Protective role of Lascorbic acid on antioxidant defense system

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Slide 1 :

BioMetals (2007) 20:177–184 Springer 2006 DOI 10.1007/s10534-006-9025-z Springer 2006 Protective role of L-ascorbic acid on antioxidant defense system in erythrocytes of albino rats exposed to nickel sulfate Kusal K. Das1*, Amrita Das Gupta1, Salim A. Dhundasi1, Ashok M. Patil2, Swastika N. Das3 & Jeevan G. Ambekar 4 1 Environmental Health Research Unit, Department of Physiology, Al-Ameen Medical College, Bijapur, 586108, Karnataka, India; 2 Department of Pathology, Al-Ameen Medical College, Bijapur, 586108, Karnataka, India; 3 Department of Engineering Chemistry, B. L. D. E. A’s. Dr. P. G. Halakatti College of Engineering and Technology, Bijapur, 586103, Karnataka, India; 4 Department of Biochemistry, Sri B.M.Patil Medical College,Bijapur,586103,Karnataka,India; * Author for correspondence (E-mail: kusaldas@yahoo.com) Received 10 April 2006; accepted 13 June 2006 © Key words: nickel sulfate, L-ascorbic acid, hematology, erythrocyte antioxidant system All correspondence: Professor Kusal K.Das, Environmental Health Research Unit, Department of Physiology, Al Ameen Medical College, Bijapur-586108,Karnataka,India. Email: kusaldas@yahoo.com

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Introduction : Nickel is a shining, light-colored metal with high electrical and thermal conductivities. Found in all soils, meteorites, emitted from volcanoes. Resistant to corrosion by air, water and alkalis but reacts with dilute oxidizing agents. Most widely used to make stainless steel. Nickel is released into atmosphere by industries : (ATSDR 2003) 1. Glass & ceramic industries 2. Nickel refineries 3. Electronic & computer equipment 5. Nickel Cadmium battery industries 6. Welding and electroplating Industries 7. Nickel as catalyst

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Typical atmospheric nickel levels for human exposure 5–35 ng/m3 By breathing air (0.1–0.7 µg/day) By drinking water (10 µg /l) Eating food (100–300 µg/day) Smoking tobacco Skin contact with nickel coins, stainless steel, jewelry Patients with artificial body part nickel: exposure pathways

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nickel : distribution, metabolism & excretion Nickel accumulates primarily in bone, liver and kidney (Das & Dasgupta, 1998; ATSDR 2003) Excreted mainly through bile and urine (Onkelinx et al, 1973) Normal urinary conc. of nickel : 0.1-13.3 µg/L Urinary conc. in welders : >188 µg/L Acts by interacting with cytoplasmic protein (ATSDR 2003) Rapidity of toxic effects is related to blood’s transport function, distributing the metal to all the body parts.

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nickel : toxicity Nickel decreases water permeability across erythrocyte membrane. Nickel decrease erythrocyte thermostability, deformability & rate of oxygen release (Tkeshelashvili et al, 1989) Actually it binds with erythrocyte membrane & plasma albumin, stimulate metallothioneins & ROS formation resulting in oxidative damage to the tissues (Sarkar et al, 1998; Das et al, 2001) It also increases lipid peroxidation in the cell (Das et al, 2001) Young erythrocytes are more prone to toxicity development. Damage the cellular integrity, alter the function of biomembrane leading to development of pathological process (Gutteridge, 1993; Das et al, 2006)

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Cellular defense mechanisms to prevent ROS buildup. Superoxide dismutase and glutathione peroxidase are natural antioxidants present in organisms which eliminate some ROS. Glutathione peroxidase catalyzes the reduction of peroxide by oxidizing glutathione (GSH) to GSSG (oxidized glutathione / GSH-disulfide). Glutathione peroxidase

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L-Ascorbic acid: An exogenous antioxidant Most important and economical antioxidant. It decrease the adverse effect of reactive oxygen species and reactive nitrogen species Protects macromolecules: Lipids, DNA, Proteins (Halliwell & Gutteridge, 1986) Nickel depletes intracellular ascorbate level (Salnikow et al, 2004) Studies showed beneficial effect of extra supplementation of ascorbate in nickel induced alterations of nucleic acid conc.of testes, lipid peroxidation and histopathology of liver (Das et al, 2001, 2006; Das & Das, 2004)

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Aims & Objectives: To evaluate the effect of nickel sulfate on various hematological parameters, lipid peroxidation and erythrocyte antioxidant defense system. II. To evaluate the role of L-ascorbic acid on the hematological parameters, lipid peroxidation and erythrocyte antioxidant defense system.

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Materials & Methods: 24 adult male Wister Rats ( 160 + 5g.b.wt) were divided into four groups: GROUP I = CONTROL GROUP II = + NiSO4 GROUP III = + L-ASCORBIC ACID GROUP IV = + NiSO4 + L-ASCORBIC ACID Dosage: NiSO4, 6 H2O ( 2.0 mg / 100 g.b.wt.;i.p.) (Bordes & Papillion, 1983) L- Ascorbic Acid ( 50 mg / 100 g.b.wt.;oral) (Hussain et al.1992) Analysis of hematological parameters (Sysmax K-4500): (Garcia-Mazano et al, 2001) Total red blood corpuscle (RBC) count Packed cell volume (PCV %) Total white blood corpuscles (WBC) count Total platelet count Clotting time Haemoglobin (Hb) (Gowenlock, 1996) Analysis of antioxidant status of erythrocyte: Malondialdehyde (MDA) (Kei et al, 1978) Superoxide dismutase (SOD) (Misra & Fridovich, 1972) Glutathione peroxidase (GSH-Px) (Paglia & Valentine, 1967) Catalase (CAT) (Aebi, 1974) Total erythrocyte glutathione (GSH) (Beutler et al, 1963)

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Results: TABLE 1 Effect of nickel sulfate on hematological parameters in rats Treatment groups: I- untreated control; II- nickel sulfate; III- L-ascorbic acid; IV- nickel sulfate + L-ascorbic acid. RBC, red blood corpuscle; PCV, packed cell volume; WBC, white blood corpuscle. In each column, values with different superscripts (a, b, c) were significantly different from each other (p < 0.05).

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RBC Hb PCV CT WBC PL Fig.1: Percent change chart of various hematological parameters of nickel sulfate treated rats. RBC, red blood corpuscles; Hb, hemoglobin; PCV, packed cell volume; CT, clotting time; WBC, white blood corpuscles; PL, platelet. E I, Group I (control) vs. Group II (+ NiSO4); E II, Group I vs. Group III (+ L- ascorbic acid); E III, Group I vs. Group IV (NiSO4 + L-ascorbic acid).

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Nickel sulfate induced anemia by decreasing RBC count, PCV % & Hb concentration. Nickel may adversely affect hematopoiesis by injuring hematopoietic stem cell Decrease in RBC count, PCV % & Hb conc. may be due to non-regenerative anemia as a result of decreased value of the above parameters (Merchant & Mod, 2004) Nickel sulfate probably induce inhibition of bone marrow activity & the number of all the types of blood cells. Simultaneous treatment with L-ascorbate decrease the toxic effects of nickel sulfate. Ascorbate facilitates the rate of production of blood cells by stimulating bone marrow. Discussion (on hematology):

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TABLE 2 Effect of nickel sulfate (2.0 mg/100 g.b.wt.;i.p) on erythrocyte antioxidant status in rats Treatment groups: I- untreated control; II- nickel sulfate; III- L-ascorbic acid; IV- nickel sulfate + L-ascorbic acid. MDA, malondialdehyde; GSH, glutathione; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase. In each column, values with different superscripts (a, b, c) were significantly different from each other (p < 0.05)

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Fig.2: Percent change chart of antioxidant status in red cell lysates of nickel sulfate treated rats.MDA, malondialdehyde; GSH, glutathione; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase. E I, Group I (control) Vs. Group II (+ NiSO4);EII, Group I vs. Group III (+ L- ascorbic acid); E III, Group I vs. Group IV (NiSO4 + L- ascorbic acid).

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Increased generation of ROS and enhanced lipid peroxidation. Nickel competes with calcium intracellularly & may cause cellular membrane disruption via lipid peroxidation by increasing MDA concentration (Kupferschmidt, 2001; Ochi et al, 1988; Shi et al, 1999) Increased GSH level & GSH-Px activity in RBC may be due to adaptive response to oxidant challenge. Increased SOD activity may be due to increased generation of O2? ¯ & OH ¯ (Kelle et al, 1999) Increased CAT & GSH-Px activity may also be due to their influence on formed H2O2 (Shaikh et al, 1999) Nickel induced depletion of cellular ascorbate level by expression of HIF-1 and HIF-1a genes (Salnikow et al, 2004) Simultaneous treatment with L-ascorbate brought erythrocyte level to near normal condition confirming its protective role on erythrocyte antioxidant defense system. L-ascorbate, a biological scavenger inhibits lipid peroxidation, oxidative DNA damage & protein damage. Discussion (on antioxidant status):

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Sources of intracellular oxidative stress and sites of antioxidant activity (Bulger and Helton, 1998) Role of Ascorbic acid as an antioxidant:

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Metal induced ascorbate depletion (Salnikow et al 2004)

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Conclusion: Nickel sulfate may induce oxidative damage in erythrocyte and depress bone marrow activity. It may elevate erythrocyte MDA & alter GSH level and GSH-Px, SOD and CAT activities. Simultaneous treatment with L-ascorbic acid may protect against toxic effect of nickel on examined parameter in rat blood.

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Acknowledgement: Director , Defence Institute of Physiology & Allied Sciences , DRDO Ministry of Defence , Govt.of India for research grant (TASK- 91, DIPAS/DRDO, 2004 ) THANK YOU The Management of Al-Ameen Charitable Fund Trust , Bangalore

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