Environ. Sci. Technol. 2005, 39, 2042-2048
Ligand Arsenic Complexation and Immunoperoxidase Detection of Metallothionein in the Earthworm Lumbricus rubellus Inhabiting Arsenic-Rich Soil C . J . L A N G D O N , * ,† C . W I N T E R S , ‡ S. R. STU ¨ RZENBAUM,‡ A. J. MORGAN,‡ J. M. CHARNOCK,§ A. A. MEHARG,| T. G. PIEARCE,⊥ P. H. LEE,⊥ AND K. T. SEMPLE⊥ Department of Environmental Management, School of Natural Resources, University of Central Lancashire, Preston, Lancashire, PR1 2HE, U.K., Cardiff School of Biosciences, Cardiff University, P. O. Box 913, Cardiff, CF10 3TL,Wales, U.K., CLRC Daresbury Laboratory, Warrington, WA4 4AD, U.K., Department of Plant and Soil Science, University of Aberdeen, Aberdeen, AB24 3UU, U.K., and Institute of Environmental and Natural Sciences, Lancaster University, Lancaster, LA1 4YQ, United Kingdom
Although earthworms have been found to inhabit arsenic-rich soils in the U.K., the mode of arsenic detoxification is currently unknown. Biochemical analyses and subcellular localization studies have indicated that As3+-thiol complexes may be involved; however, it is not known whether arsenic is capable of inducing the expression of metallothionein (MT) in earthworms. The specific aims of this paper were (a) to detect and gain an atomic characterization of ligand complexing by X-ray absorption spectrometry (XAS), and (b) to employ a polyclonal antibody raised against an earthworm MT isoform (w-MT2) to detect and localize the metalloprotein by immunoperoxidase histochemistry in the tissues of earthworms sampled from arsenic-rich soil. Data suggested that the proportion of arsenate to sulfur-bound species varies within specific earthworm tissues. Although some arsenic appeared to be in the form of arsenobetaine, the arsenic within the chlorogogenous tissue was predominantly coordinated with S in the form of -SH groups. This suggests the presence of an As::MT complex. Indeed, MT was detectable with a distinctly localized tissue and cellular distribution. While MT was not detectable in the surface epithelium or in the body wall musculature, immunoperoxidase histochemistry identified the presence of MT in chloragocytes around blood vessels, within the typhlosolar fold, and in the peri-intestinal region. Focal immunostaining was also detectable in a cohort of cells in the intestinal wall. The results of this study support the hypothesis that arsenic induces MT expression and is * Corresponding author phone: +44 1772 893496; fax: +44 1772 892903; e-mail:
[email protected]. † University of Central Lancashire. ‡ Cardiff University. § CLRC Daresbury Laboratory. | University of Aberdeen. ⊥ Lancaster University. 2042
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sequestered by the metalloprotein in certain target cells and tissues.
Introduction Soil contamination with arsenic has emerged as a matter of urgent environmental and human health concern in recent years, in some geographical areas due to geogenic sources (1), and in other areas due to the agricultural use of Ascontaining biosolids such as seaweed (2) and poultry litter (3, 4) fertilizers. Thus, it would be instructive to determine the fate and speciation of arsenic in soil-dwelling organisms with a multi-generational exposure history to high concentrations of the metalloid. Populations of Lumbricus rubellus Hoffmeister have been found inhabiting arsenic-rich soils in the U.K. (5, 6). L. rubellus from Devon Great Consols and an uncontaminated site had LC50 values of 1510 mg and 96 mg As kg-1, respectively (6), indicating that the former population has evolved the ability to resist the toxic effect of the nonessential metalloid. The mode of arsenic detoxification in earthworm tissues is presently unclear, although biochemical analyses (7) and subcellular localization studies (8) indicate that As3+-thiol complexes are probably involved. The cysteine-rich metalloprotein, metallothionein (MT), is a strong candidate thiol donator. Two MT isoforms have been characterized in L. rubellus, and polyclonal antisera have been raised against them (9, 10). It is not known whether arsenic in any of its chemical forms is able to induce MT synthesis in earthworms. However, studies indicate that arsenic induces MT-gene expression in mice (11), lending credence to the notion that at least a fraction of the accumulated arsenic burden of earthworms is sequestered by MT. Experience has shown that the protocol possesses sufficient specificity and sensitivity to detect earthworm Cd-MT (9, 10). Langdon et al. (7) showed by X-ray absorption spectrometry (XAFS) that arsenic was predominantly coordinated with -SH groups, indicative of metallothionein complexation, in isolated earthworm posterior-alimentary tissue. The dominance of As(III)-thiol complexes in the intestine agreed with Morgan et al. (8) and Yeates et al. (12), who found a close association of arsenic with S in granules within L. rubellus. It was suggested that these granules are mainly composed of As-S, not as an inorganic precipitate but probably as (unconfirmed and unclassified) organic As-thiol complexes. The first aim of the study was to determine, at the atomic level, the type of ligands complexing arsenic field-sample earthworms by using X-ray absorption spectrometry (XAS), a method comprised of X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS). The second aim was to employ a polyclonal antibody raised against an earthworm MT isoform (w-MT2) to detect and localize the metalloprotein by immunoperoxidase histochemistry in the tissues of the intestinal region of L. rubellus exposed to high arsenic concentrations in its native soil. This study aimed to link individual earthworm tissues and is considered an important initiative to establish the molecular-genetic and mechanistic bases of As-resistance in earthworm ecotypes (13), as well as to develop and optimize biomarkers of stress response for contaminated land assessments.
Experimental Section Soil Analysis. Concentrations of arsenic and copper in the soils from Devon Great Consols were determined using X-ray 10.1021/es0490471 CCC: $30.25
2005 American Chemical Society Published on Web 02/19/2005
fluorescence, using dry, powdered, pressed soil briquettes (Philips PW 1400 XRF) (14). The total cadmium and zinc in soils was determined by atomic absorption spectrophotometry (five replicates each of 0.5 g of air-dried, sieved (2 mm gauge) soil, after Aristar grade nitric acid digestion). Soil pH was determined by glass electrode using aqueous suspensions of soil that had been air-dried and sieved (2.8 mm), moisture content by oven-drying at 100 °C for 18 h, and weight loss on ignition by heating at 450 °C for 18 h (n ) 5). Earthworm Collection. Specimens of L. rubellus were collected by hand-sorting from an abandoned arsenic and copper contaminated site at Devon Great Consols near Tavistock, Devon, in southwest U.K. (Ordnance Survey Ref. SX 423736). The earthworms were identified using a standard key (15) and were kept in their parent soil until sample preparation. Earthworm Total Arsenic Tissue Concentrations. The L. rubellus were depurated for 24 h prior to analysis. After immersion in hot water, all L. rubellus were dissected dorsally and any visible particles of soil in the gut were carefully removed with a fine brush. The earthworms (n ) 10) were dried at 40 °C overnight on Whatman 540 filter papers, weighed and cut into small pieces, and placed into digestion tubes. Three samples of certified standard mussel tissue (NCS ZC 78005, GB W08571), purchased from Promochem, Teddington, Middlesex, U.K., were processed and analyzed in an identical fashion to determine the precision of the overall protocol. Ten milliliters of Aristar grade nitric acid was added to the individual tissue samples, and the digests were allowed to stand overnight prior to heating for 6 h at 90 °C in a digestion block. The digests were cooled and filtered through Whatman 540 filter papers into 100 mL acid-washed volumetric flasks, made up to volume using ultrapure water. The samples were analyzed for total arsenic on a PerkinElmer Optima 3000 ICP-OES with an As 90 autosampler and a cross-flow nebulizer with a slit width of 197.196 and plasma parameters of auxiliary 0.5, nebulizer 0.80, and a view height of 15 mm. The known detection limit of the instrument for arsenic was 5 µg kg-1. XAS Analysis. L. rubellus were maintained on moist tissue paper for 24 h (in the dark) to void their gut contents. Whole earthworm, posterior, and body wall fractions, respectively, were then frozen in liquid nitrogen prior to freeze-drying for 7 h at -30 °C at a pressure of 9 Mbar. These tissues were ground with an agate pestle and mortar, mounted onto aluminum planchettes, and secured with Cellotape. Chloragogenous tissue (a loosely liver-like tissue surrounding the alimentary canal and major blood vessels) was smeared onto Cellotape, air-dried, and mounted on an aluminum planchette. X-ray absorption spectra at the arsenic K-edge were collected on Station 16.5 at the CLRC Daresbury SRS operating at 2 GeV with an average current of 140 mA, using a vertically focusing mirror and a sagitally bent focusing Si(220) double crystal monochromator detuned to 80% transmission to minimize harmonic contamination. Data were collected with the station operating in fluorescence mode using an Ortec 30 element solid-state Ge detector. Experiments were performed at ambient temperature. Four scans were collected and summed for the whole earthworm, five scans for the body wall, six scans for the chloragogenous tissue, and three scans for the posterior section. For the soil sample, one scan was collected. XANES data were fitted using a linear combination of XANES spectra collected from model systems. The three models used were an arsenate complex (As(V) coordinated by 4 oxygens at 1.68 Å), a sodium glutathione complex (As(III) coordinated by 3 sulfurs at ca. 2.26 Å), and arsenobetaine (As(III) coordinated by 4 carbons at 1.90 Å).
Background subtracted EXAFS spectra were analyzed in EXCURV98 using full curved wave theory (16, 17). Phaseshifts were derived in the program from ab initio calculations using Hedin-Lundqvist potentials and von Barth ground states (18). Fourier transforms of the EXAFS spectra were used to obtain an approximate radial distribution function around the central arsenic atom (the absorber atom); the peaks of the Fourier transform can be related to “shells” of surrounding backscattering atoms characterized by atom type, number of atoms in the shell, the absorber-scatterer distance, and the Debye-Waller factor, 2σ2 (a measure of both the thermal motion between the absorber and scatterer and the static disorder or range of absorber-scatterer distances). The data were fitted for each sample by defining a theoretical model and comparing the calculated EXAFS spectrum with the experimental data. Shells of backscatterers were added around the arsenic, and by refining an energy correction Ef (the Fermi energy), the absorber-scatterer distance, and Debye-Waller factor for each shell, a least squares residual (the R factor (19)) was minimized. The coordination numbers were chosen on the assumption that any arsenate would be 4-coordinate and any sulfur-bound species would be 3-coordinate. The proportion of arsenic in each site was refined as a free variable, keeping the total central arsenic occupation number at 1. A third component required in the analysis of the chloragogenous spectrum was assumed to be 4-coordinate. Immunoperoxidase Histochemistry. Whole L. rubellus, collected from Devon Great Consols, were chemically fixed in 10% formal saline, wax embedded, histologically sectioned, and immunostained according to a protocol optimized, and previously described, for the detection of Cd-sequestering w-MT2 (10, 20). This protocol reveals positive w-MT2 expression as a chestnut-brown precipitate. A suite of appropriate controls was performed, and endogenous peroxidase activity was blocked by H2O2/methanol incubation prior to immunostaining. Photomicrographs of representative immunostained and appropriate control sections were digitally recorded.
Results and Discussion Soil and Tissue Analysis. The soils and earthworms collected from Devon Great Consols had high arsenic concentrations (Table 1). Soil copper concentration was also high, but the soil cadmium and zinc concentrations were low in comparison, and within the “background range” for uncontaminated soils. The bioconcentration factor was higher for arsenic than for copper in the earthworm tissues. The soil zinc was close to background levels. The maximum permissible zinc concentration value in the European Union for sewage amended soils is 300 mg kg-1 (21). The pH at Devon Great Consols is slightly acidic. XANES. The shape of the edges varied between samples (Figure 1) and showed the varying degree of oxygen and sulfur coordination. The profiles for the whole earthworm, earthworm posterior, and earthworm body wall could all be simulated reasonably well using a combination of arsenate and As:glutathione edges. However, for the chloragogenous tissue, it was not possible to get a good fit using just these two. There was a contribution to the XANES between the main As-S and As-O peaks, so addition of an arsenobetaine component, which has a main peak between arsenate and As:glutathione, improved the fit. We cannot be certain that arsenobetaine was necessarily present in the earthworm tissues; it was evident that some species other than arsenate or sulfur-bound species also contributed to the composite XANES spectra. However, arsenobetaine has been detected in earthworms collected from Devon Great Consols (7), strengthening the possibility that it was present. The percentages of each component in the fitted spectra (rounded VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. L. rubellus Tissue Arsenic Concentrations, Organic Matter Content, pH, and Arsenic, Cadmium, Copper, and Zinc Concentrations of Soils from Devon Great Consols, Mean ( SE (n ) 5 for Soils, n ) 10 for Earthworms)a soil As (mg kg-1) Cu (mg kg-1) Cd (mg kg-1) Zn (mg kg-1) organic matter (%) pH
bioconcentration factorb
multiple of reference valuesc
500 ( 56 40 ( 2.6
0.06 0.02
399 31 1 0.88
7998 ( 23 1552 ( 14 1.14 ( 0.16 177.8 ( 5.75 9.87 ( 0.34 5.25 ( 0.05
Reference Values for Metals for Soils in The Netherlandsd 20 50 1 200
As (mg kg-1) Cu (mg kg-1) Cd (mg kg-1) Zn (mg kg-1)
As (mg kg-1) Cu (mg kg-1) Cd (mg kg-1) Zn (mg kg-1)
earthworm tissue dry weight
UK Department of the Environment ICRCL Trigger Concentrations for Environmental Metalse 10 gardens and allotments 130 gardens and allotments 3 any uses where plants are grown 300 any uses where plants are grown Selected Values for the Canadian Interim Environmental Quality for Soilf soil concentration (mg kg-1)
As Cu Cd Zn
background
agricultural
residential
industrial
5 30 0.5 60
20 150 3 600
30 100 5 500
50 500 20 1500
a National soil standard values for the four inorganic residues are included to indicate the severity of contamination. b Bioconcentration factor ) earthworm tissue concentration/soil concentration. c Multiple reference values calculated from reference values for metals for soils in The Netherlands. d Data from a compilation: Table 5.25 (21). e Data from a compilation: Table 5.26 (21). f Data from a compilation: Table 5.27 (21).
TABLE 2. XANES and EXAFS Earthworm and Body Partsa XANES percentage of As in form of: sample
arsenate
As:glutathione
whole earthworm
25
75
0.0059
O S
posterior
70
30
0.0059
O S
body wall
35
65
0.0042
O S
chloragogenous tissue
10
75
0.0057
O C S
soil DGC
arsenobetaine
EXAFS
15
fit index
100
scatterer
O Fe Fe
no. of atoms 1.0 2.2 (25% O; 75% S) 2.9 0.9 (70% O; 30% S) 1.7 1.7 (43% O; 57% S) 0.6 0.8 2.0 (15% O; 20% C; 65% S) 4 1 2
r (Å) 2σ2 (Å2) R factor 1.69 2.26
0.005 0.008
22.1
1.68 2.29
0.004 0.009
27.5
1.70 2.26
0.013 0.007
27.5
1.68 1.88 2.25
0.004 0.003 0.013
36.3
1.68 2.81 3.34
0.004 0.025 0.016
29.1
a Fit index of the calculated XANES spectra with experimental XANES spectra is defined as ∑[(I 2 obs - Icalc) ]/n, where n is the no. of points in each spectrum. For EXAFS, 2σ2 is the Debye-Waller type factor, and the R factor indicates goodness of fit.
to the nearest 5%) are given in Table 2. The XANES of Devon Great Consols soil sample was very similar to that of the arsenate model and was not fitted. Metallothionein-bound arsenic would be characterized by the presence of As3+-thiol; the oxidized As5+ species has an affinity for O-donating, not S-donating, ligands. In this study described above, As3+ was detected in the earthworm samples as 65% of body wall and 75% of chloragog bound to glutathione in the As3+ form. In a previous study (7), arsenite (As3+) was detected in earthworms collected from 2044
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an As-contaminated site at Carrock Fell, Cumbria, U.K. These data suggested that arsenic was predominantly coordinated with S in the form of a -SH group, suggesting metallothionein complexation. Arsenic in earthworms is evidently (at least partly) bound to sulfydryl-containing ligands. Cavigelli et al. (22) suggested that it is As3+, not As5+, that has an affinity for -SH groups. Glutathione, a SH-containing tripetide molucule, also interacts with As3+, the interaction exerting a profound effect on the cytotoxicity of As3+ (22, 23). This observation is supported by the findings of Toyama et al.
FIGURE 2. Micrograph of an immunoperoxidase (anti-MT) stained transverse section in the intestinal region of L. rubellus collected from the arsenic/copper contaminated site at Devon Great Consols. Note the intense staining indicative of metallothionein (MT) in the chloragogenous tissue (ch). Magnification 1:500.
FIGURE 1. XANES spectra for (a) selected model compounds, As:glutathione (‚ ‚ ‚ ‚), arsenate (- - -), and arsenobetaine (-), and (b) for whole earthworm (- - -), body wall (- - - -), chloragog (‚ ‚ ‚ ‚), posterior (‚ - ‚ -), and Devon Great Consols soil (-). Vertical lines are drawn at 11 869 and 11 875 to indicate the position of absorption edge for As(III)-glutathione and As(V)-O, respectively. Labeling key: b ) body wall, bv ) blood vessels, c ) coelem, ch ) chloragogenous tissue, ie ) intestinal epithelium, g ) gut, ge ) gut epithelium, gl ) gut lumen, l ) lumen, MT ) w-MT2. (24), who demonstrated by UV absorption spectroscopy that As3+ binds to human metallothionein-2. Furthermore, the metalloid is capable, directly or indirectly, of inducing metallothionein-gene expression at least in mammalian cells (25). The immunohistochemistry conducted in this study indicated that MT is a likely donator of sulfydryl groups for arsenic sequestration in earthworm tissues. Direct biochemical confirmation of As-MT in earthworm tissues represents a formidable technical challenge because oxidizing conditions will displace the metalloid from the core of the metalloprotein. EXAFS. The parameters derived from the EXAFS fitting are presented in Table 2. For all four earthworm samples, a mixture of 4-coordinate arsenate and 3-coordinate sulfurbound arsenic gave good fits. However, the fit to the chloragogenous spectrum was improved by addition of a shell of carbon atoms at ca. 1.9 Å. The proportions of arsenic species derived from the EXAFS were in good agreement with the XANES fitting. The EXAFS of the soil sample showed arsenic surrounded by 4 oxygen atoms at 1.68 Å, indicating that all or most of the arsenic is present as arsenate. There were outer shells that could be fitted with iron backscatterers at ca. 2.8 and 3.3 Å. This was consistent with the arsenic being adsorbed onto or incorporated into an iron oxide/ hydroxide phase. Immunoperoxidase Histochemistry. w-MT2 was detected in the earthworm tissues, although its distribution was distinctly localized. w-MT2 was not detectable in the surface epithelium or in the body wall musculature (Figure 9), indicating that the primary As-uptake route may be
FIGURE 3. Micrograph of an immunoperoxidase (anti-MT) stained transverse section in the chloragogenous tissue and gut of L. rubellus collected from the arsenic/copper contaminated site at Devon Great Consols. The intense staining indicative of metallothionein (MT) is situated in the chloragogenous tissue (ch) and the gut wall (g). Magnification 1:500.
FIGURE 4. Micrograph of an immunoperoxidase (anti-MT) stained transverse section in the chloragogenous tissue of L. rubellus collected from the arsenic/copper contaminated site at Devon Great Consols. The intense staining indicative of metallothionein (MT) is situated in the chloragogenous tissue (ch). Magnification 1:500. alimentary, although fast re-allocation of As by the organism may also be possible. w-MT2 was detectable in chloragocytes around blood vessels (Figures 6 and 9), within the typhlosolar fold (Figure 5), and in the peri-intestinal region (Figure 7). However, the staining intensity in these regions was variable and patchy. It was evident in some cases that w-MT2 VOL. 39, NO. 7, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Micrograph of an immunoperoxidase (anti-MT) stained transverse section in the intestinal region of L. rubellus collected from the arsenic/copper contaminated site at Devon Great Consols. The intense staining indicative of metallothionein (MT) is situated in the chloragogenous tissue (ch). Magnification 1:500.
FIGURE 6. Micrograph of an immunoperoxidase (anti-MT) stained transverse section in the chloragogenous tissue of L. rubellus collected from the arsenic/copper contaminated site at Devon Great Consols. The intense staining indicative of metallothionein (MT) is clearly visible around the blood vessels (bv). Magnification 1:500.
FIGURE 7. Micrograph of an immunoperoxidase (anti-MT) stained transverse section in the intestinal region of L. rubellus collected from the arsenic/copper contaminated site at Devon Great Consols. The intense staining indicative of metallothionein (MT) is situated in the gut epithelium. Magnification 1:500. immunostaining within chloragocytes was focal (Figures 2-4) and reminiscent of the putative lysosomal/exocytotic-vesicle distribution of Cd-MT2 subcellular distribution (10). w-MT2 was detectable within intestinal epithelia, but the staining pattern was also focal and often intense (Figure 8). This probably indicates that a cohort of intestinal cells preferentially accumulate As-MT2 within an organelle; the staining profile certainly indicates a degree of cell specificity and intracellular compartmentalization. No immunostaining was detectable in sections of L. rubellus from a reference site (Dinas Powys, South Wales, U.K.) or in sections of L. rubellus 2046
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FIGURE 8. Micrograph of an immunoperoxidase (anti-MT) stained transverse section in the tip of the gut of L. rubellus collected from the arsenic/copper contaminated site at Devon Great Consols. The intense staining indicative of metallothionein (MT). Magnification 1:500.
FIGURE 9. Micrograph of an immunoperoxidase (anti-MT) stained transverse section in the body wall and intestinal region of L. rubellus collected from the arsenic/copper contaminated site at Devon Great Consols. The intense staining indicative of metallothionein (MT) is shown by arrows around the blood vessels in the chloragogenous tissue in the typhlosolar fold. Magnification 1:500. from Devon Great Consols, but where either the primary or the secondary antibodies were omitted (not shown). Morgan et al. (20) showed with a combination of electron probe X-ray microanalysis, immunohistochemistry, and Neutral Red labeling of acidic compartments that Cd-MT in earthworms is sequestered within an acidic organelle, probably of lysosomal origin. It would be reasonable to suppose that As-MT also accumulates in analogous organelles. For instance, Schmoger et al. (26) suggested that As-phytochelatin may be stable under the acidic conditions extant in the plant cell vacuole, a finding with profound implications in terms of the immobilization and detoxification of As3+ within the cells. Merrifield et al. (27) reported five As3+ atoms binding to metallothionein from the seaweed Fucus vesiculosus. The authors suggested that metallothionein is a clear contender as a molecular chelating agent due to the high ratio of cysteines. They also stated that, although a single role for MT has not yet been determined, the range of metals chelated suggested involvement in transport, metabolism, and detoxification of metals. Also germane are the observations of Liu et al. (28), in their study on background-matched wild-type (WT) mice and MT-null mice, that intracellular MT has an important protective role against the nephrotoxic effects of both cadmium and/or arsenic. Metalliferous soils are seldom contaminated with a single inorganic residue. Devon Great Consols soil is not an exception; it contains very high levels of copper as well as arsenic (Table 1). Although the soil copper content at Devon Great Consols is well above background levels, it has
previously been shown that the copper-inducibility of earthworm metallothionein is poor, even though it binds avidly to the metalloprotein (10, 29). Even though the bioconcentration factor in earthworm tissues is low (leading to relatively low tissue concentrations), it is not possible to eliminate the likelihood that copper is the inducer of immuno-detectable MT in the Devon Great Consols earthworms (Table 1) (30). Cadmium is a potent inducer of earthworm metallothionein gene expression (9, 10, 29), but the soil cadmium concentration at Devon Great Consols is negligible, being about 50% of the United Kingdom trigger concentration for sewage-sludge amended arable soils (21). The major zinc burden in earthworms is bound not by metallothionein, but in the phosphate-rich chloragosomes of the chlorocytes. It is possible that an unknown chemical or physical constituent in Devon Great Consols soil is the metallothionein inducer. It, should, however, be borne in mind that there is no evidence of a glucocorticoid promotor in earthworm metallothionein genes (31). This suggests an inorganic inducer, and the most likely candidate is arsenic. The immunostaining described in this study therefore probably reflects As-induced MT upregulation. The results lend support to the hypothesis that w-MT2 sequesters, and possibly detoxifies, arsenic in a cohort of intestinal epithelial cells and in the chloragocytes. It is evident that some of the (presumptive) arsenic-MT complex is compartmentalized within a hitherto unidentified organelle in the main target cells. Although the possibility should not be ignored that Devon Great Consols soil contains an inorganic or organic inducer of MT that has been overlooked, the present results lend circumstantial support for the hypothesis that MT sequesters arsenic in certain earthworm cells and tissues. Further circumstantial evidence arises from the recent observation (13) that arsenate resistance was conferred by Devon Great Consols L. rubellus parents to their laboratory bred F1 offspring; copper resistance was not heritable. Klerks and Levinton (32) found a rapid evolution of resistance to cadmium and nickel in the benthic freshwater oligochaete Limnodrilus hoffmeisteri Clapare`de from metalpolluted sites in Foundary Cove, New York. The authors reported that the elevated resistance was genetically determined, with probable MT-gene duplication, and was found to be present after two generations (F2 offspring) in clean sediment. Future research will be directed to obtain direct evidence that As is sequestered by earthworm MT, that arsenic is an inducer of MT gene or genes, and will seek to reveal genetic differences in the structure and/or functions of earthworm metallothioneins between As-resistant and non-As-resistant ecotypes. If, as seems likely, evolutionary events have resulted in earthworm populations with differential arsenic sensitivities, this would have a profound bearing on efforts to use molecular-genetic biomarkers, including metallothionein expression levels, for in situ pollution assessments.
Acknowledgments C.J.L. was supported for part of the work by NERC CASE studentship GR04/98/116/T3 and the Department of Soil Science, Reading University. The work of the Cardiff Group was supported by NERC Grants GR3/11225 and GST/02/ 1782. We would like to thank the 7th Earl of Bedford for permission to sample on his woodland estate.
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Received for review June 23, 2004. Revised manuscript received December 23, 2004. Accepted January 6, 2005. ES0490471
