Environmental Pollution 135 (2005) 275280 www.elsevier.

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Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions
Joanna Homaa, Ewa Olchawaa, Stephen R. Stu rzenbaumb, A. John Morganb, Barbara Plytycza,*
a

Department of Evolutionary Immunobiology, Institute of Zoology, Jagiellonian University, R. Ingardena 6, PL 30-060 Krakow, Poland b Cardi School of Biosciences, Cardi University, PO Box 915, Cardi Wales CF10 3TL, UK Received 15 May 2004; accepted 19 October 2004

Metals upregulate stress response proteins in earthworm coelomocytes.

Abstract This paper provides direct evidence that earthworm immune cells, coelomocytes, are exposed to bio-reactive quantities of metals within 3 days after dermal exposure, and that they respond by upregulating metallothionein (MT) and heat shock protein (HSP70, HSP72) expression. Indirect support for the hypothesis that coelomocytes are capable of tracking metals was also obtained. Coelomocytes were expelled from adult individuals of Eisenia fetida after 3-day exposure either to metal ions (Zn, Cu, Pb, Cd) or to distilled water (controls) via lter papers. The number of coelomocytes was signicantly decreased after Cu, Pb, or Cd treatment. Cytospin preparations of coelomocytes were subjected to immunoperoxidase staining with monoclonal antibodies against human heat shock proteins (HSP70 or HSP72), or rabbit polyclonal antibodies raised against metallothionein 2 (w-MT2) of Lumbricus rubellus. Applied antibodies detected the respective proteins of E. fetida and revealed that the expression of HSP70, HSP72 and w-MT2 proteins was either induced or signicantly enhanced in coelomocytes from metal-exposed animals. In conclusion, stress protein expression in earthworm coelomocytes may be used as sensitive biomarkers of metal contaminations. Further experimentation is needed for quantitative analysis of kinetics of metal-induced stress protein expression in earthworm coelomocytes. 2004 Elsevier Ltd. All rights reserved.
Keywords: Eisenia fetida; Zn, Cu, Pb, Cd; Coelomocytes; Metallothionein; HSPs

1. Introduction Earthworms are priority organisms in terrestrial ecotoxicology (Spurgeon et al., 2003). Given the pivotal role of immunocompetence in animal health, the deleterious eects of toxicants on the constitution and function of earthworm immune cells, coelomocytes,
* Corresponding author. Tel.: C48 12 663 24 28; fax: C48 12 632 72 82. E-mail address: plyt@zuk.iz.uj.edu.pl (B. Plytycz). 0269-7491/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2004.10.019

have been widely studied (Cooper and Roch, 2003; Stankiewicz and Plytycz, 1998; Kurek et al., 2002; Kurek and Plytycz, 2003; Homa et al., 2003; WieczorekOlchawa et al., 2003) yet are incompletely understood. Stu rzenbaum et al. (2001, 2004) tentatively described a previously unsuspected role of coelomocytes in the tracking of Cd from its main tissue repository in earthworms, the liver-like chloragogenous tissue, to possibly the nephridia. These observations were based on the immunohistochemical detection of a Cd-inducible, Cd-sequestering, metallothionein isoform (w-MT2)

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in a eld population of Lumbricus rubellus. Two hypotheses that are not necessarily mutually exclusive can be formulated from these observations. First, coelomocytes internalize Cd-MT exocytosed from chloragocytes into the coelomic uid. Second, coelomocytes are systemically exposed to a Cd-fraction that induces de novo expression of metallothionein in the immune cells. Moreover, at least some fraction of coelomocytes may derive from chloragenous tissue (Hamed et al., 2002). The main purpose of the present study was to test the hypothesis of coelomocytes involvement in metal tracking by exposing the standard toxicity testing species, Eisenia fetida, for 3 days to Zn, Cu, Pb or Cd salts under laboratory conditions. Metallothionein upregulation after such a short time interval would support the notion that coelomocytes had been exposed to a bio-reactive metal fraction, but would not unequivocally indicate cytotoxic damage. For this reason, coelomocytes extracted from the experimental animals were also immunohistochemically processed to enable the detection of two heat shock proteins (HSP70 and HSP72). HSPs are chaperones involved in the conformational folding of newly synthesised proteins and in the repair or disposal of damaged proteins. HSP upregulation is often considered to be general stress response (Nadeau et al., 2001). Not surprisingly, therefore, both HSP70 and HSP72 are detectable in temperature stressed earthworm coelomocytes (Kurek et al., 2001). The experiments reported herein provide a mechanistic insight (see Van Gestel and Weeks, 2004) into immunomodulatory eects of Cd and other metals in earthworms. They also provide the basis for developing novel, low-intrusion, cellular biomarkers for laboratory and eld applications in keystone soil-dwelling organisms.

protocols (Fitzpatrick et al., 1996). Heavy metal (Zn, Cu, Pb, Cd) chlorides were dissolved in double distilled water to the nal concentration of 44 mg/mL and pipetted onto the Whatman No. 1 lter paper strips (100 cm2) inserted into 50-mL plastic vials, giving the nominal heavy metal concentration of 1.32 mg/cm2. Filters soaked with a respective volume of distilled water served as controls. After 3-h fasting of a wet lter paper, washing and drying, earthworms were inserted into the vials, three per each, and exposed for 3 days via lter papers either to distilled water (controls) or to heavy metal solutions. Vials were kept horizontal in the dark wet chamber at 22  C. 2.3. Coelomocyte preparation Coelomocytes were extruded through the dorsal pores of earthworms subjected for 1 min to 4.5 V electric current as described in detail by Kurek et al. (2001). The cells of each animal were counted individually in a haemocytometer, and their viability was assessed by Trypan Blue exclusion test. Then coelomocytes from three individuals within each experimental group were pooled for cytospin preparation. Samples of coelomocytes (0.5 ! 106) were transferred to cytocentrifuge funnels, centrifuged for 5 min at 1900 rpm (Hettich Universal, Germany), xed in cold 100% methanol and stored at 20  C until use for immunocytochemistry. Experiments were repeated 35 times. 2.4. Immunocytochemistry The immunoperoxidase staining protocol was used on methanol-xed cytospin preparations as described previously (Kurek et al., 2001) after blocking of endogenous peroxidase with 3% H2O2. Monoclonal mouse anti-human anti-HSP70 (Sigma Chemical Co.) or anti-HSP72 (Amersham Pharmacia Biotech) diluted 1:1000 with 0.25% Triton X (Sigma Chemical Co.) in phosphate buer supplemented with 1% BSA (Sigma Chemical Co.) (overnight, 4  C), were followed by the Labelled Streptavidin Biotin method (DAKO LSAB Peroxidase kit). Polyclonal anti-w-MT2 antiserum was raised in rabbits challenged with the recombinant Lumbricus rubellus protein and used as primary antibody (overnight, 4  C) during present studies after dilution (1:500) in 1% BSA/0.01 M PBS, pH 7.1 (Morgan et al., 2004). The secondary antibody, i.e. peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical Co.) was diluted 1:100 (1 h, RT) in PBS/BSA, diaminobenzidine staining (10 min, RT) and hematoxylin counterstaining (30 s, RT). No-primary antibody or no-secondary antibody controls were performed.

2. Materials and methods 2.1. Earthworms Earthworms Eisenia fetida (Sav.), 0.30.4 g b.w., were collected from the stockbreeding maintained in the Institute of Zoology of the Jagiellonian University (Homa et al., 2003). The animals were kept at 22  C in plastic boxes lled with the relatively unpolluted soil from the garden of the Institute (Homa et al., 2003) and fed once a week with our. Experiments were performed during the winter. 2.2. Earthworm exposure to heavy metals on lter papers Adult sexually mature earthworms were kept in uncontaminated culturing soil (Homa et al., 2003) or exposed to heavy metals according to lter paper

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Stained cytospin preparations were imaged and photographed in a Jenamed-2 microscope (at magnication !400). 2.5. Heavy metal accumulation in the earthworm bodies After 3-day exposure to water or metals via lter papers, some earthworms (48 per group) were weighed, washed, dried at 70  C, and used for measurement of metal contents in their bodies, as described previously (Wieczorek-Olchawa et al., 2003).

Table 2 Changes of coelomocytes number, viability, and in the expression of heat shock proteins (HSP70 and HSP72) and metallothionein 2 (MT2) in coelomocytes of Eisenia fetida kept in the soil or exposed for 3 days to water or metal ions via lter papers E. fetida n exposed to: Coelomocytes Cell number ! 104 X G SE 274.5 G 27.8 243.1 G 21.9 249.8 G 10.0 80.0 G 7.6* 127.2 G 15.6* 165.3 G 16.0 Viability (%) X G SE 97.1 G 0.4 96.8 G 0.7 94.5 G 1.4 77.5 G 6.0* 76.0 G 4.6* 81.1 G 3.8* Expression of HSP70 CC CC CCCC CCC CCC CCC HSP72 C C CC CCC CCCC CCC w-MT2 C C CCC CCC C CCC

Soil Water Zn Cu Pb Cd

9 12 15 15 15 15

3. Results and discussion 3.1. Heavy metal accumulation in the animal body After 3-day contact exposure of E. fetida to Zn, the Zn content in the whole-body was similar to that in control animals. In contrast, tissue concentrations of Cu, Pb, and Cd were elevated 3-, 10-, and 58-fold, respectively (Table 1). Thus, the essential metals, Cu and Zn, were subject to physiological regulation, but the non-essential metals, Pb and (especially) Cd, were eciently accumulated. 3.2. Eects of heavy metal exposure on coelomocyte number and viability After 3-day exposure of the control individuals of E. fetida to water-soaked lters, the numbers and viability of the extruded coelomocytes were the same as those from their counterparts kept in uncontaminated culturing soil (Table 2). Also exposure to Zn ions did not aect coelomocyte number and viability. In contrast, the viability of the coelomocytes retrieved from Cu, Pb, or Cd-exposed animals was signicantly decreased. Moreover, the numbers of cells extruded from Cu and Pb exposed earthworms were also signicantly lower than those from the control animals (Table 2). In the present work no attempt was made to
Table 1 Metal concentrations and accumulation factors in Eisenia fetida exposed for 3 days either to water (W ) or to metal (M ) ions Metals Metal concentrations in MannWhitney Accumulation E. fetida [mg/kg b.w.] X G SE test factors [AF] Water exposure [W] Zn Cu Pb Cd 58.28 G 3.5 3.18 G 0.44 0.55 G 0.13 0.48 G 0.16 Metal exposure [M] 64.47 G 13.77 10.75 G 2.89 5.81 G 1.57 28.15 G 8.91 W versus M AF Z M/W

n, number of animals used for calculations of cell numbers and viability; *mean values signicantly dierent from those in soil and water ( p ! 0.05) by MannWhitney test; for immunohistochemistry cells from 35 animals were pooled; protein expression absent or negligible (C), weak (C), moderate (CC), strong (CCC), very strong (CCCC).

distinguish particular types of coelomocytes, like amoebocytes and chloragocytes. 3.3. Immunohistochemical detection of HSPs and w-MT2 The three antibodies used in the present immunohistochemical protocols were able to detect their respective E. fetida antigens even though they were raised against antigens from another earthworm species (in the case of the w-MT2 antiserum) or from a distinctly dierent taxon (in the cases of the HSPs) (Fig. 1). Cytospin preparations of the coelomocytes were consistently negative when the primary or secondary antibodies were omitted (Fig. 1A). Expression of HSPs and w-MT2 were similar on cytospin preparations of coelomocytes extruded from animals kept in soil and those exposed for 3 days to water only (Table 2). In contrast, expression of the investigated proteins was enhanced in coelomocytes from heavy metal-exposed animals in all experimental groups, except for w-MT2 in coelomocytes from animals exposed for 3 days to Pb ions (Fig. 1, Table 2). The cross-reactivity of the anti-HSP monoclonals conrms the high degree of evolutionary conservation within this stress protein super-family (Sanders et al., 1994; Marin o et al., 1999; Kurek et al., 2001; Robert, 2003). The ability of the anti-w-MT2 polyclonal to detect its antigen across the earthworm species is a new observation that, if conrmed for more species, enhances its utility in a number of cytological and applied ecotoxicology contexts. Constitutive expression of HSP70 was revealed in coelomocytes from control worms, whilst the frequency of HSP72-positive and w-MT2-positive coelomocytes

NS S S S

1.1 3.38 10.44 58.31

Dierences between W and M groups were deemed signicant (S; p ! 0.05) or not signicant (NS) by the MannWhitney test.

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Fig. 1. Immunohistochemistry on extruded coelomocytes of Eisenia fetida. (A) No-primary antibody (ab) controls (left); no-secondary antibody (ab) controls (middle); negative (pale) and positive (brownish) anti-MT2 immunoperoxidase staining (right); (B) representative anti-HSP70, anti-HSP72, anti-MT2-stained cytospin preparations of coelomocytes extruded after 3-day animal exposure either to water (controls) or to metal ions (Zn, Cu, Pb, Cd) via lter papers.

was very low in these animals. In most cases, except that of w-MT2 after Pb exposure, the expression levels of w-MT2 and of both HSPs were upregulated to some degree in coelomocyte populations extruded by metalexposed earthworms (Fig. 1). Further studies, involving double immunolabelling, should be done to investigate whether or not MT and HSPs are co-expressed in the same coelomocyte cohort(s). However, the present observations lend support to the hypothesis that earthworm coelomocytes can be exposed to functionaltering concentrations of potentially toxic metals within

relatively short exposure periods. These cells are, therefore, capable of de novo MT synthesis. This is not inconsistent with a capacity to also internalize Cd-MT released into the coelom by cells with a primary Cd-sequestering function (Stu rzenbaum et al., 2001, 2004). In the dosing regimen adopted in the present experiments, the dermal uptake route was probably dominant if not exclusive, so that the inducible chloragogenous Cd-storage sites were largely bypassed. Evidently this is not the case where uptake is mainly by alimentary transport from ingested soil (Lanno et al.,

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2004), such as in the eld-derived worms studied by Stu rzenbaum et al. (2001, 2004). Whilst the prodigious metal-sequestering capacity of the chloragogenous tissue (Morgan et al., 1993) may limit the intrusion of bioreactive metal species into the coelomocytes of free living earthworms inhabiting polluted soils, the present observations provide molecular conrmation of the vulnerability of immune cells to toxic ions. Incidentally, the early detectability of altered levels of specic geneexpression products (MT and HSPs) highlights the sensitivity and predictive possibilities of these, and similar, molecular biomarkers (Stu rzenbaum et al., 1998; Morgan et al., 1999). Table 2 indicates that there is not a consistent positive relationship between MT and HSP induction on one hand, and accumulated tissue metal burdens on the other. Despite the stable tissue Zn level, coelomocytes from earthworms exposed to this metal had signicantly enhanced levels of w-MT2, HSP70 and HSP72. Immunostaining intensities were subjectively similar in coelomocytes after Cu or Cd exposures, even though Cd concentration was biomagnied to a much greater extent relative to controls than the Cu concentration. It has previously been reported that Cu is a relatively inecient inducer of MT gene expression in L. rubellus inhabiting metal polluted soils (Marin o et al., 1998, 1999). The rapid induction of MT in coelomocytes of E. fetida after contact exposure implies either a species dierence in inducibility, or that the route of Cu uptake is an important determinant of the site(s) and kinetics of MT expression. Coelomocyte MT expression was similar in control and Pb-dosed earthworms, whilst the HSPs, especially HSP72, were strongly upregulated after Pb exposure (Table 2). These results indicate that earthworm coelomocytes are selectively sensitive to particular metal ions. The results also reinforce the notion that the value of biomarkers is higher when they are employed as suites rather than individually (De Coen and Janssen, 2003; Svendsen and Weeks, 1997; Svendsen et al., 2004; Lukkari et al., 2004).

in metal tracking, but whether they use nephridia, the dorsal pores, or some other pathway as the primary excretory route is presently unclear.

Acknowledgments This work was supported by the State Committee for Scientic Research, BW/36a/IZ (to EO), DS/IZ/ZIE (to JH and BP), the British Natural Environmental Research Council (to AJM and SRS) and the Royal Society of London (to SRS). The authors are grateful to Dr. Maria Niklinska (from Institute of Environmental Sciences, Jagiellonian University) for metal analyses.

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