ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 294, No. 2, May 1, pp. 461-4661992 Metal-Specific Induction of Metallothionein lsoforms in the Blue Crab Callinecfes sapidus in Response to Single- and Mixed-Metal Exposure’ Marius Brouwer,2 Daniel Schlenk,3 Amy Huffman Ringwood, and Thea Brouwer-Hoexum Duke University Marine Laboratory, Beaufort, North Carolina 28516 Received September 9, 1991, and in revised form December 31,199l Metallothioneins (MTs) play an important role in the metabolism of copper and zinc during the molt cycle of the blue crab. In this study we examined the hypothesis that MT expression in crabs is metal specific. Anion- exchange chromatography showed one major ZnCuMT (ZnMT-I) in control crabs, two MT isoforms in cadmium- treated crabs (CdMT-I, CdMT-II), and three forms (CuMT-I, CuMT-II, CuMT-III) in copper-treated ani- mals. Amino acid analysis of the carboxymethylated apo- MTs, purilled by reversed-phase HPLC, showed minor differences between ZnMT-I, CdMT-I, CdMT-II, CuMT- I, and CuMT-II, while CuMT-III was unique. After com- bined exposure to cadmium and copper, four MTs with differing copper/cadmium ratios were observed, equiv- alent to CdMT-II and the three CuMTs. We conclude that the blue crab has four genes that encode different MTs. Transcription of the CdMT-I gene is induced by cadmium, but inhibited by copper. CuMT-I, CdMT-II, and ZnMT- I may be the products of a single gene responsive to cop- per, cadmium, and zinc. Expression of the CuMT-II and CuMT-III genes is initiated by copper and not by cad- mium. We believe that CdMT-I and CuMT-III are im- portant in detoxification, whereas Zn/CuMT-I and CuMT-II are involved in regulatory functions. These re- sults show the importance of the use of mixed-metal ex- posures in the study of the molecular mechanisms of metal regulation and function. 0 1992 Academic Press, Inc. Class I metallothioneins (MTs)~ (1) constitute a family of cysteine-rich, low-molecular-weight metal-binding pro- teins that appear to be involved in metal homeostasis and 1 This work was supported by NIH Grant ES-04074 (M.B.). D.S. and A.H.R. acknowledge NIEHS Training Grant ES-07031. ’ To whom correspondence should be addressed. 3 Present address: University of Arkansas for Medical Sciences, De- partment of Pharmacology/Toxicology, Little Rock, Arkansas 72205. * Abbreviations used: MT, metallothionein; DEAE, diethylaminoethyl; PMSF, phenylmethylsulfonyl fluoride; MRE, metal-responsive element. 0003-9S61/92 $3.00 Copyright 0 1992 by Academic Press, Inc. AI1 rights of reproduction in any form reserved. detoxification (2). Genes coding for MT isoforms in mam- malian cell lines have been shown to be inducible and dif- ferentially regulated at the level of transcription by heavy metals (i.e., copper, cadmium, and zinc) as well as a variety of other agents (i.e., glucocorticoid hormones, pharmaceu- ticals, alcohols, cytokines, infection, food deprivation, and elevated oxygen tension) (2-6). Differential regulation of MT gene expression has also been demonstrated in rainbow trout cell lines (7). Differential regulation of class I MTs in invertebrates has not been demonstrated. Earlier studies in marine crustacea identified two Cd- inducible MT isoforms in the mud crab Scylla serrata (8) and the blue crab Callinectes sapidus (9). Recently we have found that copper induces three isoforms in the American lobster Homarus americanus (10, 11) and the blue crab C. sapidus (12). The functional relevance of the occurrence of multiple MT isoforms is not known. Our studies on the involvement of CuMT in the synthesis and degradation of hemocyanin (13-15) suggest that the MT form that sequesters copper released during hemocyanin breakdown may be different from the form that donates Cu(I), via glutathione, to the copper-free protein during hemocyanin synthesis (10, 11). These observations sug- gested the possibility that MT isoforms in marine crus- tacea may be functionally distinct, metal specific, and dif- ferentially regulated. Consequently, the purpose of this study was to compare copper and cadmium-inducible MTs from the hepatopancreas of the blue crab, C. sapidus. MT expression usually has been evaluated following exposure to a single metal. However, metal regulation within an organism is a complex process, mediated through trans- acting, DNA-binding, metal-regulatory (metallo)proteins and c&acting, metal-responsive elements (MREs) and must involve intricate interactions between multiple metals (16-18). To evaluate such interactions, MT expression in C. sapidus was studied after exposure to copper and cadmium individually and to copper and cad- mium simultaneously. 461