the detection limit for GF-AAS, the calculated detection limit for MT isoforms is 2 ng/mg protein. Therefore, this method is suitable for monitoring purposes. The implications of isoform-specific measurement for biomarker monitoring are discussed.

Seasonal and spatial comparison of metallothioneins in frog Rana ridibunda from feral populations

“…The differences in metal binding, stability, and expression between the MTs forms in vertebrate animals are also reported (Kito et al 1982;Olsson and Hogstrand 1987;Kershaw and Klaassen 1992;Muto et al 1999;Olsvik et al 2000;Lacorn et al 2001;Das et al 2002). In our study, the destruction of MT-2 upon the effect of non-favorable conditions at the U site is observed.…”

“…However, the experience of such use of MTs is mostly restricted to the estimation of their content. The diversity of MTs forms and their metal-binding function is poorly understood (Lacorn et al 2001;Das et al 2002).…”

Biomarkers of exposure to metal contamination and lipid peroxidation in the benthic fish Cathorops spixii from two estuaries in South America, Brazil

“…However, a wide range of factors such as age, sex, size, and reproductive status can also affect the induction of MT (Lacorn et al 2001). The PCA found a positive correlation among MT levels and length of C. spixii.…”

Application of two SH-based methods for metallothionein determination in mussels and intercalibration of the spectrophotometric method: laboratory and field studies in the Mediterranean Sea

“…The exact biological role of MT isoforms is not known as different isoforms of MT may accomplish different functions with different degrees of induction (Mackay et al 1993, Dallinger et al 1997, Erk & Raspor 1999, Lacorn et al 2001, Chabicovsky et al 2003. The existence of at least two interactive pools of MT in the cell's cytosol has been postulated; one, possibly MT-20, is affected by transient changes in metal concentrations in the cytosol due to environmental fluctuations; the other, MT-10, is involved in the physiological metal regulatory processes (Vasak & Hasler 2000).…”