Document k9ELyxqqMVjgYpoxo68qk5RxO

Toxicology and Applied Pharmacology xxx (2011) xxxxxx YTAAP-12059; No. of pages: 16; 4C: Contents lists available at ScienceDirect Toxicology and Applied Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p 1 Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene 2 and xylene: Attenuation by quercetin and curcumin 3 Mahendra P. Singh a,1, M. Mishra a,c, A. Sharma a,c, A.K. Shukla a,c, M.K.R. Mudiam b,c, D.K. Patel b,c, 4 K. Ravi Ram a,c, D. Kar Chowdhuri a,c, 5 a Embryotoxicology Section, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India 6 b Analytical Chemistry Section, Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India 7 c Council of Scientific and Industrial Research (CSIR), New Delhi, India 8 article info 9 10 Article history: 11 Received 10 June 2010 12 Revised 11 February 2011 13 Accepted 7 March 2011 14 Available online xxxx 1567 18 Keywords: 19 Monocyclic aromatic hydrocarbons 20 Phytochemicals 21 Midgut 22 Genotoxicity 23 Cytochrome P450 45 44 abstract Monocyclic aromatic hydrocarbons (MAHs) such as benzene, toluene and xylene are being extensively used 24 for various industrial and household purposes. Exposure to these hydrocarbons, occupationally or non- 25 occupationally, is harmful to organisms including human. Several studies tested for toxicity of benzene, 26 toluene and xylene, and interestingly, only a few studies looked into the attenuation. We used Drosophila 27 model to test the genotoxic and apoptotic potential of these compounds and subsequently evaluated the 28 efficiency of two phytochemicals, namely, quercetin and curcumin in attenuating test chemical induced 29 toxicity. We exposed third instar larvae of wild type Drosophila melanogaster (Oregon R+) to 1.0100.0 mM 30 benzene, toluene or xylene, individually, for 12, 24 and 48 h and examined their apoptotic and genotoxic 31 potential. We observed significantly (P b 0.001) increased apoptotic markers and genotoxicity in a 32 concentration- and time-dependent manner in organisms exposed to benzene, toluene or xylene. We also 33 observed significantly (P b 0.001) increased cytochrome P450 activity in larvae exposed to test chemicals and 34 this was significantly reduced in the presence of 3,4-dimethoxyflavone, a known Aryl hydrocarbon receptor 35 (AhR) blocker. Interestingly, we observed a significant reduction in cytochrome P450 activity, GST levels, 36 oxidative stress parameters, genotoxic and apoptotic endpoints when organisms were exposed simulta- 37 neously to test chemical along with quercetin or curcumin. The study further suggests the suitability of D. 38 melanogaster as an alternate animal model for toxicological studies involving benzene, toluene and xylene and 39 its potential in studying the protective role(s) of phytochemicals. 40 2011 Elsevier Inc. All rights reserved. 41 423 46 Introduction 47 Among the organic solvents, benzene, toluene and xylene are 48 extensively used in diverse chemical, industrial and commercial 49 processes (WHO, 1993). Toxicity and/or carcinogenicity of these 50 chemicals due to occupational or non-occupational exposure are of 51 concern. In the environment, these non-oxygenated MAHs are either 52 generated through processing, combustion and evaporation of 53 gasoline or found as constituents of commercial products such as 54 cleaning fluids, paints and glues (Arlien-Suborg, 1992; Indulski et al., 55 1996). In addition, these compounds are inhaled for recreational 56 purposes by young people around the globe (Greer, 1984; Kozel et al., 57 1995; Spiller and Krenzelok, 1997). Corresponding author at: Embryotoxicology Section, Indian Institute of Toxicology Research, Mahatma Gandhi Marg, Lucknow 226 001, Uttar Pradesh, India. Fax: +91 522 2628227/2611547. E-mail address: dkarchowdhuri@rediffmail.com (D.K. Chowdhuri). 1 Present address: Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, USA. Benzene-induced toxicity is related to blood disorders, including 58 bone marrow depression, and some types of cancer (Wan and Winn, 59 2004; Wetmore et al., 2008). Benzene has been shown to induce DNA 60 damage and carcinogenicity both in vitro (in HL60 cells; Kolachana 61 et al., 1993; Zhang et al., 1993) and in vivo (in mice, and in human as 62 measured through the comet assay; Hiraku and Kawanishi, 1996; Pan 63 et al., 2003; Sul et al., 2005). Recent studies from our laboratory 64 showed induction of heat shock genes (hsp70, hsp83, hsp60 and 65 hsp26), oxidative stress markers and increased ROS generation in 66 Drosophila melanogaster, exposed to benzene, toluene or xylene, 67 individually, or their mixtures, indicating the potential of these 68 chemicals to produce cellular stress (Singh et al., 2009; Singh et al., 69 2010). A few studies also suggest the DNA damaging potential of 70 toluene and xylene (Al-Ghamdi et al., 2004; Pariselli et al., 2009). 71 Addition of molecules having ROS quenching efficacy suggested that 72 mechanism underlying the DNA damage and carcinogenicity is likely 73 through ROS generation (Bellion et al., 2009; Messner et al., 2009; 74 Becatti et al., 2010). 75 Programmed cell death or apoptosis is a self-destruction of a given 76 cell due to irreparable damage. Perusal of literature shows that 77 0041-008X/$ see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2011.03.006 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 2 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 78 79 Q1 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 benzene, toluene and xylene induced apoptosis and genotoxicity in vitro models and also in vivo (Smith, 1996; Ross, 2000; Snyder, 2000; Nakai et al., 2003; Al-Ghamdi et al., 2004; Wan and Winn, 2004; 2007; Wetmore et al., 2008). Although several studies have been carried out to test the toxicity of benzene, toluene and xylene, only limited information is available on the attenuation of toxic insults of these test chemicals in the exposed organisms (Emara and El-Bahrawy, 2008). To address this, we used D. melanogaster, as an in vivo model, as it offers as an excellent alternative animal model [in accordance to European Centre for the Validation of Alternative Methods (ECVAM)] (Festing et al., 1998). Post genomic sequencing, Caenorhabditis elegans and Drosophila generated much interest to toxicologists as these show functional conservation of majority of genes present in higher mammals. Further, the availability of state-of-the-art molecular tools coupled with well-defined genetics and developmental biology places this organism better for obtaining mechanistic insights. Moreover, from a toxicological/pharmacological perspective, fly and higher mammals were shown to have similar doseresponse relationship with four monofunctional alkylating agents (Siddique et al., 2005a). Thus, we believe that laboratory-based experimental evidences using this model is useful in generating information that could be of value for their efficient extrapolation to higher organisms. Therefore, we first studied apoptosis and genotoxicity in Drosophila, induced by benzene, toluene and xylene. Subsequently, we used this model to study the cytoprotective effect(s) of two phytochemicals having anti-oxidant properties, quercetin (QC) and curcumin (CUR) (Kottke, 1998; Lodha and Bagga, 2000), against benzene-, tolueneand xylene-induced cellular toxicity in exposed organisms.We show here that QC and CUR do have the potential to protect against the benzene-, toluene- and xylene-induced toxicity in vivo. 108 Material and methods 109 Flies and maintenance 110 All experiments were performed using wild type (Oregon R+) 111 strain of D. melanogaster. The flies and larvae were reared at 23 1 C 112 on standard Drosophila food containing agaragar, maize powder, 113 sugar, yeast, nepagin (methyl-p-hydroxy benzoate salt; HiMedia, 114 India) and propionic acid. Additional yeast suspension was provided 115 for their healthy growth. 116 Chemicals used for treatment 117 All chemicals, reagents and kits were procured from Sigma, MO, 118 USA except otherwise stated. Analytical grade benzene (99.7% from 119 Ranbaxy Pvt. Ltd, India), toluene (99.5%, SRL Pvt Ltd, India), xylene 120 (mixture of o, m and p, 99.8%, SRL Pvt Ltd, India) and two 121 phytochemicals namely, quercetin (3,5,7,3,4-pentahydroxyflavone, 122 5,7,3,4-tetrahydroxyflavonol; QC) and curcumin [active ingredient 123 of the rhizome of the plant turmeric (Curcuma longa Linn); CUR] were 124 used in the study. Dimethyl sulfoxide (DMSO) (SRL Pvt Ltd, Mumbai, 125 India) was used as a solvent and final concentration of DMSO in food 126 was restricted to 0.3% (based on a previous study from this laboratory 127 Nazir et al., 2003). 128 Treatment schedule 129 Four different concentrations (1.0, 10.0, 50.0 and 100.0 mM), each 130 corresponding to different fractions of the LC50 (48 h) of benzene, 131 toluene or xylene were used. Third instar larvae were grown on 132 standard Drosophila diet, contaminated with or without different 133 concentrations of the three test chemicals in triplicates for 248 h. For 134 genotoxicity studies by alkaline and neutral Comet assay, we used 135 100.0 mM of benzene, toluene and xylene. Ethyl methanesulfonate 136 (EMS) (1.0 mM) and -radiation (40.0 Gy) using Co60 source were as used as positive controls for alkaline and neutral Comet assay, 137 respectively. In another set of experiments, 100.0 M QC or CUR 138 (based on previous studies: Balasubramanyam et al., 2003; Gupta 139 et al., 2007) was added to the food individually, or along with test 140 chemicals for exposing larvae (24 and 48 h). 141 Quantitative determination of benzene, toluene and xylene 142 The chemical burden in the larvae exposed to test chemicals was 143 quantified as described previously (Singh et al., 2009). Briefly, third 144 instar larvae were exposed to 100.0 mM benzene, toluene or xylene in 145 food with/without phytochemicals for 48 h. These larvae were 146 homogenized in deionized water in a headspace vial to obtain 147 2.0 ml homogenate (10% w/v). The vials were heated at 65 C for 148 30 min with magnetic agitation. After the equilibrium, septa were 149 pierced with SPME needle and the SPME polydimethyl siloxane 150 (PDMS) fiber was exposed to headspace for 15 min to affect the 151 adsorption of the test chemicals in the sample. SPME fiber was 152 collected and inserted directly into the injection port of Perkin Elmer 153 gas chromatography (USA) at 200 C. Total time of this chromato- 154 graphic analysis was 27 min. Blank analysis was carried out to avoid 155 any carry over phenomena and/or external contamination between 156 analyses of samples (Alegretti et al., 2004). 157 Quantitative estimation of quercetin and curcumin levels 158 We have used 125 mg of larvae exposed to QC or CUR for extraction. 159 Larvae were homogenized in 1.0 ml of 1 PBS. QC was extracted using 160 500.0 l of DMSOmethanol (1:4 v/v) following the method of Schiborr 161 et al. (2010), CUR was extracted using 95% ethyl acetate and 5% 162 methanol (v/v) and the extract was dried at 40 C and resuspended in 163 200.0-l mobile phase (49% acetonitrile, 20% methanol, and 1% acetic 164 acid (Jones et al., 1998). Subsequently, these extracts were analyzed by 165 HPLC system (Water Milford, MA, USA.) containing a reverse phase C-18 166 ODS analytical column (5 M particle size). The levels of QC were 167 estimated by injecting 20.0 l of sample (mobile phase containing 0.5% 168 aqueous solution of orthophosphoric acid and methanol) and separating 169 with a flow rate of 1 ml min-1, for a run time of 15 min, with photoarray 170 detector (PDA) at 375 nm. For CUR estimation, 20.0 l of the extract was 171 separated with a flow rate of 1.5 ml min-1, for a run time of 5 min, with 172 PDA detector at 420 nm. A blank or control (extract from larvae exposed 173 to control food) injection and the QC or CUR standards were run 174 immediately before each group of samples. 175 Measurement of oxidative stress parameters 176 In this study, we measured different oxidative stress parameters 177 such as Reactive Oxygen Species (ROS), superoxide dismutase (SOD), 178 catalase (CAT) and lipid peroxidation (LPO) measured as Malondial- 179 dehyde content (MDA) essentially following methods described 180 previously (Gupta et al., 2007; Gupta et al., 2010). 181 Preparation of microsomes 182 The method for preparation of microsomes described previously by 183 Johri et al. (2006) was followed with minor modifications. Control and 184 treated larvae were homogenized in ice-cold homogenization buffer 185 (0.25 M potassium phosphate buffer containing 0.15 M KCl, 0.25 mM 186 phenylmethanesulfonylfluoride (PMSF), 0.01 M ethylenediaminete- 187 traacetic acid (EDTA) and 0.1 mM Dithiothreitol (DTT); pH 7.25) to 188 obtain 10% homogenate. The homogenate was centrifuged at 9000 g 189 for 30 min (at 4 C) to obtain the supernatant, which in turn was 190 centrifuged at 105,000 g for 60 min to sediment microsomes. The 191 pellets were resuspended in microsome dilution buffer (0.1 M potas- 192 sium phosphate buffer, pH 7.25, 20% (v/v) glycerol, 0.25 mM PMSF, 193 0.01 M EDTA and 0.1 M DTT) and stored at -80 C until further use. 194 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 3 195 Ethoxyresorufin-O-deethylase (EROD) and methoxyresorfin-O-deethylase 196 (MROD) assay for estimation of cytochrome P450 activity. The activities 197 of EROD and MROD in the isolated Drosophila larval microsomes were 198 determined following Johri et al. (2006), with minor modifications. 199 The reaction mixture consisted of 1.0 ml of 0.1 M PBS pH 7.8, 5.0 l 200 1.0 mM ethoxy or methoxy resorufin and 25.0 l microsomal fraction. 201 The reaction was initiated by the addition of 1.0 ml 1.0 mM NADPH 202 and the mixture was incubated at 37 C for 10 min. Reaction was 203 terminated by adding 2.0 ml of methanol and the mixtures were 204 centrifuged at 2000 g for 7 min. Levels of resorufin in the supernatant 205 were measured using a Perkin Elmer LS 55 Luminescence Spectrometer 206 at excitation wavelength of 550 nm and emission wavelength of 207 585 nm. 208 209 Q2 210 211 212 213 214 215 216 217 218 219 220 221 222 Inhibition of aryl hydrocarbon receptor (AhR). The role of AhR or its homolog was examined using a potent AhR inhibitor 3,4dimethoxyflavone (DMF) (Lin et al., 2006). Larvae were exposed to food containing 100.0 M DMF together with 100.0 mM of benzene, toluene or xylene as mentioned in Treatment schedule section and subsequently, EROD activity was measured as in Ethoxyresorufin-Odeethylase (EROD) and methoxyresorfin-O-deethylase (MROD) assay for estimation of cytochrome P450 activity section. We have used larvae exposed to 200.0 ng/ml Tetrachlorodibenzo-p-dioxin (TCDD) (Cespedes et al., 2010) together with 100.0 M DMF as positive control. TCDD was used as a positive control because this is also a halogenated aromatic hydrocarbon and is known to induce cytochrome P450 1AI (CYP1A1) through AhR (Cespedes et al., 2010). Larvae exposed to 100.0 M DMF alone was used as an additional control. 223 Glutathione S-transferase (GST, EC 2.5.1.18) 224 Q3 225 226 227 228 229 230 231 Glutathione S-transferase (GST) activity was determined following Habig et al. (1974) with minor modifications. The reaction mixture consisted of 0.2 M sodium phosphate buffer, 75.0 l larval homogenate, reduced glutathione (1.0 mM) and 1-chloro 2, 4 dinitrobenzene (CDNB) (5.0 mM). An increase in absorbance (340 nm) was measured for 3 min at 30-s intervals and the enzyme activity was calculated as nmol CDNB reduced/min/mg larval protein using molar extinction coefficient of 6.25 103 M- 1 cm- 1. 232 Trypan blue dye exclusion assay 233 Tissue damage in the test chemical exposed organisms was 234 examined by trypan blue exclusion assay as described earlier (Krebs 235 and Feder, 1997). In brief, internal tissues of control and treated larvae 236 (5060/group), explanted in PSS were washed once in 50.0 mM 237 phosphate buffered saline (PBS), pH 7.4 (Dulbecco's, HiMedia Pvt Ltd., 238 Mumbai, India). They were then immersed in trypan blue stain 239 (0.2 mg/ml in 50.0 mM PBS, pH 7.4) and shaken gently for 30 min at 240 24 1 C. After staining, tissues were washed thrice in wash buffer 241 (0.1 M PBS pH 7.4) and immediately visualized and scored larvae for 242 trypan blue staining. Assay of apoptosis 253 All apoptotic end points were measured in single cells prepared 254 from midgut tissues of control and treated organisms except for 255 those of terminal deoxynucleotidyl transferase mediated dUTP nick 256 end labeling (TUNEL) assay. For TUNEL assay, we used whole midgut 257 tissues and for measuring caspase activity, we used 10% tissue 258 homogenate, to match the published standard protocols. 259 Flow cytometric determination of cellular Rpr, Hid and Grim (initiator of 260 apoptosis) levels. Previously described flow cytometric detection 261 method was followed with minor modification (Wechsler-Reya 262 et al., 1998). Cells after thorough washing in 0.1 M PBS (pH 7.4) 263 were fixed in PBS containing 0.25% paraformaldehyde for 1 h at 4 C 264 and then permeabilized by washing twice in PBS with 0.1% Triton X- 265 100 for 10 min at 24 1 C. Subsequently, cells were rinsed with PBS 266 and incubated with primary antibodies against Rpr, Hid or Grim (anti- 267 goat polyclonal IgG, 1:50 in PBS containing 2% BSA; Santa Cruz, CA, 268 USA) for 1 h at 4 C. This was followed by incubation of cells with FITC 269 conjugated rabbit anti-goat IgG secondary antibody (1:100 in 0.1 M 270 PBS, pH 7.4 containing 2% BSA) for 1 h at 4 C. The stained cells were 271 analyzed on Becton Dickinson flow cytometer (BD, NJ, USA) using Cell 272 quest software (Mac OS 8.6). For each sample, 10,000 events were 273 counted and results were expressed in terms of the percentage cells 274 expressing particular protein. 275 Phosphatidylserine (PS) externalization assay (Annexin V-FITC staining). 276 Apoptotic cells were detected using Annexin V-FITC apoptosis 277 detection kit essentially following the manufacturer's instructions. 278 Briefly, media binding reagent and Annexin V-FITC were mixed with 279 approximately 5 105 cells and incubated at 24 1 C for 15 min. 280 After removing the media, the cells were re-suspended in cold 1 281 binding buffer and stained with PI. Ten thousand events were 282 acquired per treatment group using Becton Dickinson flow cytometer 283 and data were analyzed with Cell Quest software (Mac OS 8.6). The 284 FITC signal was detected by FL1 (FITC detector) at 518 nm and Pl was 285 detected by FL2 (phycoerythrin fluorescence detector) at 620 nm. The 286 log of Annexin V-FITC and Pl fluorescence was displayed on the X- and 287 Y-axis of the data report respectively. 288 Determination of mitochondrial membrane potential (m). Depolari- 289 zation of mitochondrial membrane was analyzed following a previously 290 described method with minor modification (Vayssier-Taussat et al., 291 2001). A fluorochrome 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl 292 benzimidazolyl carbocyanine iodide (JC-1) was used for this purpose. 293 The mitochondrial membrane depolarization is associated with a shift in 294 JC-1 fluorescence emission from red to green. Cells of control and treated 295 organisms were suspended in Schneider's Drosophila medium con- 296 taining 10.0 M JC-1 (prepared in DMSO) for 30 min at 24 C. Cells were 297 then washed with 0.1 M PBS (pH 7.4) twice and finally re-suspended in 298 500.0 l PBS (pH 7.4) for FACScan analysis. Ten thousand events were 299 counted per sample in acquisition and analysis was performed using 300 cell quest software (Mac OS 8.6). The results were expressed as the 301 percentage of cells with disrupted mitochondrial membranes. 302 243 Single cell preparation 244 Midgut tissues of 15 larvae from control and treated groups were 245 incubated in collagenase (0.5 mg/ml of 1.0 M PBS pH 7.4) for 15 min 246 at 24 1 C. The dissociated cells were then passed through 80 m 247 nylon mesh to get rid of clumping. Collagenase was removed by 248 washing the cell suspension with 0.1 M PBS (pH 7.4) for at least three 249 times with gentle shaking. Viability of cells was checked by trypan 250 blue staining before the start of the experiment (Phillips, 1973). These 251 cells were processed for different end point measurements as 252 described below. Assay of DEVD- and IETD-ase activities. The assay is based on spec- 303 trophotometric detection of the chromophore p-nitroanilide (pNA) 304 obtained after specific action of different cysteine proteases involved 305 in apoptotic pathways on tetrapeptide substrates, respectively. The 306 assay was performed essentially following the manufacturer's protocol 307 (Bio Vision, Inc., CA, USA). Supernatant from the 10% tissue homogenate 308 was mixed with chilled cell lysis buffer, 2 reaction buffer (containing 309 10.0 mM dithiothreitol) and 200 M substrates. The reaction mixture 310 was incubated at 37 C for 1.5 h and absorbance of the colored product 311 was measured at 405 nm on a Cintra 20 ultraviolet spectrophotometer 312 (GBC Scientific Equipment, Melbourne, Australia). 313 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 4 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 314 Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling 315 (TUNEL) assay. TUNEL assay was performed using "In situ cell death 316 detection kit" essentially following the manufacturer's protocol 317 (Roche Molecular Biochemicals, Mannheim, Germany). Midgut 318 tissues of control and treated larvae were fixed in freshly prepared 319 2.5% glutaraldehyde and permeabilized in PBST followed by washing 320 in PBS. They were incubated in primary TUNEL mixture for 1 h at 37 C 321 and then washed with PBS for 30 min at 24 C (6 changes of 5 min 322 each) followed by incubation in converter alkaline phosphate (AP) 323 solution at 37 C. After washing, tissues were incubated in the 324 substrate solution containing nitro blue tetrazolium (NBT) and 5325 bromo-4-chloro-3-indolyl phosphate (BCIP), specific for AP, for 326 15 min in dark at 24 C. The tissues were then washed thoroughly 327 with PBS, mounted on clean glass slides using 50% glycerol and 328 coverglass was placed. The edges of the coverglass were sealed with 329 DPX and the tissues were scored for TUNEL positive cells under Leitz 330 orthoplan light microscope (Wetzlar, Germany). One hundred fifty 331 cells were examined from each group by randomized manual 332 counting (25 cells/gut tissue and 2 gut tissues/experiment and 3 333 experiments/group/time point). 334 Poly (ADP-ribose) polymerase (PARP) cleavage. We performed PARP 335 antibody staining in cells of control and treated organisms using "Anti336 PARP Cleavage Site Specific Antibody (CSSA) Assay kit" (Invitrogen, 337 USA) essentially following the manufacturer's protocol. The FITC stained 338 cells were analyzed on a Becton Dickinson flow cytometer using Cell 339 quest software (Mac OS 8.6). For each sample, 10,000 events were 340 counted and results were expressed in terms of percent cells expressing 341 PARP. 342 Evaluation of DNA damage by Comet assay 343 Single cell preparation and viability test were performed as 344 described in Single cell preparation section. Care was taken to 345 perform all the steps (from single cell preparation to staining) 346 under dim light to avoid any light-induced DNA damage. 347 Slides preparation. A previously described method for preparation of 348 slides was followed (Tice et al., 2000) wherein frosted slides (with 349 1.5 cm frosted end) were used. The slides were rinsed in methanol 350 and flame dried. The slides were then coated on non-frosted end with 351 1.0% normal melting agarose (NMA), prepared in milliQ water and 352 kept at 60 C) up to two-third of their length by dipping into molten 353 NMA and were allowed to dry for 1 h at room temperature (24 C). 354 Evaluation of DNA damage. For the entire group, slides were prepared 355 in duplicate according to the method described earlier (Siddique et al., 356 2008). All experiments were repeated three times. The cell suspension 357 (80.0 l) was mixed with 80.0 l of 1.5% low melting point agarose 358 (LMA; prepared in Ca2+ Mg2+ free PBS; final concentration 0.75%, 35 359 40 C). For each slide, 75.0 l of the above mixture was immediately 360 layered on a base slide. Cover slip was immediately placed over the 361 second layer. The slide was then placed on a chilled plate for 10 min to 362 allow solidification of agarose. This step was repeated again to place 363 another layer of LMA 0.75%. Finally, the cover slip was removed and 364 the slide was immersed for 2 h in freshly prepared, chilled lysing 365 solution (2.5 M NaCl, 100.0 mM EDTA, 10.0 mM Tris and 1.0% Triton 366 X-100, pH 10). After lysis, the slides were subjected to neutral and 367 alkaline gel electrophoresis as follows. 368 Electrophoresis for both alkaline and neutral Comet assay. For alkaline 369 comet assay, slides were placed in chilled electrophoresis buffer 370 (1.0 mM Na2EDTA and 300.0 mM NaOH, pH N 13) for 10 min for DNA 371 unwinding. Subsequently, electrophoresis was conducted in chilled 372 electrophoresis buffer (1.0 mM Na2EDTA and 300.0 mM NaOH, 373 pH N 13) for 15 min at 0.7 V/cm (300 mA/25 V) at 4 C. The slides were then washed three times with 0.4 M Tris buffer (pH 7.5) at 4 C 374 to neutralize excess alkali and then for staining as described in 375 Staining section. 376 We followed a previously described method for neutral Comet 377 assay for the detection of double-strand breaks (Fracasso et al., 2009). 378 The slides after lysis were kept in electrophoresis buffer (300.0 mM 379 CH3COONa, 100.0 mM TrisHCl, pH 8.5) for 1 h and then transferred 380 to horizontal electrophoresis unit (Life Technologies, Gaithersburg, 381 MD, USA) containing fresh buffer. Electrophoresis was carried out at 382 constant current of 60 mA for 1 h at 4 C and slides were stained 383 subsequently as described in the following section. 384 Staining. The slides were stained with ethidium bromide (20.0 g/ml; 385 75 l per slide) for 10 min in dark. After staining, the slides were dipped 386 once in chilled distilled water to remove the excess stain and cover slips 387 were placed over the slides. 388 Slide scoring. The slides were examined on a Leica DMLB microscope 389 with fluorescence attachment (Leica Germany). The images were 390 transferred to a computer through a charge coupled device (CCD) 391 camera and analyzed using Komet 5.0 software (Kinetic Imaging, 392 Liverpool, UK). One hundred fifty cells from each group (25 cells per 393 slide with two slides/experimental group in triplicates) were examined. 394 The tail length (TL) (m), tail DNA (%) (TD) and tail moment (TM) 395 (arbitrary units) were used as indicators of DNA damage as described 396 earlier (Olive et al., 1992). 397 Statistical analysis 398 Different parameters were analyzed in control and exposed third 399 instar larvae of D. melanogaster (Oregon R+). Analysis of variance 400 (ANOVA) was carried out to find out the significant differences in 401 means considering each end point as dependent variable (with or 402 without QC and CUR) and treatment (benzene, toluene and xylene); 403 concentration (1.0, 10.0, 50.0 and 100.0 mM) and duration of 404 exposure (2, 4, 6, 12, 24 and 48 h) as independent variables. Prior to 405 applying the ANOVA, homogeneity of variance was ascertained using 406 Levene's test of equality error variance. Two-way analysis of variance 407 was carried out for ROS generation, apoptotic and genotoxic markers 408 as dependent variables (Zar, 1984). SPSS 14.0 (SPSS, Mapinfo Max, 409 USA) was used to analysis the data. To identify the degree of 410 relationship between cytochrome P450 activity and oxidative stress 411 parameters analyzed in the present study with test chemical alone or 412 in combination with QC or CUR, simple linear correlation (Pearson r) 413 statistics followed significance analysis of correlation coefficient were 414 performed. 415 Results 416 We did not observe any significantly altered activities or levels of 417 all the tested parameters at 1.0 mM concentration of the test 418 chemicals throughout the treatment period and till 6 h at 10.0 mM 419 concentration of these test chemicals (data for 1.0 mM test chemicals 420 not shown). Hence, we only included data of 6, 12, 24 and 48 h 421 exposure of 10.0100.0 mM benzene, toluene and xylene. Unlike 422 apoptotic markers, Comet assay parameters did not show any 423 remarkable damage till 12 h and hence, we presented only 24 and 424 48 h data in the study. Further, data obtained from parallel control 425 (DMSO) were similar to those of controls and hence, we used the data 426 from controls for comparison. 427 Detection of chemicals in larvae of D. melanogaster 428 To ensure the larval uptake of the test chemical and its uptake is 429 not altered in the presence of QC or CUR or vice versa, we estimated 430 the chemical load in exposed organisms. We detected benzene, 431 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 5 432 toluene or xylene in larvae exposed to test chemical alone at levels 433 similar (P N 0.05) to those in combination with QC or CUR. We also 434 detected QC or CUR in organisms exposed to QC or CUR alone at levels 435 similar (P N 0.05) to those exposed to QC or CUR along with test 436 chemical (please see Supplemental Figs. S1 and S2). 437 Moderate trypan blue staining in tissues of Oregon R+ larvae exposed to 438 benzene, toluene or xylene 439 To determine if exposure to benzene, toluene or xylene results in 440 tissue damage, we analyzed trypan blue staining in tissues of 441 D. melanogaster larvae exposed to 100.0 mM benzene (Fig. 1B), 442 toluene (Fig. 1C) and xylene (Fig. 1D) for 48 h (Fig. 1). Of the larvae 443 exposed to benzene, 94% of them showed blue staining in their 444 midgut, salivary gland, gastric caeca and brain ganglia while 86% or 445 84% of the larvae exposed to toluene or xylene exhibited a pale to 446 moderate blue staining in the above mentioned tissues respectively. 447 Increased levels of Rpr, Hid and Grim (homologues of mammalian SMAC/ 448 DIABLO) in benzene-, toluene- or xylene-exposed D. melanogaster larvae 449 To examine whether apoptosis is indeed induced by the test 450 chemical, we analyzed the levels of pro-apoptotic proteins, Rpr, Hid 451 and Grim (Hay and Guo, 2006). Figs. 24 show the level of Rpr, Hid 452 and Grim in control or exposed larvae. Larvae exposed to 10.0 453 100.0 mM benzene, toluene or xylene for 24 or 48 h showed a 454 significant concentration- and time-dependent increase in Rpr 455 (Fig. 2AF), Hid (Fig. 3AF) and Grim (Fig. 4AF) levels, when 456 compared to control. Interestingly, at most concentrations, organisms 457 exposed to benzene showed a significantly higher level of Rpr, Hid and 458 Grim after 24 h whereas those exposed to similar concentration(s) 459 of toluene or xylene showed significantly increased levels of these 460 proteins only after 48 h. However, no significant difference between 461 Rpr, Hid and Grim levels was observed in toluene- or xylene-exposed 462 organisms at all the tested concentrations. The toxicity trend observed 463 for Drosophila larvae exposed to 100.0 mM benzene, toluene and 464 xylene for 48 h was as follows: benzene N toluene xylene (Figs. 24). 465 We did not observe a significant increase in levels of Rpr, Hid or 466 Grim in larvae exposed to 10.050.0 mM benzene, toluene or xylene 467 for 12 h, when compared to their controls (see also Supplemental 468 Fig. S3AC). 469 Externalization of phosphatidylserine (PS) in D. melanogaster larvae 470 following treatment of benzene, toluene or xylene 471 We examined the initiation of apoptosis in benzene-, toluene- or 472 xylene-exposed organism by quantifying the Annexin V positive cells. Fig. 2. Levels of Rpr (AF) in cells from midgut tissues of third instar larvae of D. melanogaster (Oregon R+) exposed to control food, DMSO, benzene, toluene or xylene. Panel A represents levels of Rpr in larvae exposed to 100.0 mM benzene, toluene or xylene for 6, 12, 24 or 48 h. Panel B shows levels of Rpr in larvae exposed to 10.0100 mM of benzene, toluene or xylene for 48 h. Flow cytometric panels depict level of Rpr in control (C), 100.0 mM benzene- (D), toluene- (E) and xylene- (F) treated Drosophila larvae after 48 h. Data represent mean SD of three identical experiments made in triplicates and significance in comparison to control is ascribed as *P b 0.01, **P b 0.001. B10, B50 and B100 = 10.0, 50.0 and 100.0 mM benzene; similarly T10, T50 and T100 = 10.0, 50.0 and 100.0 mM toluene and X10, X50 and X100 = 10.0, 50.0 and 100.0 mM xylene in this figure and the remaining figures in this paper. Q8 Fig. 1. Trypan blue staining in the internal tissues isolated from third instar larvae of D. melanogaster (Oregon R+) control (A) and in third instar larvae exposed to 100.0 mM benzene (B), toluene (C) and xylene (D) after 48 h. bg = brain ganglia, sg = salivary gland, pv = proventriculus, gc = gastric caeca, mg = midgut, hg = hind gut. Bar represents 100 m. Fig. 5 shows the Annexin V (AV) positive early apoptotic cells in the 473 midgut tissues of control and treated larvae. Larvae exposed to 10.0 474 100.0 mM benzene, toluene or xylene exhibited a concentration- and 475 time-dependent significant increase (P b 0.01) in AV positive cells in 476 comparison to control (Fig. 5AF). When compared to control, larvae 477 exposed to 10.0 mM benzene showed significantly higher AV positive 478 cells after 24 h whereas organisms exposed to the same concentration 479 of toluene or xylene exhibited a significantly higher AV positive cells 480 only after 48 h. Similar trend was observed even in organisms 481 exposed to higher concentrations (50.0 or 100.0 mM) of benzene, 482 toluene or xylene when compared to control: significant increase in 483 the AV positive cells by 12 h in case of 100.0 mM benzene-exposed 484 organisms (40.0% increase) and significant increase of the same in 485 toluene- or xylene-exposed organisms was only after 24 h (43.0% and 486 30.0% increase respectively) (also see Supplemental Fig. S4). 487 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 6 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx Fig. 3. Levels of Hid (AF) in cells from midgut tissues of third instar larvae of D. melanogaster (Oregon R+) exposed to control food, DMSO, benzene, toluene or Fig. 4. Levels of Grim (AF) in cells from midgut tissues of third instar larvae of xylene. Panel A represents levels of Hid in larvae exposed to 100.0 mM benzene, D. melanogaster (Oregon R+) exposed to control food, DMSO, benzene, toluene or xylene. toluene or xylene for 6, 12, 24 or 48 h. Panel B shows levels of Hid in larvae exposed to Panel A represents levels of Grim in larvae exposed to 100.0 mM benzene, toluene or 10.0100 mM of benzene, toluene or xylene for 48 h. Flow cytometric panels depict xylene for 6, 12, 24 or 48 h. Panel B shows levels of Grim in larvae exposed to 10.0100 mM level of Hid in control (C), 100.0 mM benzene- (D), toluene- (E) and xylene- (F) treated of benzene, toluene or xylene for 48 h. Flow cytometric panels depict level of Grim in Drosophila larvae after 48 h. Data represent mean SD of three identical experiments control (C), 100.0 mM benzene- (D), toluene- (E) and xylene- (F) treated Drosophila larvae made in triplicates and significance in comparison to control is ascribed as *P b 0.01, after 48 h. Data represent mean SD of three identical experiments made in triplicates and Q9 **P b 0.001. significance in comparison to control is ascribed as *P b 0.01, **P b 0.001. Q10 488 Depolarization of mitochondrial membrane potential (m) in exposed 489 third instar D. melanogaster larvae 490 Change in mitochondrial membrane potential is required for 491 caspase activation (Hay and Guo, 2006). Larvae exposed to 10.0 492 100.0 mM benzene, toluene or xylene showed a concentration- and 493 time-dependent significant (P b 0.001) depolarization of mitochon494 drial membrane (m) when compared to their respective controls 495 (Fig. 6AF). However, Drosophila larvae exposed to 10.0 mM benzene 496 exhibited significant m after 24 h, whereas toluene- or xylene497 exposed organisms exhibited significant m only after 48 h, when 498 compared to controls (also see Supplemental Fig. S5 for details). 499 Organisms exposed to (50.0 or 100.0 mM) benzene, toluene or xylene 500 showed significant m after 24 h when compared to control 501 (Fig. 6AF). In all these cases, we did not find any significant 502 difference between toluene- and xylene-treated groups. Increased IETDase, DEVDase and Poly (ADP-ribose) polymerase (PARP) 503 cleavage in benzene-, toluene- or xylene-exposed third instar 504 D. melanogaster larvae 505 To measure the activation of caspases, we have carried out IETDase 506 and DEVDase assays. The relative intensities of the cleaved chromo- 507 phore, p-nitroanilide, obtained after specific cleavage by IETDase and 508 DEVDase in the larvae exposed to benzene, toluene and xylene are 509 shown in Fig. 7. We observed a concentration- and time-dependent 510 up-regulation (P b 0.001) of IETDase activity in organisms exposed to 511 10.0100.0 mM test chemicals (Fig. 7A). Larvae exposed to 10.0 mM 512 benzene showed a significantly increased IETDase activity after 24 h 513 (2.0-fold increase as compared to control) and while those exposed to 514 toluene and xylene exhibited significantly higher activities only after 515 48 h (1.8- and 1.6-fold increase as compared to control). Larvae 516 exposed to 50.0 mM of test chemicals showed significantly increased 517 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 7 Q11 Fig. 5. Annexin V positive cells (AF) in cells from midgut tissues of control, DMSO-, benzene-, toluene- or xylene-exposed third instar larvae of D. melanogaster (Oregon R+). Panel A represents percentage of Annexin positive cells from midguts of larvae exposed to 100.0 mM benzene, toluene or xylene for 6, 12, 24 or 48 h. Percent Annexin positive cells from midguts of larvae exposed to various concentrations of benzene, toluene or xylene for 48 h is represented in Panel B. Flow cytometric panels depict cells in early apoptotic stage (Annexin V positive cells, FITC quadrant), late apoptotic stage (Annexin V and propidium iodide (PI) positive cells, FITC-PI quadrant) and necrotic state (PI positive, PI quadrant) from midgut tissues of control (C), 100.0 mM benzene- (D), toluene- and (E) xylene- (F) exposed organisms after 48 h. Data represent mean SD of three identical experiments made in triplicate and significance in comparison to control is ascribed as *P b 0.01, **P b 0.001. 518 IETDase activity after 12 and 24 h, respectively, in benzene-, toluene519 or xylene-exposed groups as compared to their respective controls. A 520 maximum (P b 0.001) 4.0-, 3.3- or 3.1-fold increase in IETDase activity 521 was observed in 100.0 mM benzene-, toluene- or xylene-exposed 522 larvae after 48 h, respectively (Fig. 7A). The DEVDase activity in the 523 test chemicals exposed organisms was similar to that of IETDase 524 activity (Fig. 7B). We observed significantly increased number of 525 PARP-FITC positive cells in organisms exposed to 100.0 mM benzene, 526 toluene or xylene for 24 and 48 h (4.3, 3.9- and 3.6-fold increase after 527 48 h respectively), when compared to their respective controls 528 (Fig. 8AE). The differences in the number of PARP-FITC positive 529 cells observed among benzene-, toluene- or xylene-exposed organ530 isms for 24 and 48 h were found to be non-significant. 100.0 mM test chemicals treated larvae after 24 and 48 h (Fig. 9). After 535 24 h, we observed significant increase in TUNEL positive cells only in 536 Drosophila larvae exposed to benzene (100.0 mM; 3.3-fold) when 537 compared to control. Organisms exposed to similar concentrations of 538 toluene or xylene showed significantly more TUNEL positive cells 539 than controls only after 48 h (7.7- and 7.9-fold increase). However, 540 even at this time point, the number of TUNEL positive cells in 541 organisms exposed to 100.0 mM of toluene or xylene for 24 and 48 h 542 was significantly lower than that in organisms exposed to benzene 543 (Fig. 9AC). 544 Increased DNA damage in the midgut cells of D. melanogaster larvae 545 exposed to benzene, toluene or xylene 546 531 Increased DNA nicking in test chemicals exposed third instar larvae of 532 D. melanogaster 533 To examine test chemical mediated DNA nicking, a key feature of 534 apoptosis, we performed TUNEL assay in midgut cells of control and To determine the extent of DNA damage, we analyzed Comet 547 parameters in organisms exposed to the test chemical. We observed 548 9598% cell viability in controls, and also in 100.0 mM benzene-, 549 toluene- or xylene-exposed groups. Larvae exposed to 1.0 mM EMS 550 (used as a positive control) showed a significant increase (P b 0.001) 551 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 8 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx Q6 Q12 Fig. 6. Alterations in mitochondrial membrane potential (m) (AF) in cells from midgut tissues of control, DMSO-, benzene-, toluene- or xylene-exposed third instar larvae of D. melanogaster (Oregon R+). Panel A represents fold increase in mitochondrial membrane depolarization in cells from midguts of larvae exposed to 100.0 mM benzene, toluene or xylene for 6, 12, 24 or 48 h. Fold change in mitochondrial membrane depolarization in cells from midguts of larvae exposed to various concentrations of benzene, toluene or xylene for 48 h is represented in Panel B. Flow cytometric panels represent alterations in m in (C) control (D) 100.0 mM benzene- (E) toluene- and (F) xylene-exposed organisms after 48 h. Data represent mean SD of three identical experiments made in triplicate and significance in comparison to control is ascribed as *P b 0.01, **P b 0.001. 552 in DNA migration (TL, % TD and TM) in their midgut cells in alkaline 553 Comet assay while those given -radiation (40.0 Gy) showed 554 significantly increased migration of DNA after neutral assay. 555 Similarly, in organisms exposed to test chemicals, we observed a 556 significant increase in DNA migration after 24 and 48 h (in the order 557 of benzene N toluene xylene), when compared to control, in both 558 alkaline and neutral Comet assays (Table 1). Interestingly, we 559 observed significantly higher (P b 0.01) migration of DNA (increased 560 TL, % TD and TM) in cells of the exposed organisms subjected to 561 alkaline Comet assay when compared to those of neutral Comet 562 assay. Increased EROD and MROD activity in larvae exposed to benzene, 563 toluene or xylene 564 Cytochrome P450 1A (CYP1A) subfamily is highly conserved and is 565 known to play a vital role in the metabolism of chemical carcinogens and 566 environmental contaminants. To estimate the CYP1A1 and CYP1A2, two 567 common isoforms of CYP1A family, we carried out ethoxyresorufin-O- 568 deethylase (EROD) and methoxyresorfin-O-deethylase (MROD) assays. 569 Fig. 10 shows EROD and MROD activities in larvae exposed to control or 570 food containing the test chemical. We observed a significant (P b 0.001) 571 6.3, 5.3 and 3.7-fold increase in EROD and 3.9, 3.3 and 2.9-fold increase 572 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 9 Fig. 9. TUNEL positive cells in midgut tissues of third instar larvae of D. melanogaster (Oregon R+) of control and benzene-, toluene- or xylene-exposed third instar larvae of D. melanogaster (Oregon R+) for 24 and 48 h. Histogram (A) depicts percent TUNEL positive cells in control and benzene-, toluene- or xylene-exposed organisms. Panels B and C represent microscopic images showing TUNEL positive cells in control and 100.0 mM benzene-exposed organisms, respectively after 48 h. Arrows indicate TUNEL positive cells. Bar represents 200 m. Data represent mean SD of three identical experiments made in triplicates and significance in comparison to control is ascribed as *P b 0.01. Q13 Fig. 7. IETDase (A) and DEVDase (B) activities in midgut tissue homogenate from control, DMSO-, benzene-, toluene- or xylene-exposed third instar larvae of D. melanogaster (Oregon R+) after 12, 24 and 48 h. Data represent meanSD of three identical experiments made in triplicates and significance in comparison to control is ascribed as *Pb 0.01, **P b 0.001. 573 in MROD activities in organisms exposed to 100.0 mM benzene, toluene 574 or xylene for 48 h, respectively, when compared to their controls 575 (Fig. 10AB). 576 Significantly decreased EROD activity in larvae exposed to benzene, 577 toluene or xylene together with DMF 578 To determine if benzene-, toluene- or xylene-mediated induction 579 of cytochrome P450 enzymes requires Aryl hydrocarbon Receptor (AhR) or its homolog, we exposed larvae to DMF, a potent inhibitor of 580 AhR, alone or in combination with test chemical. Exposure to DMF 581 (100 M) alone did not significantly decrease the EROD activity 582 compared to that in control Drosophila larvae. However, we observed 583 significantly decreased EROD activity (P b 0.001) in organisms ex- 584 posed to food containing B100, T100, X100, or TCDD (positive control) 585 along with DMF (Fig. 10C), in comparison to that observed in test 586 chemical alone treated group. 587 Effects of QC and CUR on the adverse effects of benzene, toluene or xylene 588 in exposed Drosophila 589 As we have observed increase in EROD, MROD activities, oxidative 590 stress parameters and genotoxicity in response to test chemicals, we 591 examined the effect of co-treatment with QC or CUR on the levels of 592 these various parameters. 593 Q14 Fig. 8. PARP cleavage in cells from midgut tissues of control and DMSO-, benzene-, toluene- or xylene-treated organisms for 24 and 48 h. Histogram (A) depicts fold increase in PARPFITC positive cells. Flow cytometric panels represent PARP cleavage in (B) control, (C) 100.0 mM benzene- (D) toluene- and (E) xylene-exposed groups after 48 h. Data represent mean SD of three identical experiments made in triplicates and significance in comparison to control is ascribed as *P b 0.01. **P b 0.001. Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 10 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx t1:1 Table 1 DNA migration in gut cells of D. melanogaster exposed to benzene, toluene and xylene and simultaneously with quercetin or curcumin after alkaline and neutral Comet assay. t1:2 t1:3 Groups Tail DNA (%) Tail length Tail moment (a.u.) t1:4 24 h 48 h 24 h 48 h 24 h 48 h t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 Alkaline Comet parameters Control EMS DMSO B100 T100 X100 QC control B100 + QC T100 + QC X100 + QC Cur control B100 + CUR T100 + CUR X100 + CUR Neutral Comet parameters Control -Irradiation (40 Gy) DMSO B100 T100 X100 QC control B100 + QC T100 + QC X100 + QC Cur control B100 + CUR T100 + CUR X100 + CUR 5.73 0.50 25.73 0.45* 6.13 0.64 21.58 1.22* 19.05 1.18* 17.51 0.86* 6.17 0.24 18.62 0.26* 17.23 0.15* 15.73 0.25* 6.09 0.56 15.37 0.21*$ 15.23 0.15*$ 14.11 0.08*$ 5.32 0.10 22.61 0.88* 5.67 0.33 16.42 0.21* 13.93 0.15* 13.63 0.21* 5.5 0.41 11.12 0.31*$ 11.14 0.21* 10.93 0.18* 5.62 0.51 10.21 0.16*$ 10.52 0.12*$ 10.12 0.11*$ 6.01 0.36 26.60 0.52* 5.97 0.65 24.54 1.19* 22.55 1.99* 21.05 1.45* 6.33 0.15 15.32 0.10*$ 14.93 0.51*$ 14.77 0.73*$ 6.31 0.20 14.23 0.15*$ 15.13 0.10*$ 15.01 0.20*$ 5.71 0.1 5.33 0.41 19.62 0.21* 16.51 0.30* 16.60 0.36* 6.22 0.64 10.11 0.12*$ 10.22 0.35*$ 10.13 0.58*$ 5.81 0.35 9.83 0.13*$ 9.92 0.51*$ 9.70 0.10*$ 6.68 0.57 28.28 1.27* 6.72 0.38 28.01 1.30* 26.01 0.91* 22.75 1.32* 7.09 0.21 24.47 1.28* 24.42 0.20* 21.63 0.45* 6.50 0.67 24.77 0.35* 23.80 0.35* 21.73 0.50* 3.45 0.10 13.25 0.25* 3.58 0.16 11.37 0.38* 9.60 0.10* 9.32 0.10* 3.70 0.10 9.01 0.10* 8.58 1.46* 8.02 1.12* 3.67 0.21 8.03 0.32* 8.30 0.10* 7.87 0.21* 6.95 1.00 28.59 2.60* 6.89.84 32.16 2.01* 29.03 1.19* 24.03 0.05* 7.15 0.01 15.42 0.31*$ 15.53 0.30*$ 7.12 0.36 14.93 0.15*$ 14.17 0.05*$ 14.50 0.10*$ 14.67 0.21*$ 3.60 0.05 3.72 0.11 15.47 0.12* 14.23 0.15* 14.03 0.72* 3.90 0.10 8.73 0.32*$ 8.32 0.10*$ 7.93 0.32*$ 3.72 0.10 7.93 0.38*$ 7.97 0.21*$ 7.92 0.30*$ 0.70 0.03 7.83 0.33* 0.65 0.05 8.49 0.10* 7.59 0.09* 7.16 0.11* 0.78 0.02 4.85 0.08*$ 4.14 0.08*$ 3.95 0.15*$ 0.74 0.01 3.98 0.13*$ 3.23 0.19*$ 3.27 0.03*$ 0.23 0.01 2.14 0.11* 0.25 0.04 1.24 0.03* 0.93 0.02* 0.87 0.01* 0.24 0.01 0.72 0.01*$ 0.62 0.01* 0.63 0.01* 0.26 0.01 0.58 0.03*$ 0.53 0.02*$ 0.54 0.02*$ 0.75 0.15 10.89 0.49* 0.78 0.08 10.55 0.32* 7.83 0.11* 7.69 0.09* 0.76 0.03 4.47 0.13*$ 3.90 0.22*$ 3.22 0.86*$ 0.76 0.08 3.22 0.03*$ 2.31 0.08*$ 2.41 0.05*$ 0.23 0.01 0.21 0.03 1.45 0.12* 1.18 0.02* 1.09 0.02* 0.21 0.01 0.59 0.01*$ 0.54 0.01*$ 0.52 0.02*$ 0.22 0.01 0.49 0.02*$ 0.45 0.01*$ 0.45 0.01*$ t1:36 EMS = 1.0 mM ethyl methanesulfonate, B100, T100 and X100 = 100.0 mM of benzene, toluene and xylene respectively; QC control = 100.0 M quercetin control, B100 + QC, T100 + QC and X100 + QC = 100.0 M quercetin along with 100.0 mM of benzene, toluene and xylene respectively; CUR control = 100.0 M curcumin, B100 + CUR, T100 + CUR and X100 + CUR = 100.0 M curcumin along with 100.0 mM of benzene, toluene and xylene respectively. Data presented as mean SD of three identical experiments made in triplicates and significance is ascribed as *P b 0.01 vs. control; $P b 0.01 vs. individual chemical (B100, T100 or X100). 594 Reduced EROD, MROD and GST activity 595 Larvae fed on QC or CUR along with benzene, toluene or xylene in 596 food have shown significant diminution (P b 0.001) in the EROD, 597 MROD and GST activities (Figs. 10 and 11) when compared to those 598 exposed to test chemical alone for 48 h. We observed 37%, 32%, 11% or 599 59%, 44%, 21% reduction in EROD activity in B100 + QC, T100 + QC, 600 X100 + QC or B100 + CUR, T100 +CUR, X100 + CUR, respectively, 601 when compared to that of B100, T100 or X100 exposed organisms. 602 Further, we also observed 38%, 34%, 17% or 45%, 34%, 25% reduction 603 in MROD activity in organisms exposed to B100 + QC, T100 + QC, 604 X100 + QC or B100 + CUR, T100 + CUR, X100 + CUR, respectively, in 605 comparison to organisms exposed to B100, T100 or X100 (Fig. 10AB). 606 Similarly, we observed 50%, 35%, 39% or 64%, 43%, 64% reduction in GST 607 activity in organisms exposed to B100 + QC, T100 + QC, X100 + QC or 608 B100 + CUR, T100 + CUR, X100 + CUR respectively, when compared 609 to those exposed to B100, T100 or X100 alone (Fig. 11). 610 Reduced oxidative stress levels 611 Addition of QC or CUR to the food contaminated with benzene, 612 toluene or xylene resulted in the reduction (P b 0.001) in ROS 613 generation, SOD, CAT activities and MDA content in the exposed 614 larvae when compared to those in test chemical alone treated larvae 615 after 48 h (Figs. 12 and 13). We observed a 65%, 28% and 30% reduction 616 in ROS generation in B100 + QC, T100 + QC or X100 + QC treated 617 larvae as compared to 100.0 mM of benzene-, toluene- or xylene618 exposed organisms respectively (Fig. 12) A similar reduction pattern 619 was evident when organisms were exposed to test chemicals along 620 with CUR (B100 + CUR, T100 + CUR and X100 + CUR) (Fig. 12). We 621 observed 41%, 34%, 33% or 39%, 35%, 33% reduction in SOD activity in B100 + QC, T100 + QC, X100 + QC or B100 + CUR, T100 + CUR, 622 X100 + CUR, respectively, when compared to that of B100, T100 623 or X100 exposed organisms (Fig. 13A). Further, we also observed 624 52%, 39%, 36% or 46%, 36%, 41% reduction in CAT activity (Fig. 13B) 625 and 52%, 40%, 35% or 56%, 44%, 45% reduction in MDA content 626 (Fig. 13C) in organisms exposed to B100 + QC, T100 + QC, X100 + QC 627 or B100 + CUR, T100 + CUR, X100 + CUR, respectively, in comparison 628 to organisms exposed to B100, T100 or X100. 629 Reduced apoptosis and genotoxicity 630 In organisms exposed to test chemical along with QC or CUR, we 631 observed a significantly lower IETDase activity (47%, 46% and 49% 632 lesser activity in B100 + QC, T100 +QC and X100 + QC treated 633 organisms respectively; P b 0.001; 61%, 59% and 61% reduced activity 634 in B100 + CUR, T100 + CUR and X100 + CUR exposed organisms 635 respectively after 48 h) (Fig. 14A). In these groups, DEVDase activity 636 was also significantly reduced (P b 0.001; significantly 48%, 54% and 637 52% lesser activity in B100 + QC, T100 + QC and X100+QC treated 638 organisms respectively; P b 0.001; P b 0.001; 69%, 68% and 65% reduced 639 activity in B100 + CUR, T100 + CUR and X100 + CUR exposed organ- 640 isms respectively after 48 h) when compared to those exposed to 641 test chemical alone (Fig. 14B). Similar trend was evident when we 642 measured depolarization of mitochondrial membrane potential: 36%, 643 40% and 52% reduction in B100 + QC, T100 + QC or X100 + QC exposed 644 organisms, respectively, after 48 h and significantly 45%, 43% and 57% 645 reduction (P b 0.001) in B100 +CUR, T100 + CUR and X100 + CUR 646 treated organisms, respectively, after 48 h (Fig. 15). 647 We also observed a significant reduction in DNA damage in 648 organisms exposed to B100 + QC, T100 + QC and X100 + QC and 649 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 11 Fig. 11. Glutathione S-transferase (GST) activity in larval homogenate from third instar larvae of D. melanogaster (Oregon R+) in control, DMSO and benzene, toluene or xylene alone or in combinations with QC or CUR treatment for 48 h. Data represent mean SD of three identical experiments made in three replicates. Data represent mean SD of three identical experiments made in triplicates and significance ascribed as **P b 0.001 vs. control; $$P b 0.001, reduction vs. individual chemical (B100 or T100 or X100). Fig. 10. 7-Ethoxyresorufin-O-deethylase (EROD) activity (A) and 7-methoxyresorufinO-de-ethylase (MROD) activity (B) in microsomes isolated from third instar larvae of D. melanogaster (Oregon R+) exposed to benzene, toluene or xylene alone or in combination with quercetin (QC) or curcumin (CUR). B100, T100 and X100 = 100.0 mM benzene, toluene and xylene respectively; B100 + QC, T100 + QC and X100 + QC = 100.0 M quercetin along with 100.0 mM of benzene, toluene and xylene respectively; B100 + CUR, T100 + CUR and X100 + CUR = 100.0 M curcumin along with 100.0 mM of benzene, toluene and xylene, respectively, in this figure and the remaining figures in this paper. Panel C depicts the inhibitory effect of 3,4dimethoxyflavone on EROD activity in third instar larvae of D. melanogaster exposed to 100.0 mM benzene, toluene and xylene for 48 h. Data represent mean SD of three identical experiments made in triplicates and significance is ascribed as **P b 0.001 vs. control; $P b 0.01, $$P b 0.001, reduction vs. individual chemical (B100 or T100 or X100). 650 B100 + CUR, T100 + CUR and X100 + CUR. In B100 + QC or B100 + 651 CUR exposed organisms, migration of DNA was significantly less 652 after 24 and 48 h when compared to those in organisms exposed 653 to test chemical alone, in both alkaline and neutral Comet assays Fig. 12. Levels of reactive oxygen species in cells from midguts of third instar larvae of D. melanogaster (Oregon R+) in control, DMSO and benzene, toluene or xylene alone or in combinations with QC or CUR treatments for 24 and 48 h. Histogram (A) depicts ROS generation in test chemical exposed organisms and flow cytometric panels shows the ROS generation in (B) control, (C) QC control, (D) CUR control, (E) B100, (F) B100 + QC and (G) B100 + CUR exposed organisms after 48 h. Data represent mean SD of three identical experiments made in triplicates and significance is ascribed as *P b 0.01 vs. control; $P b 0.01, reduction vs. individual chemical (B100 or T100 or X100). (Table 1). We observed a similar trend in T100 + QC, X100 + QC, 654 T100 + CUR or X100 + CUR exposed Drosophila larvae when com- 655 pared to those exposed to toluene or xylene alone (Table 1). 656 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 12 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx Fig. 14. IETDase (A) and DEVDase (B) activities in microsomes isolated from third instar larvae of D. melanogaster (Oregon R+) in control, DMSO and benzene, toluene or xylene alone or in combinations with QC or CUR treatments for 24 and 48 h. Data represent mean SD of three identical experiments made in triplicates and significance is ascribed as *P b 0.01 vs. control; $P b 0.01, reduction vs. individual chemical (B100 or T100 or X100). Fig. 13. Cu-Zn SOD (A) and CAT (B) activities and MDA content (C) in the larval homogenate from third instar larvae of D. melanogaster (Oregon R+) in control, DMSO and benzene, toluene or xylene alone or in combinations with QC or CUR treatment for 48 h. Data represent mean SD of three identical experiments made in triplicates and significance is ascribed as **P b 0.001 vs. control; $$P b 0.001, reduction vs. individual chemical (B100 or T100 or X100). 657 Discussion 658 Continuously high levels of exposure of benzene, toluene and 659 xylene to the organisms from occupational/non-occupational sources 660 cause major health hazards. These contaminants are well known to 661 disrupt various cellular processes (Gerin et al., 1998; Chambers et al., 662 2006). Previous studies showed that benzene, toluene and xylene 663 have potential to induce oxidative stress, an imbalance of the anti664 oxidant system, such as SOD, glutathione peroxidase (GP), glutathi665 one (GSH) and MDA content in mammalian cell line or petrochemical 666 workers (Croute et al., 2002; Georgieva et al., 2002). Weaver et al. 667 (2007) and Weaver and Liu (2008) demonstrated benzene-induced 668 apoptosis in epithelial cells of respiratory tracts as evidenced by 669 nicking of DNA, endonucleolytic degradation of genomic DNA as well 670 as increased caspase activity. Similar observations were made in vivo 671 using murine models (Smith, 1996; Ross, 2000; Snyder, 2000; Weaver et al., 2007). Cell death is the ultimate fate of the cell with irreparable 672 damage. DNA damage can also occur by chemicals, which if not 673 repaired, can have serious consequences leading to carcinogenesis. 674 Benzene, being classified as class I carcinogen, has been shown 675 previously to cause DNA damage both in vitro and in vivo (Sul et al., 676 2005; Weaver et al., 2007; Weaver and Liu, 2008). Singh and Winn 677 (2008) and Faiola et al. (2004) showed significant increase in 678 chromosomal breaks in K-562 cells and hematopoietic stem cells 679 (HSC) exposed to benzene and its metabolites. Similarly, in vivo 680 studies using mouse bone marrow cells showed significant increase in 681 sister chromatid exchanges and clastogenicity (Erexson et al., 1986; 682 Zhang et al., 2002). Using alkaline Comet assay, Chen et al. (2008) and 683 Galvan et al. (2008) showed increased Comet parameters in the 684 human lymphocytes and HeLa cells respectively, exposed to benzene 685 and its metabolites. Limited available information suggests toluene 686 and xylene can also induce apoptosis (Nakai et al., 2003; Al-Ghamdi 687 et al., 2004). Despite this vast knowledge on the adverse effects of 688 benzene, toluene and xylene, only a few compounds have been tested 689 for their protective roles against benzene, toluene and xylene induced 690 toxicity. In this study, we first analyzed the apoptotic and genotoxic 691 potential of these three monocyclic hydrocarbons using D. melanogaster, 692 an alternate to animal model system. Further, we examined the role of 693 potential candidates/receptors involved in mediating the benzene-, 694 toluene- or xylene-mediated toxicity. Subsequently, we utilized this 695 model to evaluate the protective effects of two well-known phyto- 696 chemicals viz. QC and CUR [quercetin (a well-known antioxidant (Chen 697 and Kang, 2005; Kanupriya et al., 2006) and curcumin (a known 698 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 13 Fig. 15. Alterations in mitochondrial membrane potential (m) in cells from midgut tissues of third instar larvae of D. melanogaster (Oregon R+) in control, DMSO and benzene, toluene or xylene alone or in combinations with QC or CUR treatments for 24 and 48 h. Histogram (A) depicts fold increase in mitochondrial membrane depolarization. Flow cytometry panels represent m in (B) control, (C) B100, (D) B100 + QC and (E) B100 + Cur exposed organisms after 48 h. Data represent mean SD of three identical experiments made in triplicates and significance is ascribed as *P b 0.01. $P b 0.01, reduction vs. individual chemical (B100 or T100 or X100). 699 antioxidant and anti-cancer agent (Becatti et al., 2010; Biswas et al., 700 2010)] against benzene-, toluene- and xylene-induced toxicity. 701 Apoptosis is a physiological mode of cellular suicide that plays a 702 vital role during embryogenesis, development, and normal tissue 703 homeostasis (Brill et al., 1999; Vaux and Korsmeyer, 1999; Fulda and 704 Debatin, 2006). The hallmarks of apoptosis include depolarization of 705 the plasma membrane, cell shrinkage, alterations in intracellular ion 706 concentrations, mitochondrial membrane depolarization, chromatin 707 condensation, and DNA fragmentation. We, therefore, examined 708 several candidates involved in the apoptotic pathway in Drosophila 709 exposed to benzene, xylene or toluene. 710 During early apoptosis, a cell loses its membrane asymmetry (Chen 711 et al., 2008). Phosphatidylserine (PS), normally present on the inner 712 cytoplasmic leaflet of the plasma membrane of healthy cells, is 713 translocated and exposed on the outer leaflet (Seigneuret and Devaux, 714 1984; Connor and Schroit, 1987). We used AV, a Ca2+-dependent 715 phospholipid-binding protein that has a high affinity for PS (Comfurius 716 et al., 1996). The significantly increased (P b 0.001) AV positive cells in 717 the present study suggested the initiation of apoptosis in benzene-, 718 toluene- or xylene-exposed organisms. 719 The process of apoptosis is controlled by a diverse range of cell 720 signals, which may originate either at extra-cellular or intra-cellular 721 level (Chang et al., 1998; Budihardjo et al., 1999; Arya et al., 2007). To 722 identify the apoptotic pathway being followed in the cells of the exposed 723 organism, we analyzed mitochondrial membrane potential, DEVDase 724 activity (caspase-3), IETDase activity, and poly ADP ribose polymerase 725 (PARP) cleavage (Chiarugi and Moskowitz, 2002). We observed 726 significant increase in DEVDase and IETDase activities, PARP cleavage 727 and depolarization of mitochondrial membrane in organisms exposed 728 to test chemicals, indicating the induction of mitochondria-mediated 729 caspase-dependent cell death pathway in Drosophila larvae exposed to 730 benzene/toluene/xylene. Similarly, a number of earlier studies also 731 documented increased IETDase and DEVDase activities in organisms/ cells exposed to organic solvents containing benzene, toluene or xylene 732 [in Jurkat cells by chlorinated biphenol (Inayat-Hussain et al., 2001; 733 Inayat-Hussain and Ross, 2005), HL60 and CD34+ cells by benzene 734 metabolites (Hiraku and Kawanishi, 1996; Moran et al., 1996)]. Caspases 735 are normally rendered inactive by Inhibitor of Apoptosis Proteins (IAPs) 736 and this inhibition is overcome by p53-mediated transcription leading 737 to increased levels of Smac/DIABLO orthologs (Hid, Rpr and Grim in 738 Drosophila) (Brenner and Kroemer, 2000; Brodsky et al., 2000; Schuler 739 and Green, 2001; Brodsky et al., 2004; Kornbluth and White, 2005; 740 Wichmann et al., 2006). The increased levels of Hid, Rpr, Grim and 741 TUNEL positive cells observed in benzene-, toluene- and xylene- 742 exposed groups suggest the activation of pro-apoptotic genes in 743 organisms leading to removal of IAP-mediated inhibition of caspases 744 and also activation of apoptotic pathway. 745 Increased PARP cleavage is usually associated with oxidative DNA 746 damage (Satoh and Lindahl, 1994; Miller et al., 2004; Babich et al., 747 2009; Deng et al., 2009). Since we also observed increased PARP 748 cleavage in benzene-, toluene- and xylene-exposed organisms, we 749 analyzed DNA damage through Comet assay, which has been adapted 750 for genotoxicity assessment (Miloshev et al., 2002; Rajaguru et al., 751 2003; Mukhopadhyay et al., 2004; Siddique et al., 2005b; Deguchi 752 et al., 2008). Of the two variants of the Comet assay, the neutral 753 version detects double-strand DNA breaks (Yasuhara et al., 2003) 754 whereas the alkaline version allows to reveal single- and double- 755 strand breaks as well as alkali labile sites (Moller, 2006). In our study, 756 we found significantly increased migration of DNA (TL, % TD and TM) 757 in the order of benzene N toluene xylene in the exposed organisms in 758 alkaline as well as neutral Comet assays. Interestingly, alkaline Comet 759 parameters showed statistically significant higher levels (P b 0.01) as 760 compared to neutral Comet parameters in the test chemical exposed 761 organisms. These results suggest benzene, toluene and xylene are 762 potentially genotoxic causing double-strand breaks to the exposed 763 larvae. These observations parallel the earlier studies on human 764 population (epidemiological data) and experimental models (both in 765 vivo and in vitro) exposed to benzene, toluene or xylene (Carere et al., 766 1995a; Carere et al., 1995b; Chen et al., 2008; Pandey et al., 2009). 767 Interestingly, the observed genotoxicity (benzene N toluene N xylene) 768 is inversely related to lipophilicity (benzene b toluene b xylene; 769 Snyder et al., 1993; ATSDR, 1997) and this is consistent with our 770 Q4 previous observations in mixture toxicity studies that the higher the 771 lipophilicity, the lower is the toxicity (Singh et al., 2010). 772 Reactive oxygen species (ROS) are generated in biological systems 773 either by normal metabolic pathways or as a consequence of exposure 774 to chemical (Iqball et al., 2002, Sun et al., 1990). Recent studies from 775 Q5 our laboratory showed induction of heat shock genes (hsp70, hsp83, 776 hsp60 and hsp26), oxidative stress markers and increased ROS 777 generation in D. melanogaster exposed to benzene, toluene or xylene, 778 individually, or their mixtures indicating the potential of these 779 chemicals to produce cellular stress (Singh et al., 2009; Singh et al., 780 2010). In addition, we observed significant induction of cytochrome 781 P450 enzymes in benzene-, toluene-, or xylene-exposed organisms 782 when compared to controls. Induction of cytochrome P450 enzymes 783 in response to benzene, toluene or xylene exposure is well 784 documented (Day et al., 1992; Nakajima and Wang, 1994; Seaton 785 et al., 1994). Further, AhR has been shown to be the key component in 786 the metabolic response to aromatic hydrocarbons, including benzene 787 (Schmidt and Bradfield, 1996; Yoon et al., 2002; Nishiumi et al., 2007). 788 AhR, a cytoplasmic bHLH-PAS transcription factor, upon binding to an 789 aromatic hydrocarbon (TCDD for example), translocates to the 790 nucleus. Within the nucleus, forms a complex with the Aryl 791 hydrocarbon receptor nuclear translocator (ARNT), another bHLH- 792 PAS protein (Hoffman et al., 1991) and together bind to xenobiotic 793 response element to control the expression of specific target genes 794 including certain cytochrome P450s (Swanson et al., 1995). Based on 795 this, we hypothesized a similar role for spineless (Drosophila homolog 796 of Aryl hydrocarbon receptor Cespedes et al., 2010) in the benzene-, 797 Please cite this article as: Singh, M.P., et al., Genotoxicity and apoptosis in Drosophila melanogaster exposed to benzene, toluene and xylene: Attenuation by quercetin and curcumin, Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.03.006 14 M.P. Singh et al. / Toxicology and Applied Pharmacology xxx (2011) xxxxxx 798 xylene- and toluene-mediated toxicity in Drosophila. To test if the 799 observed induction of cytochrome P450s is mediated by AhR 800 pathway, we exposed Drosophila larvae to test chemical together 801 with the blocker for AhR (DMF, Li, 2007) and measured the activity of 802 cytochrome P450s. The observed reduction of cytochrome P450s (in 803 particular EROD) in exposed organisms in the presence of blocker, 804 clearly indicated that like in mammals, toxicity of benzene, in part, is 805 mediated by AhR in Drosophila. In addition, our study provides an 806 evidence for the involvement of AhR also in toluene- and xylene807 mediated toxicity. 808 The other objective of our study was to look for the agents, which 809 can attenuate the toxicity conferred by benzene, toluene or xylene. As 810 cytochrome P450 enzymes, AhR and oxidative stress underlie the 811 observed benzene-, toluene- or xylene-mediated toxicity in the 812 present study, we looked for nutraceuticals that are known to act 813 on the above. We examined the efficiency of two phytochemicals, 814 namely, QC and CUR for their protective roles against benzene-, 815 toluene- or xylene-induced toxicity. QC is a polyphenolic flavonoid 816 and has protective effects on different adverse cellular events like 817 carcinogenesis through unknown mechanism of action yet reducing 818 LPO and ROS generation or having anti-proliferative activity in cells/ 819 organisms (Dihal et al., 2006; Dihal et al., 2008; Larson et al., 2010) 820 indicating its anti-oxidant and anti-cancer properties. Similarly, CUR 821 (diferuloyl methane) has been shown to possess anti-oxidative and 822 free radical scavenging properties and anti-neoplastic properties 823 (Reddy and Lokesh, 1994; Motterlini et al., 2000; Messner et al., 824 2009). In addition, both CUR and QC were found to degrade both AhR 825 and ARNT to inhibit cytochrome P450 enzymes. Based on these 826 reports (on anti-oxidative, free radical scavenging, inhibiting proper827 ties, anti-cancer properties of QC and CUR), we hypothesized that 828 addition of QC or CUR in the exposure regimen will reduce the toxic 829 effects of the test chemicals to the exposed organisms. To test this, we 830 analyzed their efficacy in modulating the cytochrome P450 and GST 831 activities, ROS generation, apoptosis and genotoxicity in the test 832 chemical exposed organism. We observed a significantly reduced 833 cytochrome P450 activities, oxidative stress, GST activity, ROS 834 generation, lower intensity of apoptosis and DNA damage in 835 organisms exposed to QC or CUR along with test chemicals in 836 comparison to those observed in test chemical alone group. Presence 837 of chemicals in organisms exposed to benzene, toluene or xylene 838 alone at levels similar to those in combination with QC or CUR 839 suggested that this reduction in assayed parameters is not due to 840 reduced intake of test chemical by the organisms in the presence of QC 841 or CUR but indeed due to the protective roles of QC or CUR. These 842 results demonstrated the potential of CUR and QC to attenuate the 843 benzene-, toluene- or xylene-mediated toxicity. Intriguingly, a 844 previous study from our group had shown that the co-treatment of 845 QC with dichlorvos (DV) enhanced the oxidative stress and apoptosis 846 compared to that of DV alone (Gupta et al., 2007). This discrepancy 847 may be attributed to the test chemical used and QC may confer 848 protection in a compound specific manner. To confirm, if this is the 849 case, we analyzed the ROS levels in organisms exposed to DV, DV + 850 QC, benzene, benzene + QC or QC alone. Interestingly, we observed 851 that ROS levels are increased in organisms exposed to DV + QC [as in 852 agreement with Gupta et al., 2007] and decreased in benzene + QC (as 853 in the present study), when compared to their respective chemical 854 controls (please see Supplemental Fig. S6). These results indicate that 855 QC may play protective roles in a compound specific manner. 856 Several molecular mechanisms by which CUR and QC may play 857 protective roles are envisaged. Given their documented roles in 858 modulating cytochrome P450 activities (Firozi et al., 1996; Thapliyal 859 and Maru, 2001) and their anti-oxidant properties (Chan et al., 2005; 860 Messner et al., 2009) they may act either at the level of metabolism or 861 alternatively they may act on the anti-oxidant defence system. During 862 Phase I xenobiotic metabolism, CUR and QC may either 1) inhibit 863 cytochrome P450 enzymes directly by acting as a competitive substrate (Appiah-Opong et al., 2007), 2) interfere with AhR mediated 864 induction of cytochrome P450 enzymes (Rinaldi et al., 2002) or 3) act 865 at the level of GSTs, which play an important role in the phase II 866 xenobiotic metabolism (Oetari et al., 1996). The reduction in 867 cytochrome P450 activities in organisms exposed to test chemical 868 along with QC or CUR and the observed involvement of in the present 869 study point to the second possibility. In addition, we observed 870 significant (P b 0.05) positive correlation (please see Supplementary 871 Table S1 for details) between activity of cytochrome P450s and GSTs, 872 ROS and other oxidative stress parameters analyzed. Based on these, 873 we believe that the reduced levels of GSTs, ROS and other oxidative 874 stress parameters might be a consequence of reduced activity of 875 cytochrome P450 in response to CUR or QC in exposed organisms. 876 These ultimately might have resulted in reduced apoptosis and 877 genotoxicity in organisms exposed to test chemical along with CUR 878 or QC. 879 Taken together, the present study suggests that MAHs particularly 880 benzene, toluene and xylene can cause genotoxicity and apoptosis in 881 the exposed Drosophila larvae in vivo, through mitochondria 882 mediated caspase dependent pathway of cell death. The study also 883 showed that AhR homologue plays a key role in the induction of 884 cytochrome P450 in response to benzene, toluene or xylene in 885 Drosophila. Among the three tested chemicals, benzene is highly 886 genotoxic (possibly due to higher uptake) while toluene and xylene 887 are less genotoxic than benzene. The study further suggests that 888 quercetin and curcumin inhibit cytochrome P450, probably influenc- 889 ing AhR mediated pathway, which eventually may result in the 890 reduction of the genotoxicity and apoptosis in test chemicals exposed 891 D. melanogaster. 892 Supplementary materials related to this article can be found online 893 at doi:10.1016/j.taap.2011.03.006. 894 Conflicts of interest 895 None. 896 Acknowledgment 897 The authors are thankful to the Director, Indian Institute of 898 Toxicology Research (IITR) for facilities. We thank Mr. N. Mathur, 899 Epidemiology Section, IITR for statistical analysis, Dr. A. Dhawan, 900 Developmental Toxicology Division, IITR for Comet assay facility, 901 Dr. M. Dixit and Mr. A.L. Viswakarma, Central Drug Research Institute, 902 Lucknow for flow cytometry facility respectively. Financial assistance 903 to MPS from UGC-SRF, New Delhi, MM from SRF-DBT, New Delhi, AS 904 from ICMR-SRF, New Delhi and DKC from Council of Scientific and 905 Industrial Research (SIP-08 and NWP-34) is thankfully acknowledged. 906 References 907 Al-Ghamdi, S.S., Raftery, M.J., Yaqoob, M.M., 2004. Toluene and p-xylene induced LLC- 908 PK1 apoptosis. Drug Chem. Toxicol. 27, 425432. 909 Alegretti, A.P., Thiesen, F.V., Maciel, G.P., 2004. Analytical method for evaluation of 910 exposure to benzene, toluene, xylene in blood by gas chromatography preceded by 911 solid phase microextraction. J. Chromatogr. 809, 183187. 912 Appiah-Opong, R., Commandeur, J.N., van Vugt-Lussenburg, B., Vermeulen, N.P., 2007. 913 Inhibition of human recombinant cytochrome P450s by curcumin and curcumin 914 decomposition products. Toxicology 235, 8391. 915 Arlien-Suborg, P., 1992. Solvent Neurotoxicity. CRC Press, Inc., Boca Raton, Florida. 916 Arya, R., Mallik, M., Lakhotia, S.C., 2007. 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