Oxidative Stress Induced By The Metallic Oxide The Copper Oxide (Cuo-Nps) On Terrestrial Snail Helix Aspersa


 Our study focused on the evaluation of the toxicity of copper oxide nanoparticles (CuO-NPs) on a bioindicator; the land snail Helix aspersa. Their effects were studied by a targeted approach in the laboratory, by evaluating the oxidative stress biomarkers in hepatopancreas and kidney (GSH, GST, GPx, CAT, and LPO). The snails were exposed to increasing concentrations (50, 100, 150, and 200 mg/kg) of CuO-NPs mixed in wheat flour during a sub-chronic treatment period of 45 days. Our results show that: CuO-NPs can induce oxidative stress, by producing reactive oxygen species (ROS), which was confirmed by the decrease in glutathione (GSH) level and reduction of its metabolizing enzyme glutathione-s-transferase (GST) in both organs, as they trigger the detoxification system resulting in increased activity of the glutathione peroxidase (GPx) and catalase defense enzyme and lipide peroxidation indices within the hepatopancreas.


Introduction
Currently, nanotechnologies and nanoscience are booming and represent a key development in the innovative materials sectors, new productions containing nanomaterials are constantly marketed (François, 2012).
Due to their nanometric size, nanomaterials have different advantageous physicochemical properties, which have already found various applications; cosmetics (sunscreen), textiles, ceramics, chemical and pharmaceutical industries (drug administration), and water treatment (Rossi, 2006).
This raised development of their uses has increased the concentrations of nanoparticles in air, water and soil, which could as a result reach ecosystems, and accentuate the risk of exposure to nanometric contaminants, all that makes the study and management of their toxicological risk di cult to establish considering that several aspects of their toxicity mechanisms are not yet well understood (Rossi, 2006;François, 2012).
To allow responsible and safe development of nanotechnologies using metallic NPs, a better understanding of their cytotoxicity mechanisms is therefore required (François, 2012). However, these unique properties make them very reactive and allow them to easily penetrate cells, resulting in possible nano toxicity to microorganisms, plants and animals (Ghosh et al. 2016). The toxicity of different types of nanoparticles is suspected to be behind the generation of reactive oxygen species (ROS). Oxidative stress is a response to cell damage, in vivo and in vitro studies have shown that nanoparticles generate more free radicals and reactive oxygen species than larger particles due to their large surface area. ROS are known for their ability to damage cells by oxidizing lipids, disrupting DNA and modifying proteins, among other interfaces. (Huang et al. 2017) CuO-NPs has potentially wide industrial use in applications such as gas sensors, antimicrobial coatings, photovoltaic cells, in catalyst applications, and in heat transfer nano uids. Changes in the physicochemical and structural properties of nanomaterials due to the decrease in particle size can lead to biological effects that are di cult to predict from other forms of the same metal. Several Studies have demonstrated that CuO-NPs are up to 50-fold more toxic than bulk CuO to crustaceans (Heinlaan et al. The aim of this study is to understanding the biological interactions between a bio indicator, known for its important bio-accumulating power in its tissues, the land snail (Helix aspersa,), with nanoparticles CuO-NPs, through a battery of enzymatic and non-enzymatic biomarkers of oxidative stress.

Chemical material
CuO used in this study is a commercial preparation, from the Center for Scienti c and Technical Research in Physico-Chemical Analysis (CRAPC)-Bou-Ismail-Tipaza, Algeria.

Characterisation of CuO-NPs nanoparticles
The X-ray diffraction (XRD) patterns were recorded on a Siemens D-5000 diffractometer with Cu-Kα radiation (λ = 1.5418 Ǻ). The morphology of sample was studied using Transmission Electron Microscopy (TEM) images.

Preparation of CuO-NPs nanoparticles
In 40 mL of distilled water, 4 mmol of CuSO 4 •5H 2 O was dissolved, then 4 mL of cyclohexylamine was added dropwise under intense stirring, and the agitation was continuously sustained for 30 minutes. Finally, the mixture was moved to an autoclave of Te on-lined stainless steel that was sealed and held for 16 h at 140 C°. The autoclave was naturally cooled down to room temperature, and the sample was puri ed and washed several times with distilled water and ethanol, and vacuum-dried for 9 h at 80 C°.

Experimental design
For 45 days, the snails were fed with 04 different concentrations of copper oxide (50, 100, 150, and 200 mg/kg) mixed in one and a half kilograms (1.5 kg) of wheat our. The control group was fed with uncontaminated our. After the treatment period, all individuals were left on an empty stomach for 48 hours to empty the contents of their digestive tract, sacri ced and dissected. The hepatopancreas and kidneys were removed and xed for antioxidants determination.

Oxidative stress biomarkers
Frozen snail tissues aliquots were thawed in crushed ice and homogenized with 10 mM ice-cold Tris-HCl buffer (pH 7.4) using a manual homogenizer. The homogenate was then vortexed and centrifuged at 10,000 g for 10 min at 4°C. Aliquots of each supernatant were used to measure total protein content and oxidative stress biomarkers by spectrophotometric methods. The protein content was measured by the Bradford dye-binding assay with bovine serum albumin as a standard (Bradford, 1973).
The GSH content was quanti ed using Elman's reagent (DTNB) as a uorescent reagent reported by Weckberker and Cory (1988). Brie y, the reaction mixture contained 500 µL of the supernatant of each tissue sample, 1.0 ml Tris-EDTA buffer (0.02 M, pH 9.6), and 25 µl DTNB (0.01 M). After thorough mixing and incubation at room temperature for 5 min, the uorescence at 412 nm was determined, and GSH concentration values were calculated from a pure GSH standard curve. The rate of GSH was expressed as nanomoles of glutathione formed per milligram of proteins (nmol GSH/mg protein).
GST activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate as described by Habig et al. (1974). Brie y, the reaction mixture containing 50 µl of each tissue supernatant, 1.05 ml of 100 mM Tris buffer (pH 7.4), 50 µl of 1 mM CDNB, and 50 µl of 1 mM GSH. The GST activity was determined by monitoring changes in absorbance at 340 nm for 2 min, re ecting the rate of CDNB conjugation with GSH, and using an extinction coe cient of 9.6 mM-1 cm-1. One unit (U) of glutathiones-transferase activity is de ned as the amount of enzyme that conjugates 1 µmole of CDNB per min. The GST activity was expressed as U/mg protein.
The enzymatic activity of glutathione peroxidase (GPx) was measured by the method of Flohé and Günzler (1984). This method is based on the reduction of hydrogen peroxide (H 2 O 2 ) in the presence of reduced glutathione (GSH), the latter is transformed into (GSSG) under the in uence of GPx. the method consists to Incubate 0.2 ml of the homogenate, 0.4 ml of GSH (0.1 mM) and 0.2 ml of the TBS buffer solution (50 mM Tris, 150 mM NaCl, pH 7.4) in a water bath at 25°C for 5 min. then, add 0.2ml of H 2 O 2 (1.3 mM) to initiate the reaction, after 10 minutes we add 1 ml of TCA (1%) to stop the reaction. the mixture is put in ice for 30 minutes, centrifuged for 10 minutes at 3000 rpm. The nal mixture contains 0.48 ml of the supernatant, 2.2 ml of the TBS buffer solution, and 0.32 ml DTNB (1 mM). the optical densities were read at 412 nm against the blank.
CAT activity was measured at 240 nm by determining the decrease in the level of hydrogen peroxide (Beers and Sizer, 1952). The reaction mixture contains 1.9 ml of 50 mM phosphate buffer (pH 7.0), 1.0 ml of 5 mM H 2 O 2 , and 100 µL of tissue supernatant. A unit of CAT activity is de ned as the quantity of enzyme catalyzing the degradation of 1 µmol of H2O2 per minute and the speci c activity corresponding to the transformation into µmoles of the substrate (H 2 O 2 ) per minute per mg of protein.
Lipid peroxidation (LPO) levels were assayed by measurement of malondialdehyde (MDA). The decomposition product of polyunsaturated fatty acids hydroperoxides was determined by the thiobarbituric acid (TBA) reaction as described by Fatima et al. (2000) with some modi cations by Zhao et al. (2009). Brie y, the assay mixture contained 1 mL tissue supernatant, 1 ml of 5% trichloroacetic acid (TCA), and 1 ml of 0.67% thiobarbituric acid (TBA). The mixture was heated at 95°C for 40 min, cooled, and centrifuged at 3000 g for 10 min. The absorbance of the supernatant was determined at 532 nm. The amount of thiobarbituric acid reactive substances (TBARS) was calculated using an extinction coe cient of 1.56 x 105 M-1 cm-1. The rate of lipid peroxidation was expressed as nanomoles of substances reactive to thiobarbituric acid (TBARS) formed per hour, per milligram of proteins (nmol. TBARS/mg protein).

Statistical analysis
The data from CuO-NPs administration and control snails were presented as mean ± S.E (n = 25, ve different animals, and each analysis from each one was repeated three different times in hepatopancreas and kidney). Groups means were compared using one-way ANOVA, when ANOVA was signi cant multiple comparisons of mean values were separated by Tukey's test. Statistical differences were determined at the P <0.05 and P <0.01 level for all analyses.

Copper oxide characterization
The Powder X-ray diffraction (XRD) pattern of CuO-NPs nanoparticles as shown in Figure 1 The diffraction peaks of this product are ascribed to the formation of the CuO-NPs (space group C2/c) monoclinic crystal phase with constants of a = 4.69 Å, b = 3.42 Å, c = 5.13 Å, (JCPDS 41-0254). In the XRD spectrum of our sample, no peaks from impurities can be detected, showing the high purity of CuO-NPs products and also, the sharp and extreme peaks in the XRD patterns of the products suggest strong CuO-NPs crystallinity.
The average particle size of the samples was calculated using the following Scherrer equation: D = 9λ / β cos θ, where β exceeds the diffraction peak distance line in radians, θ is the angle of Bragg in degrees, d is the crystal average diameter and λ is the X-ray wavelength in angstrom (Nezamzadeh-Ejhieh and Amiri, 2013). The average crystallite size of the nanoparticles of CuO-NPs is estimated about 29 nm by Scherer's formula.
The TEM image (Figure 1-B) demonstrates that the substance consists of normal morphology and narrow size distribution of spherical particles. The size of the particles shown in the TEM picture is in Strong accordance with that calculated by the XRD pattern of the Debye-Scherrer formula.

Biochemical results
A non-signi cant variation (p> 0.05), of the glutathione level, was detected at the level of the two organs in question; hepatopancreas and kidney; of Helix aspersa exposed to a commercial preparation of copper oxide.
In hepatopancreas, the highest value was noted in the control group; 5.13 ± 1.81 nmol/mg protein. This value was reduced in snails exposed to 50 mg/ kg of CuO-NPs; to 3.03 ± 0.99 nmol/mg of protein. A similarity of effects was noted for the rest of the doses (Figure 2-A, Table 1).
Stability of renal glutathione was observed following treatment with CuO-NPs (Figure 2-A, Table 2), except for the case of 150 mg/kg, a non-signi cant reduction was reported with 1.90 ± 0.03 nmol/mg of protein compared to the control 3.49 ± 1.46 nmol/mg of protein.
View the speci c relationship between GSH and its metabolizing enzyme GST; no change in glutathiones-transferase activity was reported in the hepatopancreas of either controls or snails treated with copper oxide (p> 0.05; Figure 2-B; Table 1).
Unlike the rst, the enzyme variation is signi cant in the kidneys (p <0.05; Figure 2-B; Table 2). In the control group, the enzymatic activity of GST was in the order of 2.76 ± 0.49 nmol/min/mg of protein but it was increased up to 4.04 ± 0.72 nmol/min/mg of protein in snails fed by 200 mg/kg of CuO-NPs. The only signi cant difference in the treated kidneys was noted between the two groups low (50 mg/kg) and high concentration (200 mg/kg) with a p <0.05.
Gpx and catalase are the defense enzymes that are triggered when reactive oxygen species appear.
A very highly signi cant increase (p≤0.001; Figure 2-C, Table 1) in the enzymatic activity of glutathione peroxidase was observed in the hepatopancreas following various treatments with copper oxide.
Following the 45-day diet of healthy our, the level of Gpx in the controls was 13.96 ± 5.14 nmol GSH / mg protein, while the our mixed with the different concentrations of CuO-NPs induced a signi cant increase in Gpx up to 31.86 ± 6.64 nmol GSH / mg of protein in the case of 100 mg/kg of CuO-NPs.
A slight insigni cant variation (p> 0.05; Figure 2-C, Table 2) in the defense enzyme was observed in the kidneys. The level of Gpx in controls was 17.37 ± 4.76 nmol GSH / mg protein, while in snails fed with the four concentrations of CuO-NPs the Gpx values ranged from 17 to 24 nmol GSH / mg protein.
A positive correlation (p <0.05) was established between the levels of GPx and catalase in the hepatopancreas, following the strong relationship between both enzymes in the elimination of ROS, which caused a very highly signi cant increase (p = 0.000; Figure 3-A, Table 1) of catalase level in this organ. This variation is signi cant compared to the control group (07.71 ± 1.03 µmol / min/mg of protein), and the highest value of which was reported in snails fed on our mixed with 200 mg/kg of CuO-NPs (18.34 ± 2.15 µmol / min/mg protein) Since there was no signi cant variation in Gpx in the kidney, no change in catalase enzymatic activity was noted in this organ (p> 0.05; Figure 3-A, Table 2).

One of the signs of the appearance of ROS in the cell is an increase in the lipid peroxidation index (LPO).
After 45 days of exposure of 25 snails to 04 doses of copper oxide, a signi cant induction of LPO levels was observed in both studied organs; hepatopancreas (p <0.05; Figure 2-B, Table 1) and kidney (p = 0.000; Figure 3-B, Table 2).
In hepatopancreas, the level of LPO in controls was 0.645 ± 0.176 nmol/mg protein and increased to 1.033 ± 0.016 nmol/mg protein in individuals treated with 150 mg/kg of the oxide copper.
In the kidneys; peak LPO (p <0.01) was present following exposure to 150 and 200 mg/kg CuO-NPs respectively; 2.314 ± 0.09 nmol/mg of protein and 2.315 ± 0.998 nmol / mg of protein compared to the control group 0.609 ± 0.3 nmol/mg of protein.

Discussion
Copper is an essential element used in metabolic processes for both humans and animals (Bhattacharya et al. 2016). It is needed for crosslinking connective tissue as well as in iron and lipid metabolism (Siddiqui et al. 2013). However, excessive copper is extremely toxic and can cause various pathological changes in tissues (Ozcelik et al. 2003). Besides, CuO-NPs was shown to catalyze the formation of free radical species capable of triggering lipid peroxidation (Rikans and Hornbrook, 1997;Paresh et al. 2010).
While the respiratory tract is the most common route of nanoparticle exposure, the gastrointestinal tract is also a potential route of entry. Copper nanoparticles can be exposed to the gastrointestinal tract either through direct ingestion of water and food or through the mucociliary clearance of the respiratory tract which is subsequently ingested after swallowing (Chen et al. 2006).
The main objective of this study is to evaluate the induction of oxidative stress by copper oxide on the land snail Helix aspersa exposed to mixing of the nanoparticles and the wheat our; for 45 days (subchronic exposure), with a battery of stress biomarkers (GSH, GST, GPx, CAT and LPO) Glutathione (GSH) is the most important water-soluble and low-molecular antioxidant found in cells (Deneke and Fanburg, 1989). It has multiples physiological roles; as hydrogen peroxide scavenging and preservation of SH groups in a reduced state in proteins, enzymes, and some other molecules (Meister,1985). It plays a key role in cellular defense and serves as a reservoir for the amino acid cysteine.
In the present study, GSH contents in the hepatopancreas and kidney tissues of Helix aspersa exposed to CuO-NPs showed a signi cant decrease, except those exposed to 50, 100, and 200 mg/kg in the case of kidney tissues, when compared with the control groups after 45 days. These results are similar to Xiong et al. (2011); who studied the impact of ZnO on zebra sh, Abdel-Khalek et al. (2015) who exposed Oreochromis niloticus to a series of CuO-NPs concentrations, and Anreddy (2018), who treated the Wistar rat by CuO-NPs, all teams constated that NPs suspension caused a decrease in GSH content in the hepatopancreas tissue. Redox-active metal ions such as Cu (II) readily catalyze the oxidation of GSH giving rise to thiol and hydroxyl radicals (Stohs and Bagchi, 1995). This depletion can be a result of increased binding of Cu (stabilization of Cu in the oxidative state), enhanced use of GSH's oxidizing ability (conversion into GSSG, the oxidized form of glutathione), or an ineffective GSH regeneration (Pandey et al. 2001;Parvez et al. 2003;Ahmad et al. 2005;Parvez and Raisuddin, 2006). This depletion of GSH level reduces the cellular availability to scavenge free radicals and can lead to more oxidative stressrelated damage (Elia et al. 2003).
GST is an enzyme that catalyzes in the cytosol the conjugation reaction of electrophilic xenobiotics and their metabolites with an endogenous polar ligand which is glutathione (Habig et al. 1974). It is a key detoxifying enzyme that also plays an important role in eliminating ROS and scavenging lipid peroxidation metabolite (Wang et al. 2016).
The reduction of GST activity is could be explained by the de ciency and absence of biotransformation of xenobiotics and androgenic substances. Therefore, the GST has an important role in the neutralization and detoxi cation of certain xenobiotics by increasing their water solubility and thus facilitating their elimination (Vander et al. 2003).
Induction of the GST activity has already been reported in the snail Helix aspersa, exposed to various pollutants (Belhaouchet et  GPx has a crucial role in intracellular protection against toxic compounds such as Cu and Zn (Anderson, 1997;Anderson and Luo, 1998;Mosleh et al. 2005). GPx is responsible for enzymatic defense against hydrogen peroxide and is strictly linked with the concentration of GSH, it catalyzes the reaction between glutathione and hydrogen peroxide, resulting in the formation of glutathione disul de (GSSG) (Alkaladi et al. 2013). In the present result, the GPx activity in the hepatopancreas tissues of Helix aspersa treated by copper oxide showed a signi cant increase when compared with control after 45 days. Witch is con rmed by Fahmy and Cormier (2009) who reported that CuO-NPs NPs were better able to increase the activity of GPx.
Catalase is a catalytic enzyme in the disproportionation of hydrogen peroxide (a SOD product generator) into the water and molecular oxygen (Delattre et al. 2005; Mohora et al. 2007). This hemoprotein protects tissues from hydroxyl radicals which are very reactive (Sathishsekar and Subramanian, 2005). In the present investigation, while CAT activities showed a signi cant increase in the case of hepatopancreas tissues of H. aspersa exposed to 04 concentrations of copper oxide, it showed a nonsigni cant increase in kidney tissues, when compared with matched controls.
CAT activity in invertebrates depends on the mode of exposure, species, and even within the same organism. Regoli et al. (2006) reported that vehicles' pollutants induced an increase in CAT activity in the digestive gland of Helix aspersa, however, it doesn't change when the species exposed to electromagnetic elds (Regoli et al. 2005). Some authors proposed that seasonal variations can affect catalase activity (Ramos- Vasconcelos et al. 2005). In contrast,  showed that enzyme activity was inhibited in the presence of Cadmium and Zinc in the snail Achatina fulica, in both target organs "hepatopancreas and kidney".
Lipid peroxidation was the classic result of oxidative damage and its byproduct was often formed when free radicals attacked cellular membranes (Chen et al. 2010). In the present study, the excessive level of TBA, a stable metabolite of lipid peroxidation, would further con rm the generation of oxidative damage in hepatopancreas and kidneys. All the above data suggest that nano-CuO-NPs affects the oxidationantioxidation homeostasis of both organs via increasing ROS and reducing antioxidant enzymes.

Conclusion
It appears that copper oxide (CuO-NPs-NPs) used in this study can promote oxidative stress thus activating the detoxi cation system with an induction in the activities of GST, GPx, CAT, and a decrease in the content of GSH. These modi cations are probably linked to the accumulation of ROS caused by the presence of NPs. Finally, the present study demonstrates that copper oxide nanoparticles induced hepatotoxicity and nephrotoxicity with potential enzymatic and biochemical disturbance.