Environ. Sci. Technol. 2004, 38, 6283-6289
Cadmium Detoxification in Earthworms: From Genes to Cells† STEPHEN R. STU 2 R Z E N B A U M , * ,‡ OLEG GEORGIEV,§ A. JOHN MORGAN,‡ AND PETER KILLE‡ Cardiff University, School of Biosciences, Main Building, P.O. Box 915, Cardiff, CF10 3TL U.K., and Institute for Molecular Biology, Zurich University, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Cadmium pollution has clear ecotoxicological consequences as it is readily bio-available and has a reported tendency to bio-accumulate in soil biota. Understanding the mechanisms of cadmium trafficking pathways within sentinel terrestrial invertebrates, such as the earthworm, is therefore considered to be of importance. Using X-ray microanalysis, quantitative polymerase chain reaction, and immunohistochemical techniques, we were able to demonstrate that the earthworm has the intrinsic capacity to efficiently sequester and compartmentalize cadmium via a metallothionein-mediated trafficking pathway. There is evidence that wMT-2, rather than wMT-1, is the major isoform implicated in the detoxification of cadmium and the identification of three independent wMT-2 loci (totalling over 25 kb of genomic sequence) has revealed a complex genomic organization. Complementary in silico analysis of over 6500 expressed sequence tags has identified a third metallothionein isoform, wMT-3, found to be highly enriched in embryonic tissue. In summary, this paper provides a detailed dissection of the genetic, molecular, and cellular basis of a sophisticated pathway that facilitates the uptake, accumulation, transport, and excretion of cadmium.
Introduction The world average soil cadmium concentration is 0.62 mg/ kg, but geological features and a variety of anthropogenic activities can give rise to local concentrations by several orders of magnitude (1). Cadmium is considered to be a highly toxic metal with no known biological function. The U.S. EPA has classified it as a Group B (probable human carcinogen) pollutant (2), and the European Union restricts its concentration in sewage-amended agricultural soils to 1-3 mg/kg (3-5). The ecotoxicological consequences of cadmium pollution are exacerbated by the metal’s potential bioavailability as well as its tendency to biaccumulate with a long half-life in biota (6-8). Living organisms have evolved by exploiting certain metals for structural and catalytic purposes, while discriminating against nonessential metals whose physical properties and ligand affinities resemble those of their essential analogues (9, 10). Metal selectivity and discrimination in biological systems is often weak as exem† The sequence data reported in this paper have been submitted to the EMBL/Genbank Libraries under the accession numbers AJ299434, AJ299435, and AJ299436. * Correspondence e-mail:
[email protected]; telephone: +44 2920 874119; fax: +44 2920 874305. ‡ Cardiff University. § Zurich University.
10.1021/es049822c CCC: $27.50 Published on Web 09/29/2004
2004 American Chemical Society
plified by the transport of cadmium via Ca-ATPases in a number of vertebrate cell types (11). Thus, functioning in the presence of disruptive extraneous metals is a perpetual threat faced by all cells, which is magnified in polluted environments. Earthworms are considered to exert significant direct and indirect positive effects on soil quality and fertility (12), and consequently, they are important organisms in ecotoxicity tests and in contaminated land assessments (13, 14). The modes of life, bauplan, and cellular physiology of earthworms conflate to confer on them the ability to macroaccumulate certain essential and nonessential metals (15), including cadmium. It has been documented (16, 17) that viable populations of earthworms inhabit a mixture-contaminated soil with “total” cadmium concentration exceeding the published 14-day LC50 for Cd (18). Thus, some earthworm populations appear to be relatively insensitive to cadmium (19). Moreover, earthworms excrete cadmium slowly and sequester it efficiently, such that whole-body bioconcentration factors invariably exceed unity even in heavily contaminated soils (20). Most of the cadmium body burden in earthworms is compartmentalized and bound to inducible, cysteine-rich, metalloproteins called metallothioneins (wMTs) (21-27). It has frequently been assumed that cadmium-induced MT up-regulation and cadmium binding to MT suffices to demonstrate a central role for MT in cadmium detoxification. Some authors have rightly admonished against extrapolating from phenomenon to function (28-30). However, evidence is accumulating in vertebrates that certain MT isoforms are implicated in cadmium detoxification (31), while other isoforms have distinct tissue distributions and probable functions (29, 32). Earthworms have been shown to possess two MT isoforms with different metal stabilities and inferred functions (23, 26), one of which (wMT2) is more responsive to cadmium than to copper (33). However, the function of wMT and its relationship to other proteins or ligands still remains incomplete in invertebrates in general and earthworms in particular. Metallothioneins have been much vaunted as biomarkers of metal exposure under laboratory and field conditions (34), but to harness their full potential in earthworms more knowledge is required about gene structure, induction potential and mechanisms, isoform properties, tissue distribution, and trafficking pathways. This paper aims to address these issues.
Methods X-ray Microanalysis. Freshly excised pieces (∼1 mm3) of chloragogenous tissue were hyperbarically frozen (Bazers HPM 010 high-pressure freezer) prior to thin cryosectioning at -125 °C in a Leica FC4E ultracryo chamber attached to a Reichert Ultracut E ultramicrotome. Sections were mounted on coated titanium (200 mesh) grids and freeze-dried prior to subcellular element mapping by quantitative digital X-ray microanalysis. Analysis was performed in a JEOL JEM-1210 transmission electron microscope (TEM) equipped with a LINK ATW Pentafet detector (138 eV resolution) and a LINK “ISIS” analyzer (Oxford Instruments). Quantification was based on the “Hall Continuum Normalisation Quantification Procedure” (35). Counts were corrected for signals arising from the holder, the grid, and the support film. Convoluted elemental peaks were resolved utilizing a library of spectra derived from pure salt standards. Scanning was performed in “fine probe” mode producing a narrow convergent beam of ∼100 nm diameter. The detector and the beam current were adjusted to minimize dead time to provide a count rate VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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of ∼5000 counts/s. To increase spatial resolution, the quantitative maps were background corrected and converted to single-element color qualitative maps and finally overlaid in pairs to provide information about metal and major anion (P, S) co-distribution. Soils/Earthworms. Earthworms (Lumbricus rubellus) were obtained from a commercial source (Neptune-Ecology, Ipswich, UK) and either maintained for the duration of the experiment in the supplied soil or transferred to heavy metal contaminated soil (Shipam, O.S. Grid reference ST 448573). Ten worms (all healthy, clitellate, and of similar size, 500700 mg fresh weight) were maintained in containers with perforated lids and kept for 7 weeks at 15 ( 1.5 °C in a 16-h light/8-h dark regime. Four representative soil subsamples and three individual adult earthworms were acid-digested for metal analysis as described in ref 36 and analyzed by flame atomic absorption spectrometry (Varian Spectra 30, GTA 95). Quantitative Polymerase Chain Reaction (qPCR). Three further earthworms from each of the two exposure regimes were individually homogenized and processed for quantitative PCR essentially as described in ref 37. Quantification of metallothionein transcription was determined using primers that amplify all metallothionein isoforms (23). Expression values were calculated from three sample measurements from each individual earthworm and normalized against actin, a housekeeping gene that has previously been determined to be invariant within the experimental scenario used (38). Expressed Sequence Tag (EST) Database. The relational sequence database LumbriBASE (see ref 39 for method and http://www.earthworms.org for access) was queried for sequence annotations containing the word metallothionein. All hits were individually re-analyzed, and only full-length sequences (containing the entire coding region) were taken further for phylogenetic analysis using the EMBL-EBI ClustalW software. Genomic Sequence. Genomic DNA was isolated from eight individual earthworms according to standard methods, however followed by at least 10 extensive phenol washes and a final RNase treatment step. DNA was digested and ligated into λ arms and packaged according to the manufacturers guidelines (Stratagene). The genomic library was grown in Q359 cells, and the final titer exceeded 500 000 plaque forming units (pfu). The entire library was plated onto four large plates and screened using a wMT-2 probe. In detail, the probe was obtained using primers initially designed to amplify 170 bp within the second half of the wMT-2 coding region (5′-ACATGTGCCTGCTCCAAATG-3′ and 5′-TCACCACAGCATCCTTTCTTG-3′). Using genomic DNA as a template, it was possible to amplify a 1163 bp fragment containing a 993 bp intron, surrounded by the wMT-2 flanking sequence. Using standard protocols, the genomic library was replica blotted onto filters to which the Nick column (Pharmacia) fractionated probe mix (wMT-2 probe, random nucleotides,32P, enzymes and buffers in Church Hybridization solution) was added. The filters were incubated overnight at 65 °C under continuous rotation. The filters were washed four times for 30 min at 70 °C and exposed to photographic film for 7 h. After development, 32 putative wMT-2 positive plaques were identified, excised, and eluted at room temperature (shaking in 500 µl SM buffer) and individually plated onto 90-mm Petri dishes. In a secondary screen (this time using duplicate filters), eight positive excisions could provisionally be re-confirmed. λ DNA was subsequently isolated from all eight positives, and a final Southern experiment identified three final wMT-2 positive isolates. The sequence was determined by direct sequencing via primer walking, with a coverage exceeding 3-fold, and the contig aligned using the Sequencher software (Gene Codes Corporation). 6284
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FIGURE 1. Spatial distribution of selected elements within part of a freeze-dried cryosection of a chloragocyte. Single pseudo-colors represent qualitative locations of calcium, phosphorus, sulfur, and cadmium determined by X-ray microanalysis. Note that final overlays (right-hand panels) indicate that calcium and phosphorus are colocalized within discrete compartments that are distinct from the compartments containing sulfur and cadmium. Immunohistochemistry. A polyclonal antibody raised against wMT-2 (see ref 26 for isolation procedure) was applied to formalin-fixed, wax-embedded, earthworm sections using an established immunoperoxidase-staining protocol (22, 27). Sections were imaged and recorded with an Olympus BH2RFL light microscope and Fuji HC-300Z digital camera system.
Results and Discussion Earthworms inhabit some of the world’s most heavily contaminated sites. In doing so they are exposed to, and are obliged to deal with, xenobiotics considered to be highly toxic. Cadmium is a prime example as it is a heavy metal that is abundant, non-essential, and toxic; hence its detoxification and immobilization is of utmost importance to the animal. Previously, it has been shown that a high proportion of the cadmium burden accumulates in the intestinal region of earthworms, particularly in the chloragogenous tissue coating the coelomic surfaces of the gut (40). This could be substantiated using X-ray microanalysis on anhydrous cryosectioned chloragogenous tissue. Two distinct metalsequestering compartments could be identified, namely, an electron dense granule that co-localizes calcium, phosphorus, lead, and zinc (the latter two not shown for reasons of simplicity) and a vesicular structure containing cadmium and the bulk of detectable sulfur (see Figure 1). The codistribution of sulfur and cadmium within distinct compartments provides initial, though only circumstantial, evidence that a sulfur-cadmium thiol complex, such as metallothionein, may be involved. We have previously been able to isolate two metallothionein isoforms from the earthworm that are capable of several orders-of-magnitude transcriptional up-regulation (23). These
TABLE 1. Nitric Acid Extractable Heavy Metal Content of Commercial Soil Supplied with the Earthworms (“Native Soil”) and Soil Sampled from Shipham Commona
a
soil
cadmium
copper
lead
zinc
“native” soil Shipham soil
>0.001 ( 0.0 279.0 ( 78.4
17.7 ( 3.9 36.3 ( 4.6
14.6 ( 1.5 7080 ( 1321
94.1 ( 25.7 35265 ( 4007
Values are expressed as µg/g dry weight ( standard deviation from four replicate samples.
TABLE 2. Nitric Acid Extractable Heavy Metal Content in Earthworm Samplesa pre-transfer post-transfer metal concentration factor transcriptional up-regulation
cadmium
copper
lead
zinc
MT
0.001 ( 0.0 1 129 ( 66.8 4.9
8.0 ( 3.5 11.3 ( 2.3 0.07
5.0 ( 3.0 982 ( 320 0.14
714 ( 78.0 3223 ( 457 0.18
52.0 ( 4.2 40 299 ( 1984 775
Values are expressed as µg/g dry weight ( standard deviation from three individual worms previously not exposed to heavy metals (pretransfer) or after 7-week exposure to Shipham soil (post-transfer). Concentration factors were calculated as ([cw]post -transfer - [cw]pre-transfer)/ ([cs]post-transfer - [cs]pre-transfer), where cw ) worm metal concentration and cs ) soil metal concentration. Metallothionein (MT) transcription was determined using the Lightcycler. Values are expressed as molecules per microliter of reverse transcribed and actin normalized mRNA from triplicate measurements of three individual samples. a
preliminary data have been exploited to assess accumulation of metals and transcriptional up-regulation of “total” metallothionein levels over a 7-week exposure experiment. In detail, naı¨ve control earthworms (with no past contact with significant levels of toxic heavy metals) were obtained (with their native soil) from a commercial source. Acid-extractable heavy metal concentration was negligible in both the reference soil (Table 1) and earthworms (Table 2). In contrast, soil obtained from Shipham, a mine in southwest England, contained significant amounts of cadmium (279 µg/g dry soil), lead (7080 µg/g dry soil), and zinc (35265 µg/g dry soil) (Table 1). Naı¨ve earthworms increased their heavy metal body burden after a 7-week exposure period to Shipham soil (Table 2). Interesting is the fact that the concentration factor remained below “1” (the benchmark value that indicates an equilibrium between earthworm concentration and soil concentration) for the elements copper, lead, and zinc. In sharp contrast, an almost 5-fold bioaccumulation was observed in the case of cadmium, with average tissue levels exceeding 1100 µg/g Cd (Table 2). To escape a toxic (if not lethal) response, a highly effective protection mechanism is essential. Previously, we have been able to confirm that metallothionein is the major protein involved in the protective sequestration of cadmium (26), hence explaining the measured induction of metallothionein expression of some 775fold over the 7-week exposure period (Table 2). A comparable induction (314-fold) was previously observed in earthworms exposed to cadmium as a single metal ion over a 42-day period (33). This indicates that cadmium as a single metal can directly induce metallothionein transcription. Although it has been shown that copper alone is not able to significantly induce metallothionein expression (22), there is a possibility that other metals (such as zinc) may contribute to the observed transcript induction within complex mixtures. Nevertheless, the order of transcriptional up-regulation observed is impressive by any standard and supports the notion of a highly efficient detoxification mechanism. In an ongoing effort to unravel the genetic basis of what constitutes an earthworm, we have created a database of Expressed Sequence Tag (EST) that is fully searchable by sequence and/or annotation through a web-based interface (http://www.earthworms.org). Since our recent publication (39) that described the methodology and bioinformatic analysis of the first 577 sequences, the database has grown to include 6548 sequences generated from a control library (1498 ESTs), an earthworm cocoon library (2141 ESTs), and cadmium- (1478 ESTs) or fluoranthene- (1431 ESTs) exposed
libraries. Data mining of all 6548 sequences returned 27 true earthworm metallothionein hits, of which 22 were full-length sequences (i.e. encoding for the entire coding region) and included in the phylogenetic analysis. A ClustalW alignment of all full-length clones returned a three branched tree with 27% (6 ESTs) clustering into a wMT-1 like subgroup, 50% (11 ESTs) into a wMT-2 subgroup, and 23% (5 ESTs) into a previously unidentified novel subgroup henceforth designated wMT-3 (Figure 2A). Given that ESTs are short single read sequences that are randomly derived from cDNA libraries, one can assume that EST number/source is a reflection of actual mRNA levels. It is therefore not surprising that 77% (17 ESTs) originated from the library synthesized from earthworms exposed to cadmium. In contrast, only one metallothionein EST (CF798856), a member of the wMT- 2 cluster, was isolated from the fluoranthene library and none from the control library, although similar quantities of total sequences were obtained from all. All four metallothioneins obtained from the embryonic earthworm cocoon library assembled together to form a new class, wMT-3. Given that one further wMT-3 was identified from the cadmium library, it is not possible to conclude that wMT-3 is exclusively expressed only in cocoons; however, it indicates that this isoform is highly abundant and functionally active during early embryonic development. These observations underscore the fact that the EST sequencing strategy provides valuable information on mRNA expression characteristics and on specific gene involvement in fundamental metabolic events. Furthermore, it emphasizes the necessity to incorporate numerous libraries to avoid the misrepresentation of EST sequences that arise through redundancies of sequences that are highly expressed with strong stage, age, and specificities. Within the three clusters, the overall sequence identity is predictably high, bar a few base pair discrepancies (which may be accounted for by sequencing errors). For completeness, it should be mentioned that the consensus primary sequence of wMT-1 is not identical to the previously published sequence (NCBI accession number AJ005823). Nevertheless, the three isoforms are unquestionably highly conserved. Notable differences between wMT-1, wMT-2, and wMT-3 are their size (81 aa, 77 aa, and 86 aa, respectively) and the variable N-terminal and central linker regions; the former containing an additional cysteine in the case of wMT-3 (Figure 2B). The presence of variable linker regions indicate the location of a potential post-translational modification or proteolytic cleavage similar to those observed in another earthworm species, Eisenia fetida (24), and the springtail VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Earthworm metallothionein Expressed Sequence Tags (ESTs). Panel A depicts the phylogenetic clustering of all 22 full-length metallothionein ESTs obtained by searching the freely accessible relational database LumbriBASE (http://www.earthworms.org). In addition to the previously characterized wMT-1 and wMT-2, a new isoform (wMT-3) was identified. Most sequences were derived from a cadmiumexposed cDNA library (solid lines, clear boxes). However one sequence originated from earthworms exposed to fluoranthene (dashed line, clear box) and four sequences from an embryonic library (black boxes) all of the latter matched the new wMT-3 cluster. Panel B shows the deduced amino acid alignment of all three isoforms. Orchesella cincta (41, 42). However, in neither study was it possible to confirm whether the post-translational modification can be regarded as a deliberate cellular mechanism or rather a result of unspecific enzymatic attack due to the isolation procedure (25). No publication has previously dealt with the genomic structure of earthworm genes. This shortcoming has been rectified. With the aim to identify the genomic structure of the earthworm metallothionein isoforms, we focused on wMT-2, the isoforms previously shown to be especially responsive to cadmium (26). Genomic DNA was extracted, purified, and used as a template for a series of genomic PCR reactions, ultimately resulting in an amplification of 1163 bp of the wMT-2 locus spanning two exons and one intron (Figure 3). This fragment was used as a probe in a genomic library screen (considered to be close to saturation) and yielded over 25 kb of high quality genomic sequence encompassing three wMT-2 loci. The genomic scaffolding is, in principle, identical within the three loci, designated wMT-2a, wMT2-b, and wMT-2c (namely, four exons and three introns). Once spliced, the coding regions encompass 234 bp or 77 aa. At the amino acid level wMT-2a and wMT-2b 6286
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display an identity of 100%, and wMT-2a and wMT-2c display an identity of 94%. However, the four amino acids that differ in wMT2c are unlikely to influence its primary structure or overall function as they are neutral, nonpolar substitutions distributed evenly over the entire protein at aa positions 5, 21, 35, and 76. It should be noted that the last intron of the wMT-2c database entry contains a perfect 1331 bp IS-element that encodes for a 402 aa bacterial transposase, an artifact likely to originate from the construction of the bacterial artificial clones. No further up- or down-stream genes could be identified within the 5′ and 3′ regions of all three isoforms. However, an (AGACn) repeat element was identified within the distant 3′ region of wMT2c, providing a promising target for microsatellite analysis. Detailed investigations regarding this matter are currently being performed in our laboratory. In silico analysis of the promoter regions identified three putative conserved metal responsive elements (MREs, with the consensus sequence: TGCRCNC) within the landmark region of 400 bp upstream of the start codon (Table 3). The presence of putative MREs within close proximity initially indicated that the mode of transcriptional regulation of earthworm metallothioneins might be analogous to the
FIGURE 3. Genomic organization of wMT loci. The upper panel is a representative scheme of the intron-exon architecture of three wMT2 isoforms that were isolated by genomic screens using a gene-specific genomic probe. Note the three conserved putative metal regulatory elements (MREs) upstream of the start codon. The lower panel shows the size of the isolated DNA that was sequenced, the proportion encoding the gene, and the size of the exons and introns. All numbers are base pairs (bp).
TABLE 3. Conserved Metal Regulatory Element (MRE) Cluster in Close Proximity to the Transcriptional Initiation Sitea isoform wMT2a wMT2b wMT2c
regulartory element
orientation
sequence (5′ to 3′)
location (from ATG)
MRE MRE MRE MRE MRE MRE MRE MRE MRE
+ + + -
TGCACGC TGCACAC TGCACGC TGCACGC TGCACAC TGCACGC TGCACGC TGCACAC TGCACGC
-208 -265 -360 -211 -268 -371 -200 -257 -349
a Orientation, sequence, and upstream distance (bp) from the start codon are given.
regulatory mechanism of human, mouse, fish, and fly metallothioneins (i.e., via the transcription factor MTF-1) (43-47). However, after extensive experimentation, we are not able to identify a member of the otherwise conserved MTF-1 family in earthworms. Techniques included a PCRbased approach utilizing over 20 degenerate primer combinations within the conserved MTF-1 zinc finger regions as well as cDNA and genomic screens using Drosophila (dMTF1) and mouse (mMTF-1) as a probe. The putative MREs were also used in a band shift/gel shift exercise using nuclear earthworm extract and the three conserved MREs. Although positive results were obtained with dMTF-1 and mMTF-1 extracts, we could not demonstrate that an earthworm protein is capable of binding to any of the earthworm putative MREs (data not shown). In consequence, we are not able to confirm (nor categorically exclude) that earthworm metallothioneins are driven by an MTF-1 mediated pathway. To some extent this circumstantial evidence supports data from the nematode Caenorhabditis elegans. Although two MT isoforms (mtl-1 and mtl-2) have been identified and thoroughly studied in C. elegans (48), their promoters not only lack MREs but extensive analysis of the fully sequenced genome has failed to identify MTF-1 homologues. Indeed, transgenic lines of the nematode C. elegans that contained an earthworm promoter GFP construct were shown to be cable of being
“turned on” upon exposure to cadmium (49). This is even more surprising given that superficial inspection of the earthworm and C. elegans promoters could not identify distinctive transcription recognition sites (49). All this taken together suggests that an alternative mode of transcriptional activation has evolved in C. elegans and possibly other invertebrates, such as the earthworm. If this proves to be correct, a fundamental divergence in the mechanism of eukaryotic control of heavy metal mediated transcriptional activation might be evident, a statement clearly warranting further investigation. So far this paper has dealt with individual facets revolving around cadmium and the architecture and transcription of metallothionein genes. But can these components be translated to provide meaningful information regarding the detoxification and trafficking of cadmium at a protein level? Using a polyclonal antibody, raised against wMT-2, we have previously been able to highlight specific cellular and subcellular locations of the putative cadmium-metallothionein complex (23, 27). On the basis of extensive immunohistochemical evidence, this information has now been taken one step further to provide a summary of events that constitute the metallothionein-mediated transport of cadmium in earthworms (Figure 4). The major route of cadmium uptake is through the gut epithelium (Figure 4A) following food ingestion rather than an intake via the external body surface. The highest metallothionein levels have been recorded in the chloragogenous tissue enclosed within the fold of the typhlosole (Figure 4B) and in chloragogenous tissue coating the coelomic surfaces of the gut (Figure 4C) in the posterior alimentary region, thus confirming this tissue to be the primary site for metal sequestration and detoxification. Similarly, wMT expression is high in chloragocytes around blood vessels and to some extent near the calcium transporting epithelia within the calciferous glands in the oesophageal region (27). Coelomocytes, free floating and encapsulated in seminal vesicles, are both targeted sites of systemic/cellular transport of the Cd-MT complex (Figure 4D). Direct physical elimination of cadmium by the extrusion of coelomocytes via dorsal pores in the body wall remains a possible excretory route that was not the subject of this present study. Intense immunostaining of wMT in the nephridial wall (Figure 4E) indicates that urine is a candidate excretory pathway of cadmium. Alternatively, VOL. 38, NO. 23, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Cadmium trafficking in earthworms. Immunohistochemistry with a polyclonal antibody raised against wMT-2 shows that metallothionein is strongly expressed in the gut epithelium (A) and the typhlosole (B), both components of the alimentary surface and thus likely to be major routes of cadmium uptake from ingested soil. In addition, strong positive immuno-staining was detected in the apexes of the chloragocytes (C), cells with probable cadmium-storage function. Finally, strong metallothionein expression was detected in coelomocytes (D) and the nephridia (E), indicating a possible route of cadmium elimination. Note that the immunopositive staining is only observed in cadmium-induced earthworms and not in unexposed control earthworms (see refs 26 and 27 for more details). if metallothionein is not involved in the trans-tubular transport of cadmium, nephridial accumulation may well indicate an inefficient excretion via urine. If this is the case, then there is scope for investigating the possibility that cadmium exerts pathological effects on the earthworm nephridia analogous to renal damage in vertebrates (50). Either way, the immunohistochemical investigations summarized in Figure 4 graphically illustrate that the earthworm 6288
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is not merely a simple tubular structure but a sophisticated organism that has evolved effective mechanisms facilitating the uptake, accumulation, transport, and excretion of cadmium. In summary, we can now describe the entire pathway, from genes to cells, by which an earthworm handles cadmium, enabling it to accumulate a body burden in excess of 1/1000th of its total dry body weight and yet capable of successfully completing its life cycle.
Acknowledgments The authors wish to thank the U.K. National Environmental Research Council and the Royal Society for continuous support. Furthermore we are grateful to Prof. Dr. Walter Schaffner at the Department of Molecular Biology (Zurich, Switzerland) for his enthusiasm, encouragement, and collaborative efforts. Likewise we thank Dr. David Spurgeon (CEH, Monks Wood) for the supply of earthworms used in the EST project, Dr. Jennifer Chasely for the synthesis of cDNA libraries, Carole Winters and Dr. Paul Monaghan for assistance with cryopreparation and electron probe analysis, and finally Drs. Mark Blaxter and John Parkinson and Ms. Ann Hedley for bioinformatic building of LumbriBASE.
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Received for review February 3, 2004. Revised manuscript received July 20, 2004. Accepted August 4, 2004. ES049822C
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