Amelioration of oxidative stress-mediated cytotoxicity and genotoxicity induced by copper and ubendiamide in-vivo and in- vitro by potent antioxidants

Present study was designed to assess the toxicity of copper @ 33 mg/kg and ubendiamide @ 200 mg/kg in vivo in male Wistar rats orally once daily for 90 days and protective effect of α-tocopherol, resveratrol, curcumin and catechin and in vitro cyto-genotoxicity in primary cell culture of thymocytes. In vivo study showed signicant (p<0.05) increase in AST, total bilirubin and uric acid, creatinine and BUN levels while decrease in total proteins, GSH, SOD and GST levels and increased LPO and GPx with severe degenerative changes were observed in liver and kidney tissues in intoxicated groups. In vitro thymocytes were exposed to 40 µM concentration of ubendiamide and/or showed signicant increase in TUNEL+ve cells, micronuclei, DNA shearing, and comet formation per 100 cells. Concurrent treatment with α-tocopherol in xenobiotics intoxicated groups showed almost normal values of the biochemical parameters and decreased LPO production and improved antioxidant enzymes activities and histoarchitecture of liver and kidney tissues suggest ameliorative potential of α-tocopherol whereas, resveratrol, curcumin, catechin or α-tocopherol in vitro decreased TUNEL+ve cells, micronuclei induction and comet formation and effect of antioxidants was concentration-dependent and their order of potency on equimolar concentration (10 µM) basis is: curcumin > resveratrol >catechin = α-tocopherol. potential 31-33 Curcumin, α-tocopherol (α-TOH), resveratrol catechinarewell biologically active constituents possessing anti-oxidant, anti-inammatory, anti-apoptotic potential preserved in 10% buffered formalin saline were processed employing the standard procedure. Tissue sections of 5-6 µm thickness were cut using microtome (Lieca, Germany) and stained with haematoxylin-eosin as per the method of Luna (1968) 55 . Slides were thoroughly examined under light microscope to observe the histoarchitectural changes, if any. Interactive Index. Mansour’s formula 56 was used to determine the type of interaction between the copper and ubendiamide in the groups where used in combination in terms of interaction index. fatty acids in membrane lipids when generation of ROS exceeds antioxidant defence mechanisms 82,71 and produces histopathological abnormalities in different tissues due to ROS generation and DNA damage 83 . The results of this study showed increased ROS levels and decreased GSH content and reduction in the activities of SOD and GST while increase in GPx activity. Present ndings are in line with the previous toxicity studies of copper and copper nanoparticles 84,85,67,77,72 and ubendiamide toxicity studies 86-88 in liver and kidneys. copper might not have reciprocally and signicantly affected the metabolism of each other by altering their activation and/or their detoxication mechanism. Similar results have also been reported following combined exposure to arsenite + parathion 119 and malathion+arsenite 120 . However, combined effect of copper and ubendiamide on kidneys tissue oxidative stress biomarker (LPO, GSH, SOD, GST and GPx) was observed potentiating in interactive index analysis while catalase and total protein was shown was additive and antagonistic, respectively, suggest kidneys was more vulnerable to toxic insult than liver following copper and ubendiamide in combination. The results of present study indicated moderate to marked and/or signicant alterations in biochemical parameters, oxidative stress biomarkers and histology in liver and kidneys and drug metabolizing enzymes activity in liver of the rats following exposure to the test xenobiotics suggest that the test xenobiotics induces oxidative stress which is ultimately responsible for hepatic- and renal-insults as evident from the correlation and association data between the oxidative stress markers and liver and kidney function test markers. However, hepatic degenerative changes observed in rats of ubendiamide-treated group were similar to those reported in JMPR following 13 weeks exposure of rats to ubendiamide 6 . Although ubendiamide is a category III compound having low order of toxicity 5 but hepatotoxic and nephrotoxic effects of ubendiamide, as observed in the present study, seem to be of great concern, therefore, further studies are warranted. Dexamethasone-induces apoptosis in thymocytes by causing mitochondrial dysfunction, caspase-3 activation, reactive oxygen species (ROS) production and oxidative damage 128 whereas ROS generated interact with cellular DNA and produced comet formationinducing single-and double-strand breaks of DNA. Similar increase in DNA fragmentation following exposure to copper has been reported by Vidyashankar and Patki 129 in HepG2 cells. In the present study, oxidative stress in liver and kidneys could be the reason behind DNA damage and comet formation in thymocytes. Similar results of DNA damage and comet formation has been reported in our previous study conducted on spleen cells 18 . Micronuclei induction assay detectsclastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage and chromosome loss 130,131 and is used for evaluation of the genotoxic potential of xenobiotics 132 . Compared to 1.1% micronuclei formed in control group and 4.63% in dexamethasone-exposed group, signicant increase in number of the micronuclei formation in thymocytes of copper (17.1%) and ubendiamide (3.63%) groups suggest genotoxic potential of copper and ubendiamide, however thymocytes have been found to be more prone to cyto-genotoxic effects. Cyto-genotoxic potential of copper have been reported in mice 133 and other tissues 134 and rat splenocytes 18 . However, comparison of the micronuclei data of thymocytes (present study) and of splenocytes reported recently from our laboratory 18 revealed that splenocytes were more susceptible to micronuclei formation following exposure to ubendiamide while thymocytes were more susceptible to copper-induced micronuclei formation. Thus the ndings on cell viability, micronuclei and comets formation data obviously suggest differential susceptibilities of different cells to cyto-genotoxic effects of different xenobiotics. Overall cyto-geno-protective potential of different natural antioxidants at equimolar concentration (10 µM) against ubendiamide and copper was found to be curcumin > resveratrol >catechin = α-tocopherol. Based on the present ndings, it is evident that natural polyphenols and α-tocopherol hold promising potential in preventing cyto-genotoxic effects of xenobiotics. Therefore, their inclusion in functional foods or their use as nutraceutical seems to hold promising potential in preventing the deleterious effects of environmental pollutants including ubendiamide and copper.


Introduction
Indiscriminate and non-injudicious use of insecticides/pesticides in agricultural practices and waste disposal of heavy-metals and chemical industries in water bodies are entering the food chain unintentionally producing direct impact on human and animal health 1,2 . Flubendiamide (Pthalmic acid diamide) is a new class of insecticide used extensively as a substitute for organophosphate and organochlorine pesticide.
Flubendiamide, a ryanodine class insecticide stimulates ryanodine-sensitive calcium release channels (RyR) of insects 3,4 and disrupt muscle functions without mammalian ryanodine receptors 3,5 .Use of ubendiamide in agriculture practices has crossed its threshold and resulting in environmental contamination due to its slow degradation and persistent behaviour and thus adversely affect human and animal health [6][7][8][9][10] .
Though, the United State Environmental Protection Agency (USEPA) has cancelled the registration of all ubendiamide products due to unreasonable adverse effects on the aquatic environment 11 , however, it is still in use in several countries including India for control of Lepidopteron sp. pests in agricultural operations 12 . Flubendiamide has been reported to primarily affect liver followed by thyroid and haemopoietic system 5, 13 but does not produces genotoxicity effects on bone marrow cells 14,15 . Whereas, in our previous studies we have reported ubendiamide induced oxidative stress in spleen and testicular tissues with slightly altered haematological parameters in vivo in rats [16][17][18] and genotoxic behaviour on primary cell culture of rat splenocytes following in vitro exposure 18 .
Contaminated food and water bodies from industrial e uents contain both essential and non-essential/trace elements that may also produce health hazard. Mn, Fe, Co, Cu and Mo are essential trace metals participate in wide range of redox reactions and their toxicity can be attributed to accumulation of excess concentration of these metal ions in speci c tissues or organs leading to generation of free radicals and consequent oxidative stress is one of the important mechanisms of cell death 19 .Copper, although biologically essential, used for control of bacterial and fungal infections as weedicide, fungicide in agricultural practices and as molluscicide in domestic lakes and ponds 20 can be highly toxic to biota (plants, animals and human beings) if present in higher concentrations 21 . In India, river, ponds and lakes are commonly used for irrigation and drinking purposes can be a major source of contamination and intoxication where, riverwaterhas been reported to have more than 300 µg/ml 22 while WHO recommends not more than 2mg/L. Thus, human beings and animals are being continuously exposed to copper through drinking water. Long term chronic exposure to copper causes hepatic and neurotoxic effect in rats 23,24 and neurodegenerative disorders (Alzheimer's disease and Parkinson's disease) in humans and dogs [25][26][27] .Copper has also been reported to produce cyto-genotoxic effects 28-30 .
Food-derived componentshavereceived great attention in last two decades particularly due to their antioxidant and anti-in ammatory potential [31][32][33] . Curcumin, α-tocopherol (α-TOH), resveratrol and catechinarewell studied biologically active constituents possessing anti-oxidant, anti-in ammatory, anti-apoptotic potential having ability to chelate metal ions and upregulate the antioxidant enzymes have been reported to produce cytoprotective, cardioprotective, antiapoptotic effects in metal-and pesticide-induced oxidative and DNA damage 33, 34-40 .
Assessment of single chemical toxicity on an individual basis does not re ect actual conditions in environment or in human and animal health where the target is exposed to various chemicals at a same time 41 . Thus, the concept of combined exposure to multiple chemicals and assessment of their toxicity has been evolving over decades. Therefore, interaction toxicity studies between two or more toxicants have become important and an emerging area of research in modern toxicology where interaction may lead to synergistic, antagonistic, inhibitory or potentiated toxic effects 42 . Paucity of any information on toxicity of these xenobiotics where ubendiamide is claimed to be safest insecticide and copper as essential micro-mineral prompted us to take up the present study to assess their combined effect and interactive behaviour on biochemical indices, oxidative stress biomarkers, drug metabolizing enzymes and histological changes in liver and kidneys in vivo in rats and Preparation of microsomes. In brief, liver was homogenized in 3 volumes (1:3) of ice-cold (1-4 0 C) homogenization buffer containing 1.15% KCl in 50 mM potassium phosphate buffer solution (pH 7.4). The homogenates were centrifuged at 15000 g for 30 min using ultra centrifuge (Himac CS120GX II, Hitachi, Japan.).The mitochondrial supernatant (8ml) spun at 105000 g for 1 h in ultracentrifuge to sediment the microsomal pellet. Cytosolic fractions were collected from middle separating the lipid layer on top in a separate Eppendorftube. The microsomal pellet was then washed by adding 8 ml of resuspension buffer (homogenization buffer containing 0.05 mM EDTA) and spun again 105000 g for 15 min. The microsomal pellet, so obtained, was resuspended in 4 ml of resuspension buffer and 1 ml aliquots were immediately stored at -80 0 C until assayed for drug metabolizing enzymes.
Phase I drug metabolizing enzymes. Hepatic microsomal pellets prepared were used for assay of phase I drug metabolizing enzymes, namelycytochrome P 450 and CYP b5 activities measured as per the protocol described by Omura and Sato 52 .Aminopyrine-N-demethylase and aniline phydroxylase activities were estimated according to the method of La Du et al. 53 and hepatic microsomal protein was determined by the method described by Lowry et al. 46 .
Phase II drug metabolizing enzymes. Hepatic microsomal and cytosolic glutathione sulfotransferase were estimated by determining the rate of increase in optical density (OD) at 340 nm at 25 0 C due to formation of 1-chloro-2, 4-dinitrobenzene (CDNB) conjugate of glutathione as described by Habig et al. 50 and UDP-glucuronosyltransferase activity was assessed as per the method of Dutton and Storey 54 .
Histopathological studies. Pieces of liver and kidneys preserved in 10% buffered formalin saline were processed employing the standard procedure. Tissue sections of 5-6 µm thickness were cut using microtome (Lieca, Germany) and stained with haematoxylin-eosin as per the method of Luna (1968) 55 . Slides were thoroughly examined under light microscope to observe the histoarchitectural changes, if any.
Interactive Index. Mansour's formula 56 was used to determine the type of interaction between the copper and ubendiamide in the groups where used in combination in terms of interaction index. Finally, I.I. of all parameters were summarized in tabulated form and used to draw the conclusion of type of interaction between compounds when they are present together in body of living beings.
In vitro study. Twenty healthy male Wistar rats weighing 80-100 g were used for in vitro study and kept in the Departmental Laboratory Animal House for acclimatization period of one week before the start of the experiment and provided ad lib feed and water.
Isolation of thymocytes. Rats were sacri ced by cervical dislocation and whole thymus was collected aseptically. Brie y, thymus was quickly disintegrated into small pieces in chilled phosphate buffered saline (PBS) by scissor and nely minced by vigorous pipetting using glass pipette and suspension was transferred to 15 ml sterile centrifuge tube. Tubes were allowed to stand on ice for 15 min. Top 12 ml of the suspension was collected and pelleted by centrifugation at 1500 rpm for 10 min. The cell pellet was re-suspended in PBS and centrifuged again at 1500 rpm for 10 min. The resultant pellet was then treated with 5 ml of RBC lysis buffer (4.15 g NH 4 Cl; 0.5 g NaHCO 3 ; 0.0186 g Na 2 -EDTA; 200 ml DW) and kept for 10 min in ice and centrifuged at 1500 rpm for 10 min. Cells were cleared off the lysis buffer by washings the cells twice with PBS. Further, the cells were re-suspended in1.0 ml Roswell Park Memorial Institute (RPMI-1640) medium with 10% foetal calf serum (FCS), antibiotic and antimycotic solution. Cell countand their viability was determined employing 0.1% Trypan blue dye exclusion test 57 . Cells with more than 90 % viability were used for this experiment and the cell density was adjusted to obtain 10 6 cells/mlas per the method standardized previously 18 . Determination of median lethal concentration (LC 50 ) of copper and ubendiamide. LC 50 values of copperand ubendiamidein primary cell culture of rat thymocytes were calculated by Probit analysis using Graph Pad Prism8 software as described earlier 18 . Cyto-genotoxicity studies. LC 50 calculated for copper and ubendiamide @ 40 µM were used for exposure to 10 6 cells/ml of primary culture of rat thymocytesfor further in vitro studies. Antioxidants viz. curcumin (5 and 10 µM), α-tocopherol (5, 10 and 20 µM), resveratrol (5 and 10 µM) and catechin(10 and 20 µM) were used for treatment to assess their ameliorative potential. For each treatment, samples were run in triplicate.
Induction of apoptosis. To determine the comparative protective e cacy of curcumin, α-tocopherol, resveratrol and catechin against copper and ubendiamide-induced apoptosis in primary cell culture of rat thymocytes, 10 6 thymocytes/ml per well were taken in 24 well culture plates and intoxicated with 40 µM/ml concentration of copper and/or ubendiamide in the presence of different concentrations of test antioxidants. After 12h of incubation in CO 2 incubator at 37°C percent apoptotic cells were determined as per the protocol described in TUNEL Assay Kit (Invitrogen, USA). Apoptotic cells, which undergo extensive DNA degradation during the late stages of apoptosis,were examined under blue lter of the uorescent microscope and cells which uoresced brightly were considered as apoptotic.
Micronuclei-induction assay. Freshly isolated thymocytes (10 6 cells/ml/well) were seeded in 24-well plate and exposed to both toxicants and treated with all four test antioxidants except control and DMSO exposed group. Micronuclei-induction assay was performed as per the method described by Hayashi et al. 58 Three hundred cells, both mononuclear and binucleated were examined in each treatment group in blind mode under green lter of the uorescent microscope to determine the frequency of micronuclei formation. DNA fragmentation assay. Genomic DNA from rat thymocytes was isolated by using standard phenol-chloroform DNA isolation protocol 59 . Comet assay. Comet formation in thymocyteswas determined by employing the protocol described by Dhawan et al. 60 . Individual cell/comets were observed and images were captured at 40X magni cation using green lter of uorescent microscope (Microscan 20 PFM, Nitco) and two slides per treatment were observed and at least 50 cells from each slide were scored for comets formation i.e. total of 100 cells/treatment were scored.
Statistical analysis. Data obtained in in vivo studies for blood-biochemical indices, oxidative stress biomarkers,drug metabolizing enzymes and micronuclei formation, were subjected to statistical analysis using One Way analysis of variance(ANOVA)followed by Tukey's Bmultiple comparison Post-hoc test and presented as Mean±SE of the six observations in each treatment group. The Statistical Package for Social Sciences (SPSS 20.00 Software for Windows) was used for all the statistical analysis.Comparisons were made between the values in control groupsand those treated with xenobiotics alone and also between the xenobiotics alone treated and those treated concurrently with αtocopherol. Median lethal concentrations (LC 50 ) of copper and ubendiamidewasdetermined by Probit analysis method using Graph Pad Prism8 software.

In vivo study
Absolute and relative weights, liver and kidneys functions tests. Observations of the absolute and relative weights of liver and kidneys are presented in Table-1. Perusal of data revealed mild to moderate decrease in absolute weights of liver and kidneys, while relative weight of liver only in copper exposed group of rats. However, ubendiamide alone and in combination with copper and other treatment groups were almost comparable with control and vehicle control group of rats. Similarly, liver function biomarkers were evident of non-signi cant difference among different treatment groups where non-signi cant increased serum levels of albumin and decrease in globulin was recorded in combined group instead individual exposure groups compared to control groups. Signi cant (p<0.05) increase in total and direct bilirubin and enzymatic activities of AST, where copper alone and in combination with ubendiamide have also signi cantly increased AST activity compared to control and vehicle control groups (Table-2). AST/ALT ratio were recorded to be highest in copper followed by its combination with ubendiamide, αtocopherol treated groups and ubendiamide alone. Surprisingly, ratio was found to be increased in vehicle treated group also.
Kidney function biomarkers viz. BUN, creatinine and uric acid in copper and ubendiamide combination group were found to be altered with higher values but lower than their alone exposed groups as compared to control and vehicle control, however values obtained were signi cantly higher in intoxicated groups than control group. α-tocopherol treatment has signi cantly reduced these markers with exception of creatinine in ubendiamide alone combined exposure groups (Table 3).
Oxidative stress biomarkersin liver and kidneys. Perusal of data on oxidative stress markers of liver revealed non-signi cant change in the levels of lipid peroxidation while mild to moderate decrease in GSH content in rats exposed to copper and was signi cant (p<0.05) in ubendiamide alone and combined groups as compared to control and vehicle control groups. The enzymatic activity of SOD was reduced appreciably in copper alone and combined group while it was signi cantly (p<0.05) in ubendiamide alone exposed group of rats, whereas a signi cant reduction was recorded in GST activity in combination group than alone exposed groups. Mild to moderate but non-signi cant increase in CAT and signi cant increase in GPx activities in copper and ubendiamide alone, respectively compared to control and vehicle groups ( Fig. 1 a-g).
Oxidative stress biomarkers data of rat kidneys are presented in Fig. 1a-g. Signi cant (p<0.05) increase in lipid peroxidation level and decrease in GSH content were evident in copper and/or ubendiamide exposed groups compared to control and vehicle control groups, however, levels of LPO and decrease in GSH content was relatively lower in combined than alone exposed groups. Signi cant (p<0.05) reduction in SOD and GST activities was recorded in copper and ubendiamide alone compared to control and combination groups. Increased GPx activity was comparable among copper and ubendiamide alone exposed groups while combined group was almost comparable with control groups, however activity of CAT remains unaltered in different intoxicated groups. Following simultaneous treatment with α-tocopherol improved the antioxidant status by restoring enzymatic antioxidant activity and GST content in both liver and kidneys.
Drug metabolizing enzymes. The data on hepatic drug metabolizing enzymes are presented in Fig. 2 a-d. CYP 450 , CYP b5 and APH, UGT and GST activities were observed to be reduced moderately to signi cantly in the groups intoxicated with copper alone whereas, increment in the activities of these enzymes were recorded in ubendiamide alone and in combination with copper compared to copper alone but was comparable to both control group of rats. α-tocopherol have also been observed to produce similar effects as copper when used alone, while simultaneous treatment has increased their activities. Activity of ANDM did not show any signi cant changes across the groups.
Histopathological examination of liver and kidneys. Histopathological examination of liver sections of the rats exposed to copper alone exhibited degenerative changes in hepatocytes, vacuolations in hepatic parenchyma, coagulative necrosis and ruptured hepatic cord ( Fig.   3b) and ubendiamide produced congested central vein with rari cation of hepatic parenchyma along with degenerative changes (Fig. 3c). Combined exposure group has been also observed to produce congestion of central vein, mild degenerative changes in hepatocytes and vacuoles of different sizes indicating mild fatty changes (Fig. 3d). Treatment with α-tocopherol in copper alone exposed group revealed moderate congestion of central vein, cloudy swelling of hepatocytes and mild changes in liver parenchyma (Fig. 3e), similarly ubendiamide alone and combined groups and simultaneous treatment with α-tocopherol showed milder fatty changes in hepatocytes and cloudy swelling in some areas compared to intoxicated groups (Fig. 3g).
Histopathological ndings of kidney tissues in rats exposed to copper revealed severe cellular swelling in renal epithelium along with areas of congestion and desquamation of renal tubular epithelium (Fig. 4b). Flubendiamide exposure leads to degenerated glomerulus along with desquamation of renal tubular epithelium ( Fig. 4c) whereas, combined exposure group revealed degenerated renal tubular epithelium, compressed glomeruli with increase in periglomerular space and diffused cloudy swelling in renal tubular epithelium (Fig. 4d). Compared to the alone and combination exposed group of rats along with α-tocopherol has shown to restoration of the histoarchitecture of kidney tissues with mild degenerative changes in renal tubular and mild cellular swelling in renal tubular epithelium, mild renal tubular damage with few areas of congestion along with decreased areas of periglomerular space ( Fig. 4e-g). Findings of histopathological examination have clearly suggested ameliorative potential of α-tocopherol against toxic insults produced by copper and ubendiamide.
Interactive index. Based on the ndings of biochemical parameters, oxidative stress indices and drug metabolizing enzymes in present in vivo study, combined interactive index (I.I.) analysis study was carried out and the data are summarized in Table 4. Out of thirty-one (31) parameters evaluated, seventeen (17) parameters showed antagonism. Twelve (12) parameters showed potentiation and two (2) parameters were predictive of additive effect. Combined effect of copper and ubendiamide on biochemical parameters viz. total and direct bilirubin, AST, BUN, creatinine and uric acid levels were antagonistic while albumin, globulin and ALT showed potentiation and on total protein it was additive. In liver, LPO, GSH, GPx and total protein showed antagonistic while SOD, GST and CAT showed potentiation. In kidney, LPO, GSH, SOD, GST and GPx showed potentiation while CAT and total protein were additive and antagonistic effect, respectively. CYP 450, ANDM, APH, microsomal protein content, GST, UGT were observed with antagonist effect while CYP b5 showed potentiation.
In vitro study Median lethal concentrations (LC 50 ) of ubendiamide and copper. Flubendiamide and copper (1.0-80 µM) produced concentration-dependent inhibitory effect on viability of thymocytes as evident from the data summarized in Table 5. Based on Probit analysis of the live-dead thymocytes count data, the median lethal concentration (LC 50 ) values of ubendiamide and copper for thymocytes were found to be 43.45 µM (R 2 = 0.98) and 40.78 µM (R 2 = 0.93), respectively. As both these values were almost very close to 40µM, therefore, 40µM was considered as the approximate LC 50 of both toxicants for in vitro studies in primary cell culture of rats thymocytes.
Comparative anti-apoptotic e cacy of resveratrol, curcumin, catechin and α-tocopherol. Copper and ubendiamide @ 40 μM/ml alone treated wells showed higher number of TUNEL positive thymocytes compared to the control and DMSO-treated thymocytes. TUNEL positive thymocytes in different treatment groups, namely copper and/or ubendiamide along with antioxidants used curcumin, α-tocopherol, resveratrol and catechinhave been shown in Fig. (5 and 6). Signi cantly lesser number of TUNEL positive thymocytes compared to the xenobiotics-alone treated groups and decrease in TUNEL positive cells was concentration-dependent. Based on comparative apparent e cacy of these antioxidants at 10 μM concentration, the order of e cacy was resveratrol>curcumin ≥ catechin ≥α-tocopherol against ubendiamide, whereas to copper it was curcumin >α-tocopherol≥ resveratrol>catechin.
Micronuclei induction. Copper (17.1±1.09), ubendiamide (3.63±0.14) and dexamethasone (4.63±0.20) exposure showed signi cantly (p<0.05) higher percentage of micronuclei formation compared to 1.10±0.05 % and 1.80±0.05% in negative and DMSO treated cells, respectively ( Table  6, Fig. 7 and 8) whereas, simultaneous treatment with antioxidants resulted in signi cant (p<0.05) decrease in the number of micronuclei formation except in ubendiamide+curcumin (5 µM) treated group ( Table 6). The order of e cacy of these natural antioxidants at equimolar concentrations of 10 µM against ubendiamide: α-tocopherol > resveratrol >curcumin=catechin, however decrease in number at 20 µM of catechin and α-tocopherol in ubendiamide treated cells. Similarly, tested antioxidants signi cantly (p<0.05) reduced the number of micronuclei in copper treatment and the comparative e cacy at 10 µM concentration of each of these against copper was: curcumin > α-tocopherol >catechin ≥ resveratrol, however, α-tocopherol (20 µM) resulted drastic reduction in percent micronuclei formed compared to 10 µM of the same (Table 6; Fig. 8). Further, based on the number of micronuclei formed, copper was found to be more genotoxic than ubendiamide. DNA fragmentation. Fig.9 and 10 illustrate the DNA fragmentation of rat thymocytes following in vitro exposure to copper and/or ubendiamide and simultaneous treatment with test antioxidants for 12 h. DNA from the copper, ubendiamide and dexamethasone exposed thymocytes showed severe shearing of DNA than control groups where shearing was not observed. Simultaneous treatment with curcumin at both concentrations has been observed to be more protective on DNA damage than other test antioxidants.
Comet assay. Observations of per cent comet formation of different treatment groups are summarized in Table 6. Perusal of the data revealed that DMSO did not induce comet formation while dexamethasone conspicuously promoted comet formation as these values were found to be 5% and 29.57%, respectively compared to control (4.06%). Copper and ubendiamide exposure resulted in 28.57 % and 23.52 comets, respectively (Table 6; Fig. 11). Concurrent treatment with antioxidants signi cantly reduced number of comets formed irrespective of toxicants (Table 6) and the comparative e cacy order of these natural antioxidants at equimolar level (10 µM) against ubendiamide was curcumin> resveratrol>catechin> α-tocopherol. But at 20 µM concentration level, catechin was comparatively more effective than α-tocopherol. For copper the order of e cacy at equimolar concentration (10 µM) was curcumin>resveratrol>α-tocopherol≥catechin. However, at 20 µM, α-tocopherol was more effective in decreasing copper-induced comet formation.

Discussion
Copper is an essential trace element required for numerous biological activities responsible for normal development/growth and are building blocks of cuproproteins like ceruloplasmin, tyrosinase and superoxide dismutase and supports the function of numerous cellular enzymes 61 .
However, overe-xposure at sublethal concentration for several weeks may produce detrimental effects in kidney 62 , spleen and thymus 63 and if the concentration exceeds the body tolerance level, may exert hepatotoxicity 62,64,65 . Liver is regarded as the main target organ where hepatocytes are the target cells for copper toxicity 65 . In presentstudy, signi cant decrease in absolute weight of liver in rats exposed to copper only. In contrast, no appreciable changes in absolute and relative weights of liver and kidneys in rats exposed to copper and/or ubendiamide were recorded. Elevated serum levels of total and direct bilirubin, AST, AST:ALT ratio in copper and/or ubendiamide group compared to control and vehicle control groups. In harmony with our ndings, Manna et al. 66 , Kumar et al. 67 Mohammadyari et al. 68 , Arafa et al. 69 , El-Magd et al. 70 , Abdelazeim et al. 71 have also demonstrated elevated levels of serum total bilirubin, AST, and AST:ALT ratio in copper oxide nanoparticles after oral administration to rats. Increased AST:ALT ratio in copper intoxicated group indicates liver damage leading to cellular leakage. In our previous study, we have reported marked increase (15.15 % and 27.27%) in hepatic copper concentration in copper alone and in combination with ubendiamide groups 64 , this could be a reason of suppressing hepatic development. Besides, our results also demonstrated degenerative changes in hepatocytes, vacuolations in hepatic parenchyma, coagulative necrosis, ruptured hepatic cord, and congestion of central vein in the rats exposed to copper (Fig. 6b and 6d). Thus, the liver toxicity is clearly evident with increased serum bilirubin and transaminases 65,67 .
Simultaneously elevated BUN, creatinine and uric acid suggested kidney dysfunction and is substantiated by degenerative changes, desquamation of renal tubular epithelium, proximal-tubule necrosis and swelling of tubular epithelium in present study after histopathological examination con rms nephrotoxic potential of copper and ubendiamide. The above manifestations induced by copper were consistent with the oral ingestion of copper sulphate 72,73 , copper nano-particles 74,66,75 and slight increase in levels of serum and urinary creatinine 77,67 .Increased levels of serum albumin and decrease in globulin is an indicator of dehydration 17 and liver/kidney damage with decreased synthesis, nephrotic syndrome where there is renal protein loss, respectively in copper and/or ubendiamide groups. On the other hand, toxicity data of ubendiamide is scanty however reduced serum albumin and signi cant increase in total and direct bilirubin suggested marked liver damage and loss of function leading to hepatic insu ciency in contrast to the previous studies 5, 13,16 who have reported mild toxic effects on liver, thyroid gland, haemopoietic system and testes.
Long term exposure of copper, their nano-particles and copper saltshave been reported to produce hepatotoxicity and nephrotoxicity in rats and mice 69,77,73,78,65 by inducing oxidativestress which is directly linked to apoptosis and genotoxicity 71 . Numerous mechanisms have been proposed to explain cellular toxicity of copper exposure, however most accepted one is stimulation of ROS production which results in oxidative stress 79 . Hosseini et al. 80 explained that excess of deposited copper increases lipid peroxidation where excess deposition of copper in liver has already been reported in our previous study 64 . Oxidative stress is an important mechanism of toxicity for number of heavy metals and insecticide toxicity. Copper is a redox heavy metal and its redox identity contributes potential toxicity 81,77 which induces oxidative stress by increased production of reactive oxygen species (ROS) and depletion of cellular antioxidant capacity affecting cellular integrity by causing peroxidative degeneration of polyunsaturated fatty acids in membrane lipids when generation of ROS exceeds antioxidant defence mechanisms 82,71 and produces histopathological abnormalities in different tissues due to ROS generation and DNA damage 83 . The results of this study showed increased ROS levels and decreased GSH content and reduction in the activities of SOD and GST while increase in GPx activity. Present ndings are in line with the previous toxicity studies of copper and copper nanoparticles 84,85,67,77,72 and ubendiamide toxicity studies [86][87][88] in liver and kidneys.
Orally administered copper gets absorbed through intestineand reaches liver which is suggested to be the primary storage site, where transition metal copper undergoes redox cycling and promotes ROS production and in presence of GSH and superoxides, Cu ++ gets converted to Cu + which further leads to formation of hydrogen peroxides and hydroxyl radicals 89,90 . Further, Cu interacts with GSH and forms Cu(I)-[GSH] (2) complex and gives superoxide and by action of SOD superoxide gets converted to H 2 O 2 91,90,92 . Reduced glutathione is very important for survival of cells as it provides protection against free radicals-induced cellular damage. Following exposure of rats to copper or ubendiamide in the present study, signi cant decrease in GSH levels in liver and kidneys of rats indicates its increased utilization in conjugation with xenobiotics and it is facilitated by glutathione-S-transferase. Thus, there may be detoxi cation of the xenobiotics which are converted to less reactive chemical moieties or it prevents interaction of xenobiotics with cellular proteins and nucleic acids 93,94 . GSH also binds with Cu + and physiologically regulates intracellular copper levels 95 and protects against cellular damage induced by GSH-copper complexation and Cu ++ reduction. Signi cant decrease in glutathione-S-transferase (GST) activity in liver and kidneys may be responsible for comparatively better detoxifying ability of this organ and thus comparatively lesser damage to liver compared to kidneys where moderate decrease in GST activity and marked decrease in GSH level was observed in rats of the ubendiamide-treatment group. Our ndings are in agreement with the observations of other researchers who have also reported reduction in GST activity following exposure of mice and/or rats to cypermethrin, dimethoate, deltamethrin and pronil [96][97][98] . This might be the one possible reasonfor the reduced GSH level of copper-exposed animals. Further, copper exposure signi cantly raised catalase and GPx activities suggest the activation of defensive mechanisms to protect against copperassociated free radical generation. During oxidative stress conditions increase in SOD, GST, CAT and GPx activities are well documented, however overall comparison of the alterations in the values of different oxidative stress biomarkers observed in liver and kidneys of the rats of groups III, IV and V revealed that kidney is comparatively more vulnerable to oxidative stress-induced insult than liver, may be due to better regenerative and reparative ability of liver 99 . Also, increased GPx activity in liver and kidneys following exposure to copper and ubendiamide is in agreement with the toxicity studies of pesticides and Cu, which it may be due to increased formation of hydroperoxides in kidneys 100 and increase in GPx and catalase activities in liver and/or kidneys of the rats of ubendiamide or copper exposed groups in the present study might be due to adaptive response of the body against the generated free radicals and increase levels of iron in liver of ubendiamide alone and in combination with copper 64 . However, reduced SOD activity in kidney and liver tissues in present study could be due to excessive accumulation of Cu in the liver 77 .
Drug metabolizing enzymes of phase-I and II reactions have been reported to protect liver from dysfunctions caused by xenobiotics. Signi cant alterations in the activity of different DMEs in the rats exposed to copper and ubendiamide alone and/or in combination groups in the present study revealed decrease in CYPs (CYP 450 and CYP b 5 ) activities and impaired function of NADPH-P 450 reductase in rats exposed to copper could be due to oxidative stress induced generation of reactive oxygen species which inhibit synthesis of heam-prosthetic group 101,102,103, 104 Kim et al. 105 reported the inhibitory effect of Cu 2+ on the P 450 -catalyzed reactions due to inability of an e cient electron transfer from NPR to P 450 and conformational changes in the P 450 . However, inhibition of CYP systems can be effective in protecting liver against xenobiotic toxicity 106 .
On the other hand, ubendiamide promotes binding to the lipophilic site of cytochrome P 450 and induces signi cant induction in activity of the cytochrome-P 450 and cytochrome b 5 is in agreement with the ndings of Motoba 107 .The increased cytochrome-P 450 activity might be due to increased de novo synthesis of proteins could be due to increased gene transcription and further decrease in rate of degradation of enzymes 108 . Simultaneous treatment with α-tocopherol, activities of cytochrome-P 450 and cytochrome b 5 were almost comparable to control groups suggesting the reparative potential of α-tocopherol. Thus, inhibition of metabolism by α-tocopherol is due to xenobiotics-antioxidant interactions and occurs by two basic mechanisms 1) competition of copper for access to the catalytic site of the relevant drug-metabolizing enzyme and 2) formation of a catalytically inactive, covalently bound complex between a metabolite of the substrate xenobiotics and the P 450 enzyme 109 , because compounds are metabolized by a variety of different enzymic reactions, and the simultaneous ingestion of two or more compounds may lead to their competition for various enzymic pathways, leading to decreased rates of metabolic transformation.
Aniline-P-hydroxylase (APH) and aminopyre-N-demthylase (ANDM) activities did not alter signi cantly in any of the treatment groups except in copper-intoxicated group where insigni cant decrease in APH activity was observed compared to those in rats of control group, shows that detoxi cation of copper and ubendiamide through ANDM may not be of considerable biological signi cance. Glutathione-S-Transferase and UDP-glucuronosyltransferase (UGT) are generally responsible for the detoxi cation of xenobiotics. Toxic effects of a given xenobiotic are the net result of a balance between activating and detoxifying pathways and factors modulating the overall biotransformation capacity are of paramount importance. Glutathione-S-transferases (GST) and UDP-glucuronosyltransferase (UGT) activities were found to be signi cantly decreased in copper alone group and concurrent treatment with α-tocopherol did not signi cantly improve the GST and UGT activities are in agreement with the ndings of Nakahama et al. 115 who reported heavy metal-induced inhibition of UGT. UGT prevents the accumulation of potentially toxic compounds and/or their subsequent bioactivation to more toxic intermediates. The suppression in GST and UGT activities in copper-treated group is due to increased lipid peroxidation, generation of super oxide by copper (Fenton and/or Haber-Weiss reactions) which disorganizes the membrane lipid bilayer and modify activation state of UGT. However, in contrast to present ndings, Rajpoot et al. 116 observed an increase in UGT activity in rats following exposure to xenobiotics and ascorbic acid.
Simultaneous exposure of rats to copperand ubendiamide, the toxic effects were not found to be signi cantly augmented in the present study; therefore, our observation is contrary to the ndings of other researchers who have reported augmented toxic effects of agrochemicals and metals on concurrent exposure [116][117][118] . Co-exposure of copper and ubendiamide did not have any strong impact on the activities of CYP 450 , ANDM, APH, GST and UGT and further microsomal or cytosolic protein contents in rat liver due to antagonistic effect observed in interactive index study analysis suggesting that the effect produced by ubendiamide was not appreciably affected by copper and vice-versa, and ubendiamide and copper might not have reciprocally and signi cantly affected the metabolism of each other by altering their activation and/or their detoxication mechanism. Similar results have also been reported following combined exposure to arsenite + parathion 119 and malathion+arsenite 120 . However, combined effect of copper and ubendiamide on kidneys tissue oxidative stress biomarker (LPO, GSH, SOD, GST and GPx) was observed potentiating in interactive index analysis while catalase and total protein was shown was additive and antagonistic, respectively, suggest kidneys was more vulnerable to toxic insult than liver following copper and ubendiamide in combination. The results of present study indicated moderate to marked and/or signi cant alterations in biochemical parameters, oxidative stress biomarkers and histology in liver and kidneys and drug metabolizing enzymes activity in liver of the rats following exposure to the test xenobiotics suggest that the test xenobiotics induces oxidative stress which is ultimately responsible for hepatic-and renal-insults as evident from the correlation and association data between the oxidative stress markers and liver and kidney function test markers. However, hepatic degenerative changes observed in rats of ubendiamide-treated group were similar to those reported in JMPR following 13 weeks exposure of rats to ubendiamide 6 . Although ubendiamide is a category III compound having low order of toxicity 5 but hepatotoxic and nephrotoxic effects of ubendiamide, as observed in the present study, seem to be of great concern, therefore, further studies are warranted.
Thymus is a primarywhile spleen is the secondary lymphatic organ and both play a vital role in building the immune system. T-lymphocytes from thymus mature into functional defence cells like T effector cells, T-killer cells, T helper cells etc. and are responsible for coordinating the innate and the adaptive immune system. In view of the different nature and functions of thethymocytes and splenocytes of thymus and spleen, respectively are likely to have differential susceptibility to toxic effects of xenobiotics. In our previous study, we have reported ubendiamide and copper induced-cytogenotoxic effects in splenocytes 18 . Copper-induced activation of both the intrinsic and extrinsic apoptotic pathways and mitochondrial ROS generation in redox cycling are responsible for cytotoxic and DNA strand breaking effects 121,122 .Similar oxidative stress induced DNA damage have been reported following exposure to pesticides, metals and environmental pollutants 123,124,125 .Therefore, in this study, we evaluated the vulnerability of thymocytes to cyto-genotoxic effects of both these xenobiotics using TUNEL assay, DNA fragmentation assay and micronuclei induction assay with an endeavour to establish if there is any difference in cyto-genotoxic effects onthymocytes and splenocytes.
TUNEL assay is a gold-standard for cellular self-destruction by apoptosis and signi cant increase in number of TUNEL + ve thymocytes following exposure to ubendiamide and copper evidently suggest the involvement of apoptotic signaling cascades in cytotoxic effects. Flubendiamide and copper possibly interact with double-stranded DNA (dsDNA) and induce cellular damage in thymocytic DNA and TdT binds with 3'OH label blunt ends of dsDNA and serve as a marker of apoptosis. Lu et al. 126 and Tufan et al. 127 have also reported signi cant increase in number of TUNEL + ve fragmented DNA in brain and hippocampus of copper-treated mice and rats respectively. Similar increase in TUNEL + ve cells has been recently reported in splenocytes as well following exposure to ubendiamide and copper 18 .
DNA fragmentation assay in the present study also revealed that the pattern of DNA shearing in copper and ubendiamide-treated thymocytes was similar to that observed in dexamethasone-treated group.DNA damaging effect of ubendiamide and copper in thymocytes in the present study is also substantiated by Comet assay data wherein number of comets formed in thymocytes treated with ubendiamide (23.52%) and copper (28.57 %) were almost similar to those observed in the positive control group (29.57%) treated with dexamethasone in the present study and also reported earlier in our study withsplenocytes 18 . Thus suggesting that thymocytes and splenocytes are almost equally susceptible to ubendiamide and copper induced genotoxic effects.
Dexamethasone-induces apoptosis in thymocytes by causing mitochondrial dysfunction, caspase-3 activation, reactive oxygen species (ROS) production and oxidative damage 128 whereas ROS generated interact with cellular DNA and produced comet formationinducing singleand double-strand breaks of DNA. Similar increase in DNA fragmentation following exposure to copper has been reported by Vidyashankar and Patki 129 in HepG2 cells. In the present study, oxidative stress in liver and kidneys could be the reason behind DNA damage and comet formation in thymocytes. Similar results of DNA damage and comet formation has been reported in our previous study conducted on spleen cells 18 .
Micronuclei induction assay detectsclastogens and aneugens as well as mitotic delay, apoptosis, chromosome breakage and chromosome loss 130,131 and is used for evaluation of the genotoxic potential of xenobiotics 132 . Compared to 1.1% micronuclei formed in control group and 4.63% in dexamethasone-exposed group, signi cant increase in number of the micronuclei formation in thymocytes of copper (17.1%) and ubendiamide (3.63%) groups suggest genotoxic potential of copper and ubendiamide, however thymocytes have been found to be more prone to cyto-genotoxic effects. Cyto-genotoxic potential of copper have been reported in mice 133 and other tissues 134 and rat splenocytes 18 . However, comparison of the micronuclei data of thymocytes (present study) and of splenocytes reported recently from our laboratory 18 revealed that splenocytes were more susceptible to micronuclei formation following exposure to ubendiamide while thymocytes were more susceptible to copper-induced micronuclei formation. Thus the ndings on cell viability, micronuclei and comets formation data obviously suggest differential susceptibilities of different cells to cyto-genotoxic effects of different xenobiotics.
Precise mechanism of cyto-genotoxic effects of ubendiamide is still not clearbut possibly the cytotoxic effects on thymocytesis due to ROSinduced oxidative stress and apoptosis due to activation of caspases, certain kinases and other cell signaling molecules 135 . Few reports have suggested that several metals and insecticides induces ROS generation which leads to increased cytosolic free Ca 2+ concentration and further impairs Ca 2+ clearance system, as a result mitochondrial depolarization and ultimately cell dysfunctions and apoptosis 136,137 . Our observations on thymocytes in the present study are in agreement with ubendiamide-induce doxidative stress in liver and kidneys of rats and also on acute exposure in Daphnia magna 138 .
Plants-based polyphenols (resveratrol, curcumin, and catechin) and α-tocopherol have been reported to possess immunomodulatory 139,140 and strong antioxidant activities 141 , promotion, progression and cellular proliferation 142,143 , and cytotoxicity induced by metals and insecticides 144,145,118,146 . Concurrent treatment with polyphenols (resveratrol, curcumin, and catechin) or α-tocopherol in present study showed decrease in TUNEL + ve cells, per cent micronuclei and comet formation in thymocytes evidently suggest the ameliorative potential against ubendiamide and copper-induced toxic effects. Observations are in agreement with our earlier report against splenocytes 18 . However, all the tested antioxidants failed except curcumin in preventing thymocytic DNA shearing exposed to xenobiotics.
Cyto-and geno-protective potential of resveratrol and curcumin against ubendiamide and/or copper toxicity in thymocyte sreduced the TUNEL + ve cells, comet formation, micronuclei-induction and reduction in DNA fragmentation might be due to its unique structure which enables the lipids to conjugate with 3' position of curcumin 147 , radical trapping ability as a chain-breaking antioxidant, inhibition of lipid peroxidation 148 . Antioxidant action of resveratrol is due to scavenging of hydroxyl, superoxide and other [149][150][151] and prevents lipid peroxidation in cell membranes and DNA lesions and fragmentation [151][152][153] . Thus resveratrol and curcumin can reduce the amount of reactive oxygen species (ROS) in response to oxidative stress 154 . Similar, cyto-protective and ant-apoptotic effects of curcumin against other xenobiotics like copper, arsenic, uoride induced DNA damage have also been reported [155][156][157][158]118 . Although in the present study, we have not researched the precise pathway of cyto-and geno-protective effect of any of the natural phytoconstituents, but possibility of modulation of different above mentioned mechanisms/pathways involved in cyto-and geno-protective effect of resveratrol cannot be ruled out.
Besides these, catechin has ability to chelate copper (II) and decreases expression of proapoptotic genes (Bax, Bad, Mdm2), induces antiapoptotic genes (Bcl-2, Bcl-w, Bcl-x L ) 159 and free radical scavenging activity by forming stable semiquinone free radicals which prevent the deaminating ability of free radicals. Possible involvement of similar mechanism(s) in protective effect of catechin against ubendiamide and copper-induced cyto-genotoxicity cannot be ruled out as both these xenobiotics are known to cause oxidative stress; therefore, further studies on the mechanistic pathways of geno-protective effect of catechin against these xenobiotics are required 160 .
α-tocopherol (α-TOH) is known to protect cellular membranes and lipoproteins from peroxidation and results in formation of an unreactive αtocopheroxyl radical 161 that can be recycled back to active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol 162 . Lipophilic nature of α-tocopherol facilitates its entry through membrane and thereby quenches the free radical species, terminates lipid peroxidation chain reaction and interferes with initiation and progression of xenobiotics-induced oxidative damage 163  These results along with histological examination and drug metabolizing enzymes activity evidently suggest the ameliorative potential of αtocopherol against hepato-and nephrotoxicity especially induced by copper, however, it was not found to be so effective against ubendiamideinduced tissue-insults. Our nding on protective and reparative activity of α-tocopherol corroborates well with the observations of several other researchers against certain pesticides 171,172,16,18 and copper 21,173,16,18 due to its chain breaking antioxidant activity and free radicals scavenging activity.