Toxicology Mechanisms and Methods

Toxicology Mechanisms and Methods ISSN: 1537-6516 (Print) 1537-6524 (Online) Journal homepage: https://www.tandfonline.com/loi/itxm20 Triflumuron induces genotoxicity in both mice bone marrow cells and human colon cancer cell line Rim Timoumi, Ines Amara, Yossra Ayed, Intidhar Ben Salem & Salwa AbidEssefi To cite this article: Rim Timoumi, Ines Amara, Yossra Ayed, Intidhar Ben Salem & Salwa AbidEssefi (2020): Triflumuron induces genotoxicity in both mice bone marrow cells and human colon cancer cell line, Toxicology Mechanisms and Methods, DOI: 10.1080/15376516.2020.1758981 To link to this article: https://doi.org/10.1080/15376516.2020.1758981 Accepted author version posted online: 21 Apr 2020. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=itxm20 Triflumuron induces genotoxicity in both mice bone marrow cells and human colon cancer cell line Rim Timoumi1, 2, Ines Amara1, 2, Yossra Ayed1, Intidhar Ben Salem1 and Salwa Abid-Essefi1* 1 University of Monastir, Faculty of Dental Medicine, Laboratory for Research on Biologically Compatible Compounds (LRSBC),LR01SE17, 5019 Monastir, Tunisia; University of Monastir, Higher Institute of Biotechnology of Monastir, Avenue Taher Hadded, Monastir 5000, t 2 us cr ip Tunisia an *Corresponding author: M Pr. Salwa ABID. Laboratory for Research on Biologically Compatible Compounds (LRSBC), Faculty of Dental Medicine, University of Monastir. Rue Avicenne, 5019 ce pt e Tel.: +216 73 42 55 50 d Monastir, Tunisia. Fax: +216 73 42 55 50 Ac E-mail address: salwaabid@yahoo.fr Abstract Triflumuron (TFM) is an insect growth regulator (IGR), an insecticide commonly used over the world. It is known for its several toxic manifestations, such as reprotoxicity, immunotoxicity and hematotoxicity, which could affect public health. However, studies that reveal its toxic effects on mammalians are limited. To reach this purpose, our study aimed to elucidate the eventual genotoxic effects of TFM in mice bone marrow cells and in HCT 116 cells after a short term exposition. TFM was administered intraperitoneally to Balb/C male ip t mice at doses of 250, 350 and 500 mg/kg bw for 24 h. Genotoxicity was monitored in bone cr marrow cells using the comet test, the micronucleus test and the chromosome aberration us assay. Our results showed that TFM induced DNA damages in a dose-dependent manner. This genotoxicity was confirmed also in vitro on human intestinal cells HCT 116 using the comet an test. It was then asked whether this genotoxicity induced by TFM could be due to an oxidative stress. Thus, we found that TFM significantly decreased HCT 116 cell viability. In addition, it M induced the generation of reactive oxygen species (ROS) followed by lipid peroxidation as d revealed by the increase in the malondialdehyde (MDA) levels. Similarly, the activation of the ce pt e antioxidant enzymes (catalase and superoxide dismutase) was also observed. Our results indicated that, in our experimental conditions, TFM had a genotoxic effect on bone morrow cells and in HCT 116 cells. Moreover, we demonstrated that this Ac genotoxicity passes through an oxidative stress. Key words: Triflumuron, Genotoxicity, Comet assay, Micronucleus test, chromosome aberration, bone morrow cells, HCT 116 Cells. Introduction Currently, pesticides still play an important role in vector control and agriculture protection crops. However, some of pesticide such as organophosphates and pyrethroids had been hampered due to resistance (Reynolds 1987; Mulla et al. 2003; Tunaz and Uygun 2004; Fontoura et al. 2012). That’s why, the appearance of new products is needed (Ansari et al. 2006; Martins et al. 2008). t Among this new category of synthetic products, we found Insect Growth Regulators ip (IGRs) insecticides. This family is used to control agrochemical products, crops and public cr health (Lowden et al. 2007). IGRs compounds have been extremely studied since their us discovery in the 1970’s (Farnesi et al. 2012). They inhibit chitin synthesis which is a molecule presents in the insect’s life stages and which plays a crucial structural role in many eukaryotic an mammals. It forms also, the external cuticle of insects. There, by acting with chitin, these M compounds may alter the formation of the cuticle or cause the death of the insect by famine (Lowden et al. 2007). d Triflumuron (TFM), 2-chloro-N-[[[4-(trifluoromethoxy) phenyl] amino] carbonyl] ce pt e benzamide which belong to IGRs, is known commercially by the name of Starycide 480 Sc (Batra et al. 2005). TFM is commonly used over the world to protect crops (apple, tomato, fruit, soybeans, vegetables, forest trees, cotton, potato and soya bean) and domestic animals Ac (horse, sheep and chicken) against a broad spectrum of parasites which causes animal and human diseases (Lowden et al. 2007). TFM had distinct mechanisms of action and is more selective, in comparison with classic pesticides such as organophosphate and synthetic pyrethrinoids. Indeed, TFM interfere with the insect moult causing then the death of the insect in the next moult (Wilson and Cryan 1997; Vasuki 1992 a, 1992b). TFM acts by inhibiting chitin synthesis. It prevents the insertion of Nacetylglucosamine into the biosynthesis of chitin. Indeed, chitin is a linear polymer composed of N-acetyl-glucosamines. The biosynthesis of this polymer involves several enzymes to convert different sugars (glucose, trehalose) into UPD-N-acetyl-glucosamine (Merzendorfer 2011). The last step is synthesized by a very important enzyme, chitin synthase. Thus, by blocking the transport of N-acetylglucosamine across the epithelial membrane, TFM could therefore act as a general stressor making the insect more susceptible to diseases. This ip t facilitates the entry of pathogenic fungi into the insect through the weakening of their cuticle cr which accelerates their death (Tunaz and Uygun 2004). us Such as all other pesticides, TFM is known by its several effects on both mammalian and environment. In fact, it exhibits acute toxicity particularly in aquatic organism and an invertebrate animals following oral, dermal, inhalative, intraperitoneal and subcutaneous administration. Moreover, the investigation of the acute toxicity of TFM was evaluated in M rats, dogs and rabbits, successively, after oral, inhalative and dermal administration of this d compound (EFSA 2011; Waller and Lacery 1986). ce pt e Benzoylurea insecticides are known by their ovicidal acticity. Indeed, the treatment of insects of the genus Aedes albopictus with TFM inhibits the occlusion of eggs. Also, an abnormal morphology of the egg shell has developed (Suman et al. 2013). In addition, TFM Ac acts on the offspring of insects, especially at the time of pupal formation. Like most IGRs, it causes malformations of pupae from treated females (Ouédrago 1998) and death of the embryo (Itard 1986). It provokes high rates of abortion in infected insects (Langley 1995). Insects from the pupae of treated females die during hatching or in a few days later (Ouédrago 1998). Moreover, oral administration of TFM to male Wistar rats for 28 days resulted in elevated hemoglobin accompanied by an increase in the number of reticulocytes (Tasheva and Hristeva 1993). Recently, TFM has been shown to promote the metastasis of liver cancer cells (Hep G2) by interfering with hypoxia-inducible factor 1α (Ning et al. 2018). To our knowledge, the genotoxicity of TFM was evaluated neither in Balb/c male mice bone morrow cells nor in HCT 116 cells. Although previous reviews showed no mutagenic or carcinogenic effects using CHO cells, human lymphocytes and rat hepatocytes (EFSA, 2011), the aim of our current study was t (i) to evaluate the eventual genotoxic effect of TFM in both bone morrow cells and in HCT ip 116 cells and (ii) to determine the involvement of oxidative stress in the possible genotoxic cr effect of TFM. For this purpose, we measured DNA damage in experimental mice bone us morrow cells and in HCT 116 using the comet assay. Also, the micronucleus (MN) test and the chromosome aberrations test were carried out. Oxidative stress involvement was assessed an by the measurement of ROS generation, MDA level, and some anti-oxidative enzymes Ac ce pt e d M activities. Materials and Methods Chemicals Triflumuron (TFM) known chemically as, 2-chloro-N-[[[4-(trifluoromethoxy) phenyl] amino] carbonyl] benzamide, (CAS Registry Number: 64628-44-0 and purity > 98 %) was supplied by Sigma-Aldrich (St. Louis, MO, USA). 3-4,5-Dimethylthiazol-2-yl, 2,5diphenyltetrazolium bromide (MTT), cell culture medium (RPMI 1640), foetal calf serum t (FCS), phosphate buffer saline (PBS), trypsin-EDTA, penicillin and streptomycin mixture ip and L-glutamine (200 mM) were from GIBCO-BCL (UK). 2,7-Dichlorofluoresce diacetate cr (DCFH-DA) was supplied by Molecular Probes (CergyPontoise, France). Low melting point us agarose (LMA) and normal melting point agarose (NMA) were purchased from Sigma (St. Louis, MO, USA). All other chemicals used were of analytical grade. an Biological material M Animals d We carried out our study on Balb/C male mice weighting between 25 and 30 g and ce pt e having an age of 8 weeks. These mice were purchased from the central pharmacy (SIPHAT, Tunis, Tunisia). Before starting our experiment, mice stayed one week under adequate laboratory conditions: a temperature of 22 ± 3 ° C, a relative humidity of 55 ± 5 %, and a dark light cycle of 12 h. The animals received a normal diet (SICO, Tunis, Tunisia) and drink tap Ac water ad libitum. The experimental procedures were carried out according to the National Institute of Health Guidelines for Animal Care and approved by the local Ethics Committee. After acclimatizing the mice, they were subdivided into four groups of 18 mice each. TFM at doses of 250, 350 and 500 mg/kg bw was administered intraperitoneally (i.p.) respectively to animals of groups 2, 3 and 4. These doses are corresponding respectively to 5, 7 and 10 % of the LD 50 (The Good Scents Company Information 1980). Group 1, which is the control group, received equivalent amount of vehicle: Ethanol/Water (1:1, v: v). A single administration by intra-peritoneal injection of 100 µl of solutions was performed. 24 h after treatments, animals were sacrificed by cervical dislocation. Under these conditions, we have a compromise between a moderate concentration of TFM and observable toxic effects. Animals within different treatment groups were divided into 3 subgroups: A (for the comet assay), B (for the MN assay), and C (for the chromosome aberration assay) (6 animals per subgroup) and received their respective treatment. All animals were sacrificed by cervical ip t dislocation. cr Cell culture and treatment us Human colon carcinoma cells HCT 116 were cultured in RPMI, supplemented with 10 % FBS, 1 % L-glutamine (200 mM), 1 % of mixture penicillin (100 IU/ml) and streptomycin an (100 g/ml), at 37 °C with 5 % CO2. TFM was dissolved in DMSO. The different M concentrations of TFM (100 to 1000 μM) were added to the cell medium when the cells were ce pt e Comet assay in vivo d in the exponential phase of growth. After sacrificing the animals of the subgroup A, both of femurs and tibias were removed and the content was directly flushed out with the help of a 24-gauge needle into a microcentrifuge tube. The cell suspension was prepared in PBS. Bone marrow cell Ac suspensions (60 µl) were embedded in 60 µl of 1 % low melting point agarose and spread on pre-coated slide with a layer of 1 % (w/v) normal melting point agarose prepared in PBS (Singh et al., 1988). Then, cells were then lysed in a buffer containing 2.5 mol/l NaCl, 100 mmol/l EDTA, and 10 mmol/L Tris (pH = 10.0) with freshly prepared 1 % Triton X-100 and 10 % dimethyl sulfoxide (DMSO) for 24 hours at 4°C. After lysis, slides were rinsed 3 times in deionized water to remove salt and detergent. Slides were placed in a horizontal electrophoresis unit and DNA was allowed to unwind for 20 minutes in alkaline solution containing 300 mmol/l NaOH and 1 mmol/l EDTA, pH > 13. The DNA was electrophoresed for 15 minutes at 300 mA and 25 V (0.90 V/cm). The slides were neutralized with 0.4 mol/L Tris (pH 7.5), stained with ethidium bromide (20 µg/ml) before examination with a Nikon Eclipse TE 300 fluorescence microscopes (Nikon, Tokyo, Japan). A total of 150 comets on each randomly coded slide were visually scored according to ip t the relative intensity of fluorescence in the tail and classified as belonging to 1 of 5 classes. cr Each comet class was given a value of 0, 1, 2, 3, or 4 (from undamaged, 0, to maximally us damaged, 4) as described previously by Collins and his team (1996). The total score was calculated by the following equation: (percentage of cells in class 0 × 0) + (percentage of cells an in class 1 × 1) + (percentage of cells in class 2 × 2) + (percentage of cells in class 3 × 3) + M (percentage of cells in class 4 × 4). Consequently, the total score ranges from 0 to 450. 2.4. Mice bone marrow micronuclei assay d Immediately after the animals were sacrificed, femur and tibia of the mice in the ce pt e subgroup B were freed from adherent tissues and were dissected out. The bone marrow was sampled by injection of filtered foetal calf serum using a syringe. The collected cells were centrifuged at 2000 g for 5 min, a little volume of supernatant was discarded and the cells Ac were re-suspended in the remaining fluid. A small drop of the re-suspended cell pellet was spread on a glass slide, fixed in absolute methanol for 5 min and air-dried for conservation at room temperature. Air dried slides were stained for 15 min in phosphate buffered saline (0.15 M; Ph7.4) containing 10 µg / ml of acridine orange (freshly prepared), rinsed in the same buffer for 15 min and allowed to dry in the dark at room temperature. The slides were scored immediately under 1000 magnification using a fluorescence microscope (Nikon Eclipse E 400). Two thousand polychromatic erythrocytes (PCE) were examined from each animal and the number of micro-nucleated polychromatic erythrocytes (PCEMN) was recorded. PCEMN appear red with one or more yellow-fluorescent corpuscles, which are micronuclei (MN). Scoring of micronuclei was performed according to criteria described by Hayashi and coworkers (1983). These criteria are based essentially on the diameter and the shape of the MN. The number of PCEMN among 2000 PCE per mouse sample was determined to appreciate the induction of micronuclei. t Chromosome aberration assay ip Bone-marrow preparation cr Bone-marrow cells were obtained according to the technique of Yosida and Amano us (1965). Briefly, femur and tibia of mice in the subgroup C were removed immediately after animal sacrifice and bone marrow was flushed out with a KCl solution (0.075 M, 37 ◦C) by an use of a syringe. The bone-marrow cell suspension was incubated for 20 min at 37 ◦C and M centrifuged at 1200×g for 10 min. The supernatant was discarded, the pellet was resuspended in 5 ml of fixative (acetic acid/methanol, 1:3, v/v), centrifuged (1200×g for 10 min) and the d supernatant was discarded again. This step was repeated three times in order to clean the ce pt e pellet. Finally, the pellet was re-suspended in 1 ml of the above fixative solution used for chromosome preparation. Chromosome preparation Ac The preparation of the chromosome was carried out according to Evans and co- workers (1960) with some modifications. Indeed, the cell suspension was drained on a glass slide thus giving smears on a flame for 5 s. Then the blades were dried at the room temperature and then they were either kept at room temperature or they were colored directly by the Giemsa. The Giemsa working solution was freshly prepared (4 ml in 100 ml) in phosphate buffer (0.15 M, pH 7.2). Slides were left for 15 min in the staining solution, then rinsed with water and allowed to dry at room temperature. Slide analysis The analysis of the slides was carried out using an optical microscope (Carl Zeiss, Germany) at a magnification of 100 times. Of the five hundred metaphases, the anomalies were analyzed for each group. These abnormalities may be gaps, rings, chromosome breaks t and centric fusions and have been expressed as percentages of total metaphases per group. ip Cell toxicity assay cr MTT assay us The MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay (a an tetrazolium salt reduction assay) was carried out. Indeed, this test provides sensitive measurements of the normal metabolic status of cells particularly that of the mitochondrion, M where measurements reflect early cellular redox changes (Mosman 1983). HCT-116 cells d were seeded in 96-well plates at 2.5 × 104 cells/well and were treated with the different ce pt e concentrations of TFM ranged from 100 to 1000 µM for 24h at 37 °C. Wells containing untreated cells served as a negative control. After treatment, cells were incubated with the MTT solution for 3h. In contact with DMSO, The dark-blue formazan crystals appeared in living cells will be dissolved. Thus, a microplate with a spectrophotometer reader (Bioteck, Ac Elx 800) allowed measuring the absorbance at 570 nm. The results were expressed as the percentage of MTT reduction relative to the absorbance’s measured from negative control cells. All assays were performed in triplicate. TFM induces DNA damages in Human intestinal carcinoma cells HCT116 To measure DNA damage, caused by a toxic agent, in individual mammalian cells, the comet test was considered. For this, 6-well plates were used to seed HCT 116 cells at 7.5 × 105 cells/well. After 24 h of incubation, cells were treated with different TFM concentrations. H2O2 (20 μM) served as a positive control. Cells already recovered in PBS were mixed with Low Milting Agarose and the mixture was then spread on a microscope slides covered with Normal garose. The rest of the steps were already detailed above Reactive oxygen species determination and oxidative stress status Reactive oxygen species (ROS) are markers of oxidative status. Indeed, in case of t cellular stress, it has an increased production of these ROS that interact with DNA, lipids and ip as well as proteins. The intracellular amounts of ROS were measured by a fluorometric assay cr with 2,7- dichlorofluorescein diacetate (DCFH-DA) used extensively to monitor oxidation in us biological systems as a well-established compound to detect and quantify intracellular produced such as superoxide radical, hydroxyl radical, and hydrogen peroxide (Cathcart et al. an 1983; Chen and Wong 2009). M The conversion of the non-fluorescent (DCFH-DA) to the highly fluorescent 2,7dichlorofluorescein product (DCF) (λmax = 522 nm) happens in many steps. The fluorescent d probe, after diffusing in the cell membrane, is hydrolysed by intracellular esterases to non- ce pt e fluorescent dichlorofluorescein (DCFH), which is trapped inside the cells then oxidized to fluorescent DCF through the action of peroxides in the presence of ROS (LeBel et al. 1992). HCT 116 cells were seeded on 24-well culture plates (Polylabo, Strasbourg, France) at 105 Ac cells/well for 24 h of incubation. After, cells were incubated with different concentrations of TFM, for 24 h at 37 °C. H2O2 (20 μM) was used as a positive control. After incubation, cells were treated with 20 μM DCFH-DA. Intracellular production of ROS was measured after 30 min incubation at 37 °C by fluorometric detection of DCF oxidation on a fluorimeter (Biotek FL 800×) with an excitation wavelength of 485 nm and emission wavelength of 522 nm. The DCF fluorescence intensity is proportional to the amount of ROS formed intracellularly. Lipid peroxidation measurement The measurement of lipid peroxidation was determined according to Ohkawa et al. (1979) by the measurement of the level of malondialdehyde (MDA) which is an ultimate fragment of the degradation of the polyunsaturated fatty acids of the lipidic membrane. To do this, cells were plated in 6-well plates at 7.5 × 105 cells/well and treated with the different concentrations of TFM for 24h of incubation. A positive control using H2O2 (20 μM) t have been realized. Then, Cells were collected in coded test tubes. Samples were mixed with ip 0.1 ml of 1.15 % KCl, 0.2 ml of 8.1 % SDS, 1.5 ml of 20 % acetic acid adjusted to pH 3.5 and cr 1.5 ml of 0.8 % thiobarbituric acid. The obtained mixture was vortexed and heated at 95 °C us for 2 hours. After cooling to room temperature, a volume of 5 ml of mixture of n-butanol and pyridine (15:1, v: v) was added to each sample and the mixture was shaken vigorously. A an centrifugation (4000 rpm for 10 min) allowed isolating the supernatant fraction. Finaly, the M absorbance was measured at 546 nm. The concentration of MDA was determined according ce pt e Protein extraction d to a standard curve. To obtain cell lysates, HCT 116 cells were seeded in 6-well plates (106 cells/well) and then treated with TFM for 24 h. Thus, cells were recovered in PBS, centrifuged and collected in cold lysis buffer solution for 30 min. A centrifugation makes it possible to obtain the Ac protein extracts. Protein concentrations were determined in cell lysates using the Bradford assay (Bradford 1976). Measurement of superoxide dismutase (SOD) activity Superoxide dismutases (SOD) are ubiquitous metalloenzymes that catalyze the disproportionation of superoxide ions into hydrogen peroxides and molecular oxygen. The measurement of SOD activity was carried out according to the method described by Markland and Marklund (1979), by following the autooxidation and illumination of pyrogallol at 440 nm for 3 min. One unit of SOD activity was calculated as the amount of protein that caused 50 % pyrogallol autooxidation inhibition. The SOD activity is expressed as U/mg protein. Measurement of catalase (CAT) Catalases are enzymes that intervene in the defense of the cell against oxidative stress by eliminating oxygen species (H2O2). To measure the activity of this enzyme, 780 µl of t phosphate buffer solution, 20 µl of each sample and 200 µl of H2O2 (the substrate of the ip enzyme) were placed in a quartz cuvette. Then, the optical density was measured at 240 nm cr for an interval of time of 1 min (Aebi 1984). The activity of catalase was calculated using the us molar extinction coefficient (0.04 mM-1cm-1). The results were expressed as mmol/ min/mg protein an Statistical analysis M Data were expressed as the mean ± standard deviation (S.D.) of the means. In all cases p < 0.05 was considered statistically significant. Pearson’s correlation coefficient was used to Ac ce pt e d measure the linear association between two variables. Results TFM induced DNA damages in mice bone marrow cells Comet assay DNA damage results, in mice bone marrow cells, were assessed by the comet assay. Indeed, this test was considered as a cells-DNA detecting method characterized by its sensitivity, rapidity and simplicity (Singh et al. 1988). Figure 1 A showed the results of this t test. Indeed, mice treated with TFM indicated a higher DNA damages level, in comparison ip with the untreated mice. This increase of DNA damages caused by TFM in mice bone marrow cr cells was in a dose - dependent manner (P < 0.05). (B) Different classes of DNA damages us quantified by the comets test and visualized using fluorescent microscope. Then cell were an photographed using a digital camera (original magnification ×200). Micronuclei test M Our results showed that, compared to the control group, the number of Poly Chromatic d Erythrocyte (PCE) content micronucleus (MN), was significantly increased in mice exposed ce pt e to the different doses of the TFM. This number increased from 40.6 ± 4.52 in the control group to 80.6 ± 5.033, 120.33 ± 4.04 and 183.33 ± 54.62 respectively in animals treated with TFM at 250, 350 and 500 mg/kg bw (Figure 2 A). On the other hand, this test can tell us about the cytotoxicity of the tested substance. In this context, we counted the number of Ac PCE/1000 (NCE + PCE). We noticed that this number decreased from 129 ± 16.7 in the control group to 107.33 ± 5.68, 84.66 ± 9.29 and 30.33 ± 1.25 respectively in animals receiving TFM at the three tested doses (Figure 2 B). We observe that TFM at 500 mg/kg bw provides the most induction of MN. (Figure 2 C) Different classes of NCE (appear in red) and PCE (appear in green) quantified by the micronucleus test and visualized using fluorescent microscope. Then cell were photographed using a digital camera (original magnification ×200). Chromosome Aberration test In this study, the different treatments are able to induce alterations in all chromosomes. Then, we enumerated structural aberrations having a special emphasis on centric fusions, breaks, rings and gaps (Figure 3). Table 1 represented the frequency of these four types of abnormalities in both control and treated animals. We founded that, in mouse bone marrow cells, TFM is an inductor of Chromosome t Aberration at the doses of 250, 350 and 500 mg/kg bw. In comparison with the control group, ip we showed that TFM was able to increase the percentage of chromosome aberration. Indeed, cr this percentage passed from 9.5 ± 2.12 in control group to 29 ± 4, 33 ± 1.41 and 37.5 ± 0.7 us respectively in animals treated with TFM at 250, 350 and 500 mg/kg bw. an TFM indices cytotoxicity and genotoxicity in HCT 116 cells TFM induces cell death in HCT116 M After treating HCT 116 cells with the increasing concentrations of TFM (100, 200, 400, d 600, 800 and 1000 µM) for 24 h, the MTT test which is a cell viability test has been carried ce pt e out. We showed that TFM decreased significantly cell viability (p<0.05) with a value of IC 50 around 400 μM (Figure 4). DNA damages quantification Ac Results of this test were presented in Figure 5 A. We have shown that the concentrations used (100, 200, 400 and 600 μM respectively representing ¼, ½ IC50 and 3/2 IC50) were able to damage the DNA. Indeed, the total score passed from 53 ± 8.48 in the control cells to 166.5 ± 0.7 when cells were treated with TFM at 400 µM. H2O2 (20 μM)treated cells (positive control) induced 298.4 ± 14.24 of the total score of DNA damage. Moreover, these damages are classified in 5 classes from 0 to 5 (Figure 1B). Measurement of reactive oxygen species (ROS) production The concentration range already used (100, 200, 400 and 600 µM) was tested to verify the proportion of the oxidative stress induced by TFM in the HCT116 cells. Thus, the generation of ROS was measured via the production of fluorescent DCF. Our results showed that TFM is able to increase the level of ROS in the cellular model used in a dose-dependent manner (Figure 6). t Induction of lipid peroxidation ip After exposure of HCT116 cells to different concentrations of TFM for 24 h, the MDA cr assay was performed. Our results showed that TFM is able to induce lipid peroxidation. us Indeed, the level of MDA increased from 0.057 ± 0.0069 µmol MDA/mg of protein in the untreated cells to 0.125 ± 0.0081 µmol MDA/mg of proteins in cells treated with TFM at 400 an µM (Figure 7). M Effect of TFM on Antioxidants enzymes activities d To evaluate the effect of TFM on the activities of antioxidant enzymes, we measured ce pt e the activity of superoxide dismutase (SOD) and catalase (CAT). Our results showed that TFM increased the activities of these enzymes in HCT 116 cells, in a dose-dependent manner. Indeed, in the untreated cells, SOD activity increased from 113.80 ± 4.28 (USOD/ mg of proteins) to 223.70 ± 6.61 (USOD/ mg of proteins) when cells were intoxicated with TFM Ac (400 µM). CAT activity increased also, from 64.26 ± 3.58 (mmol/min/mg of proteins) in the untreated cells to 125.23 ± 1.62 (mmol/min/mg of proteins) in cells treated with TFM at 400 μM (Figure 8A and 8B). Discussion Developing countries, particularly, has become among the regions that use pesticides excessively in increased doses. This intensive use has become a major contamination threat to public health and the environment. Consequently, all the populations are in daily exposure to these products through their inevitably use in many areas (Boussema et al. 2012). In the current study, we worked with Triflumuron (TFM) which is an insecticide t belonging to the benzoylureas family commonly used around the world. ip The mode of action of TFM was not yet well discussed but it seems that it inhibits the cr synthesis of chitin in the insect (Lowden et al. 2007). Indeed, TFM blocked the transport of us the N-acetylglucosamine, which represents the basic pattern polymer chitin through the epithelial membrane (Tunaz and Uygun 2004). It could therefore act as a general stressor by M accelerates their death (Irigary et al. 2003). an facilitating the entry of pathogenic fungi in the insect by the weakening of their cuticle which Many studies have demonstrated that TFM is known by its several toxic effects ce pt e d especially in insects. However, data that reported its potential genotoxicity in vivo and in vitro were limited. That’s why our work aimed to quantify the DNA-damage level, the frequency of micronuclei and the percentage of chromosome aberrations in bone marrow cells of mice treated with TFM at different doses for 24 h. Certainly, intra-peritoneal (IP) route is not the Ac natural route for Triflumuron exposure. However, naturel exposure occurs by inhalation or ingestion of contaminated products. In this case, the toxic effect will depend according to the exposure doses and period. Generally, natural intoxication occurs over the years by the accumulation of frequent xenobiotic exposures. In this study, our purpose was to investigate the genotoxic effects induced by Triflumuron after one unique exposure. Therefore, our IP approach is preferred in order that the major part of the injected product will reach the circulation and the target organs. Additionally, to avoid losses and exposure variability through the gastrointestinal tract (Do Nascimento et al. 2017). Moreover, Iwanie and Tumer (2013) showed that, the use of the IP injections in acute toxicological studies is more efficient than the other types of injections. Considering the ADI value as identified by EFSA (0.014 mg/kg body weight (bw) per day) as the ‘extreme worst case’ for exposure and the almost complete absorption by the gastrointestinal tract (EFSA, 2011), the blood circulating concentration in an adult of 60 kg bw (and a total Volume of distribution of about 13 L) would correspond to 0.33 μM. ip t The toxicity of TFM to the non-target species and especially to humans and animals could be cr due to its accumulation in grain and crops. Interestingly, several studies have detected the us presence of TFM residues in different plant matrices. Using high-performance liquid chromatography (HPLC) coupled to a UV detector. Miliadis et al. (1999) founded from 0.004 an to 0.005 mg of TFM/kg of analyzed apples. Another variety of HPLC using photo-irradiation with fluorescence detection was used to detect the presence of benzoylureas insecticides M particularly, TFM, in tomatoes with value ranged from 0.5 to 2.1 μg/kg (Martinez-Galera et d al. 2001). ce pt e For the study of the genotoxicity of chemicals, we monitored the comet assay which is one of the standard methods for assessing DNA damages including single and double-strand DNA breaks (Collins et al. 1996). We found that TFM is an inductor of DNA damages. Ac Thus, the micronucleus test was done. This test can estimate the level of delayed necrosis, apoptosis, mitosis and different chromosome alterations such as chromosome loss, breakage and non-disjunction (Fenech 1993; Iwaniec and Tumer 2013). Our results demonstrated that TFM is an inductor of micronuclei in mice bone marrow cells. Then we can expect that TFM was a clastogenic compound after it’s DNA-bending. Moreover, the treatment of animals with TFM reduced significantly the number of PCE/1000 cells which is known as an element of cytotoxicity. This reduction could be explained either by the direct cytotoxic effects of TFM, its capacity to form micronuclei or by causing DNA damages leading then to cell death. It was known that oxidative stress can induce many kinds of negative effects including membrane peroxidation, protein cleavage, and DNA strand breakages, which could lead to cancer (Mittler 2002; Collins and Harrington 2002). Oxidative DNA damage is the most frequently occurring damage and includes oxidized bases, DNA single- and double-strand breaks, abasic sites, and DNA–protein cross-links (Cadet et al. 2003a, b; Marnett 2000; ip t Bjelland and Seeberg 2003). Some of the forms of DNA oxidation, when persistent during cr replication, can lead to mutations that’s up to the stop of DNA replication and therefore can DNA via the production of ROS (Ayed et al., 2012). us cause cell death (Hadi, 2004 ). Moreover, many chemicals are known to induce damage to an The other test for the detection of genotoxicity and mutagenicity of some toxic products is the chromosome aberration assay. Indeed, we found that the TFM could affect M chromosomes in a dose dependent manner. Some cells can survive with a few numbers of d chromosome’s abnormalities, but these alterations can be inherited by the next generation ce pt e through the stem cells or the clonal expansion of the somatic cells (Rjiba et al. 2013). Then, compared to the control group, we found that the TFM increased significantly the percentage of chromosome aberrations. Ac Thus, in this present study, we demonstrated the genotoxic character of TFM in mice bone marrow cells using the comets assay, the micronuclei and the chromosomal aberrations tests. Also, the genotoxicity of TFM was evaluated using the comet assay in another cell model: the HCT 116 cells, which are the intestinal human cells that represent the first barrier that have a contact with toxic substances. Thus,this genotoxicity was confirmed by the significant DNA damage caused by the exposure of HCT 116 cells to increased concentrations of TFM Indeed, the present results showed that the TFM induced DNA damages in mice and human cell carcinoma, in a dose-dependent manner. These data were in disagreement with previous studies that have shown that TFM is unable to induce DNA damages using the comet assay, the micronuclei test and the chromosomal aberration assay in vivo, in mice and some amphibian species, and in vitro in bacterial and mammal cells (Merzendorfer 2011). In other side, our results agree with those made by Herrera and his co-workers (Herrera et al. 2013) in which they studied the genotoxicity of Buprofezin, an insecticide belongs to the same family ip t as the TFM, in vitro in embryonic cells of syrian hamster. Indeed the exposure of these cells cr to growing concentrations from 12.5 pmol to 100 pmol has engendered a notified increase in the micronuclei number. In the same context, another insecticide belonging to the TFM family us was tested for its genotoxic effects. Indeed, the treatment of Chinese Hamster Ovary Cells an (CHO K1) with the Flumexuron for three hours induced a significant increase in the percentage of chromosome aberrations (Meyer 1991). M Our results showed that TFM is able to induce DNA damage, in bone morrow mice d cells, quantified by the comets assay, the micronuclei and the chromosomal aberrations tests. ce pt e This genotoxicity was also confirmed using human intestinal cells. It was then asked whether this genotoxicity was occurred through oxidative stress. To do this, we evaluated some of oxidative stress biomarkers in vitro, which were the ROS generation, the MDA level and the Ac measurement of the activity of some antioxidant enzymes. To study the involvement of the oxidative stress in the genotoxicity process induced by the TFM, we measured the level of ROS production in HCT 116 cells. Indeed, when there is an oxidative stress, there is an overproduction of ROS which can subsequently alter cell structure and functions. We have previously shown that TFM is an oxidizing agent in the liver and kidneys of mice and this through the generation of ROS and free radicals by the increase of carbonyl protein and MDA levels. Also, the activities of the antioxidant enzymes were altered (Timoumi et al. 2019). Our results indicated that the exposure of HCT 116 cells to increased concentrations of TFM induced a dose-dependent cell death as revealed by the MTT test. Besides, we found that TFM-induced cell death was associated with a significant increase in the ROS generation after 24 h of treatment. t When ROS interact with the lipidic membrane, the oxidation of polyunsaturated fatty ip acids isproduced and by consequence,the lipid peroxidation occurs (Rice-Evens and Burdon cr 1993). Indeed, MDA is the ultimate fragment of the polyunsaturated fatty acid degradation of us the lipid membrane that serves as a reliable marker of oxidative stress (Draper et al. 1993; Dotan et al. 2004). an So, the MDA level was examinated in HCT 116 cells, the results showed that the M exposure of cells to different TFM treatments increased significantly the MDA levels. Oxidative stress is the result of an imbalance between the production of ROS and ce pt e d their disappearance by the intermediary action of the antioxidant enzymes (Haliwell 1994). Thus in this present work we measured the activities of two antioxidant enzymes, SOD and Catalase (CAT). SOD represents the body's first line of defense against oxidative damage (Fridivich Ac 1995). SOD catalyzes the disproportionation of O2 .- into O2 and H2O2 (Jiang and Zang, 2002). Catalase is an enzyme mainly located in peroxisomes, liver and red blood cells converting H2O2 to H2O and O2 (Maté et al. 1999). Our work demonstrated that TFM increased the activity of these two enzymes in a dose-dependent manner. Then, we can conclude that TFM was genotoxic as revealed by the comet assay, the micronucleus test and the chromosome aberrations test in bone morrow mice cells. Moreover, we confirmed this genotoxicty using another Cell line model; the HCT 116 cells. Thus this genotoxicity resulted from an over production of ROS. 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Values are expressed as mean ± SD of six mice in each group. *p < 0.05, t statistically, significant compared to the control group. **p < 0.01, statistically significant ip compared to the control group. (B) Different classes of DNA damages quantified by the comets test cr and visualized using fluorescent microscope. Then cell were photographed using a digital camera us (original magnification ×200). Figure 2 A. Number of micronucleated polychromatic erythrocytes (PCEMN) in 2000 an polychromatic erythrocytes (PCE) in mice bone morrow cells receiving TFM at 250, 350 and M 500 mg/Kg bw. Each group contains six mice, and it expressed values as mean ± SD. * p <0.05 versus control. d Figure 2 B. Number of Polychromatic erythrocytes (PCE) in 1000 polychromatic ce pt e erythrocytes and normochromatic erythrocytes (PCE + NCE) after mice treatments with TFM at 250, 350 and 500 mg/kg bw. Each group contains six mice, and it expressed values as mean ± SD. A group treated with ethanol 1:1 (v/v) served as a control. **p < 0.01, statistically Ac significant compared to the control group. ***p < 0.001, statistically significant compared to the control group . Figure 2 C. Different classes of NCE and PCE quantified by the micronucleus test and visualized using fluorescent microscope. Then cell were photographed using a digital camera (original magnification ×200). Figure 3. Different types of all structural chromosome aberrations types in mouse bonemarrow cells obtained after coloration with the Giemsa and observed using optic microscope. Figure 4. Cytotoxic effect of Triflumuron on HCT116 cells after 24 h-treatment. Cells were treated with different concentrations. Cell viability was determined using the MTT assay and expressed as percentages of viability. Data are expressed as the mean ± S.D. of three independent experiments. Values are significantly different (p < 0.05) from control. Figure 5. Induction of DNA damages in HCT116 cells, following treatment with Triflumuron at 100, ip t 200, 400 and 600 μM. H2O2 (20 μM) was used as a positive control. DNA stand breaks were detected by the standard comet assay. Data are expressed as the mean ± S.D. of three independent experiments. cr Values are significantly different (p < 0.05) from control. *p <0.05, statistically significant compared us to the control group. **p <0.01, statistically significant compared to the control group. an Figure 6. Levels of relative fluorescent DCF production after exposure of HCT116 cells to different Triflumuron concentrations for 24 h. H2O2 (20 μM) was used as a positive control. Data are expressed M as the mean ± S.D. of three independent experiments. Values are significantly different (p < 0.05) from control. *p <0.05, statistically significant compared to the control group. **p <0.01, statistically ce pt e control group. d significant compared to the control group. *** p <0.001, statistically significant compared to the Figure 7. Induction of Lipid peroxidation in HCT116 cells, after 24 h of incubation with Triflumuron measured by the production of malondialdehyde (MDA). H2O2 (20 μM) was used as a positive control. Ac Data are expressed as the mean ± S.D. of three independent experiments. Values are significantly different (p < 0.05) from control. *p <0.05, statistically significant compared to the control group. **p <0.01, statistically significant compared to the control group. *** p <0.001, statistically significant compared to the control group. Figure 8. Effects of Triflumuron on antioxydant enzymes activities such as catalase (A) and Superoxyde Dismutase (B), after incubation of HCT116 cells with the tested concentrations of TFM for 24 h. Data are expressed as the mean ± S.D. of three independent experiments. Values are significantly different (p < 0.05) from control. *p <0.05, statistically significant compared to the Ac ce pt e d M an us cr ip t control group. **p <0.01, statistically significant compared to the control group. Ac d ce pt e t ip cr us an M Ac d ce pt e t ip cr us an M Ac d ce pt e t ip cr us an M Ac d ce pt e t ip cr us an M t ip cr us an M d ce pt e Legend to table Table 1. Percentage of chromosome aberration types (total of all types of chromosome Ac aberration), in bone-marrow cells, of treated mice after acute exposure.