Quantification of Metallothionein by Differential Pulse Polarography

Oct 22, 2008 - If metallothionein concentrations in invertebrates are to be used as biomarkers for metal contamination in the aquatic environment, ...
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Environ. Sci. Technol. 2008, 42, 8426–8432

Quantification of Metallothionein by Differential Pulse Polarography Overestimates Concentrations in Crustaceans KNUD L. PEDERSEN,† SØREN N. PEDERSEN,† JENS KNUDSEN,‡ A N D P O U L B J E R R E G A A R D * ,† Institute of Biology and Institute of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark

Received May 16, 2008. Revised manuscript received August 7, 2008. Accepted September 1, 2008.

If metallothionein concentrations in invertebrates are to be used as biomarkers for metal contamination in the aquatic environment, it is imperative that the methods used for quantitative analysis are reliable. A review of the literature concerned with quantification of crustacean metallothionein shows that utilization of differential pulse polarography generally results in higher concentrations than any other method. The obvious discrepancies were investigated by experimental comparison of three different methods (enzyme linked immuno sorbent assay (ELISA), a spectrophotometric assay, differential pulse polarography) for determination of metallothionein concentrations in the shore crab Carcinus maenas. Application of an ELISA to cytosolic tissue extracts of unexposed crabs gave basal metallothionein levels of approximately 180 and 80 µg g-1 dw in midgut gland and gill, respectively; the levels increased 14-fold and 11-fold after exposure to 2 mg l-1 Cd for 3 weeks. The spectrophotometric assay generally gave 2-fold higher results than the ELISA in unexposed crabs and similar results in Cd-exposed crabs. The determination of metallothionein by differential pulse polarography (successfully applied in vertebrate tissue) was found to be unsuitable for crustacean tissues due to unidentified interfering compounds which led to 5- to 20-fold overestimation of metallothionein levels. The method should not be used unless thoroughly validated in the group of organisms in question.

Introduction The use of metallothionein (MT) concentrations in aquatic invertebrates as biomarkers for metal contamination in the environment has been suggested by several reviewers (1, 2) and during the latest decade a number of papers on metallothionein concentrations in mainly decapod crustaceans (3-11, 11-26) but also a number of nondecapod crustaceans (17, 27-37) have been published. For proper use of metallothionein as a biomarker it is imperative that the methods used for quantitative analysis are reliable. A number of methods for determination of metallothionein in vertebrate species exist: Indirect assays exploiting the metal-binding capacity of metallothionein include * Corresponding author e-mail: [email protected]. † Institute of Biology. ‡ Institute of Biochemistry and Molecular Biology. 8426

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quantification of bound metals (38, 39) and displacement assays using high affinity metals such as Cd (40), Hg (41), or Ag (42). Another strategy has been to measure the thiol contents of pretreated tissue supernatants by polarographic (43, 44) or spectrophotometric methods (45). Specific assays include high performance liquid chromatography (46) and the immuno-chemical techniques such as radioimmuno assays and enzyme linked immuno sorbent assays (ELISA) (47-49). Several comparative evaluations of different methods for the quantification of metallothionein in vertebrates have been published (45, 50). The polarographic methods used for determination of metallothionein concentrations in tissue homogenates from decapod crustaceans have largely been adopted from the methods used in vertebrates (43, 51). In their validation of the method for use in crustaceans, Thompson and Cosson (52) used purified metallothionein (from the crab Scylla serrata) and not tissue homogenates. It is apparent from Table 1 that metallothionein concentrations in gills and midgut glands of decapod crustaceans determined by means of polarographic methods generally appear to be 5-10-fold higher than metallothionein concentrations determined by other methods, although some exceptions are seen. The objective of the present work therefore was to compare different methods for determination of metallothionein concentrations in tissues of decapod crustaceans and for this purpose an ELISA for determination of C. maenas metallothionein was developed and validated. Results obtained by means of the ELISA method, differential pulse polarography, and spectrophotometry were compared.

Materials and Methods Exposure of Shore Crabs. Shore crabs, Carcinus maenas (L.) were caught in Egensedybet, Odense Fjord, Denmark in October and acclimated for a few days in large tanks supplied with running seawater with a salinity of 15-22 ppt until experiments began. For the experiments, mature green male crabs of 60-70 g body weight were selected and kept in groups of five in well-aerated 10 L polystyrene aquaria at a constant temperature of 15 °C. Sea-water was changed twice a week. The animals were not fed during the experiment and were assigned randomly to four groups of 10 individuals each, either unexposed (control) or exposed to 0.1, 1, or 2 mg L-1 of Cd, respectively. The cadmium was added as CdCl2 after each change of water. After 21 days of exposure, the crabs were killed. Midgut glands and gills were dissected out immediately, and collected in liquid nitrogen. Tissues were frozen in liquid nitrogen and stored at -80 °C until used. Determination of Metallothionein. Metallothionein concentrations in midgut gland and gills were determined by means of the newly developed ELISA, by spectrophotometry according to ref 53 and differential pulse polarography according to ref 43. Detailed descriptions of the development, validation, and procedure used for the ELISA is given in the Supporting Information together with details of the spectrophotometric and polarographic methods. The specificity of the differential pulse polarography response for metallothionein was tested on gel filtration fractions (from a Superdex 75 column) of supernatants from midgut glands of unexposed crabs without the final heat precipitation step (further details are given in the Supporting Information). The correct use of the pulse polarographic method for determination of metallothionein concentrations in vertbrate tissues was validated on rainbow trout liver (54) (described in the Supporting Information). 10.1021/es8013584 CCC: $40.75

 2008 American Chemical Society

Published on Web 10/22/2008

TABLE 1. Metallothionein Concentrations Reported in Gills (A) and Midgut Gland (B) of Decapod Crustaceans or Various Other Crustaceans (C) not Exposed to Metals in the Laboratory; Organisms Have Been Collected from Metal Contaminated Sites in Some of the Studiesa species

method

value

reference

Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus aestuarii Pachygrapsus marmoratus Eriocheir sinensis Charybdis japonica Nephrops norvegica Penaeus indicus Litopenaeus vannamei

A: Gills ELISA Spectro Spectro DPP DPP metal Spectro DPP Ag saturation Hg saturation DPP DPP Ag saturation

9.3 27-103 20 500 76 5-48 23-27 483-1318 40 1f 19c 700b 100-300e

this study (3) this study (4) this study (5) (9) (7) (58) (10) (12) (11) (13)

Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus maenas Carcinus aestuarii Pachygrapsus marmoratus Pachygrapsus marmoratus Eriocheir sinensis Charybdis japonica Nephrops norvegica Procambarus clarkii Procambarus clarkii Procambarus clarkii Procambarus clarkii Penaeus indicus Penaeus vannamei Litopenaeus vannamei Homarus americanus Macrobrachium rosenbergii

B: Midgut Gland ELISA Spectro Spectro Spectro Spectro metal metal DPP DPP DPP Spectro DPP Spectro DPP DPP Ag saturation Hg saturation DPP Ag saturation Ag saturation Ag saturation DPP DPP DPP Ag saturation ELISA Cd saturation

43 110-200b 61 228-301 112 100-200b 165 2000 1,000,000c 1.4c 250-350 1233 278 2000 1677-3244 350 2-5f 56c 120 120 110 17c 3250b 2500 250-300e 210 400

this study (6) (15) (16) this study (6) (59) (4) (18) (19) (14) this study (9) (4) (7) (58) (10) (12) (17) (21) (22) (20) (11) (23) (13) (24) (8)

Gammarus locusta Gammarus locusta Gammarus locusta Gammarus pulex Orchestia gammarellus Rimicaris exoculatag Mirocaris fortunatag Palaemon elegansg Palaemonetes variansg Palaemonetes pugio Tigriopus brevicornis Artemia parthenogenetica Artemia Daphnia magna Daphnia magna Daphnia magna Daphnia magna Daphnia longispina

C: DPP DPP DPP DPP DPP DPP DPP DPP DPP SDS-page DPP Ag saturation Ag saturation Ag saturation Ag saturation DPP DPP DPP

325b 67-307b 155-260b 1250-3250b 975b 1825d 318d 1085d 413d 0.07-0.22c 0.15-0.35 10-20 280 13-30 20-30 720 1250-2500b 1000-4500b

(29) (28) (27) (31) (32) (25) (25) (25) (25) (26) (33) (34) (17) (36) (35) (30) (37) (37)

a Metallothionein concentrations determined by ELISA, spectrophotometric methods (Spectro), i.e., according to ref 53, Cd, Cu, and Zn content in the metallothionein peak after gel filtration (Metal) according to ref 38, differential pulse polarography (DPP) according to, i.e., refs 43, 52. or metal saturation assays (67). Values are given as µg metallothionein g-1 wet weight. Some of the values given in the table are recalulated from the footnotes below. In the recalculation, wet:dry weight ratios for midgut gland (55) and whole organisms of 4 have been used. Wet:dry weight ratio for gills (55): 8. Twenty-five percent of the dry weight assumed to be soluble protein (66, 68). b Dry weight data. c µg MT mg-1 protein. d µg MT mg-1 wet weight protein. e mg Ag bound g-1 tissue wet weight. f nmol Hg mg-1 protein. g Whole organism except exoskeleton and gills.

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TABLE 2. Carcinus maenas. Concentrations of Cadmium in Midgut Glands and Gills of Control Crabs and Crabs Exposed to Cadmium for 3 Weeksa

midgut gland gill

control

0.1 mg Cd L-1

1 mg Cd L-1

2 mg Cd L-1

3.6 ( 1.3

18 ( 14

97 ( 37

359 ( 208

2.4 ( 3

35 ( 10

144 ( 39

298 ( 53

Values are given as µg Cd g-1 dry weight (means(SD). n ) 10 for the control, 0.1 and 1 mg Cd L-1 groups, and n ) 6 for the 2 mg Cd L-1 group. Average wet: dry weight ratios were 4.1 in midgut and 7.6 in gills. a

Metal Analysis. Tissue cadmium was determined by atomic absorption spectrophotometry on a Perkin-Elmer 2380 atomic absorption spectrophotometer as described in ref 55. Data Analysis. Data on metallothionein concentrations did not fulfill the assumptions for ANOVA and were tested nonparametrically using Kruskal-Wallis and nonparametrical Tukey-tests. Except for the latter multiple comparison, all statistics were calculated using SYSTAT. Statistical significance was accepted at the R ) 0.05 level of confidence.

Results Cadmium concentrations in midgut gland and gills (Table 2) of crabs exposed to cadmium for 3 weeks increased almost linearly with exposure concentration. In unexposed crabs, metallothionein determination by means of the ELISA method resulted in basal levels of metallothionein of 180 and 80 µg metallothionein g-1 dw in midgut and gill, respectively (Figure 1). The same tissue samples subjected to the spectrophotometric method gave basal levels of 440 and 160 µg metallothionein g-1 dw in midgut and gill, respectively (Figure 1), approximately twice the amount found by the ELISA method. Both methods showed a significant increase in the metallothionein concentrations in the midgut gland of crabs exposed to 1 or 2 mg Cd L-1 (Figure 1) and in the gills exposed to 2 mg Cd L-1 (Figure 1); the ELISA method also showed induction of the metallothionein synthesis in gills of crabs exposed to 1 mg Cd L-1 (Figure 1). The levels determined by the spectrophotometric method were correlated in both midgut glands and gills with the metallothionein values obtained by the ELISA method (r ) 0.92, p < 0.001, and r ) 0.88, p < 0.001, respectively) (Supporting Information Figure S1). Basal metallothionein levels determined by differential pulse polarography were approximately 10-20-fold (midgut gland, Figure 1A) and 4-8 fold (gills, Figure 1B) higher, respectively, than levels measured by spectrophotometry and ELISA. When measured by differential pulse polarography, cadmium exposure did not significantly increase the metallothionein levels in the midgut gland, whereas an increase from the basal metallothionein level (600 µg metallothionein g-1 dw) was observed in gill tissues upon exposure to 1 mg Cd L-1 for 3 weeks (Figure 1b); metallothionein levels measured by differential pulse polarography showed no significant increase in crabs exposed to 2 mg Cd L-1. No correlation was found between metallothionein levels determined by differential pulse polarography and corresponding values measured by ELISA or spectrophotometry (results not shown). When applied to rainbow trout livers, the differential pulse polarography technique gave values (Supporting Information Table S2) similar to those previously published by ref 54, indicating that the discrepancy in the crustacean samples was not caused by incorrect use of the differential pulse polarography technique in our laboratory. 8428

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FIGURE 1. Carcinus maenas. Comparison of metallothionein concentrations determined spectrophotometrically (open bars), immunochemically (ELISA, cross hatched bars) and by differential pulse polarography (solid bars) in midgut glands (A) and gills (B) of shore crabs exposed to 0, 0.1, 1, or 2 mg L-1 cadmium for 3 weeks. Values are given as mean + SEM (n ) 10 for all groups except the 2 mg L-1 group where n ) 6). An asterisk denotes that a group is significantly different from the control group with p < 0.05. The specificity of the differential pulse polarography response toward metallothionein in crab midgut gland tissues was investigated by differential pulse polarography analysis of individual fractions obtained from chromatographic separation of supernatants ((heat treatment) from unexposed crabs (Figure 2). After heat treatment, differential pulse polarography still responded to compounds of both higher and lower molecular weight than metallothionein, although most of the high molecular weight material absorbing at 254 nm had been precipitated (Figure 2B). An ELISA screening of the differential pulse polarography responsive fractions was only positive for the sample from the metallothioneinpeak (Figure 2B). The differential pulse polarography response from nonmetallothionein fractions comprised approximately 80% of the total response regardless of whether the supernatant had been heat treated or not. Also acetone (60% v/v) failed to precipitate the high molecular weight compounds responding to differential pulse polarography (results not shown).

Discussion The results of this investigation show that metallothionein concentrations in tissues of the shore crab Carcinus maenas are overestimated when they are detemined by pulse polarography compared to both ELISA and a spectrophotometric method; this is true for both midgut gland and gills, although the overstimation seems somewhat smaller in the gills than in the midgut gland. As pointed out by Dieter et al. (45), one of the main reasons for discrepancies between reported estimates of metallothionein levels in the same tissues, is a lack of standard reference material and proper calibration of standard curves. In order to compare three different methods, special attention

FIGURE 2. Carcinus maenas. Differential pulse polarography (bars) and UV-response (solid line) of gel filtration fractions obtained from supernatants of 1 g midgut gland from an unexposed crab. (A) Supernatant loaded directly on the column after homogenization and centrifugation. (B) Supernatant prepared as in A, but heat treated at 95 °C for 5 min before chromatography. Diamonds denote fractions screened for metallothionein with the ELISA assay. ), No ELISA response; solid diamond, positive ELISA reponse. Arrow, metallothionein fraction. was therefore paid to the accurracy in the present study. The standard curves for ELISA and differential pulse polarography were made from purified (described in the Supporting Information) metallothionein from the same species as investigated and further calibrated by amino acid analysis and calculation from the known protein sequence (56). Values obtained with the spectrophotometric method were converted to metallothionein concentrations from a standard curve of glutathione as described elsewhere (53). The overestimation demonstrated in the present investigation for C. maenas is in accordance with the general trend in the literature values summarized in Table 1, showing that for most decapod crustaceans, the concentrations of metallothionein are generally reported to be 5-20-fold higher in investigations where pulse polarography has been used than when any of the other techniques have been applied. The polarographic methods used for determination of metallothionein concentrations in tissue homogenates from decapod crustaceans have largely been adopted from the methods used in vertebrates. In their development of the method for use in crustaceans, Thompson and Cosson (52) used purified metallothionein (from the crab Scylla serrata) and not tissue homogenates; thus, potential interference from compounds present in the crustacean tissues would not have been noticed. In the present study, the differential pulse polarography method was validated by estimating metallothionein concentrations in livers from control- and cadmium-injected trout. Using the same protocol as Olsson et al. (54), almost identical levels of metallothionein were

obtained (Supporting Information Table S2) and this indicates that genuine differences exist between vertebrates and decapod crustaceans concerning interference in the determination of metallothionein concentrations by pulse polarography. The exact nature of the interference from the crustacean tissue is not known. Heat treatment of supernatants has proved to be a sufficient pretreatment for polarographic analysis of extracts from many species (57). However, in the present study, 80% of the response in midgut glands from control crabs was shown to come from nonmetallothionein material even after heat treatment (Figure 2). The fact that the differential pulse polarography-responsive material found outside the metallothionein-fractions was not recognized by the antimetallothionein antibodies proved that the material was not metallothionein that had been polymerized by the heat treatment. The remaining 20% of the total differential pulse polarography-response found in the metallothionein-fraction still comprised 2-4 times the amounts found by ELISA and spectrophotometry, probably due to nonmetallothionein compounds eluting in the metallothionein fraction. Differential pulse polarography-analysis of gel filtration fractions is the commonly used evidence for specificity of the differential pulse polarography-response (57) but our results might indicate that the method is not sufficient. A few of the pulse polarographic determinations of metallothionein in gills and midgut glands (11, 12) of decapod crustaceans do not give higher values (Table 1) than those obtained by some of the other methods, i.e., metal saturation assays (8, 13, 58) and spectrophotometric methods (9, 14, 16). An examination of the experimental procedures used by refs 11, 12 does not give any indication of the reason underlying this. Even if the apparently overestimated results from the differential pulse polarographic determinations are disregarded, the range of basal metallothionein concentrations reported for various crustaceans and tissues (Table 1) appears broader than one would expect from ordinary biological variation between species. Some authors obviously present errors in their units for metallothionein (17 mg metallothionein mg-1 protein as a basal level (18)), but generally there seems to be a need to consider if the results on absolute metallothionein contents obtained are realistic when compared across different methods, species and tissues. In some of the investigations in which crustacean metallothionein has been determined by means of differential pulse polarography, a low metal to metallothionein ratio strongly suggests that the metallothionein concentrations have indeed been overestimated. Legras et al. (4) present very detailed information about soluble concentrations of copper, zinc and cadmium in gills and midgut gland of crabssincluding C. maenasstogether with differential pulse polarographic determinations of metallothionein. Using average values for both the concentrations of soluble metals (Cd, Cu, Zn) and metallothionein, the calculation (from Figure 3 in ref 4, assuming wet to dry weight ratios of midgut and gills of 4 and 8 (55), respectively) shows that each molecule of metallothionein would only contain approximately 1.2 and 1 metal atom in midgut gland and gills, respectively. This calculation furthermore assumes that all soluble copper, zinc, and cadmium is bound in metallothionein and this is known not to be the case (59); in reality, the metal:metallothionein ratio is probably below one metal atom per two molecules of metallothionein. Given the normal metal binding capacity of crustacean metallothionein (six Zn/Cd atoms or nine Cu atoms (38)), this indicates either an unusually low degree of metal saturation on the metallothionein, a very large proportion of apo-metallothionein VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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or more likelysin view of the results of the present investigationsthat the differential pulse polarography determination of metallothionein has overestimated the real metallothionein concentrations by a factor of approximately 10. The problems with the pulse polarographic determination of metallothionein in crustacean tissues seem to extend to other invertebrate groups as well (60). Large, nonspecific differential pulse polarography responses from material that could not be heat precipitated were found in the digestive gland of the marine mollusc Littorina littorea (61) and metallothionein concentrations in the mussel Mytilus galloprovincialis were between 9 and 38 times higher when determined by pulse polarography than by spectrophotometry (62). The fairly high metallothionein concentrations reported in the sea urchin Strongylocentrotus droebachiensis (63) and the sea star Asterias rubens (64) indicate that also in echinoderms the use of pulse polarography should be carefully validated. In the midgut gland, differential pulse polarography was unable to detect an increase in metallothionein levels as a function of the large increase in accumulated cadmium in the present study (Table 2). In some investigations in which pulse polarographic determinations of crab metallothionein have been used, positive correlations have been identified between copper, zinc, and cadmium concentrations on one hand and, on the other hand, metallothionein concentrations in crabs collected from metal contaminated sites (4, 7), and in other studies metallothionein concentrations have been reported to increase upon laboratory exposure to metals in water or sediment (18, 19, 65). Since the exact nature of the confounding responses in the crustacean tissues is not known, it is difficult to say if these correlations are the result of real increases in metallothionein concentrations, increases in the concentrations of the confounding compounds upon exposure to metal, or a combination of both. The metallothionein levels measured by ELISA compared favorably with those obtained by the spectrophotometric method (Figure 1). Both ELISA and the spectrophotometric method gave similar results in midgut glands and gills from cadmium exposed crabs and values for individual animals obtained by the two different methods were correlated (Supporting Information Figure S1). The latter confirmed that the large variances found within each group were not due to experimental error, but reflected the large interindividual differences in metal handling known to exist in C. maenas (66). However, in unexposed crabs the spectrophotometric method gave approximately 2.5 and 2.0 times the concentration found by ELISA in midgut gland- and gill tissues, respectively (Figures 1 and 2). Exactly the same differences were reported in rat liver cytosols by (45) when comparing an immunochemical technique (RIA) with a spectrophotometric assay. The overestimation by the spectrophotometric assay is probably due to high levels of nonmetallothionein thiol groups relative to the metallothionein concentrations expressed under unexposed conditions. The spectrophotometric method has previously been used to estimate concentrations of metallothionein in midgut glands of C. maenas from the heavily contaminated Fall Estuary, UK (5); in this investigation, the elevated metallothionein levels (400-500 µg g-1 dry weight) were confirmed by measurements of metals associated with metallothionein fractions. In conclusion, although the differential pulse polarography method seems to be specific for metallothionein in most vertebrates after heat precipitation of tissue extracts, this pretreatment appears inadequate in decapod crustaceans. In crustaceans, nonspecific responses were found in metallothionein-fractions even after chromatographic separation which makes the method highly questionable for quantification of metallothionein in these species. The 8430

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spectrophotometric method proved suitable for quantification of crustacean metallothionein from exposed animals, but care should be taken when working with very low levels of metallothionein.

Acknowledgments We thank Bente Frost, Vibeke Eriksen, Christina Madsen, and Leif Hansen for tecnical assistance and help in keeping the animals. This work was funded by the Danish Environmental Research Programme and Danish Natural Science Research Council grants to P.B. and a PERC grant to J.K.

Supporting Information Available A detailed description of the development and validation of the ELISA method for quantifying Carcinus maenas metallothionein is available together with detailed information about the polarographic and spectrophotometric methods. This material is available free of charge via the Internet at http://pubs.acs.org.

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