1.       Introduction

Heavy metals are pollutants generated by human activity and have a high toxicological impact. Toxic metals are numerous but most notably arsenic, cadmium, lead and mercury. They have impacts on plants, consumer products and on humans [1]. The metallic elements are in various forms always present in the environment. In the state of traces, they are necessary even essential to the living being [2]. Heavy metals do not all present the same risk due to their effects on the organisms, their chemical, physiochemical and biological properties. their toxicity is very variable and their impact on the environment is very different [3].The metals transferred to humans can be the cause of an oxidative stress which represents one of the factors potentiating the genesis of plurifactorialy diseases such as cardiovascular diseases, diabetes, rheumatism asthma, cancer and diseases neurodegenerations diseases [4].In fact, the risk on human health is to abort associated with properties of heavy metals has pollute water, the atmosphere food and soil and dependent also the state chemical their chemical form, of their concentration the environmental context, the possibility of passage in the chain living [5].

Toxicology, they can be defined as metals cumulative nature (often in tissues fat) having essentially effects very adverse living organism. Nutrition and agronomy, they can even be assimilated to trace elements essential for organizations especially by their catalytic action at the metabolism [6,7]. Nickel (atomic number 28, the atomic weight 58.69) is a metal which belongs to the group VIIIB of periodic table. The oxidation states the largest nickel is 2, while the oxidation states +3 and +4 are also known [8]. The nickel compounds are important in modern industry and are used in electroplating and for the production of battery nickel cadmium and electronic equipment. The nickel alloys, as stainless steel, are used in the production of tools, machinery arms and devises, they are also used to flow coins and produce jewel and prostheses medical [9]. The pure nickel can be polished, forged, welded, rolled and inert corrosion by the air, water, acids, alkalis and many organic solvents non-oxidizing [10]. Salts of nickel are used in electroplating, ceramics, pigments and feedstock (e.g.: catalysts, the formation of other nickel compounds) [11]. Nickel is commonly used in many industrial processes and implications Eco toxicological important [12]. Rabbits of different varieties have been used to establish experimental models that are very useful in various spheres of biomedical research (embryology, toxicology, virology) and they are frequently used routinely in serology because the readily produce antibodies against a multitude of antigens [13]. In our work, we tried to demonstrate the effect of nickel chloride at two concentrations 250and 500 ppm on rabbits.

2.       Material and Methods

2.1.  Animals

For our experiments, we chose to work on rabbits Oryctolagus cuniculus. All rabbits were males weighing between 1,5 - 1,8Kg. Animals were kept under constant conditions of temperature environ 25 ± 3^°C and humidity 35 ± 5%. The total body weight was daily recorded before and during the experiments. There was a gain in body weight and increase of food consumption indicating the good conditions of laboratory.

2.2.  Experimental Design

We have handled 15 rabbits. These rabbits were divided on 2 lots of 5 rabbits and we kept five rabbits as control. The treatments began 15th day (adaptation period of rabbits) all treatments are per os (p.o) as follows:

Lot1: controls no treatments

Lot2: treated with NiCl₂ at 250ppm for 90 days of treatment

Lot3: treated with NiCl₂ at 500ppm for 90 days of treatment

After 90 days of treatment, the rabbits were sacrificed. The liver was recovered, collected, weighed and stored for the determination of certain biochemical metabolites and certain oxidative stress parameters (GSH, GPx, CAT, MDA, GST).

2.3.  Dosing Methods

2.3.1. Extraction and dosage of proteins

The method used for protein assay is Bradford (1976) [14], using BSA (Bovine Albumin Serum) as the standard.

2.3.2. Determination of glutathione GSH

The determination of glutathione realized by Weckbeker and Cory (1988) [15]. the principle of this determination is based on measuring the absorbance of 2nitro-5-mercapturique.

2.3.3. Determination of Glutathione Peroxidase (GPx)

The enzymatic activity of GPx was measured by the method of Flohe and Gunzler (1984) [16], using H2O2 as substrate. 

2.3.4. Determination of Glutathione S-Transferase Activity (GST)

The measuring of glutathione s transferase activity was determinate by the method of Habit et al. (1974) [17]. It is based on the conjugation reaction between GST and a substrate.

2.3.5. Determination of activity Catalase (CAT)

The spectrophotometric determination of catalase (CAT) activity is carried out according to the method of Chakma and Horst (1991) [18].

2.4.  Statistical Analysis

The significant differences between the control and the treated groups were determined by the Student's t test.  Statistical calculations were carried out using Minitab 17.1 statistical package and the Excel 16.0 (Microsoft, Inc.).

3.       Results and Discussion

The results are expressed by the mean ± (standard deviation) of n experiments. The differences are considered: significant when P≤0.05, very highly significant when P ≤ 0.001, highly significant when P ≤0.01. The purpose of this study was to demonstrate the potential toxicity of NiCl₂ to certain biochemical, enzymatic and non-enzymatic parameters of Oryctolagus cuniculus rabbits as a biological model. The Oxidative stress is typically defined as an imbalance in the balance between antioxidant defense systems and the production of ROS in favor of these systems [19]. In our work, we have shown an increase in the level of liver proteins. This result confirms the study of the exposure of rabbits to environmental stress (Cadmium, Copper and Zinc) can modify the metabolism of proteins and their syntheses at the level liver [20]. These results are confirmed by [21,22] which showed a correlation between the disruption of the total protein level and the xenobiotic toxicity. This effect is explained on the one hand by the induction of protein synthesis Stress related to the phenomenon of bio activation / biotransformation and on the other hand by the lipid peroxidation generated by the ROS. Metallothioneins (MTs) are proteins capable of trapping free radicals and chelating metals, which is confirmed by the work of [23,24] who found a significant increase in metallothionein levels in the liver of rabbits treated with cadmium. The Exposure of rats to environmental stress (Ni) can alter the metabolism of amino acid proteins and their synthesis in the liver [25].

The Phagocyte nickel by the cell increases the activity of oxygen radicals, catalyzes the transformation of DNA and can cause cancer, the intervention of endogenous and exogenous antioxidants stops these reactions [26-29]. The organism can defend and adapt these stresses by its endogenous antioxidants and its stress proteins such as metallic thiamine’s encountered in humans and many animals [30]. In our study, the administration of nickel chloride in rabbits caused oxidative damage. Nickel with cadmium and chlorine, are considered as oxidative stresses [31]. The Oxidative stress in the cell can be induced either by increased production of ROS or inhibition of the antioxidant defense system. This balance between the production and catabolism of oxidants is essential for the maintenance of the biological integrity of the tissues [32].

MDA is a secondary product generated during the oxidation of polyunsaturated fatty acids [33]. Our results show an increase in the level of hepatic MDA in rabbits treated with a high dose of NiCl₂ (150 mg / kg / day) compared to control rabbits, which is a marker of lipid peroxidation. Our results confirm those of [34], who found an alteration in antioxidant status in nickel-treated rats. This alteration is accompanied by an increase in lipid peroxidation and a decrease in cellular GSH level. MDA is also a by-product of prostaglandin biosynthesis [35]. Our results are in agreement with those of Viareggio et al. Which studied the toxic effects of heavy metals on lipid peroxidation in Mytillusgalloprovinciallis. They had demonstrated a significant level of MDA after exposure to copper [36].

For Glutathione S-Transferase (GST), this enzyme plays an important role in the detoxification of xenobiotic and / or in protecting against harmful metabolites generated after degradation of macromolecules following exposure to oxidative stress [37]. According to our results, an increase in GST in the liver [38].  Report an increase in hepatic and cerebral GST activity following an injection of cadmium to guinea pigs. The results obtained confirm the ability of the metal to generate cellular radical effects at various metabolic levels. Glutathione also intervenes at a second level in anti-radical defense by its involvement in detoxification reactions catalyzed by Glutathione-S-transferase [39]. The response of GST activity depends on several factors such as xenobiotic type, concentration, exposure time and species [40].

GSH is a crucial element of the antioxidant defense mechanism, functions as a direct reactive free radical sensor. In our experimental conditions [41], we notice a statistically significant decrease in GSH levels, which can be explained by an adaptive response to oxidative stress. The decrease in GSH can be explained by several assumptions: First, GSH plays a key role in the detoxification of free radicals and heavy metals [42] and in the case of nickel, the latter interacts directly with high affinity to the GSH thiol (-SH) groups, second, glutathione Can also interact with the free radicals generated by this metalloid [43-46], third, nickel inhibits glutathione synthetize, and glutathione reductase [47], so little GSH is produced. The effect of nickel chloride on the concentration of the hepatic GSH and the activities of the antioxidant enzymes (GPx, GST) is accompanied by an increase in the free radical’s ROS. The latter initiated the lipid peroxidation [48].

Glutathione peroxidase, superoxydedismutase and catalase are mutually supportive defense against ROS [49,50]. Our results showed an increase in GPx, CAT and hepatic GST activity in rabbits treated nickel. GPx is a key antioxidant enzyme that regulates the level of ROS (GPx is able not only to reduce hydrogen peroxide to water, but also hydro peroxides resulting from the oxidation of unsaturated fatty acids) and thus protects the cells Against the damage caused by nickel [51]. The antioxidant enzymes (SOD, GPx and CAT) limit the effects of oxidizing molecules in tissues and play a role in defense against cellular oxidative damage being free radical scavengers [52].

Catalase plays an important role in protecting the body against oxidative stress damage [53]. Zikic et al report an increase in the activity of both enzymes (SOD and CAT) in erythrocytes and tissue Pisces.

4.       Conclusion

 In this study, we investigated the effect of nickels on the oxidative stress biomarkers of the liver of rabbits Oryctolagus cuniculus. We can conclude that this species is sensitive to the presence of metals, this sensitivity was manifested by the effects of induced oxidative stress and the enzymatic mechanisms involved. 

Figure 1: Variation of protein (mg) in the control and treated rabbits after 90 days of treatment.

Figure 2: Variation of MDA (μM/mg pot) in the control and treated rabbits after 90 days of treatment.

Figure 3:  Effect of NiCl₂ on the activity of GST (μM/min/mg) after 90 days of Treatment.

Figure 4: Variation of GSH (μM/mg prot.) in the control and treated rabbits after 90 days of treatment.

Figure 5: Effect of NiCl₂ on the activity of GPx (μM/min/mg) after 90 days of Treatment.

Figure 6:  Effect of NiCl₂ on the activity of Catalase (μ M/min/mg) after 90 days of treatment.

1.       Benedetto Di (1997) Spectrometric methods of analysis and characterization, dossier SAM, LES METAUX LOURDS. Axe " Process Engineering ", centre SPIN, Ecole des Mines de Saint-Etienne 55.

2.       Mohan D, Pittman CU Jr, Steele PH (2006) Single binary and multi component absorption of copper and cadmium from aqueous solutions on Kraft lignin a biosorbent. Journal of Colloid Interface Sci 297 : 489-504.

3.       Reddad Z, Gerente C, Andres Y, Cloirec P (2002) Absorption of several metal ions onto a low cost biosorbente: kinetic and equilibrium studies. Environ. Sci. Technol 36: 2067-2073.

4.       Nzengue Y (2008) Comparison of Redox Toxicity Mechanisms of Cadmium, Copper and Zinc: Place of Metallo Thionines and PhD Thesis, Joseph Fourier University, Grenoble.53.

5.       El Hraiki A, Kessabi M, Sabhi Y, Bernard P, Buhler DR (1992) Contamination by cadmium, chromium, mercury and lead of Moroccan fishery products taken from the Mediterranean Sea. Rev Med Vet 143 : 49-56.

6.       Adriano, DC (1986) Trace elements in the environment. Springer-Verlag Berlin Heidelberg GmbH; New York 1.

7.       Fergusson JE (1980) Heavy metals pollution by traffic in Choustchurch, New Zealand: Lead and cadmium content of dust, soil, and plants. New Zealand journal of science 23:2830.

8.       Tundermann JH, Tien JK, Howson TE (2005) Nickel and nickel alloys. In: Kirk-Othmer Encyclopedia of Chemical Technology. 17.

9.       Garrett RG (2000) Natural sources of metals to the environment. In: Centeno JA, Collery P, Fernet G, Finkelman RB, Gibb H, Etienne J-C, editors. Metal ions in biology and medicine, Paris: John LibbeyEurotext 6: 508-510.

10.    Falbe, J, Regitz M. Editors. (1999) RO MPP Kompakt: BasislexikonChemie. Stuttgart, NY: Georg Thieme,1647 -1651.

11.    USGS. Nickel. In: 2006 Minerals Yearbook. Reston, VA: US Geological Survey (2008).

12.    Sunderman WF Jr (1986) Nickel. In Handbook on Toxicity of Inorganic Compounds (Edited by Siegel H. and Seiler H. G.) Marcel Dekker, New York 453 - 468.

13.    Russell RJ, Schilling PW (1973) The Rabbit. Aero med. REUS. USAF School of Aerospace Medicine, Brooks Air Force Base, San Antonio, TX 21: 6.

14.    Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Analytical Biochemistry 72: 248-254.

15.    Weckbecker G, Cory JG (1988) Ribonucleotide reductase activity and growth of glutathione-depleted mouse leukemia L 1210 cells in vitro. Cancer Letters 40: 257- 264.

16.    Flohé L, Günzler WA (1984) Analysis of glutathione peroxidase. Methods Enzymol 105 : 114-121.

17.    Habig WH, Pabst MJ, Jakoby WB (1974) The first enzymatic step in mercapturic acid formation. J Biol Chem 249: 7130-7139.

18.    Cakmak ET, Horst (1991) Effects of aluminum on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiologia plantarum 83: 463-468.

19.    Favier A (2003) Oxidative stress: Conceptual and experimental interest in understanding the mechanisms of diseases and therapeutic potential. Biochemical mechanisms. Chemical News 108-115.

20.    Harabawy AS, Mosleh YY (2014) The role of vitamines A, C, E and selenium as antioxydants against genotoxicity and cytotoxicity of cadmium, copper, lead and zinc on erythrocytes of Nile tilapia, Oreochromis niloticus. Ecotoxicol Environ Saf 104: 28-35.

21.    Redhouane Salah S (2004) Effects of metallic discharges of acidiaries from the Elhadjard'annaba iron and steel complex on a marine cellular model: tetraselmissuecica. Science et technologie 121-124.

22.    Rouabhi R, Djebar-Berrebbah H, Djabar MR (2006) Effect of a pesticide, diflubenzuron on fresh water macroinvertebrate (Tetrahymena pyriformis). Chin J Appel Environ Biol 12 : 514-517.

23.    Anicka K, Cempei M (2001) lipid peroxidation and selected antioxidants in rat liver after oral exposure to nickel (II) chloride Bromate. Chem Toksykol 34: 291.

24.    Michael S, Waalkes, John A, Tomass, John U (1982) Induction of epaticmétallothionine in the rabbit cadmium exposure. Toxicology and Applied pharmacology 62: 211-218.

25.    Stinson TJ, Jaw S, Jeffery EH, Plewa MJ (1992) The relationship between nickel chloride-induced peroxidation and DNA strand breakage in rat liver. Toxicology and Applied Pharmacology 117: 98-103.

26.    Huang X, Thuang Z, Frenkl K, Klein CB, Coster M (1994) The role of nickel and nickel mediated reactive oxygen species in the mechanism of nickel carcinogenesis. Environ Health Perspect 102: 281-284.

27.    Fletcher GG, Rossetto FE, Turnbull JD, Nieboer E (1994) Toxicity, uptake, and mutagenicity of particulate and soluble nickel compounds. Environ. Health. Perspect 102: 69-79.

28.    Costa M, Salnikour K, Cosentino S, Klein CB, Huang X, et al. (1994) Molecular mechanism of nickel carcinogenesis. Environm Health Perspect 102 : 127-130.

29.    Landolph JR (1994) Molecular mechanisms of transformation of C3H/10T 1/2 C1 mouse embryo cells and diploid human fibroblasts by carcinogenic metal compounds. Environ Health Perspect 102: 119-125.

30.    Kagi JHR. (1993) Evolution structure and chemical activity of class I metallothioneins: an overview in: metallothionein III, Biological roles and medical implications. Suzuki K.T., ImuraN., Kimura M.Eds. Berlin, BirkhauserVertag 29-56.

31.    Frenkel K, Karkoszka J, Cohen B, Barański B, Jakubowski M, et al. (1994) Occupational exposures to Cd, Ni, and Cr modulate titers of autoxidized DNA base autoantibodies. Environ Health Perspect 102 : 221- 225.

32.    Fatima S, Mahmood R (2007) Vitamin C attenuates potassium dichromate-induced nephrotoxicity and alterations in renal brush border membrane enzymes and phosphate transport in rats. Clin Chim Acta 386: 94-99.

33.    Misra M, Rodriguez RE, Kasprzak KS (1991) Nickel induced lipid peroxydation in the rat : correlation with nickel effects on antioxydant systems. Toxicol Lett 57 : 269-281.

34.    Xie J, Funakoshi T, Shimada H, Kojima S (1995) Effects of chelating agents on testicular toxicity in mice cause by acute exposure to nickel. Toxicology 103 : 147 -155.

35.    Coeurdassier M (2001) Use of terrestrial (Helix aspersa) and aquatic (Lymnia stagnalis and Lymnia palustris) pulmonary gastropod molluscs as indicators of pollution by metallic elements and xenobiotics. PhD thesis, University of Franche-Comté, Besançon 22.

36.    Viarengo A, Canesi L, Pertica M, Poli G, Moore MN, et al. (1990) Heavy metal effects on lipid peroxidation in the tissues of Mytilus galloprovincialis Lam. Comp Biochem Physiol 97: 37-42.

37.    Messarah M, Klibet F, Boumendjel A, Abdennour C, Bouzerna N, et al. (2012) Hepatoprotective role and antioxydant capacity of selenium on arsenic-induced liver injury in rats. Exp Toxicol Pathol 64 : 167-174.

38.    Iscan M, Coban T, Eke BC (1994) Differential combined effect of cadmium and nickel on hepatic and renal Glutathionne-S-transferases of the Guinea pig. Environ health perspect 102 : 69-72.

39.    Barillet, S. (2007) Toxicokinetics, chemical and radiological toxicity of uranium in zebrafish (Danio rerio). PhD thesis, Paul Verlaine University of Metz, 98.

40.    Oruç EÖ, Üner N (2000) Combined effects of 2, 4-D and azinphosmethyl on antioxidant enzymes and lipid peroxidation in liver of Oreochromis niloticus. Comp. Biochem Physiol C 127: 291-296.

41.    Romão PRT, Tovar J, Fonseca SG, Moraes RH, Cruz AK, et al. (2006) Glutathione and the redox control system trypanothione /trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity. BrazJ Med Biol Res 39: 355-363.

42.    Hultberg B, Anderson A, Isakson A (2001) Interactions of metals and thiols in cell damage and glutathione distribution: potential of mercury toxicity by dithiothreitol. Toxicology 156: 93-100.

43.    Anicka k, Cempei M (2001) lipid peroxidation and selected antioxidants in rat liver after oral exposure to nickel (II) chloride Bromate. Chem Toksykol 34: (in Polish) 291.

44.    Ito H, Okamoto K, Kato K (1998) Enhancement of expression of stress proteins by agents that lower the levels of glutathione in cells. Biochim Biophys Acta 1397: 223-230.

45.    Whanger PD (1973) Effects of dietary nickel on enzyme activities and mineral content in rats. Toxicol Appl Pharmacol 25: 323-331.

46.    Weischer CH, Kordel W, Hochrainer D (1980) Effects of NiCl₂ and NiO in Wistar rats after oral uptake and inhalation exposure, respectively. Zentral Bakteriol Mikrobiol Hyg (B) 171: 336 -351.

47.    James CW et al. (2006) Nickel-Seleniuinteraction-Time Dependent Biochemical Alterations and Metal Decorporation in Rats. Lam Environmental Pollution 144: 790-801.

48.    Upadhyay AK, Mathur R, Bhadauria M, Nirala SK (2009) Therapeutic influence of Zinc and ascorbic acid against lead induced biochemical alterations. Therapie 64:  383-388.

49.    Ahmed RS, Suke SG, Seth V, Chakraborti A, Tripathi AK, et al. (2008) Protective effects of dietary Ginger (Zingiber officinales Rosc.) on lindane-induced oxydative stress in rats. Phytother Res 22 : 902-906.

50.    Soudani N, Sefi M, Ben Amara I, Boudawara T, Zeghal N (2010) Protective effects of Selenium (Se) on Chromium (VI) induced nephrotoxicity in adult rats. Ecotoxicol Environ Saf 73: 671-678.

51.    Arnaudabe J, Arnaultc N, Rousselbde AM, Bertraisc S, Ruffieuxa D, et al. (2007) Relationship between selenium, lipids, iron status and hormonal therapy in women of the SU.VI.M.AX cohort. Journal of Trace Elements in Medicine and Biology 21: 66-69.

52.    Gutteridge JM (1995) Lipid peroxydation and antioxydants as bio markers of tissue damage. Clin Chem 41 :1819-1828.

53.    Ballesteros ML, Winderlin DA, Bistoni MA (2009) Oxidative stress responses in different organs of Jenynsia multidentata. Ecotoxicol Environ Saf 72 : 199-205.