Page 1 of 15 Articles in PresS. Physiol Genomics (May 8, 2007). doi:10.1152/physiolgenomics.00018.2007
A Drosophila systems approach to xenobiotic metabolism Jingli Yang1, Caroline McCart2, Debra J. Woods3, Selim Terhzaz1, Karen G. Greenwood3, Richard ffrench-Constant4, Julian A.T. Dow1 1
Division of Molecular Genetics, University of Glasgow, G11 6NU, UK;
Department of Biochemistry & Biology, University of Bath, Bath, BA2 7AY UK;
Veterinary Medicine Research and Development, Pfizer Animal Health, Ramsgate Road, Sandwich, Kent
CT13 9NJ, UK; and 4
School of Biological Sciences, University of Exeter in Cornwall, Penryn, TR10 9EZ UK.
Corresponding Author: Professor Julian Dow, Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, UK. Phone +44 141 330 4616 FAX +44 141 330 4878 Email [email protected]
Running head : Xenobiotic metabolism in Drosophila
Yang et al.
Copyright © 2007 by the American Physiological Society.
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Abstract Insecticide resistance is a major problem for both medicine and agriculture, and is frequently associated with over-expression of metabolic enzymes that catalyse the breakdown of pesticides, leading to broad-spectrum resistance. However the insect tissues within which these metabolic enzymes normally reside remain unclear. Microarray analysis of 9 adult tissues from Drosophila melanogaster reveals that cytochrome P450s and glutathione-S-transferases show highly tissuespecific expression patterns; most were confined to one or more epithelial tissues, and half showed dominant expression in a single tissue. The particular detoxifying enzymes encountered by a xenobiotic thus depend critically on the route of administration. In particular, known insecticide metabolism genes are highly enriched in insect Malpighian (renal) tubules, implicating them in xenobiotic metabolism. The tubules thus display, with the fat body, roles analogous to the vertebrate liver and immune system, as well as its acknowledged renal function. To illustrate this, when levels of a single gene, Cyp6g1, were manipulated in just the Malpighian tubules of adult Drosophila, the survival of the whole insect after DDT challenge was altered, whereas corresponding manipulations in the nervous system or the fat body were without effect. This shows that, although detoxification enzymes are widely distributed, baseline protection against DDT resides primarily in the insect excretory system, corresponding to less than 0.1% of the mass of the organism.
Introduction Insects are major vectors of transmissible disease and pests of major crops. Effective insect control is thus vital, and insecticide resistance is a continuing problem, both in developed and developing worlds. Insecticide resistance across many species has been attributed to up-regulation of enzymes associated with xenobiotic detoxification and metabolism (12), whereas the unusual sensitivity of the honey-bee to insecticides may reflect under-representation of these genes in its genome (8). In Drosophila melanogaster several different cytochrome P450s have been implicated in conferring resistance to DDT and a range of more recent insecticides such as the neonicotinoids; for example CYP6G1, CYP6A2, CYP12D1 and CYP12A4 (1, 10). In the case of the P450 Cyp6g1, the insertion of an Accord transposable element into the 5’ end of the gene has led to its elevated expression (16). DDT resistance in mosquitoes has also been associated with up-regulation of glutathione-S-transferases (12), such as GSTE2 (18). These broadspecificity mechanisms produce cross-resistance to other novel classes of insecticide, such as pyrethroids (12) and the recently introduced neonicotinoids (10). However, despite the extensive literature on the number and types of P450s and GSTs expressed in resistant strains, there is little information on the tissues in which xenobiotic metabolism is effected. Given the large number of P450 and GST encoding genes in the Drosophila genome and the large number of studies implicating different genes in resistance in different Drosophila strains, there is a lively debate as to the relative importance of single versus multiple genes in insecticide resistance (5, 10-12, 16, 17). Despite the observation that single metabolic genes are up-regulated in resistant strains recently isolated from the Yang et al.
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field, it is a matter of biochemical fact that several of the candidate enzymes are capable of detoxifying xenobiotics, and so it is possible that different genes play key roles in different circumstances. For example, different tissues might provide the first line of defence against ingested or topical insecticides, and if detoxifying enzymes are tissue-specific in their distribution, then apparently conflicting results might be obtained. It is thus critical to obtain a clear view of gene expression in multiple tissues. Although the biochemistry of insecticide resistance is well understood, the physiology is much less so. Is insecticide metabolism a ‘housekeeping’ role found in all cells, or are specific tissues (like the nervous system) responsible for local or global defence against xenobiotics? Such data could affect strategies for efficacious and environmentally-sparing insecticide use. Previously, attention has focused on the midgut, fat body and Malpighian tubules for metabolism of both insecticides and plant secondary metabolites. For example phenobarbitol administration induced Cyp6a2 over-expression in Drosophila midgut, the pericuticular fat bodies and the Malpighian (renal) tubules (4), whereas the detoxification of furanocoumarin by Cyp6b1 and Cyp6b3 depended on midgut and fat body (22). These data are consistent with a classical view that the fat body performs as an insect ‘liver’. Re-analysis of a detailed microarray study of gene expression in adult Drosophila Malpighian (renal) tubule (26) revealed substantial up-regulation of several members of the cytochrome P450 and glutathione-S-transferase families (data not shown). In particular, the known insecticide resistance genes Cyp6g1 and Cyp6a2 were found to be upregulated 9.4 fold and 8 fold respectively, in tubule compared with whole fly (26). These data clearly suggest that on an organismal scale, xenobiotic metabolism may be primarily a renal function, and so a more detailed investigation was undertaken.
Results and discussion Expression profiles of xenobiotic metabolism gene families Recently, a comprehensive microarray-based atlas of adult gene expression in multiple Drosophila tissues has become available (http://flyatlas.org) (6), and so it is possible to establish whether detoxification and metabolism genes are indeed widely-expressed “housekeeping” genes, or have more specific patterns of expression. The results (Table 1), provide a uniquely comprehensive overview of the P450 and GST gene families (those mainly implicated in xenobiotic metabolism); and, while confirming previous reports of expression in midgut and Malpighian tubules, provide detailed evidence that the majority of the genes in each family are expressed in defined subsets of tissues. Remarkably, none of the P450 family, and only a few of the GST family (for example gstd1) show ubiquitous, “housekeeping”-like expression patterns; a very few show widespread expression except for a particular tissue (for example Cyp6d5, which is very abundant in all tissues except ovary); the large majority are confined to epithelial tissues (midgut, hindgut, Malpighian tubules); and about half are extremely specific, showing overwhelming expression in a single tissue. For example, Cyp6g2 is unique to brain and head, whereas Cyp6d4 is predominantly expressed in midgut, Cyp6a18 in tubules and Cyp6a19 in ovary.
Yang et al.
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Unusual P450s The P450 gene family has been described as having a relatively large number of pseudogenes, given that such genes are relatively rare in the genome as a whole (13). However, as the Drosophila genome sequence has been successively refined, many gene calls have been revised, leading to several pseudogenes being reannotated as expressed genes. Intriguingly, one of the P450 pseudogenes (Cyp6a16psi) shows strong tissuespecific expression in tubule (Table 1), although the other pseudogenes are transcriptionally almost silent. Although still annotated in Flybase as a pseudogene with at most a 392 aa ORF (with a truncated match to the pfam00067 p450 conserved domain), Swissprot accession Q9VMN8 reports a 496 aa ORF for cyp6a16 that includes a full-length match to the pfam p450 domain. It seems likely that Cyp6a16 is a genuinely transcribed gene; its highly specific expression pattern may have militated against its earlier identification in EST projects. By contrast, no expression was detected for the pseudogenes Cyp6t2psi or Cyp6a15psi. The position with respect to Cyp9f3psi remains unclear; it is clearly expressed, both from our data and from BDGP embryonic in situ data, but it lacks the C-terminal heme domain, and abuts so closely between its neighbours that there is little space in which to discover such a domain. It is also extremely close (GFP reporter construct showed highest levels of expression in tubule (Fig. 1). In addition, CG16936, the Drosophila gene most similar (BLASTP e value 1e-58) to Aedes GSTE2 (another insecticide resistance locus (11) is also most abundant in tubule. Several lines of evidence thus suggest that the tubule may thus be the dominant tissue for xenobiotic metabolism in the adult. This has implications for whole-organism microarray studies of insecticide resistance, as such studies are biased to follow concerted changes in expression across multiple tissues, and so are predicated on detecting global changes in expression of widely expressed genes (6).
A major role for tubule? Given that several genes and multiple tissues might impinge on insecticide detoxification, how can the importance of the tubule be tested experimentally? We decided to adopt a systems approach in Drosophila, as it is possible to modulate expression levels of particular genes in a cell specific fashion (2). Thus, if Cyp6g1 expression in the Malpighian tubule is limiting for insecticide susceptibility to xenobiotics, then small perturbations in levels of Cyp6g1 in just the tubule should impact detectably on survival of the whole organism. Such experiments are possible in Drosophila using the GAL4/UAS binary expression system (2). Several transgenic Drosophila UAS-RNAi lines were generated, and further lines obtained from the NigFly project (http://shigen.lab.nig.ac.jp/fly/nigfly/). They were crossed to the c42 GAL4 driver line that drives expression specifically in tubule principal cells (3), and screened by quantitative PCR for effective suppression of Cyp6g1 expression. One such line (UAS-Cyp6g1-RNAi-I) proved highly effective; when driven in Malpighian tubules with c42, a principal-cell specific GAL4 driver line (3, 23, 24), Cyp6g1 mRNA expression (measured by qPCR) was 0.64% of the level in parental UAS-Cyp6g1-RNAi-I flies. Such knockdown flies are significantly (P