Some methodological aspects of metallothionein evaluation

The specificity of the methods used for metallothioneins (MTs) determination in both field and laboratory studies is crucial to a relevant interpretation of the results. The technique applied in the present study is commonly used, but several potential problems may limit its validity: (i). the stability of the metal-SH links during heating and the possibility of metal exchanges between MTs and others compounds; (ii). the presence of heat-stable thiol compounds with high molecular weight or very low molecular weight, which may subsist in the supernatant and interfere during global analysis of MTs; (iii). the subsistence of metals not bound to MT in the supernatant after heating; and (iv). the impact of reducing agents such as mercaptoethanol on the metal-binding ligands. Our investigations were conducted mainly on oysters (Crassostrea gigas) and polychaetes (Hediste/Nereis diversicolor) sampled, respectively, in two French metal-rich sites, the Gironde estuary and Boulogne harbour. This study, which allowed us to clarify most of the potential problems noted above, indicates the importance of dealing with methodological difficulties.