SUMMARY Insects have long been known to excrete toxins via the Malpighian (renal) tubules. In addition, exposure to natural or synthetic toxins is commonly associated with increases in the activity of detoxification enzymes such as the P450 monoxygenases (P450s) and the glutathione-S-transferases (GSTs). We examined the links between mechanisms for detoxification and excretion in adult Drosophila melanogaster using functional assays and measurements of changes in gene expression by quantitative reverse transcriptase PCR in response to dietary exposure to compounds known to alter activity or gene expression of P450s and GSTs. Dietary exposure to phenol, which alters gene expression for multiple GSTs after seven to 10 generations, was also associated with an increase (more than twofold) in secretion of the organic anion methotrexate (MTX) by isolated tubules. Dietary exposure to the insecticide synergist piperonyl butoxide (PBO) was associated with reduced expression of two P450 genes (Cyp4e2, Cyp4p1) and two GST genes (GstD1, GstD5) in the tubules, as well as increased expression of Cyp12d1 and GstE1. Thin layer chromatographic analysis of fluid secreted by isolated tubules indicated that dietary exposure to PBO resulted in increased levels of an MTX metabolite. In addition, exposure to PBO altered the expression of transporter genes in the tubules, including a Drosophila multidrug resistance-associated protein, and was associated with a 73% increase in MTX secretion by isolated tubules. The results suggest that exposure of Drosophila to toxins evokes a coordinated response by the Malpighian tubules, involving both alterations in detoxification pathways as well as enhanced transport.

No abstract

Rosmarinus officinalis L. is an aromatic plant belonging to the Lamiaceae family widely distributed in the Mediterranean area. The interest on this species is related to the multiple uses of the plant as a food ingredient, in the pharmaceutical and cosmetic industries. The chemical composition of essential oil (EO) from 5 accessions of R. officinalis L., collected monthly through a full year in Sardinia, has been studied by gas chromatography (GC) and GC-mass spectrometry technique. The EO ranged from 0.29% to 0.89%. The qualitative determinations revealed the presence of 27 compounds belonging to 6 chemical groups (hydrocarbon monoterpene, alcohols, aldehydes, ketones, esters, hydrocarbon sesquiterpene). Overall the GC-flame ionization detector analysis showed the presence of 7 major compounds: α-pinene (26%-28%), camphene (5%-8%), 1,8-cineole (15%-25%), borneol (5%-11%), camphor (3%-12%), verbenone (6%-15%), and bornyl acetate (4%-7%). Chromatographic data were also subjected to a chemometric approach that evidenced discrimination of the samples according to the site of collection.

An earlier study has shown that RNAi knock-down of a single organic anion transporter (OAT) gene in the principal cells of Drosophila Malpighian tubules is associated with reductions in the expression of multiple, functionally related genes. In this study, we measured the rates of secretion of four fluorescent ions by tubules isolated from flies expressing targeted RNAi knock-down of specific OAT genes. Droplets secreted by isolated tubules set up in the Ramsay assay were collected in optically flat capillary tubes and the concentrations of fluorescent ions were determined by confocal laser scanning microscopy. Reductions in the expression of organic anion (OA) transporting polypeptide 58Dc (OATP; CG3380) were associated with reduced secretion of the OAs fluorescein and Texas Red. Reduction in the expression of Drosophila multidrug resistance associated protein (dMRP; CG6214) was correlated with reduced secretion of the P-glycoprotein substrate daunorubicin. Secretion of the organic cation quinacrine was unaffected by reduced expression of OATP, dMRP, or a multidrug efflux transporter (MET; CG30344). The results highlight the difficulties of assigning a rate-limiting role in transport of a specific OA to a single membrane transporter.

The effects of dietary exposure to organic anions on the physiology of isolated Malpighian tubules and on tubule gene expression were examined using larvae of Drosophila melanogaster. Acute (24 h) or chronic (7 d) exposure to type I organic anions (fluorescein or salicylate) was associated with increased fluid secretion rates and increased fluxes of both salicylate and the type II organic anion methotrexate. By contrast, chronic exposure to dietary methotrexate was associated with increased fluid secretion rate and increased flux of methotrexate, but not salicylate. Exposure to methotrexate in the diet resulted in increases in the expression of a multidrug efflux transporter gene (MET; CG30344) in the Malpighian tubules. There were also increases in expression of genes for either a Drosophila multidrug resistance-associated protein (dMRP; CG6214) or an organic anion transporting polypeptide (OATP; CG3380), depending on the concentration of methotrexate in the diet. Exposure to salicylate in the diet was associated with an increase in expression of dMRP and with decreases of MET and OATP. Exposure to dietary salicylate or methotrexate was also associated with different patterns of expression of heat shock protein genes. The results suggest that exposure to specific type I or type II organic anions has multiple effects and results not only in increased organic anion transport but also in increased rates of inorganic ion transport, which drives osmotically-obliged fluid secretion. Increased fluid secretion may enhance secretion of organic anions by eliminating diffusive backflux from the tubule lumen to the hemolymph.