3.Introduction

The late twentieth and early twenty-first centuries saw the emergence of a new kind of materials: nanoparticle, that is to say a particle size of less than 100 nm. We will not discuss in this study nanoparticles of natural origin, from volcanoes or fires, which have existed for millennia, but manufactured nanoparticles intentionally by humans [1].

Thanks to their special properties due to their small size, nanomaterials are used in numerous consumer applications (cosmetics, nanostructured materials, ...). However, the increasing use of such materials today raises public health issues. Indeed, the interaction of nanoparticles with biological systems and their potential toxicity, are the little-known today. The associated health risk is therefore difficult to evaluate [2].

The snails have been widely used as a model system to reveal biomarkers of environmental pollution, the central model of this study is the small gray snail Helix aspersa, known for its important to be bio accumulative in its tissues.

The objective of this work is to study the toxicological effects induced by manufactured nanoparticles (CdSe) on an organism bioaccumulation, land snail Helix aspersa.

4. Material and Method
5. 1. Biological Material

Description Helix aspersa [3]. Helix aspersa, better known under the name snail gardens or Little gray and used in our experiments is a gastropod mollusk pulmonate stylomatophore belonging to the family of Helicidae.

1. 2.  Breeding Snails

Sampling of the snail Helix aspersa was performed at Beccaria in the wilaya of Tebessa Raising Snails Helix aspersa at the cellular toxicology laboratory during the period 3 months.

Snails are raised in the following conditions that are considered optimum: photoperiod of 18 h light / 24 h, temperature 20 ± 2^°C, humidity of 80 to 95%; feeding the wheat flour. Snails are distributed in transparent plastic boxes with perforated lid. Each box contains a wet sponge to retain moisture [4,5].

1. 3. Chemical Equipment
2. 1. 1. Definition

The semiconductor nanocrystals (CdSe) are objects of size between 1 and 100nm and formed of a few tens of thousands of atoms arranged in a crystalline order. They are made by chemical synthesis by mixing in solution the elements (e.g., carbon cadmium and selenium) that make up the semi-conductive material. At temperatures of some hundred degrees, the different elements fit together to start the nucleation and growth of the material.

1. 1. 2. Synthesis of Nanoparticles CdSe

There are several methods to get nanoparticles CdSe. The synthesis of CdSe nanocrystals was made by standard chemical techniques to air. A mixture of CdO (1.8 mmol, 0.2311 g), oleic acid (OA, 6.0 mmol) and diphenyl ether (8 ml) was heated to 18^°C for 2 hours. Then a solution of 3.2 g of selenium TOP, which contains 0.32 g, 4.0 mmol of selenium was injected very rapidly in this hot solution [6].

1. 4. Procedure

Treatment of animals was performed by injecting skin with a micro syringe We selected two doses and a control medium. The doses that have been used are 3.6µg/g/2j (Jackson et al. 2012) and 7.2µg/g/2j.

• Preparation and sacrifice of animals After the treatment period, the snails are placed fasting for 48 hours to empty their digestive tract, the animals are then sacrificed by freezing at -20^°C and then dissected,
• The extraction and protein quantification: The proteins are quantified by the method of Bradford (1976) [7].
• The catalase activity: The catalase activity was determined in the hepatopancreas calorimetrically according to [8]. H₂O₂ disappearance rate is monitored by observing the rate of decrease in absorbance at 240 nm.
• The activity of GST: The measurement of the activity of Glutathione S-Transferase (GST) is determined according to the method of Habit et al. [9] following the formation of 1-glutathione-2,4-dinitrobenzene resulting from the conjugation between the substrate (1-Chloro-2,4-Di Nitro Benzene (CDNB) with reduced glutathione [9].
• The GSH: Glutathione level is determined according to the method of Weckbeker and Cory [10].
• The MDA levels: The assay of MDA is carried out according to the method of Ester Bauer. MDA may be detected by a colorimetric reaction with Thio Barbituric Acid (TBA).
• The AChE activity: The assay of AChE activity was conducted according to the method of which comprises providing the enzyme (AChE) an analogue artificial substrate, acetylthiocholine, which will be hydrolyzed to acetic acid and thiocholine.

The latter in the presence of DTNB (5'-dithio-bis-2-nitrobenzoic acid) yields a yellow product TNB (acid 5-thio-2-nitrobenzoic acid) which is determined at a wave length of 412 nm [11].

5.  Results

The results are expressed as mean ± (standard deviation) of n experiments (where n represents the number of animals used with n = 10), differences are considered: significant when p ≤ 0.05, very highly significant when P ≤ 0001, highly significant when P ≤0,01.

6. Discussion

The purpose of this study is to understand how organisms like snails adapt to toxicity against cadmium selenide. The production of reactive oxygen species is considered as a key factor of the adaptive response or reaction to a toxic event, it is whether the latter is characterized by an increase in antioxidant defenses [13].

Current knowledge of the toxic effects of nanoparticles are relatively limited. Available data indicate that some insoluble nanoparticles can cross different protective barriers, is distributed in the body and accumulate in various organs. Toxic effects have been documented at the pulmonary, cardiac, reproductive, renal, skin cell and as nanoparticles can be distributed throughout the body, including inside the cells. Significant accumulations were demonstrated in the lungs, brain, liver, spleen and bone [12].

The toxicity of certain quantum wells may be related to the release of cytotoxic ions to oxidative mechanisms (14). .and other phenomena less well understood [15].

The total protein content is a test often used to highlight a stress in a bio-indicator organism [16]. Indeed, when environmental stresses (drought stress, thermal, oxidative, exposure to pollution, infection by pathogens ...) are strong, most proteins undergo denaturation [17].

 In our work, we have demonstrated a significant increase in protein levels at the hepatopancreas, these results point in the same direction as those of Grana., [18] which highlight also increased protein levels total at hepatopancreas snails treated with heavy metals. On the other hand, Koehler, et al. [19] suggest early induction of the synthesis of the storage structures (MTs and granules) bonded to toxic kinetics after exposure of the slug D. reticulum at high concentrations of Cd, Pb and Zn simultaneously with the induction of stress proteins HSP 70. Our results support this work, as we have also shown an increase in the protein levels in the presence of cadmium nanoparticle.    

The exposure of snails CdSe resulted in a very highly significant increased activity of catalase (CAT) in the hepatopancreas, this increase is dose-dependent on the concentration of CdSe. Exposure Experiments with TiO₂ NP in the terrestrial invertebrate Poercellio saber revealed sub lethal effects such as induction of catalase [20]. Our results are in perfect agreement with those of Buffet et al. [21] who observed induction of CAT in the bivalve Spline and annelid Divers H color exposed to Cu NPs. The CAT also induced in Spline exposed to AT NP [22,23] also observed an induction of CAT in magna D exposed to TiO₂ NP.

S. PAIN-DEVIN, et al. a studied the effect of three nanoparticles (nAg, nTiO₂, nCeO₂) on two models of aquatic invertebrates (Dreissena polymorph and Grammars spa), they reduced exposure concentrations of 1000 mg / L (nTiO₂) 0.5 mcg / L (nAg) and increased exposure time of 24 to 21 days (nCeO₂). A large battery of biomarkers was used to evaluate the impacts of exposure, such as lysosomal and antioxidant defenses (catalase, GPx, MDA). they showed some effects on some parameters evaluated, such as an increase in antioxidant activity, their results are struggling to clearly demonstrate the harmful effect of nanoparticles at lower concentrations.

Our results have shown, very highly significant levels of MDA in the digestive gland in Helix aspersa treated with cadmium selenide. These results are consistent with those of Dutta, et al. [24] have shown that elevated levels of Malondialdehyde, a byproduct of lipid peroxidation, correlated to the production of ROS by the zinc oxide nanoparticles placed under illumination. Moreover, Sewell, et al. [25] showed a significant increase in the rate of MDA in the marine snail Linnaean natalensis exposed to environmental pollutants.      

The decrease in the rate of GSH could be explained by a reaction / CdSe direct bond with glutathione, indeed the carboxyl groups of the glutathione (amine group, sulfhydryl group (-GH) as well as two peptides) are combined with the xenobiotic This interaction takes place through the intervention of the GST that allows the conjugation of xenobiotic or its metabolites with GSH in phase II metabolism.

 GST is in phase II biotransformation in the conjugation reaction. Our results show a highly significant dose-dependent increase and GST at the digestive gland. Induction of GST was observed in Spline and Divers H Color Cu exposed to NP [26] and Spline to the NP [22] this biomarker was also boosted by galloprovincialis Exposed to SiO₂ NP [27] and D. magna exposed to TiO₂ NP [23].

The AChE has a role in the transmission of nerve impulses because it hydrolyzed the neurotransmitter acetylcholine. Its inhibition is a biomarker of neurotoxicity. The decrease in its activity is often associated with the presence of organic or metal contaminants in the medium.

In our study we demonstrated a dose-dependent decrease and very highly significant activity of AChE in treated snails, these results are confirmed by the work of Wang, et al. [20] which showed that different types of nanoparticles could pose neurotoxic properties and propose using AChE as a biomarker for NP. Neurotoxic effects evidenced by the inhibition of AChE was demonstrated after only 15 days of exposure to M. galloprovincialis exposed to Cu NP [28]. showed inhibition of cholinesterase activity during exposure of carp Cyprinids carpi juveniles to Cu NP, and also suggest their potential neurotoxic power. In vitro and in vivo studies in mammals, instability or biodegradation of the nanoparticles CdSe leads to the release of toxic metal ions Cd₂ + [29]. The specific chemical properties of metals, including their ability to bind to electron donor atoms (O, S, N), or participate in reactions of oxidation-reduction, can lead to deleterious effects on the cell with including the occurrence of oxidative stress, structural alterations or physiological / enzymatic dysfunction. The mechanisms involved in the toxicity of metals have recently been summarized in a review Limier, et al. [30].

7. Conclusion

In this study, we investigated the effect of oxidative stress on CdSe biomarkers hepatopancreas of the snail Helix aspersa. We can conclude that the species Helix aspersa is sensitive to the presence of semiconductor materials CdSe, this sensitivity was demonstrated by the effects of oxidative stress induced and enzymatic mechanisms involved.  

Figure 1: Breeding snails Helix aspersa (staff photo).

Figure 2: Production of ROS by the CdSe [12].

Figure 3: Variation of tissue protein content (mg) in control and treated snails after 90 days of treatment.

Figure 4: Change catalase activity (nmol / min / mg port) in hepatopancreas in control snails and treated after 90 days of treatment.

Figure 5: Change of GSH (mol / mg port) in hepatopancreas in control snails and treated after 90 days of treatment.

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Figure 6: Variation in the activity of GST (µmol / min / mg port) in the hepatopancreas snails in control and treated after 90 days of treatment.

Figure 7: Change in tissue MDA content (nmol / mg port) in the hepatopancreas snails in control and treated after 90 days of treatment.

Figure 8: Variation of the activity of AChE hepatopancreas (in nmol / min / mg protein) of the control snails and treated after 90 days of treatment.

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