A drosophila model for the screening of bioavailable NADPH oxidase inhibitors and antioxidants

Mol Cell Biochem (2011) 352:91–98 DOI 10.1007/s11010-011-0743-3 A Drosophila model for the screening of bioavailable NADPH oxidase inhibitors and antioxidants Nguyen Thi Tu Anh • Maiko Nishitani • Shigeharu Harada • Masamitsu Yamaguchi Kaeko Kamei • Received: 16 October 2010 / Accepted: 28 January 2011 / Published online: 11 February 2011 Ó Springer Science+Business Media, LLC 2011 Abstract NADPH oxidase is the major source of nonmitochondrial cellular reactive oxygen species (ROS), and also is reported to be a major cause of various diseases including atherosclerosis and hypertension In order to screen a new curative reagent that can suppress NADPH oxidase activity, we developed a Drosophila melanogaster fly that would overexpress human Dual oxidase (hDuox2), a member of the NADPH oxidase family, as a screening model These flies (GMR-GAL4/UAS-hDuox2) had a high generation of ROS in the posterior region of the eye discs along with an easily recognizable rough-eye phenotype, which is an ideal and convenient marker for further screening steps Moreover, the hDuox2-induced rough-eye phenotype can be rescued by feeding with a culture medium containing mulberry leaves (MLs), which reportedly have an antimetabolic effect Some commercially available antioxidants such as quercetin-3-O-D-glucoside or quercetin-3-O-glucose-600 -acetate, or the naringin contained in MLs and other herbs, also have shown a similar suppressing effect on the rough-eye phenotype Our results suggest that flavonoid glycoside is absorbed from the intestine and functions in the body of D melanogaster as it does in mammalian models such as rats Thus, the GMR-GAL4/UAS-hDuox2 fly line is a promising model for the screening of novel drugs such as NADPH oxidase inhibitors and/or antioxidants N T T Anh Á M Nishitani Á S Harada Á M Yamaguchi Á K Kamei (&) Department of Applied Biology, Kyoto Institute of Technology, Matsugasaki Sakyo-ku, Kyoto 606-8585, Japan e-mail: kame@kit.ac.jp Keywords NADPH oxidase Á Drosophila melanogaster Á Metabolic syndrome model Á Screening Á Antioxidant Á NADPH oxidase inhibitor Introduction Reactive oxygen species (ROS) play a critical role in the oxidative stress that causes various diseases, including atherosclerosis and hypertension [1–3] In recent years, it has become clear that ROS are produced by specific enzymes, including NADPH oxidase, by transferring one electron from NADPH to molecular oxygen [4] The NADPH oxidase family exists, with conserved functions, in a wide variety of cell types from many organisms, including mammals, nematodes, fruit flies, green plants, fungi and slime molds [5] Inappropriate activation of NADPH enzymes may contribute to tissue dysfunction such as damage to the liver and pancreatic b-cells that leads to progression from the metabolic syndrome to Type diabetes [6] Recently, Sugimoto et al discovered that inhibition of ROS generation via the suppression of NADPH oxidase expression in white adipose tissues and liver by the administration of mulberry leaves (MLs) ameliorated metabolic disorders in a mouse model [7] As well, several studies have revealed that MLs contain many antioxidants, particularly quercetin-3-(6-malonyglucoside), which is the most abundant flavonoid glycoside that has attenuated atherosclerotic lesion development in LDL receptor-deficient mice [8] These studies indicated that NADPH oxidase is an important target of antimetabolic drugs that may be present in MLs In order to screen new antimetabolic substances via the suppression of NADPH activity using MLs and other substances, the development of a metabolic syndrome model is needed Drosophila 123 92 melanogaster might be a viable candidate for studies of metabolic syndrome because it shares most of the same basic metabolic functions with vertebrates Many analogous organ systems that control nutrient uptake, storage and metabolism in humans are present in fruit flies [9] In addition, their rapid growth, inexpensive costs of breeding, and small genome size facilitates screening steps Human dual oxidases (hDuox1 and hDuox2), members of the NADPH oxidase family, are expressed in the thyroid, the airway epithelial cells and in a wide variety of tissues such as brain, cerebellum, mammary gland, muscle, nervous, placenta, and testis The genes encoding for these two enzymes are located on chromosome 15, and share 83% sequence similarity [10] In the thyroid gland, Duox enzymes produce hydrogen peroxide, which is essential for the oxidation step of iodide in thyroid hormone production The important role of Duox2 in this process was proven by the identification of patients who suffer from hypothyroidism due to mutations in the Duox2 gene [11] Besides, both in human and other organisms, dual oxidases are novel H2O2 sources that are indispensable in host defense against infection and extracellular matrix modification [12–14] Recently, the gut antimicrobial activities of flies, in which Drosophila NADPH oxidase (dDuox) had been silenced, could be markedly ameliorated by the introduction of the hDuox2 gene, indicating that hDuox2 is functional in Drosophila [13] Therefore, in order to construct an in vivo screening system for new antimetabolic substances via the suppression of NADPH activity, we utilized the GAL4-UAS expression system to provide ectopic expression of hDuox2 in D melanogaster [15] GAL4 is a yeast transcriptional activator that can activate the transcription of any gene after its introduction into Drosophila—if the gene is preceded by a GAL4 upstream activating sequence (UAS) that consists of five optimized GAL4 binding sites [16] In this study, a transgenic fly with a GMR (glass multiple reporter)-GAL4 gene on chromosome X was crossed with a fly with a UAS-hDuox2 gene on chromosome Since the GMR element causes high-level expression of GAL4 protein in cells from the posterior to the morphogenetic furrow, we were able to obtain an expression of hDuox2 in the posterior area of the eye discs This system has the advantage of allowing evaluation of hDuox2 activity by simple inspection of the eye phenotype Furthermore, we evaluated the use of flies that overexpress hDuox2 as models for the screening of antimetabolic substances The evaluation was carried out by feeding MLs and their flavonoids to transgenic Drosophila Mulberry leaves are reported to have an antimetabolic effect due to their content of flavonoids with antioxidant activities The effectiveness of MLs and these substances was evaluated based on their ability to suppress the hDuox2-induced rough-eye phenotype 123 Mol Cell Biochem (2011) 352:91–98 Materials and methods Materials Quercetin was purchased from Nacalai Tesque (Kyoto, Japan) The rutin, epigallocatechin gallate, and naringin were acquired from Wako Pure Chemical (Osaka, Japan) Quercetin-3-O-D-glucoside, quercetin-3-O-glucose-600 -acetate, and kaempherol all were acquired from Extrasynthese (France) Mulberry leaves The mulberry trees were cultured in a mulberry plantation at the Center for Bioresource Field Science, Kyoto Institute of Technology, using a standard Japanese method The ‘‘Shin-Ichinose’’ mulberry (Morus alba L.) race was used Mulberry leaves were harvested and immediately dried by air flush at 180°C for s The average diameter of the dried powder was 20 lm Fly stocks Fly stocks were maintained at 25°C The UAS-hDuox2 fly line that carries the human Duox2 gene on second chromosome was kindly provided by Dr Won-Jae Lee, of Ewha Woman’s University, South Korea [13] The GMR-GAL4/ UAS-DREF line was used as a control strain [17] The transgenic fly line carrying GMR-GAL4 on the X chromosome (strain number 16) has been described previously [17] Establishment of transgenic flies Virgin females were used for every crossing step to insure purity To construct GMR-GAL4/Y; UAS-hDuox2/?, virgin female GMR-GAL4 flies containing two copies of the GMR-GAL4 gene on the X chromosome were mated with male UAS-hDuox2 flies containing two copies of the UAShDuox2 gene on the second chromosome In the next generation, all male flies carry a single copy of both GMRGAL4 and hDuox2 (GMR-GAL4/Y; UAS-hDuox2/?) To construct the GMR-GAL4; UAS-hDuox2 line, virgin female GMR-GAL4 flies containing two copies of the GMR-GAL4 gene on the X chromosome and a secondbalancer chromosome CyO (GMR-GAL4; Sp/CyO) were crossed with male UAS-hDuox2 flies containing two copies of the UAS-hDuox2 gene on the second chromosome The second chromosome balancer CyO suppresses recombination with their homologs, carries dominant markers (curly-wing phenotype), and causes a lethal effect when it is homozygous In the next generation, all male Mol Cell Biochem (2011) 352:91–98 flies surely contain one copy of GMR-GAL4 on the X chromosome These male flies were further selected by curly-wing phenotype to get one copy of UAS-hDuox2 and one second chromosome balancer, CyO (GMR-GAL4/Y; UAS-hDuox2/CyO) These flies crossed again with female GMR-GAL4; Sp/CyO Subsequently, we could select female GMR-GAL4; UAS-hDuox2/CyO and male GMRGAL4/Y; UAS-hDuox2/CyO These two fly lines were mated together to finally establish fly lines containing two copies of GMR-GAL4 on the X chromosome and two copies of UAS-hDuox2 on the second chromosome (GMRGAL4; UAS-hDuox2) Culture medium The medium buffer contained 1% yeast extract and 0.5% agar in phosphate-buffered saline (PBS) ML-supplemented culture medium for feeding was prepared by mixing 750 ll medium buffer with 0.15 g instant medium Formula 4–24Ò (Carolina Biological Supply Co., USA) and 10, 20, or 30% (w/w) of MLs Antioxidant stocks were prepared by being dissolved in 99% ethanol Antioxidantsupplemented mediums were prepared by dissolving antioxidant stocks into medium buffer before mixing with instant medium Formula 4–24Ò to obtain a final 3% (w/w) concentration A blue coloring agent contained in the instant medium facilitated recognition of whether the larvae consumed the tested medium Feeding method Three males and three females of the control (GMR-GAL4; UAS-DREF) or the hDuox2-overexpressing flies (GMRGAL4; UAS-hDuox2) were, respectively, mated to lay eggs on culture mediums containing various concentrations of mulberry powder or antioxidants for day Newborn larvae were continuously fed on the same mediums for 10 days at 25°C to reach the adult phase Scanning electron microscopy Adult flies were anesthetized, mounted on stages, and observed using a VE-7800 (Keyence Inc., Osaka, Japan) scanning electron microscope in high vacuum mode In every experiment, the eye phenotype of at least five adult flies of each line was simultaneously examined by scanning electron microscopy, and these experiments were repeated three times In the experiments, no significant variation in eye phenotype among the five individuals was observed The rough area of the eyes was circled as an index of the effect on the rough-eye phenotype 93 In vivo ROS detection Eye discs from third instar larvae were dissected in PBS, and then incubated with 10 lM CM-H2DCFDA (5-(and6)-carboxy-20 , 70 -dichlorodihydrofluorescein diacetate, acetyl ester) (Molecular Probes, Invitrogen) for After washing with PBS, samples were fixed in 1% paraformaldehyde for min, washed three times with PBS, and then mounted in Vectashield Mounting Medium (Vector laboratories) Preparations were examined under a fluorescence BX-50 microscope (Olympus, Tokyo, Japan) equipped with a cooled CCD camera (ORCA-ER; Hamamatsu Photonics K.K., Shizuoka, Japan) Immunohistochemistry Third instar larvae were dissected in PBS and eye discs were fixed in 4% paraformaldehyde–PBS for 30 at 25°C After washing with 0.3% Triton X-100 in PBS (PBST), samples were blocked for 30 at 25°C with 0.15% PBST containing 1% bovine serum albumin Samples were then incubated with rabbit anti-hDuox2 antibody (Abcam) at a 1:100 dilution and 4°C for 16 h After extensive washing with PBST, the samples were incubated with goat anti-rabbit IgG Alexa FluorTM 488 (Molecular Probes, Invitrogen) at a 1:400 dilution for h at 25°C, further washed with PBST and PBS, then mounted in Vectashield Mounting Medium (Vector laboratories) Preparations were examined under a fluorescence BX-50 microscope (Olympus, Tokyo, Japan) equipped with a cooled CCD camera (ORCA-ER; Hamamatsu Photonics K.K., Shizuoka, Japan) Results Overexpression of human Duox2 disrupted normal eye development The hDuox2 transgenic flies were successfully used to rescue the defect in antimicrobial infection activity in the Drosophila NADPH oxidase (dDuox) RNAi strain, indicating that hDuox2 is functional in Drosophila [13] In this study, we utilized the GMR-GAL4 driver strain to provide ectopic expression of hDuox2 from the posterior to the morphogenetic furrow of a Drosophila eye [17] Under scanning electron microscope, the eyes of flies carrying a single copy of GMR-GAL4 (GMR-GAL4/Y; ?) appeared as a normal phenotype (Fig 1a), while those of flies carrying a single copy of both GMR-GAL4 and hDuox2 (GMR-GAL4/Y; UAS-hDuox2/?) had a mild rough-eye phenotype (Fig 1b) When the copy number of both GMRGAL4 and hDuox2 were increased to two (GMR-GAL4; 123 94 Mol Cell Biochem (2011) 352:91–98 Fig Adult compound eyes observed by scanning electron microscopy a The eyes of flies carrying a single copy of GMR-GAL4 (GMR-GAL4/Y; ?) appeared as a normal phenotype; b flies carrying a single copy of both GMR-GAL4 and hDuox2 (GMR-GAL4/Y; UAS-hDuox2/?) showed a mild rough-eye phenotype; c flies carrying two copies of GMR-GAL4 (GMR-GAL4; ?) had many fused ommatidia; d flies carrying two copies of both GMR-GAL4 and hDuox2 (GMR-GAL4; UAS-hDuox2) showed a more severe rougheyes phenotype Magnification is 2009 for left panels and 7009 for right panels Flies were reared at 25°C UAS-hDuox2, Fig 1d), the compound eyes of these flies had many fused ommatidia and became more severely rough than those of flies containing two copies of GMRGAL4 only (GMR-GAL4; ?, Fig 1c) To further confirm the overexpression of hDuox2 in the compound eyes, we performed immunostaining of eye imaginal discs from third instar larvae using anti-hDuox2 polyclonal antibody Only in GMR-GAL4; UAS-hDuox2, an ectopic hDuox2 signal from the posterior region to the morphogenetic furrow in eye discs was detected (Fig 2b) The GMR-GAL4 line that was used as the negative control showed no signal (Fig 2a) Immunostaining of eye imaginal discs of both GMR-GAL4; ? and GMR-GAL4; UAShDuox2 using only goat anti-rabbit IgG Alexa FluorTM 488 were also carried out to further exclude background effects (Fig 2c, d, respectively) GMR-GAL4 or hDuox2-overexpressing flies, as shown in Fig 3a and b, respectively In contrast, upon treatment with substrate CM-H2DCFDA, hDuox2-overexpressing flies showed stronger fluorescent signals from the posterior region to the morphogenetic furrow in eye discs (Fig 3d) as compared with that of the control GMR-GAL4 fly line (Fig 3c) This indicates that ROS generation was induced by the overexpression of hDuox2 in eye discs Overexpression of hDuox2 increased ROS generation Overexpression of hDuox2 can lead to an increased generation of ROS To detect intracellular ROS in hDuox2overexpressing flies, we used the non-fluorescent substrate CM-H2DCFDA, which can be oxidized by ROS to become an intracellular green fluorescent product With no treatment with substrate CM-H2DCFDA, no detectable signal was observed in eye imaginal discs for either control flies 123 Mulberry leaves could suppress the hDuox2-induced rough-eye phenotype In previous research, metabolic disorder was ameliorated in obese db/db mice fed MLs, at least partially by the suppression of oxidative stress by both antioxidative flavonoid scavenging of ROS and by the inhibition of NADPH oxidase expression [7, 18] Therefore, in this study, to evaluate the use of GMR-GAL4/UAS-hDuox2 as a metabolic syndrome model, we fed these flies 10, 20, and 30% (w/w) MLs To exclude effects of MLs other than on hDuox2 activity, flies overexpressing DREF, a transcription factor, by GMR-GAL4 with approximate rough-eye phenotype were used as a control [17] As shown in Fig 4a–d, MLs had no effect on the eye phenotype of DREF-overexpressing flies, indicating that MLs exerted no effect on GAL4 expression However, the rough-eye phenotype of Mol Cell Biochem (2011) 352:91–98 95 Fig Immunostaining of eye imaginal discs with anti-hDuox2 antibody Eye imaginal discs were reacted with rabbit anti-hDuox2 antibody followed by anti-rabbit IgG Alexa FlourTM 488 antibody (a, b) a Immunostaining of eye discs of GMR-GAL4; ? showed no detectable signal; b notably, in GMR-GAL4; UAS-hDuox2, an ectopic hDuox2 signal from the posterior region to the morphogenetic furrow in eye discs was detected Immunostaining of eye imaginal discs of both GMR-GAL4; ? (c) and GMR-GAL4; UAS-hDuox2 (d) with only the anti-rabbit IgG Alexa FlourTM 488 antibody showed no detectable signal Flies were reared at 28°C The arrowheads indicate morphogenetic furrows in the eye discs GMR-GAL4/UAS-hDuox2 was suppressed effectively when feeding with a culture medium containing 20% MLs (Fig 4g) The extent of suppression was more increased as the concentration of MLs was increased up to 30% in the medium (Fig 4h) The suppressive effects can be realized by comparing the rough areas of eyes marked by dot circles in Fig These results suggest that the GMR-GAL4/UAShDuox2 fly is a potential model for the screening of new antimetabolic substances glucose-600 -acetate, and naringin, were tested for the ability to suppress the hDuox2-induced rough-eye phenotype The oral administration of quercetin, rutin, kaempherol, and epigallocatechin gallate did not affect the rough-eye phenotype of hDuox2-overexpressing flies (Fig 5b–e) However, flavonoid glycosides such as quercetin-3-O-Dglucoside, quercetin-3-O-glucose-600 -acetate, and naringin could significantly suppress the hDuox2-induced rough-eye phenotype (Fig 5f–h) Antioxidants could suppress the hDuox2-induced rough-eye phenotype Discussion Some phenolic compounds that have antioxidative activities, including quercetin, rutin, kaempherol, epigallocatechin gallate, quercetin-3-O-D-glucoside, quercetin-3-O- In this study, we crossed UAS-hDuox2 with GMR-GAL4 driver to establish a fly line overexpressing hDuox2 The eyes of these flies appeared severely rough compared with 123 96 Mol Cell Biochem (2011) 352:91–98 Fig Detection of ROS in eye imaginal discs of third instar larvae Without substrate CMH2DCFDA, there were no detectable signals in either GMR-GAL4; ? (a) or GMRGAL4; UAS-hDuox2 flies (b) However, by incubation with substrate CM-H2DCFDA, a weak fluorescent signal in control flies (GMR-GAL4; ?) (c) was detected Remarkably, a strong fluorescent signal was detected in hDuox2overexpressing flies (GMRGAL4; UAS-hDuox2) (d) Flies were reared at 25°C The posterior of the discs are on the right The arrowheads indicate morphogenetic furrows in the eye discs Fig Effect of MLs on the hDuox2-induced rough-eye phenotype Scanning electron micrographs of adult compound eyes at 2009 magnification of GMR-GAL4; UAS-DREF (a–d) and GMR-GAL4; UAS-hDuox2 (e–h) GMR-GAL4; UAS-DREF flies fed culture mediums containing 10% (b), 20% (c), or 30% (d) MLs showed no effect on the rough-eye phenotype as compared to flies not fed MLs 123 (a) However, compared to flies not fed MLs (e), the rough-eye phenotype of hDuox2-overexpressing flies was suppressed when fed culture mediums containing 10% (f), 20% (g), or 30% (h) MLs The suppressive effects can be realized by comparing rough areas of the eyes marked by dot circles Flies were reared at 25°C Mol Cell Biochem (2011) 352:91–98 97 Fig Effect of flavonoids on the hDuox2-induced rough-eye phenotype Scanning electron micrographs of adult compound eyes at 2009 magnification of GMR-GAL4; UAS-hDuox2 Untreated GMR-GAL4; UAS-hDuox2 flies showed a severe rough-eye phenotype (a) Treatment with 3% (w/w) quercetin (b), rutin (c), kaempherol (d), and epigallocatechin gallate (e) showed no detectable changes in compound eyes However, treatment with 3% (w/w) quercetin-3-O-D-glucoside (f), quercetin-3-O-glucose-600 -acetate (g), and naringin (h) strongly suppressed the hDuox2-induced rough-eye phenotype Flies were reared at 25°C GMR-GAL4 flies By immunohistochemical analysis with anti-hDuox2 antibody, we proved that these flies expressed an abundant amount of hDuox2 protein in the posterior region to the morphogenetic furrow in eye discs Subsequently, the production of ROS in this region also increased indicating that the hDuox2 enzyme functioned in these flies These results showed that GMR-GAL4/UAShDuox2 flies induced high oxidative stress in the posterior region of the eye discs that led to an easily recognizable rough-eye phenotype in adults We continued to feed this fly line with MLs and several antioxidative flavonoids to evaluate the ability of the line to be used as a model for the screening of antimetabolic substances The results show that, with increasing concentration of MLs in the culture medium, the suppressive effect of MLs became more prominent Similar results were obtained when culture medium containing 3% (w/w) flavonoid glycoside, quercetin-3-O-D-glucoside, quercetin3-O-glucose-600 -acetate, or naringin was fed to the fly However, it is noteworthy that flavonoids without a glycosidic ring, quercetin, rutin, kaempherol, and epigallocatechin gallate exerted no effect on the hDuox2-induced rough-eye phenotype This differential effect could be explained by the differences between flavonoids and their glycosides in intestinal absorption and bioavailability due to chemical structures and water solubility raised from attached glycoside rings that had been described in previous human and rat studies [19–21] Our results might indicate that the absorption of antioxidants in the intestine of D melanogaster is similar to mammalian models such as rats In this study, we used flavonoids as ROS scavengers to evaluate the fly as a screening tool, and, as well, the transgenic fly can be used to screen for NADPH oxidase inhibitors However, our screening system cannot distinguish between ROS scavengers and NADPH oxidase inhibitors Therefore, further ROS scavenging assay using chemicals and/or kinetic assay of ROS generation by NADPH oxidase should be carried out Despite this limitation, Drosophila has many advantages as a screening 123 98 system for new drugs Drosophila is easier to inexpensively rear in the laboratory, has a shorter generation time, and produces much more progenies when compared with mice or rats Therefore, the use of our model is recommended for the first simultaneous screenings of a great number of samples In addition, because of the similar basic metabolic functions and analogous organs between Drosophila and vertebrates [9], the GMR-GAL4/UAShDuox2 fly line constructed in our study is a promising model for the screening of bioavailable novel drugs Acknowledgments We are grateful to Dr Won-Jae Lee, Ewha Woman’s University, South Korea for the UAS-hDuox2 stock References Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I (2004) Increased oxidative stress in obesity and its impact on metabolic syndrome J Clin Invest 114(12):1752–1761 Ando K, Fujita T (2009) Metabolic syndrome and oxidative stress Free Radic Biol Med 47(3):213–218 Houstis N, Rosen ED, Lander ES (2006) Reactive oxygen species have a causal role in multiple forms of insulin resistance Nature 440(7086):944–948 Quinn MT, Gauss KA (2004) Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases J Leukoc Biol 76(4):760–781 Kawahara T, Quinn MT, Lambeth JD (2007) Molecular evolution of the reactive oxygen-generating NADPH oxidase (Nox/Duox) family of enzymes BMC Evol Biol 7:109 Guichard C, Moreau R, Pessayre D, Epperson TK, Krause KH (2008) NOX family NADPH oxidases in liver and in pancreatic islets: a role in the metabolic syndrome and diabetes? 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References Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I (2004) Increased oxidative stress in obesity and its impact on metabolic... our screening system cannot distinguish between ROS scavengers and NADPH oxidase inhibitors Therefore, further ROS scavenging assay using chemicals and/ or kinetic assay of ROS generation by NADPH