The two Caenorhabditis elegans metallothioneins (CeMT-1 and CeMT-2) discriminate between essential zinc and toxic cadmium Sukaina Zeitoun-Ghandour 1 , John M. Charnock 2 , Mark E. Hodson 3 , Oksana I. Leszczyszyn 4 , Claudia A. Blindauer 4 and Stephen R. Stu ¨ rzenbaum 1 1 School of Biomedical & Health Sciences, King’s College London, UK 2 School of Earth, Atmospheric and Environmental Sciences, University of Manchester, UK 3 Department of Soil Science, University of Reading, UK 4 Department of Chemistry, University of Warwick, Coventry, UK Introduction Metal pollution in the environment is a matter of con- cern. Many studies have focused on the use of terres- trial biomonitors to determine how organisms, in particular invertebrates, control and tolerate increased exposure to elevated levels of metals [1–7]. Responses may include avoidance, excretion, chelation or Keywords affinity; C. elegans; cadmium; metal speciation; metallothionein; zinc Correspondence S. Stu ¨ rzenbaum, School of Biomedical & Health Sciences, Pharmaceutical Science Division, King’s College London, 150 Stamford Street, London SE1 9NH, UK Fax: +44 2078484500 Tel.: +44 2078484406 E-mail: stephen.sturzenbaum@kcl.ac.uk (Received 22 January 2010, revised 23 March 2010, accepted 30 March 2010) doi:10.1111/j.1742-4658.2010.07667.x The nematode Caenorhabditis elegans expresses two metallothioneins (MTs), CeMT-1 and CeMT-2, that are believed to be key players in the protection against metal toxicity. In this study, both isoforms were expressed in vitro in the presence of either Zn(II) or Cd(II). Metal binding stoichiometries and affinities were determined by ESI-MS and NMR, respectively. Both isoforms had equal zinc binding ability, but differed in their cadmium binding behaviour, with higher affinity found for CeMT-2. In addition, wild-type C. elegans, single MT knockouts and a double MT knockout allele were exposed to zinc (340 lm) or cadmium (25 lm) to investigate effects in vivo. Zinc levels were significantly increased in all knockout strains, but were most pronounced in the CeMT-1 knockout, mtl-1 (tm1770), while cadmium accumulation was highest in the CeMT-2 knockout, mtl-2 (gk125) and the double knockout mtl-1;mtl-2 (zs1). In addition, metal speciation was assessed by X-ray absorption fine-structure spectroscopy. This showed that O-donating, probably phosphate-rich, ligands play a dominant role in maintaining the physiological concentration of zinc, independently of metallothionein status. In contrast, cadmium was shown to coordinate with thiol groups, and the cadmium speciation of the wild-type and the CeMT-2 knockout strain was distinctly different to the CeMT-1 and double knock- outs. Taken together, and supported by a simple model calculation, these findings show for the first time that the two MT isoforms have differential affinities towards Cd(II) and Zn(II) at a cellular level, and this is reflected at the protein level. This suggests that the two MT isoforms have distinct in vivo roles. Abbreviations EXAFS, extended X-ray absorption fine structure; ICP-OES, inductively coupled plasma optical emission spectrometry; XANES, X-ray absorption near-edge structure. FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2531 immobilization of metal ions, or activation of general stress response mechanisms ⁄ proteins [8,9]. A promi- nent response pathway involved in the chelation of metal ions involves metallothioneins (MTs). These are proteins of low molecular mass that are characterized by a high cysteine content [15–30%], high heat stability and lack of aromatic amino acids (including histidine) [10,11]. Although the discovery of MTs dates back to 1957 [12], their precise physiological functions are still debated. It has become evident that a single function does not exist for this heterogeneous superfamily of proteins, and that they are ‘multipurpose’ proteins [13], with roles in protection against cadmium toxicity [14], essential Cu(I) and Zn(II) homeostasis [15], and response to oxidative stress [16]. There is growing evidence that the existence of mul- tiple MT isoforms is associated with functional differ- entiation, for example in snails [17], earthworms [18], plants [19] and vertebrates [16]. So far, studies have focused on the discrimination between monovalent Cu(I) and divalent Zn(II) and Cd(II) [20]. As the coor- dination geometries of mono- and divalent metal ions are very distinct (digonal or trigonal planar versus tet- rahedral), it is easily conceivable that the steric require- ments imposed by binding of these metal ions will differ, and this offers a straightforward mode of dis- crimination. In contrast, discrimination between the essential Zn(II) and toxic Cd(II), which have relatively similar coordination chemistry, presents a major challenge for organisms that are exposed to both metal ions. The soil nematode Caenorhabditis elegans is a case in point [21,22], and offers a unique biological system for the study of MT isoform specificity, because its fully sequenced genome contains only two metallothioneins CeMT-1 and CeMT-2 [23]. The encoded proteins bear the hallmarks of metallothioneins, i.e. they are small and cysteine-rich, and their expression is induced by metals [24]. More recently, RNA interference (RNAi) and chromosomal deletion of the C. elegans MT loci have highlighted an increased sensitivity of mutant strains to metal toxicity, reflected by reduced growth, brood size and lifespan [23,25]. In addition, phytochel- atins, which are small, non-ribosomally synthesized, Cd-binding peptides, play a prominent role in protec- tive responses to Cd exposure [26–28]. Significantly, the two MT isoforms show differential expression profiles [24]. CeMT-2 is only induced in intestinal cells in the presence of cadmium, but CeMT-1 is also constitutively active in three cells of the lower pharyngeal bulb [24]. These studies provided the first evidence that CeMT-1 and CeMT-2 may have distinct in vivo functions, but although additive sensitivity towards cadmium was observed in C. elegans metallo- thionein knockout alleles, isoform-specific in vivo effects have not been observed to date, even by detailed meta- bolomic profiling analysis [28]. At the protein sequence level, CeMT-1 and CeMT-2 display intriguing differences, and are more different from one another than vertebrate MT isoforms. CeMT-1 contains a 15 amino acid insert with two additional histidines and one cysteine [23,24,29], with a further histidine at position 54 (see Fig. S1A for sequence alignment). Recent in vitro characterization of recombinantly expressed CeMT-1 and CeMT-2 by ESI-MS and CD spectroscopy has begun to determine the differences in metal binding properties of the two isoforms [30]. A clear preference for divalent metal ions was discovered, but, most significantly, this study suggested that CeMT-1 and CeMT-2 show differential metal preferences, with CeMT-1 biased towards Zn(II) and CeMT-2 biased towards Cd(II). In the present study, we explore whether these quali- tative findings are reflected by overall in vivo metal accumulation and speciation of metallothionein- mutated C. elegans strains, as well as the in vitro metal ion affinities of the two isoforms under metal-replete and metal-excess conditions. Results Metal-binding properties of recombinant metallothioneins For characterization and quantification of the metal- binding properties of CeMT-1 and CeMT-2, an expres- sion strategy was adapted that avoids the use of fusion tags, as we have previously observed that tags can influ- ence the metal binding properties of recombinantly expressed metallothioneins [31]. In contrast to most expression tag systems, our protocol also allows the expression of proteins with no additional residues at the termini. Careful chemical precipitation followed by gel filtration chromatography yielded pure proteins (> 95%, as judged by ESI-MS analysis) with no addi- tional species (see Fig. S1B,C for expression and purifi- cation, respectively). Both metallothioneins were expressed in the presence of either Zn(II) or Cd(II) in the culture medium. The metal ion stoichiometry of the purified proteins was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) and ESI-MS (Fig. 1 and Table 1). Consistent with previous work [30,32], CeMT-2 expressed in the presence of Cd(II) had six Cd(II) ions bound. We found the same stoichiometry for Zn(II), with no discernible peaks for metal-depleted C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al. 2532 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS species in the mass spectrum, and corresponding ICP- OES results. Consistent with recent findings [30], our analysis also confirmed that CeMT-1 binds seven metal ions, with Zn 7 -CeMT-1 the only species observed in mass spectra at neutral pH for Escherichia coli cells grown in Zn(II)-supplemented medium (Fig. 1). To allow quantification of metal affinities and their comparison, it was very important to obtain clearly defined homo-metallic species, and the data compiled for Zn 6 -CeMT-2, Cd 6 -CeMT-2 and Zn 7 -CeMT-1 in Fig. 1 and Table 1 show that this was achieved by expression in the presence of the desired metal ion. However, the CeMT-1 form isolated from Cd(II)-sup- plemented cultures was Cd 6 Zn-CeMT-1 (Fig. S2), and incorporation of seven cadmium ions was only possible by reconstitution of metal-free CeMT-1 with rigorous exclusion of Zn(II), using an established protocol [33]. Although the Cd 7 -CeMT-1 species was the major form in this preparation, we observed a loss of definition in metal binding stoichiometry despite extensive gel filtra- tion and washing, as Cd 8 -CeMT-1 and Cd 9 -CeMT-1, as well as a very small amount of Cd 6 Zn 1 -CeMT-1 me- talloforms, were present as minor species (Fig. 1B). The contribution of these over-metallated species is also apparent in the stoichiometry determined by ICP-OES given the larger than expected stoichiometry for cadmium-bound CeMT-1. The overall in vitro affinities of CeMT-1 and CeMT-2 towards Zn(II) and Cd(II) were determined by comp- etition experiments using the metal chelator 5F-BAPTA [34] and 19 F-NMR spectroscopy under conditions that allow direct comparison with literature values. The stability constants obtained for the homo-metallic zinc and cadmium complexes of CeMT-1 and CeMT-2 are given in Table 2, and represent means over all six (CeMT-2) or seven (CeMT-1) binding sites. As expected for predominantly thiol coordination, the sta- bility constant for cadmium binding in CeMT-2 was significantly larger than that for zinc binding, and was close to the value for human MT-2 measured under similar conditions [34]. Remarkably, this was not the case for CeMT-1. Although both isoforms displayed identical affinities for Zn(II), cadmium binding in the CeMT-1CeMT-2 Relative Intensity 8000 8400 8800 9200 [–Met] [–Met] [–Met] [–Met] [–Met] C D 6600 7000 7400 7800 0 50 100 50 100 A B [–Met] [+Met] Cd 6 Cd 6 Zn 6 Zn 7 Cd 6 Zn Cd 8 Cd 7 Cd 9 [–Met] Mass (Da) Fig. 1. Deconvoluted ESI mass spectra of the various metalloforms of Caenorhabd- itis elegans MTs. Holo zinc (A) and cadmium (B) species of CeMT-1; zinc (C) and cad- mium (D) species of CeMT-2 obtained at neutral pH (10 m M ammonium acetate, 10% methanol). Samples (A), (C) and (D) result from expression in the presence of the respective metal ion; sample (B) was obtained by expression in presence of Zn(II) and reconstitution of the apoprotein with Cd(II). )Met and +Met annotations refer to the absence or presence of the N-terminal methionine residue for each species. The peaks in (A), (C) and (D) to the right of the main peaks correspond to Na + adducts. Table 1. Metal to protein stoichiometries for CeMT-1 and CeMT-2 metalloforms determined by mass spectrometry and elemental analysis. Theoretical and observed mass are given for the major species in each mass spectrum. )MET, without Met; +Met, includ- ing Met. Metalloform Mass spectrometry Stoichiometry (ICP-OES) Theoretical mass (Da) Observed mass (Da) Zn Cd CeMT-1 Zn 7 8402.7 ()Met) 8401.9 ± 0.7 6.6 ± 0.7 8.8 ± 0.8 Cd 7 8731.9 ()Met) 8731.5 ± 0.5 CeMT-2 Zn 6 6843.1 ()Met) 6843.3 ± 0.7 5.6 ± 0.6 6.0 ± 0.6 Cd 6 7256.5 (+Met) 7257.0 ± 0.6 S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2533 Cd 7 -CeMT-1 complex was dramatically weaker than that in Cd 6 -CeMT-2 and other MTs (Table 2), even when the effect of the over-metallated species is taken into account. To date, the overall stability of cadmium binding to CeMT-1 is the lowest value reported for any MT. However, it is important to note that, despite this, the overall affinity of CeMT-1 towards Cd(II) is still an order of magnitude larger than that for Zn(II), so it would be inappropriate to claim that CeMT-1 is a Zn(II)-specific MT, although one particular site does indeed appear to have an absolute preference for Zn(II). In vivo metal speciation in wild-type C. elegans: excess zinc and cadmium are handled differently To investigate the native organism-wide responses to cadmium and zinc exposure, the wild-type (N2) C. ele- gans strain was grown on supplemented media, and the collective ligand environment of intracellular cad- mium and zinc was analysed by X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy. Because the low-energy photoelectrons have a long mean free path, XANES spectroscopy is strongly affected by multiple scattering, which means that it is very sensitive to differences in geometry as well as coordination number and oxidation state. Although this complexity complicates the analysis of XANES data, it is valuable as a ‘fingerprint’ technique, com- paring unknowns with model compound spectra. Indeed, XANES and EXAFS spectroscopy have previ- ously been used successfully on rat liver samples to distinguish different binding modes in Cd–S clusters and metallothionein [35]. The cadmium XANES spec- tra (Fig. 2A) show that the edge shape and position are distinct from Cd–O-bonded complexes, and display features that are more similar to S-coordinated cad- mium models (Cd–S and rat Cd 7 -MT). The EXAFS results and associated Fourier transforms, together with the best possible fits, are shown in Fig. S3, and indicate a single major transform peak at R+D = 2.5 A ˚ (other fits gave higher residuals, data not shown). When modelled, the cadmium EXAFS data produce a best fit with one shell of four sulfur scatterers at 2.49 A ˚ (Table 3). However, due to the small size of the nematodes, even 300 000–500 000 syn- chronized nematodes generated only a dilute sample that, although sufficient for analysis, produced a short data range and had a poor signal-to-noise ratio, thus precluding fitting of further shells of scatterers. Although the EXAFS data were admittedly noisy, they Table 2. Zinc and cadmium binding affinities for MTs in Caenor- habditis elegans and other species. Log K binding constants for zinc and cadmium metalloforms of C. elegans MTs were deter- mined and compared to those of other MTs. The log K for cadmium binding was determined by competition between protons and metal ions for complexed thiolate ligands [33]. In all cases, the log K of zinc binding was measured by competition for metal ions between the MTs and 5F-BAPTA (ionic strength 4 m M and pH 8.1) [34,48,53]. MT isoform log K Zn log K Cd CeMT-1 (C. elegans) 12.0 ± 0.1 a 13.1 ± 0.3 CeMT-2 (C. elegans) 12.0 ± 0.1 15.0 ± 0.1 MT-2 (human) [34] 11.5 14.8 MT-3 (human) [34] 10.8 14.3 SmtA (bacterial) [53] 13.0 – E C (plant) [53] 10.6 – a Recalculating this value to account for over-metallation yields a log K value of 13.4. Wildtype Cd-S Cd-MT Cd(OH) 2 CdSO 4 26 680 26 84026 80026 76026 720 Energy (eV) Normalised signal Wildtype Zn-S Zn foil ZnSO 4 . H 2 O Zn 3 (PO 4 ) 2 Normalised signal 9600 9700 9800 9900 10 000 Ener gy (eV) A B Fig. 2. XANES profiles in wild-type nematodes and standards. Cd XANES spectra (A) and Zn XANES spectra (B). For cadmium, a minor monochromator drift during data collection made it necessary to correct the edge position using reference spectra, therefore the error in the absolute position of the edge was marginally larger than the station benchmark. C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al. 2534 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS are of sufficient quality to justify the conclusion that sulfur coordination gives the best single shell fit, which is also consistent with XANES results. Previously recorded Zn(SO 4 )ÆxH 2 O, ZnS and Zn 3 (PO 4 ) 2 spectra were used to model the zinc XANES spectra of the wild-type (N2) nematode. The spectra show that, of all reference compounds, the wild-type spectrum displayed features most similar to those of the zinc phosphate standard (Fig. 2B). This was corroborated by EXAFS spectra (Table 3 and Fig. S4), which indicated the best fit to be four oxygen atoms surrounding zinc in the first coordination shell, with a mean Zn–O distance of 1.97 ± 0.03 A ˚ . This is consistent with the tetrahedral coordination of zinc phosphate [36]. Although, it may not be technically possible to distin- guish between N ⁄ O ⁄ F or between P ⁄ S ⁄ Cl as a scatterer, the difference between O and S is substantial. Therefore, these data suggest that the mechanisms to deal with zinc and cadmium employed by C. elegans are separate and distinct, as accumulated cadmium is predominantly S-bound and zinc is predominantly O-bound. C. elegans metallothioneins are not the only players in metal detoxification and homeostasis The effects on the ligand environments of cadmium and zinc upon deletion of metallothioneins were inves- tigated by comparative analysis of XANES spectra (Fig. 3) and EXAFS data (Table 3, Figs S3 and S4). Cadmium XANES spectra (Fig. 3A) for the MT knockout strains do not show features significantly dif- ferent from those observed for the wild-type (N2), which suggests that the cadmium ions are still predom- inantly coordinated by sulfur atoms. However, a broader edge and lower starting energy (1.5–2 eV) were observed in spectra of the CeMT-1 KO and the double knockout, both were observed in spectra of the CeMT-1 knockout mtl-1 (tm1770) and the CeMT-1 Table 3. Cd ⁄ Zn EXAFS parameters. Best fit of the Cd ⁄ Zn K-edge data for Caenorhabditis elegans wild-type and metallothionein knockout strains, where r is the absorber–scatterer distance in A ˚ (± 0.02 A ˚ , inner shell; ± 0.05 A ˚ , outer shell), N is the number of scatterers around the central atom, 2d 2 is the the Debye–Waller factor in A ˚ 2 , ± 25%, and the R factor is the least-squares residual, which indicates goodness of fit. Strain Cadmium Zinc Scatterer Nr(A ˚ )2d 2 (A ˚ 2 ) R factor Scatterer Nr(A ˚ )2d 2 (A ˚ 2 ) R factor Wild-type S 4 2.49 0.026 74.3 O 4 2.00 0.010 39.1 CeMT-1 KO S 4 2.53 0.017 48.6 O 4 2.00 0.015 30.9 O 1 1.98 0.015 40.5 S 3.3 2.52 0.013 37.6 CeMT-2 KO S 4 2.48 0.028 78.8 O 4 1.97 0.014 P 2 3.27 0.008 S 4 2.51 0.020 67.6 O 4 1.98 0.012 26.8 Double KO O 1 1.98 0.016 61.6 S 3.3 2.49 0.015 Energy (eV) Normalised signal 26 700 26 780 26 820 26 860 26 740 26 700 26 710 26720 Wildtype CeMT-1 KO CeMT-2 KO Double KO Wildtype CeMT-1 KO CeMT-2 KO Double KO 9630 9670 9710 9750 9790 Energy (eV) 9657 9658 9659 9660 Normalised signal A B Fig. 3. Metal speciation in Caenorhabditis elegans strains. Compari- son of Cd XANES spectra (A) and Zn XANES spectra (B) obtained for C. elegans wild-type and metallothionein knockouts. The inserts show the cadmium and zinc energy shifts between samples. S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2535 and CeMT-2 double knockout mtl-1;mtl-2 (zs1). Such features are characteristic of a Cd–O phase. This observation was supported by EXAFS analysis, for which data fitting was improved by addition of a shell of oxygen scatterers and refining the distances, the De- bye–Waller factors, and the ratio of S-bound to O- bound cadmium (Table 3). The refined Cd–O distance of 1.98 A ˚ is arguably very short compared to crystallo- graphic values for Cd–O in phosphates, carbonates, etc. However, this may be due to the Cd being four- coordinate rather than six-coordinate, or may reflect a larger than normal error in the EXAFS distance due to weaker scattering of the oxygen than of the sulfur, making the Cd–O contribution to the total EXAFS spectra much smaller than the Cd–S contribution. Nevertheless, this confirms that the cadmium coordina- tion environments of the CeMT-1 KO and double KO differ from those of the wild-type (N2) and the CeMT-2 KO, although it should be emphasized that the majority of the cadmium remained bound to sulfur in all strains (including the metallothionein deletion stains) (Table 3). The absence of either or both MT(s) had no observa- ble effect on zinc speciation. All XANES spectra (Fig. 3B) were similar, and EXAFS data analysis (Table 3 and Fig. S4) identified a common first shell scatterer peak at 1.97 A ˚ , characteristic of O-coordina- tion. Adding a second shell of phosphorus scatterers improved the fit for all four spectra, but this shell was statistically significant only in the case of mtl-2 (gk125). Although superbly fitted Zn and Cd XANES and EXAFS data have previously illustrated that isolated mammalian metallothioneins bind metals [37,38], the data presented here reveal that the MT status of the nematode does not significantly alter the overall speci- ation of zinc and cadmium in cells, as the principal ligand environment for both metals is similar to that of the wild-type (N2) strain. Nevertheless, the data provide insights about the ultimate fate of each metal ion. As Cd–S bonds were maintained in the double knockout strain, it is clear that the Cd–S spe- cies observed do not correspond to metallothionein- bound Cd. Instead, it is likely that phytochelatins dominate Cd speciation. Excess zinc in C. elegans is clearly not MT- or phytochelatin-bound, but may be sequestered through other means such as deposition in phosphate-rich granules [39], possibly synonymous to those found in earthworms [40,41]. However, these facts do not preclude a role for MTs in metal handling, as binding of zinc and cadmium by MTs may be transient, particularly as MTs are capable of releasing metal ions relatively rapidly [42–44], possi- bly to molecules downstream in the detoxification pathway. We therefore next address the question of whether MTs in C. elegans influence overall zinc and cadmium levels at all. Metal levels in metal-exposed worms: CeMT-2 is important with regard to cadmium accumulation In the wild-type (N2) strain, low levels of cadmium accumulation were observed when nematodes were grown (from L1 larval to pre-adult stage L4) on Cd-supplemented medium (Fig. 4A and Table S1). An equivalent cadmium body burden was also observed in the CeMT-1 knockout. This suggests that, upon cad- mium exposure, the CeMT-1 KO strain responds ‘as wild-type’, and the mechanism of this response is not hindered by lack of CeMT-1 in the cytosol. In con- trast, the CeMT-2 KO strain shows an approximately twofold increase in cadmium levels compared to the wild-type (N2) strain, indicating that one of the mech- anisms by which C. elegans normally responds to cad- mium exposure has been disrupted. This is exacerbated in the double knockout strain, in which the cadmium burden is significantly increased. These data suggest that (a) if CeMT-2 is expressed, then CeMT-1 does not play a significant role in the cadmium response, (b) if CeMT-2 is absent, then CeMT-1 can fulfil the role carried out by CeMT-2, but not as effectively, and (c) if both metallothioneins are absent, the ‘normal’ and ‘back-up’ MT-mediated pathways of dealing with cadmium exposure are impaired, leading to hyperaccu- mulation of cadmium compared to the wild-type strain. Both CeMT-1 and CeMT-2 are important in maintaining physiological zinc levels Under control (non-metal-supplemented) conditions in the wild-type (N2), zinc was maintained at basal physiological levels (Fig. 4B and Table S1). For the CeMT-2 and double mutant strains, no significant dif- ference from wild-type (N2) was observed; however, the CeMT-1 mutant accumulated slightly more Zn(II). Under Zn-supplemented conditions, all three knockout strains accumulated significantly more zinc compared to the wild-type (N2) strain. Of the single knockout strains, deletion of CeMT-1 resulted in accumulation of the highest zinc concentration; however, deletion of CeMT-2 also led to a moderate increase in zinc levels. The double knockout did not differ significantly from the CeMT-1 knockout. This indicates that (a) CeMT-1 has a more significant role than CeMT-2 in the regula- tion of zinc levels, (b) both CeMT-1 and CeMT-2 are required to maintain physiological zinc levels, as lack C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al. 2536 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS of CeMT-2 also disrupts the mechanism that prevents zinc accumulation, and (c) CeMT-1 and CeMT-2 oper- ate in a synergistic manner in zinc trafficking. Figure 4B also includes data for Zn(II) levels after Cd exposure, and these data offer further interesting insights. In the CeMT-1 knockout, which showed only basal Cd levels, Zn levels were depressed, but were ele- vated under Zn exposure. This observation can only be rationalized if we consider that the two isoforms are regulated differently, and that Cd(II) strongly induces CeMT-2. Hence, in the CeMT-1 knockout, induction of CeMT-2 may have led to enhanced excre- tion (or reduced uptake) of not only Cd(II), but also some of the basal Zn(II), possibly mediated by the same CeMT-2-dependent pathway. No difference in Zn levels was observed for the CeMT-2 knockout mutant, indicating that zinc homeostasis functioned normally even in the presence of Cd(II). Finally, in the double knockout, a significant increase in Zn(II) levels was observed, indicating significant disruption of Zn(II) homeostasis. CeMT-1 and CeMT-2 provide a system for discrimination between essential Zn(II) and toxic Cd(II) The question of how cells select the correct metal ions is of current interest [45]. One emerging concept holds that it is not the absolute but the relative affinity of various metal-trafficking proteins towards various metal ions in a common cytosol that governs metal ion selection and distribution. The in vitro and in vivo data presented here are consistent with this concept, and allow development of a framework that helps to understand the discrimination between Zn(II) and Cd(II) by the two metallothioneins in C. elegans,as well as at a more general level. To illustrate this idea, we have used the in vitro (Table 2) and in vivo (Fig. 4) data to approximate the proportion of metal ions bound to CeMT-1 and CeMT-2 if presented with Zn : Cd ratios as encountered by C. elegans. Using a Cd : Zn ratio of 33 : 1 [21 nm Cd(II) and 0.7 lm Zn(II)] and 0.1 lm of CeMT-1 and CeMT-2 each, and the stability constants given in Table 2, it can be calculated that 98.6% of Cd(II) is bound to CeMT-2, and only 1.4% to CeMT-1. Zn(II) is more evenly distributed (45 : 55%) between CeMT-1 and CeMT-2. When equimolar amounts of Zn(II) and Cd(II) are used (0.65 lm each), 93% of Zn(II) is bound to CeMT-1, and 85% of Cd(II) is bound to CeMT-2. With a 10-fold excess of MTs and the same metal con- centrations, 98.4% of Cd are bound to CeMT-2, and the Zn(II) distribution is 57 : 43% for CeMT-1 : CeMT-2. These numbers have been calculated based on two relatively crude simplifications: first that all binding sites in CeMT-1 and CeMT-2 are equivalent, and second that no other competing ligands are present. It is conceivable that the overall reduction in Cd(II) affinity is to a con- siderable extent, but not exclusively, due to weaker binding to the histidine-rich site. It is therefore likely that the difference in affinities for binding to the Cadmium Concentration (ng·L –1 )Concentration (ng·L –1 ) Zn-exposed a a b c ND ND ND ND ND ND ND ND WT CeMT-1 KO CeMT-2 KO Double KO 0 500 1500 1000 2000 2500 3000 c b a a a b c b a 3500 Zinc a a a * ** * * ** ** ** Control 0 Cd-exposed 20 40 60 80 100 Double KO CeMT-2 KO CeMT-1 KO WT A B Fig. 4. Metal accumulation in nematodes. Levels of cadmium (A) and zinc (B) were quantified by ICP-OES in Caenorhabditis elegans wild-type and metallothionein deletion strains cultured in the pres- ence or absence of cadmium (25 l M) or zinc (340 lM). Values are the means ± SEM of five replicates. Different letters above bars indicate statistical significance compared with each other. *P < 0.05;**P < 0.01. ND, not detectable (below detection limits). S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2537 all-thiolate sites in the two proteins is < 1.9 orders of magnitude. However, even a difference of only 0.3 log units would achieve a 66 : 34% distribution of Cd(II) in CeMT-1 and CeMT-2, and, given the presence of fur- ther mechanisms, this sorting level may be sufficient to ensure tolerable management of both Zn(II) and Cd(II) in C. elegans. Our simplistic model demonstrates that, even though both MTs show an overall preference for Cd(II) over Zn(II), as expected for predominantly thio- late coordination, the decrease in affinity of Cd for CeMT-1 may allow segregation of Cd(II) into predo- minantly CeMT-2 in a common cytosol. Discussion Like other soil-dwelling organisms, C. elegans nema- todes are constantly exposed to and ingest varying lev- els of essential and toxic metal ions present in the surrounding medium. Consequently, such organisms require mechanisms that capture and redistribute the correct amounts of biologically essential metal ions whilst preventing the accumulation of harmful levels of toxic metal ions. Within this framework, mechanisms must exist that allow the cell to distinguish between closely similar essential and toxic metal ions, such as Zn(II) and Cd(II), respectively. The predominating Cd–S and Zn–O forms observed by X-ray absorption analysis suggest that separate pathways exist for traf- ficking of these two metal ions. These pathways do not appear to be MT-mediated, and the negligible effect on in vivo speciation for either Cd(II) or Zn(II) in knockout mutants has excluded the possibility that MTs function as metal storage proteins in C. elegans. In contrast, the reduced accumulation, or excretion, of cadmium and zinc is MT-mediated, as there was a large effect on the levels of accumulated zinc and cadmium when CeMT-1 and CeMT-2 were deleted. We interpret this observation as an indication that some processes, possibly excretion of excess zinc and cadmium, do not function normally in the double knockout strain. Furthermore, and most importantly, the extents to which these MT- mediated processes are disrupted are isoform- and metal-ion specific. We have shown that CeMT-2 plays a more significant role in preventing hyperaccumulation of cadmium. Conversely, both CeMT-1 and CeMT-2 are important in maintaining physiologically acceptable zinc levels, and the lack of CeMT-1 had a more deleteri- ous effect. These metal-specific preferences at the cellu- lar level are mirrored in the relative affinities of the individual CeMT-1 and CeMT-2 proteins towards Zn(II) and Cd(II). The thermodynamic data suggest that, when presented with both MT isoforms, cadmium ions preferentially bind to CeMT-2, thus leaving CeMT- 1 to deal with zinc. The origin of this differential affinity is most likely rooted in the structure of the two isoforms. It is conceivable that the differences in specificity are, at least to a considerable extent, associated with the four additional metal ligands in CeMT-1, particularly the his- tidine residues (see Fig. S1A). Previous studies on both zinc fingers [46] and metallothioneins [47–50] have dem- onstrated that an increasing number of histidine residues in a metal binding site shifts the preference towards Zn(II). Further studies, including determination of 3D structures for CeMT-1 and CeMT-2, are required to determine the precise cause of the observed metal speci- ficities. In conclusion, the nematode C. elegans exhibits both MT-mediated and non-MT-mediated pathways to deal with cadmium and zinc. We have shown for the first time that the responses to cadmium and zinc ions at the cellular level are isoform-specific, and that this specificity is reflected at the protein level. Experimental procedures Cloning of MT constructs Total RNA was isolated from nematodes using TRI reagent (Sigma, St Louis, MO, USA) and reverse tran- scribed into cDNA from 1 lg RNA using oligo(dT) primers and MMLV reverse transcriptase (Stratagene, La Jolla, CA, USA), all according to the supplier’s protocols. C. elegans metallothioneins were amplified by PCR from cDNA using isoform-specific primers containing SalI and NdeI restriction site extensions (mtl-1_fwd: 5¢-TATACAT ATGGCTTGCAAGTGTGACTGC-3¢; mtl-1_rev: 5¢-AGC TTGTCGACGTTAATGAGCCGCAGCAGTTCCC-3¢; mtl-2_fwd: 5¢-TATACATATGGTCTGCAAGTGTGACT GC-3¢ and mtl-2_rev: 5¢-AGCTTGTCGACGTTAATGA GCAGCCTGAGCACAT-3¢), generating DNA fragments of 247 and 211 bp for isoform 1 and isoform 2, respec- tively. The purified PCR products, as well as the plasmid pET29a, were digested using SalI and NdeI (Promega, Madison, WI, USA) at 37 °C for 3 h, and ligated overnight at 4 °C using T4 ligase (Promega). The ligations were trans- formed into DH5a-competent cells (Invitrogen, Carlsbad, CA, USA) and positive clones were identified by PCR screening. The identity of the insert was confirmed by sequencing both strands of the cloned inserts. In vitro protein expression and purification Plasmids containing the respective metallothionein isoform were transformed into E. coli Rosetta TM2 (DE3)pLysS (Merck, Nottingham, UK) using standard molecular clon- ing techniques. Expression cultures (1 L) selective for kana- mycin and chloramphenicol (50 and 34 lgÆmL )1 , C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al. 2538 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS respectively) were induced using isopropyl b-d-1-thiogalac- topyranoside (500 lm final concentration). Following induction, ZnSO 4 or CdCl 2 (both Sigma) were added to a final concentration of 500 lm. Protein expression was per- formed for up to 6 h at 30 °C before harvesting the cells by centrifugation at 5000 g. Cell pellets were resuspended in ice-cold sonication buffer (50 mm Tris ⁄ Cl, 0.1 m KCl, 3mm dithiothreitol, 1 mm ZnSO 4 , pH 8.5), and ruptured by sonication. This was followed by centrifugation at 45 000 g for 45 min to remove cell debris. The resulting lysate was subjected to a chemical fractionation similar to that described by You et al. [32]. Briefly, strepto- mycin (10% w ⁄ v solution) was added to the lysate at 0.375 mLÆg )1 wet cell weight. To this mixture, one volume of chilled ethanol ⁄ chloroform (100 : 8) solution was added dropwise with continuous stirring. The mixture was centri- fuged for 5 min at 5000 g. A further three volumes of the ethanol ⁄ chloroform solution was added dropwise to the resulting supernatant, and this mixture was stored over- night at )20 °C. The precipitate was collected by centrifu- gation at 5000 g, resuspended in 20 mm ammonium bicarbonate (pH 7.8), and purified by gel filtration (16 ⁄ 60 HiLoadÔ 75 SuperdexÔ prep grade, GE Healthcare, Little Chalfont, UK). MT-containing fractions were pooled and concentrated by ultrafiltration (Amicon Ultra; Millipore, Billerica, MA, USA). The isolated proteins either retained or did not retain the N-terminal methionine. The cleavage efficiency of the E. coli Met aminopeptidase appeared to be dependent on the metal ion supplied, such that MTs expressed in the presence of Cd(II) mostly retained the initi- ation methionine. Preparation of Cd 7 -CeMT-1 Cd 7 -CeMT-1 was prepared using a modified procedure based on the method reported by Vas ˇ ak [33]. Briefly, an ali- quot of Zn 7 -CeMT-1 (50 mm Tris, 50 mm NaCl, pH 7.4) was incubated at room temperature with dithiothreitol (approximately 10 mm) for 1 h. This mixture was acidified to a pH of approximately 1 using 2 m HCl, and applied to a gel filtration column (Sephadex G25, PD10, Amersham Biosciences). The demetallated protein was eluted under nitrogen gas using 0.1 m HCl. CdCl 2 (7.5 molar equiva- lents) was added to the eluate, and the pH was increased to > 7.0 via addition of 2 m Tris base. Extensive washing by ultrafiltration ensured removal of unbound metal ions. Mass spectrometry All isoforms (20 lm) were buffer exchanged into 10 mm ammonium acetate (pH 7.2) by ultrafiltration. Prior to the analysis, methanol was added to a final concentration of 10% v ⁄ v. Samples were infused directly via a syringe pump operating at a rate of 250 lLÆh )1 . Analyses were performed using either ESI-TOF (MicrOTOF; Bruker, Bremen, Germany) or ESI-ion trap (HCT-UltraTM Dis- covery System; Bruker) mass spectrometers. Data were acquired for 1.5 min in the positive mode over the m ⁄ z range 500–3000 Th. Using data analysis software supplied by Bruker Daltonics, smoothing and baseline subtraction were applied to averaged data, which were subsequently deconvoluted. 19 F-NMR spectroscopy A sample of each C. elegans metalloform, approximately 480 lm with respect to metal ion concentration, was pre- pared in 10 mm Tris ⁄ Cl (pH 8.1, 10% D 2 O). Accurate determinations of metal ion content were performed using ICP-OES (Optima 5300 DV ICP-OES; PerkinElmer, Cambridge, UK). 5F-BAPTA [1,2-bis(2-amino-5-fluoro- phenoxy)ethane-N,N,N¢,N¢-tetraacetic acid; 4 mm final con- centration] was added to the sample, and incubated overnight at room temperature. Direct observe 1D 19 F-NMR spectroscopy was performed using a DRX400 spectrometer (Bruker) fitted with a quadruple nuclei probe (QNP) probe head operating at 375.91 MHz for 19 F nuclei. Chemical shifts are reported with respect to the signal for CCl 3 F [51]. Spectra were acquired at 298 K with a spectral width of 50 p.p.m., an acquisition time of 3.48 s and a relaxation delay of 1.0 s, with 12 288 scans. Fre- quency Induction Decay (FID)s were apodized using squared-sine bell functions, Fourier-transformed using 65 536 complex data points, and baseline-corrected. Spectra were processed using topspin version 2.1 software (Bruker Biospin). The value for K Cd(BAPTA) (at 30 °C and I = 138 mm) was corrected for temperature (25 °C) and ionic strength (4 mm) as described by Hasler et al. [34] to give a log K value of 11.75. Calculations of apparent stabil- ity constants for metal–MT complexes were performed using a published procedure [34]. Sample preparation for in vivo studies Wild-type (N2) and the CeMT-2 knockout strain mtl-2 (gk125) were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota, Minneapolis, MN, USA, and the CeMT-1 knockout strain mtl-1 (tm1770) was obtained from the Mitani Laboratory at the Tokyo Women’s Medical University School of Medicine, Japan. The metallothionein double knockout mtl-1;mtl-2 (zs1) was generated previously [25]. Each strain was syn- chronized (bleach prepped), and 300 000–500 000 L1 nema- todes were cultured on nematode growth medium containing sub-lethal concentrations of either CdCl 2 (25 lm) or ZnCl 2 (340 lm). A maximum of 2000 nematodes were cultured per plate (90 mm diameter), and grown at 20 °C to pre-adult stage (L4), then harvested and quench- frozen by immersion in liquid nitrogen. S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between metals FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS 2539 Metal quantification Nematodes were digested in 1 n concentrated nitric acid, and metal concentrations were quantified by inductively coupled plasma optical emission spectrometry (ICP-OES) using standard methods [52]. X-ray absorption spectra collection and analysis The samples were ground to a fine powder under liquid nitrogen, and stored as fully hydrated deep-frozen samples at )80 °C. The X-ray absorption spectra at the cadmium K-edge (approximately 26 710 eV) and the zinc K-edge (approximately 9660 eV) were collected on station 16.5 of the Synchrotron Radiation Source (now closed) at the Science and Technology Facilities Council Daresbury Labo- ratory, Warrington, UK. The ring operated at 2 GeV with a mean current of 140 mA: the station was equipped with a vertically focusing mirror and a flat Si (220) double crystal monochromator detuned to 70% transmission to minimize harmonic contamination. The monochromator was cali- brated at each energy value using a 15 lm cadmium foil or a10lm zinc foil. Data were collected with the station operating in fluorescence mode using an Ortec 30 element solid-state Ge detector. The samples were mounted onto aluminium sample holders, and X-ray absorption spectros- copy measurements were performed at cryogenic tempera- ture (approximately 20 K) using an Oxford Instruments helium closed-cycle cryostat. The standard samples were prepared by grinding in an agate pestle and mortar, diluted with boron nitride to give an edge step of approximately 1, and mounted in 1 mm thick aluminium sample holders with Sellotape windows. Single scans were collected for the model compounds in the transmission mode, and 16–23 scans were collected and summed for each experimental sample. Background subtraction and analysis of EXAFS spectra were performed as described previously [36]. Acknowledgements This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC grant BB ⁄ E025099), the Science and Technology Facilities Council (STFC grant BB ⁄ E05099), an Altajir Trust PhD studentship (to S.Z G.), and the Royal Society (Olga Kennard Fellowship to C.A.B.). The X-ray absorption spectroscopy was performed at the Dares- bury Synchrotron Radiation Source (station 16.5), managed and kindly assisted by Mr Bob Bilsborrow. We wish to acknowledge Dr Suresh Swain (King’s College London) and Dr Samantha Hughes (King’s College London, now at Oxford University) for valu- able advice and resources provided throughout the project, and finally the Caenorhabditis Genetics Centre (CGC), which is funded by the National Institutes of Health National Centre for Research Resources, for the supply of Caenorhabditis elegans wild-type (N2) and mtl-2 ( gk125) and Escherichia coli OP50, and the Mitani Laboratory at the Tokyo Women’s Medical University School of Medicine, Japan, for the supply of mtl-1 (tm1770). References 1 Van Straalen NM & Roelofs D (2008) Genomics technology for assessing soil pollution. J Biol 7, 19. 2 Timmermans MJTN, de Boer ME, Nota B, de Boer TE, Marie ¨ n J, Klein-Lankhorst RM, Van Straalen NM & Roelofs D (2007) Collembase: a repository for springtail genomics and soil quality assessment. BMC Genomics 8, 341. 3 Bundy JG, Sidhu JK, Rana F, Spurgeon DJ, Svendsen C, Wren JF, Stu ¨ rzenbaum SR, Morgan AJ & Kille P (2008) Systems toxicology approach identifies coordi- nated metabolomic responses to copper in a terrestrial non-model invertebrate, the earthworm Lumbricus rubellus. BMC Biol 6, 25. 4 Owen J, Hedley A, Svendsen C, Wren JF, Jonker M, Hankard KP, Lister L, Stu ¨ rzenbaum SR, Morgan AJ, Spurgeon DJ et al. (2008) Transcriptome profiling of developmental and xenobiotic responses in a keystone soil animal, the oligochaete annelid Lumbricus rubellus. BMC Genomics 9, 226. 5 Menzel R, Swain SC, Hoess S, Claus E, Menzel S, Steinberg CE, Reifferscheid G & Stu ¨ rzenbaum SR (2009) Gene expression profiling to characterize sediment toxicity – a pilot study using Caenorhabditis elegans whole genome microarrays. BMC Genomics 10, 160. 6 Galay-Burgos M, Spurgeon DJ, Weeks JM, Stu ¨ rzenbaum SR, Morgan AJ & Kille P (2003) Developing a new method for soil pollution monitoring using molecular genetic biomarkers. Biomarkers 8, 229–239. 7 Spurgeon DJ, Stu ¨ rzenbaum SR, Svendsen C, Hankard PK, Morgan AJ, Weeks JM & Kille P (2004) Toxico- logical, cellular and gene expression responses in earth- worms exposed to copper and cadmium. Comp Biochem Physiol C Toxicol Pharmacol 138, 11–21. 8 Newman MC & McIntosh AW (1991) Metal Ecotoxi- cology: Concepts and Applications. Lewis Publishers, Chelsea, MI. 9 Rainbow PS & Dallinger R (1986) Strategies of metal detoxification in terrestrial invertebrates. In Ecotoxicolo- gy of Metals in Invertebrates (Dallinger R & Rainbow PS eds), pp. 245–290. Lewis Publishers, Boca Raton, FL. 10 Hamer DH (1986) Metallothionein. Annu Rev Biochem 55, 913–951. C. elegans metallothioneins discriminate between metals S. Zeitoun-Ghandour et al. 2540 FEBS Journal 277 (2010) 2531–2542 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... 183–197 The Royal Society of Chemistry, Cambridge, UK 24 Freedman JH, Slice LW, Dixon D, Fire A & Rubin CS (1993) The novel metallothionein genes of Caenorhabditis elegans Structural organization and inducible, cell-specific expression J Biol Chem 268, 2554–2564 C elegans metallothioneins discriminate between metals 25 Hughes S & Sturzenbaum SR (2007) Single and double ¨ metallothionein knockout in the. .. elements and transcription factors responsible for cell-specific expression of the metallothionein genes from Caenorhabditis elegans J Biol Chem 42, 29655–29665 22 Williams PL, Ma H, Glenn TC, Jagoe CH & Jones KL (2009) A transgenic strain of the nematode Caenorhabditis elegans as a biomonitor for heavy metal contamination Environ Toxicol Chem 10, 496 23 Sturzenbaum SR (2009) Earthworm and nematode ¨ metallothioneins. .. P & Sturzenbaum SR (2009) The metabolomic responses of ¨ Caenorhabditis elegans to cadmium are largely independent of metallothionein status, but dominated by changes in cystathionine and phytochelatins J Prot Res 8, 3512–3519 29 Swain SC, Keusekotten K, Baumeister R & Sturzenbaum SR (2004) C elegans metallothioneins: ¨ new insights into the phenotypic effects of cadmium toxicosis J Mol Biol 341, 951–959... analysis of the cadmium coordination geometry in cadmium thiolate clusters in metallothionein Inorg Chem 44, 4923–4933 36 Fomina M, Charnock JM, Hillier S, Alexander IJ & Gadd GM (2006) Zinc phosphate transformations by the Paxillus involutus ⁄ pine ectomycorrhizal association Microb Ecol 52, 322–333 37 Jiang DT, Heald SJ, Sham TK & Stillman MJ (1994) Structures of the cadmium, mercury, and zinc thiolate... Liu J & Diwan BA (2009) Metallothionein protection of cadmium toxicity Toxicol Appl Pharmacol 238, 215–220 15 Maret W (2009) Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals Biometals 22, 149–157 16 Hidalgo J, Aschner M, Zatta P & Vasak M (2001) Roles of the metallothionein family of proteins in the central nervous system Brain Res Bull 55, 133–145... Purification and characterization of recombinant Caenorhabditis elegans metallothionein Arch Biochem Biophys 375, 44–52 33 Vasak M (1991) Metal removal and substitution in vertebrate and invertebrate metallothioneins Methods Enzymol 205, 452–458 34 Hasler DW, Jensen LT, Zerbe O, Winge DR & Vasak M (2000) Effect of the two conserved prolines of human growth inhibitory factor (MTIII) on its biological activity and. .. elegans reveals cadmium dependent and independent toxic effects on life history traits Environ Pollut 145, 395– 400 26 Vatamaniuk OK, Bucher EA, Ward JT & Rea PA (2001) A new pathway for heavy metal detoxification in animals: phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans J Biol Chem 276, 20817–20820 27 Clemens S, Schroeder JI & Degenkolb T (2001) Caenorhabditis elegans. .. function relationship for metallothioneins: histidine coordination and unusual cluster composition in a zinc- metallothionein from plants Proteins 68, 922–935 Supporting information The following supplementary material is available: Fig S1 Amino acid sequence and expression ⁄ purification of recombinant Caenorhabditis elegans MTs Fig S2 Deconvoluted ESI mass spectra of Caenorhabditis elegans Cd6Zn-CeMT-1... Cd6Zn-CeMT-1 species (obtained using the method described for Cd7-CeMT-2 in Experimental procedures) Fig S3 Cd K-edge EXAFS spectra and Fourier transforms of Caenorhabditis elegans strains Fig S4 Zn K-edge EXAFS spectra and Fourier transforms of Caenorhabditis elegans strains Table S1 Total Cd and Zn contents in acid-digested nematodes This supplementary material can be found in the online version of this article... Romagosa M, Domenech J, Atrian S & Capdevila M (2009) Caenorhabditis elegans metallothionein isoform specificity – metal binding abilities and the role of histidine in CeMT1 and CeMT2 FEBS J 276, 7040–7056 31 Blindauer CA, Harrison MD, Robinson AK, Parkinson JA, Bowness PW, Sadler PJ & Robinson NJ (2002) Multiple bacteria encode metallothioneins and SmtA-like zinc fingers Mol Microbiol 45, 1421– 1432 32 You . The two Caenorhabditis elegans metallothioneins (CeMT-1 and CeMT-2) discriminate between essential zinc and toxic cadmium Sukaina Zeitoun-Ghandour 1 ,. knockouts. The inserts show the cadmium and zinc energy shifts between samples. S. Zeitoun-Ghandour et al. C. elegans metallothioneins discriminate between