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Does an ARYL HYDROCARBON RECEPTOR (AHR)-like molecule exist in earthworms? Some implications for immunity

Posted on: Tuesday, 2 March 2004, 06:00 CST

Summary

Strong pathogenic effects, including immunotoxicity and carcinogenesis of xenobiotics like TCDD and PCBs, in mammals are mainly mediated by the aryl hydrocarbon receptor (AHR). AHR- homologs have recently been shown for Caenorhabditis e/egans (Nematoda), Dmsophila melanogaster(Diptera) and Mya arenaria (Bivalvia). Since immune modulating effects have been described in earthworms after exposure to xenobiotics such as PCBs this investigation focused on the existence of an AHR-like molecule in Eisenia fetida. Our data revealed two proteins in tissue extracts, and one protein in coelomocyte lysates, of fetida that cross- reacted with an affinity purified AHR-antibody by western blot analysis. Reverse transcription of poly (A)+RNA from E. fetida and amplification of cDNA with the degenerate primers, designed for AHR detection in invertebrates as well as aquatic vertebrates, produced two independent bands of approximately 550 bp and 250 bp. Efforts to identify an AHR cDNA homolog in fetida with RT-PCR analysis using poly (A)+RNA were not successful. Further studies using different primers and/or specific tissues and developmental stages are needed to substantiate whether or not an AHR-like molecule exists in E. fetida.

Key words: AHR, earthworms, invertebrate immunity, RT-PCR, xenobiotics

Introduction

Many organisms have to deal with an increasing amount of xenobiotics released in the environment. In mammals, the toxicity of dioxin-like compounds (including non-ortho-substituted PCBs) is mainly mediated by the aryl hydrocarbon receptor (AHR). There the AHR exists in the cytoplasm as a complex of two heat shock protein (hsp 90) molecules and the AHR interacting protein (Hankinson 1995; Zhang et al. 1996). After ligand binding, the AHR is translocated to the nucleus and dissociation of the two hsp 90 molecules occurs, leading to AHR transformation. The activated receptor dimerizes with the AHR nuclear translocator (ARNT), interacts as AHR-ARNT complex with xenobiotic responsive enhancer (XRE) and results in gene transcription. AHRhomologs have recently been shown also for arthropods, mollusks and nematodes (Hahn et al. 1997; Po well- Coffman et al. 1998; Emmons et al. 1998; Butler et al. 2001). As soil dwelling organisms earthworms come into intimate contact with xenobiotic contaminants. The immune modulating effects of xenobiotics like PBCs have actually been shown in earthworms (RodriguezGrau et al. 1989; Cooper & Roch 1992; Suzuki et al. 1995; Ville et al. 1995). Therefore, the purpose of the present study was to determine wether an AHR-like molecule occurs in E.fetida. The existence of such a receptor would provide a mechanistic basis for the previously described toxicity of dioxin-like compounds.

Materials and Methods

Eisenia fetida was purchased from Carolina Biological Supply Company (USA). Earthworms were either stored in plastic boxes on moist paper towels at 160C and fed on Gerber oatmeal baby cereal or immediately frozen in liquid nitrogen for RNA preparation.

Tissue extract preparation: Earthworm coelomocyte- and tissue- donors were maintained without food for at least three days to avoid contamination during harvesting procedure. Coelomocyte lysate was obtained as described previously (Kauschke et al. 2001). For tissue extract preparation, deep frozen tissue (100 mg/ml) was placed in lysis buffer supplemented with protease inhibitors (aprotinin 100 U/ ml, PMFS 100 M, leupeptin 50 M), transferred into a chilled glass homogenizer and manually homogenized on ice. Ice cold homogenate was sonicated and then centrifuged (12.000 rpm; 4C; 15 min) to pellet debris. For RNA preparation, earthworm tissue was deep frozen in liquid nitrogen, kept at-80C and frozen mechanically homogenized following the instruction for using RNA STAT-60 (Tel-Test B, Inc.) see below.

SDS-PAGE was performed using a Bio-Rad MiniProtean II Dual Slab Cell. A 7.5 % resolving gel was topped by a 4 % stacking gel. Coelomic fluid or coelomocyte lysate (50 1;100 g protein) mixed with sample buffer (0.5 M Tris-HCl, pH 6.8; 10 % glycerol; 2 % SDS; 0.05 % w/v bromphenol blue) in a 4:1 ratio were applied per well. Electrophoresis was performed under constant current of 30 mA per gel.

Western Blotting: Protein transfer onto nitrocelluose (NC) was carried out using a Bio-Rad mini-tank blotting device. After transfer, nonspecific binding sites were blocked by blocking buffer [4 % (v/v) goat donor serum in TBS (80 g NaCl; 2 g KCl; 30 g Tris base in 1 l dH^sub 2^O; pH 7.4) plus 0.1 % Tween-20, 5 % (w/v) nonfat dry milk powder] and NC sample sheets then incubated with an affinity purified anti-AHR polyclonal antibody (1:5000) for 1 h at RT, washed 3 10 min before the secondary antibody [Goat anti Rabbit, Fc specific, HRP coupled (PIERCE), 1:20.000] was added (1 h, RT). Antibody binding was visualized after intensive washing (65 min) by using the ELC (enhanced chemiluminescence) detection solution (Amersham) followed by film exposure. HEPA1 cell extract was used as positive control. The affinity purified anti-AHR antibody was a kind gift of Dr. O. Hankison (UCLA). The A-IA (murine) AHR-antibody and the rt-84 (trout) ARNT-antibody were kindly provided by Dr. R. Pollenz (University of South Florida).

RNA preparation: Total RNA was isolated from deep frozen tissue ofE.fetida with RNA STAT-60 (TelTest B, Inc.). RNA STAT-60 (1 ml) was used for homogenization of 100 mg frozen tissue. After 5 min incubation at room temperature chloroform was added (0.2 ml), shaken vigorously for 15 seconds, incubated for further 2-3 min at room temperature and then centrifuged at 12.000 rpm for 15 min at 4 C. The upper aqueous phase was recovered and used for RNA precipitation. Therefore isopropanol (0.5 ml/ml RNA STAT-60) was added for 10 min at room temperature. The RNA pellet was obtained after centrifugation at 12.000 rpm for 10 min at 4 C, washed twice in ethanol (75 %), shortly air dried and then dissolved in a small volume of DEPEC-dH^sub 2^O. RNA was quantified by UV^sub 260/280^ absorption spectroscopy. The quality of total RNA was verified on ethidium bromide stained formaldehyde agarose gel. Total RNA (1 mg) was used for poly(A)+RNA isolation by affinity chromatography using an oligo(dT) spin columns kit (5 Prime 3 Prime, Inc.).

RT-PCR analysis was performed with degenerate primers designed for the detection of AHR homologs in invertebrates and aquatic vertebrates, slightly modified from those described previously (Karchner & Hahn 1996; Butler et al. 2001): (QF: 5'-AAC CCI TCI AAG MGI CAY MG-3'; INBO: 5'-TCI GGY TTI SWR TTY TTR TA-3'; INA2: 5'-TAY CTI GGITTY CAY TG-3'; A2F: 5'-TAY YTI GGI TTY CAY CAR-3'; A2R: 5'RTC IGA YGR TGR AAI CC-3'; AHR-Al: 5'-CAR GCI CTS AAY GGITT-3'; AHR-Bl: 5'-CATICC RCT YTC ICC IGT YTT-3'). Primers were synthesized by Life Technologies, Inc., USA and Integrated DNA Technologies, Inc., USA, and used in a concentration of 1 M in PCR reaction. Poly(A)+RNA was used to synthesize the first strand cDNA by RT-PCR. The general procedures followed those used previously for cloning AHR homologues (Hahn et al. 1997; Karchner et al. 1999).

DNA-sequencing was performed at the University of Maine, ME, USA.

Results

Western blot analysis:

Tissue extract and coelomocyte lysate of E.fetida contained proteins that cross-reacted with an affinity purified AHR-antibody as revealed by western blot analysis (Fig. 1). Whereas for coelomocyte lysate one band was visualized by western blotting, two bands were detected for tissue extract. Nevertheless, both proteins were of lower molecular weight than AHR-positive controls Hepa-1 cell extract.

By using an A-I A- (murine AHR) antibody, which recognizes amino acids 1-416 (nearly the entire bHLH/PAS region) and the rt-84 (trout) - ARNT antibody, bands were visible that correspond in molecular weight to the putative AHR and ARNT in Hepa-1 cells. In general, a stronger signal was shown for earthworm whole-tissue homogenate than for coelomocyte lysate using both A-IA and rt-84 antibodies in western blotting (Fig 2A-D).

Fig. 1. Western blot analysis for an AHR-like molecule in tissue extract (1), coelomic fluid (2), coelomocyte lysate (3) of E, fetida and a HepaA1 positive control (4, 5) using an affinity purified anti- AHR antibody

Fig. 2. Western blot analysis of earthworm homogenate (A and C) and cytosol (B and D) for AHR and ARNT by using an A-1A(murine)AHR antibody (A and B) and the rt-84 (trout) ARNT antibody (C and D). Wild type Hepa-1 cells (wt Hepa) were used as controls. In A and B earthworms were kept on wet paper (lane 1-3) or on wet paper plus TCDD (lane 4-6). In C and D all earthworm samples were kept on wet paper (lane 1-6)

RT-PCR:

Reverse transcription of poly (A)+RNA from E.fetida and amplification of cDNA with the degenerate primers Of/INBO and INBO/ INA2 produced two independent single bands of approximately 550 bp and 250 bp (Fig. 3). Both fragments obtained were cloned and sequenced. A BLASTX algorithm was used for sequence comparison with Genbank database (Fig. 4). The translated deduced amino acid sequence of the amplified 550 bp fragment was mostly related to parts of paramyosin from Mytilus galloprovincialis, Echinococcus granulosus and Taenia solium with 38 %, 37 % and 36 % amino acid identity, respectively.

The translat\ed amino acid sequence of the amplified RT-PCR product of 250 bp was mostly related to parts of cytochrome b from Lumbricus terrestris and Chironomus crassiforceps, with 71% and 56% amino acid identity, respectively.

Efforts to identify a AHR cDNA homolog in E. fetida with the RT- PCR analysis using poly (A)+RNA have not been successful so far.

Fig. 3. Ethidium bromide-stained agarose gel showing ~550 bp (1) and ~250 bp (2) PCR products amplified with specific designed primers (see material and methods; 3 bp marker)

Fig. 4. Deduced amino acid partial sequence of paramyosin and cytochrome b from E. fetida obtained by RT-PCR with degenerated primers designed for the detection of AHR homologs in invertebrates and aquatic vertebrates

Discussion

Knowledge about the toxic potential and mode of action of xenobiotics is of great importance in evaluating their risk to humans as well as to the environment in general. Most ecological screening methods reflect acute toxic processes due to xenobiotics, but the immune system is centrally important in individual protection. The molecular and cellular biology of immune mechanisms are well known. Therefore, certain immune functions, analyzable in vitro, are seen as particularly suitable for investigating effects and risks of xenobiotics. That applies to vertebrates and invertebrates, since immune modulating effects of xenobiotics have been shown for both. Any compromise of the immune system resulting from contamination may lead to serious effects in aquatic and terrestrial organisms, altered immune responses are indicators of exposure and potential harm. Assays that reveal immune function are available with respect to transfer of an effect from its interaction with a specific receptor molecule to ecological effects (Rodriguez- Grau et al. 1989; Cooper & Roch 1992; Suzuki et al. 1995; Ville et al. 1995).

Strong pathogenic actions including immunotoxicity, thymic atrophy and carcinogenesis evoked by xenobiotics like TCDD and PCBs (Rowlands & Gustafsson 1997; Schmidt & Bradfield 1996; Hankinson 1995) in mammals are mainly mediated by AHR. AHR- and ARNT- homologs have recently been shown for three invertebrates, C. elegans, D. melanogaster and M. arenaria (Hahn et al. 1997; Powell-Coffman et al. 1998; Emmons et al. 1998; Butler et al. 2001). Vertebrate AHR and ARNT and AHR- and ARNT-homologs in C. elegans and D. melanogaster dimerize and recognize a specific DNA-sequence in the mammalian XRE. Moreover, the AHR-homolog in C. elegans shares sequence motifs with vertebrate AHR, which are responsible for XRE- binding (Ikuta et al. 1998; Butler et al. 2001).

Although western blot analysis revealed proteins binding to an anti-AHR and to an ARNT (rt-84, trout) -antibody in E. fetida, our data did not identify a AHR cDNA homolog using RT-PCR. Further studies using different, newly developed primers and/or specific tissues and developmental stages are needed to substantiate whether or not an AHR-like molecule definitely exists in E. fetida.

In contrast to the AHRs of vertebrates, the interactions of AHR- homologs with ARNT-homologs of invertebrates appear to be completely TCDD independent. Identification of natural ligands for AHR- homologs in invertebrates should, therefore, be of primary interest, since they may display different activation pathways for mediated processes, and may provide further insights into the physiological function of these molecules, both in invertebrates and vertebrates.

Acknowledgements. The authors like to thank Dr. O. Hankinson (School of Medicine, University of California Los Angeles) and Dr. R. Pollenz (University of South Florida) and their laboratories for kindly providing the anit-AHR- and anit-ARNT-antibodies as well as for assistance with the western blot analysis.

Data presented here are part of a research project, granted by scholarships of the country Mecklenburg-Vorpommern Germany, the German Academic Exchange Service (DAAD) and the German Society of Immunology (DGfI), awarded to Mr. Lutz Wiesner as well as partly funded by United States National Institute of Health (ES06272), awarded to Dr. M. E. Hahn. Dr. E. L. Cooper was partly supported by the Alexander von Humboldt Foundation, Germany.

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Lutz Wiesner1, Mark E. Hahn2, Sibel I. Karchner2, Edwin L. Cooper3 and Ellen Kauschke4*

1 Professor Dennert Strae 5, D-53173 Bonn, Germany

2 Department of Biology, Woods Hole Oceanographic Institutions, Massachusetts, MA 02543, USA

3 Laboratory of Comparative Immunology, Department of Neurobiology, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095-1763, USA

4 Institute of Anatomy, Ernst-Moritz-Arndt-University Greifswald, F.-Loeffler-Strae 23c, D-17487 Greifswald, Germany

Submitted September 6, 2002 . Accepted November 6, 2003

*E-mail corresponding author: kauschke@uni-greifswald.de

Copyright Urban & Fischer Verlag 2003

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