[
Gene,
2001]
The aryl hydrocarbon receptor (AHR) mediates numerous toxic effects following exposure of vertebrate animals to certain aromatic environmental contaminants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To investigate possible effects of TCDD on invertebrates, a cDNA encoding an AHR homologue was cloned from the soft-shell clam, Mya arenaria. The predicted amino acid sequence contains regions characteristic of vertebrate AHRs: basic helix-loop-helix (bHLH) and PER-ARNT-SIM (PAS) domains and a glutamine-rich region. Phylogenetic analysis shows that the clam AHR sequence groups within the AHR subfamily of the bHLH-PAS family, in a clade containing AHR homologues from Drosophila melanogaster and Caenorhabditis elegans. AHR mRNA expression was detected in all tissue types tested: adductor muscle, digestive gland, foot, gill, gonad, mantle, and siphon. The in vitro-expressed clam AHR exhibited sequence-specific interactions with a mammalian xenobiotic response element (XRE). Velocity sedimentation analysis using either in vitro-expressed clam AHR or clam cytosolic proteins showed that this AHR homologue binds neither [(3)H]TCDD nor [(3)H]beta-naphthoflavone (BNF). Similarly, in vitro-expressed D. melanogaster and C. elegans AHR homologues lacked specific binding of these compounds. Thus, the absence of specific, high-affinity binding of the prototypical AHR ligands TCDD and BNF, is a property shared by known invertebrate AHR homologues, distinguishing them from vertebrate AHRs. Comparative studies of phylogenetically diverse organisms may help identify an endogenous ligand(s) and the physiological role(s) for this protein.
[
International Worm Meeting,
2003]
Cytochromes P450 (CYP) are a super family of heme containing and NADPH dependent monooxygenases which catalyze the oxidative metabolism of many exogenous and endogenous compounds. While the investigation of CYP induction pathways in mammals has made considerable progress, very little is known about P450s from invertebrates, such as the nematode Caenorhabditis elegans. C. elegans possesses, however, a remarkably high number of 80 different cytochrome P450 genes. The functions of the encoded proteins are almost unknown. In a previous study it could be demonstrated that the five different CYP forms 35A1-A5 are strongly inducible by PAHs, PCBs and different drugs [1]. In order to study this xenobiotically induced gene expression different transgenic C. elegans lines expressing GFP under control of the CYP35A1-5 promoter regions were constructed. With this tool it was possible to follow the GFP production in vivo depending on the applied inducer. Induction with -naphthoflavone resulted in all cases in an expression of the green fluorescent protein exclusively in the worm intestine, identifying this compartment as the likely tissue of P450 35A localization. For the CYP35A2 and A5 forms promoter-deletion experiments were performed revealing a 400 bp and a 250 bp fragment, respectively, to be sufficient to induce GFP production. While the 35A2 promoter caused GFP production in all developmental stages, the 35A5 based gene expression was mainly detectable in eggs and in L1/L2 larvae. In particular in case of the 35A2 promoter region a striking correspondence to mammalian xenobiotic response elements (XRE) has been found. To obtain more information about the biological functions of the five different CYP35A forms dsRNA producing E. coli strains for RNAi by feeding experiments were produced. Surprisingly, embryonic lethality was observed as the loss of function phenotype for all CYP35A forms with exception of 35A2. This might indicate that at least one or more CYP35A forms play an essential role in the embryogenesis of C. elegans. Using semi-quantitative RT-PCRs the cross-reactivity level of the individual RNAi constructs was determined. Future efforts will be focused to characterize the blocked embryonic development more in detail.[1] Menzel R., Bogaert T. and Achazi A. (2001) A systematical gene expression screen of the Caenorhabditis elegans cytochrome P450 genes revealed CYP35 as strong xenobiotic inducible. Arch. Biochem. Biophys. 395, 158-168.