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[Indian Journal of Experimental Biology (2005): 43 (September), 773-781]

Effect of Spirulina and Liv.52 on Cadmium induced Toxicity in Albino Rats

Jeyaprakash K and Chinnaswamy P Department of Biochemistry, Dr. N.G.P. Arts & Science College,

Kovai Medical Center Research & Educational Trust, Coimbatore, India Oral administration of cadmium (6 mg/kg body weight/day) as cadmium chloride (CdCl2) for 30 days resulted in a significant increase in thiobarbituric acid reactive substances (TBARS) level and a decrease in the levels of copper, zinc, iron, selenium, glutathione, superoxide dismutase, catalase, glutathione peroxidase when compared to normal control. Administration of either Liv.52 alone or in combination with spirulina produced a well pronounced protective effect in respect to these parameters in cadmium intoxicated rats. The protective effect of spirulina and Liv.52 in respect to biochemical changes were also confirmed by histopatholgoical study in the liver and kidney sections. Keywords: Cadmium chloride (CdCl2), Liv.52, Spirulina, Thiobarbituric acid reactive substances (TBARS) Cadmium, a heavy metal well known to be highly toxic to both human and animals, is distributed widely in the environment due to its use in various industries. Some of the toxic effects of cadmium exposure are testicular atrophy, renal dysfunction, hepatic damage, hypertension, central nervous system injury and anemia1. Cadmium may induced oxidative damage in different tissues by enhancing peroxidation of membrane lipids in tissues and altering the antioxidant systems of the cells. The peroxidative damage to the cell membrane may cause injury to cellular components due to the interaction of metal ions with the cell organelles2. Cadmium depletes glutathione and protein bound sulfhydryl groups resulting in enhanced production of reactive oxygen species such as superoxide ions, hydroxyl radicals and hydrogen peroxides. These reactive oxygen species result in increased lipid peroxidation3. Biological compounds with antioxidant properties contribute to the protection of cells and tissues against deleterious effects of reactive oxygen species and other free radicals. Protective agents from plant origin with antiperoxidative and antioxidant properties play an important role in protecting the liver against toxicity4. Although, high incidence of low level exposure to cadmium takes place, proper therapeutic intervention remains obscure. Most of the synthetic antidotes in the management of cadmium toxicity have met with limited success due to their inherent toxicity and nonspecificity5. Traditional medicines are effective in certain disorders and are based on experience in the use of plant products in amelioration of common diseases. Liv.52, an Ayurvedic multiherbal formulation is widely used in various hepatic disorders6,7. Spirulina is a microscopic, multicellular filamentous blue green algae (cyanobacterium). It is unique among blue green algae because of it has a long history of safe use8. The antioxidant properties of spirulina have attracted the attention of researchers recently. The antioxidant effect of spirulina against carbon tetrachloride9 and mercury induced toxicity10 in rats have been studied. However, the antioxidant and protective effect of spirulina against cadmium induced toxicity in respect to lipid peroxidation, antioxidant status, trace elements in tissues remains unexplored.

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Therefore, the present study has been undertaken to delineate its antioxidant and protective role against cadmium induced toxicity in rats. MATERIALS AND METHODS Chemicals: Cadmium chloride (CdCl2), thiobarbituric acid (TBA), nitroblue tetrazolium (NBT), reduced glutathione (GSH), 5,51-dithio-2-nitrobenzoic acid (DTNB) were purchased from Sigma Chemical Co. (St. Louis, Mo, USA). Other chemicals used were of analytical grade and purchased locally. Liv.52 tablets (500 mg each) were obtained commercially from The Himalaya Drug Company, Bangalore, India. Each Liv.52 tablets is composed of Capparis spinosa (65 mg), Cichorium intybus (65 mg), Solanum nigrum (32 mg), Cassia occidentalis (16 mg), Terminalia arjuna (32 mg), Achillea millefolium (16 mg), and Tamarix gallica (16 mg). Spirulina tablets (100 mg each) were obtained commercially from Parrys Neutraceuticals Ltd., Chennai, India. Animals and treatment: Male albino rats (Wistar strain) weighing 150-175 g were obtained from animal breeding centre, P.S.G. Institute of Medical Sciences & Research, Coimbatore, Tamilnadu, India. They were housed in KMCH College of Pharmacy, Coimbatore, Tamilnadu, India, in controlled temperature (27o ± 2oC), humidity (55 ± 10%) and light with 12:12 hr L:D cycle. Animals were fed with standard pellet (Hindustan Lever Ltd., India). They were given a week time to get acclimatized with laboratory condition. Ethical clearance for the handling of experimental animals was obtained from the committee constituted for the purpose (685/02/a/CPCSEA/2002). After acclimatization the animals were divided into the following 8 groups of 6 rats each: Gr A: normal control; Gr B: cadmium (6 mg/kg body weight/day) as CdCl2 orally for 30 days; Gr C: Spirulina (500 mg/kg body weight/day) orally for 30 days; Gr D: Cadmium as in group B + spirulina as in group C orally for 30 days; Gr E: Liv.52 (500 mg/kg body weight/day) orally for 30 days; Gr F: Cadmium as in group B + Liv.52 as in group E orally for 30 days; Gr G: Spirulina as in group C + Liv.52 as in Group E orally for 30 days; and Gr H: Cadmium as in group B + Spirulina as in group C + Liv.52 as in group E orally for 30 days. At the end of the experimental period, the rats were deprived of food overnight and sacrificed by light ether anaesthesia. Serum samples were collected to estimate the amount of copper11, zinc12, iron13 and selenium14. Liver and kidney were removed and cleaned in normal saline. A known weight of these tissues was quickly weighed and then homogenized (10% w/v) in ice cold phosphate buffer (0.1 M, pH 7.4) using potter Elvehjem tefton homogenizer. The homogenate was centrifuged at 5000 rpm at 4oC for 30 minutes and supernatant obtained was used for the assay of various enzymes. Lipid peroxidation was estimated by the method of Das et al.15. Superoxide dismutase (SOD; EC.1.15.1.1)16 Catalase (CAT: EC.1.11.1.6)17, reduced glutathione (GSH)18 and glutathione peroxidase (GPx: EC.1.11.1.9)19 were assayed. Protein was quantified as per Lowry et al.,20, using bovine serum albumin as standard. Histopathology: Small pieces of liver and kidney tissues were fixed in 10% formalin solution, dehydrated with 90% ethanol, embedded in paraffin, cut into thin sliced section (5 µm thickness), and stained with haematoxylin-eosin dye21. Phytochemical analysis: Preliminary quantitative measurement of total phenols22, flavonoids23, vitamin C24, vitamin E25 and glutathione (GSH)18 was done in spirulina and Liv.52 samples. Mineral analysis in spirulina and Liv.52: Spirulina and Liv.52 samples (0.5 g each) were digested with 5 ml perchloric acid and nitric acid mixture (1:3) in a microjeldhal digestion flask. The digestion was continued till no more brown fumes evolved and the solution in the flask become colourless. The digested mixture was transferred to a 100 ml standard flask and made upto 100 ml

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with double distilled water. The aliquots of the sample was used for the estimated of copper26, zinc27, iron28 and selenium29. Statistical analysis: Statistical analysis was performed by one way analysis of variance (ANOVA). Critical difference (CD) was calculated at 1% level according to the method of Gomez et al.30 and results were expressed as mean ± SD of six rats in each group. RESULTS AND DISCUSSION Spirulina and Liv.52 are good sources of flavonoids, total phenols, vitamin C, vitamin E, reduced GSH, copper, zinc, iron and selenium (Table 1).

Table 1: None enzymic antioxidants & antioxidant minerals in spirulina and Liv.52

Samples Flavonoids (mg/g)

Total phenols (mg/g)

Vit C (mg/g)

Vit E (mg/g)

GSH (nm/g)

Copper (µg/g)

Zinc (mg/g)

Iron (mg/g)

Selenium (mg/g)

Liv.52 6.92 12.96 1.30 0.838 67.30 302 6.80 1.26 0.02 Spirulina 8.98 9.65 0.21 0.150 123.80 12 1.50 30 1.50 In rats treated with cadmium (Gr.B), there was a significant decrease (p<0.01) in the level of these trace elements (copper, iron, zinc and selenium) as compared to normal control (Gr. B) (Table 2). This may be due to interference of cadmium on absorption and transport of these trace elements, which would have resulted in the depletion of these metals in this group of rats. Cadmium may inhibit zinc activities at many stages, interfering with absorption, distribution and transport of zinc into cells or into several intracellular structure31. Bannis et al.32 have suggested that cadmium impairs iron absorption and utilization or both. Cadmium induced decrease in the level of copper33 and selenium34 have been reported. Co-administration of either spirulina (Gr. D) or Liv.52 (Gr. F), or both Liv.52 and spirulina (Gr. H), along with cadmium, caused a significant increase (p<0.01) in the level of these trace elements in serum as compared to cadmium intoxicated rats (Gr. B). This may be due to interaction and antagonistic action of these trace elements, which are present in spirulina and Liv.52 with cadmium and resulted in elevated level of these trace elements in these groups of rats. Studies have shown that supplementation of zinc35, copper36, selenium37, iron38 offered a protective effect against toxic effects of cadmium.

Table 2: Levels of trace elements in serum of different experimental groups of rats (Values expressed in µg/dl are mean ± SD of 6 rats in each group)

Groups Treatment Copper (µ/dl) Zinc (µg/dl) Iron (µg/dl) Selenium (µg/dl) A Normal control 102.18 ± 0.32a 94.21 ± 0.41a 156.35 ± 1.19a 74.31 ± 0.15a B Cadmium treated 42.55 ± 0.50b 31.62 ± 0.22b 80.20 ± 0.82b 36.24 ± 0.66b C Spirulina alone 102.35 ± 0.62a 94.62 ± 0.76c 157.15 ± 0.98c 74.81 ± 0.32a D Cadmium + Spirulina 81.40 ± 0.45c 76.78 ± 1.05c 122.12 ± 1.12c 62.42 ± 0.46c E Liv.52 alone 103.10 ± 1.16a 95.20 ± 0.80a 158.17 ± 0.59a 74.63 ± 1.10a F Cadmium + Liv.52 94.31 ± 0.93d 83.24 ± 0.52d 143.53 ± 1.23d 70.12 ± 0.52d G Spirulina + Liv.52 103.66 ± 0.65a 95.81 ± 0.91a 159.75 ± 0.72a 75.14 ± 0.12a

H Cadmium + Spirulina + Liv.52 96.72 ± 0.49d 96.06 ± 0.21a 146.79 ± 1.10d 71.37 ± 0.71d

Values with same superscript did not differ significantly (p<0.01). In cadmium treated rats (Gr. B), there was a significant increase (p<0.01) in the level of TBARS in both liver and kidney, as compared to normal control (Gr. A) (Tables 3 and 4). This could be possibly due to excessive formation of free radicals, which leads to deterioration of biological macromolecules3. Manca et al.39, have reported that lipid peroxidation is considered as a sensitive index of cadmium exposure. Simultaneous administration of either spirulina (Gr. D) or Liv.52 (Gr. F), or both Liv.52 and spirulina (Gr. H), along with cadmium, caused a significant decrease (p<0.01) in the level of TBARS when compared to rats treated with cadmium alone (Gr. B). This

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may be attributed to the presence of phenolic compounds, flavonoids and antioxidant vitamins (Vitamins C and E) in Liv.52 and spirulina. Vitamins E and C are potent free radical scavengers and prevent oxidative damage by utilizing the free radicals40. Flavonoids and phenolic compounds have long been recognized as excellent scavengers of superoxide, hydroxyl ion and peroxyl radicals and as potent inhibitors of lipid peroxidation41. Suja et al.42 reported that administration of Liv.52 reduced the peroxidative effects of hydrogen peroxide and inhibited the deleterious effects of lipid peroxidation by enhanced supply of reduced GSH. In cadmium treated animals (Gr. B), there was a significant decrease (p<0.01) in the activities of SOD and CAT when compared to normal control (Gr. A) (Tables 3 and 4). This may be due to either the antagonistic effect of cadmium with copper and zinc, which are important metals for the activity of SOD molecule43 or inactivation of SOD by cadmium induced lipid peroxidation44. Whanger et al.45, reported that the decrease in catalase activity may reflect decreased absorption of iron, an essential trace element required for the activity of catalase. These findings support the results of the present study. Co-administration of either spirulina (Gr. D) or Liv.52 (Gr. F) or both spirulina and Liv.52 (Gr. H) along with cadmium significantly (p>0.01) increased the activities of SOD and CAT when compared to rats treated with cadmium alone (Gr. B). This could be attributed to the presence of nonenzymic antioxidants (vitamin C, vitamin E, phenols, and flanoids) and antioxidant minerals (copper, zinc, selenium and iron) in spirulina and Liv.52.

Table 3: Effect of spirulina and Liv.52 against cadmium induced toxicity in liver (Values are mean ± SD of 6 rats)

Gro

ups

Treatment TBARS (nm/mg protein)

SOD* (Units/mg protein)

CAT# (Units/mg protein)

GSH (µg/mg protein)

GSx$ (Units/mg protein)

A Normal control 0.66 ± 0.01a 6.99 ± 0.40a 70.68 ± 1.99a 5.29 ± 0.14a 6.83 ± 0.64a B Cadmium treated 2.12 ± 0.02b 3.75 ± 0.15b 34.61 ± 0.81b 3.18 ± 0.16b 3.41 ± 0.40b C Spirulina alone 0.65 ± 0.04a 7.08 ± 0.033a 71.11 ± 0.66a 5.42 ± 0.13a 6.99 ± 0.53a D Cadmium + Spirulina 1.09 ± 0.11c 5.70 ± 0.13c 63.18 ± 0.98c 4.46 ± 0.10c 4.78 ± 0.33c E Liv.52 alone 0.64 ± 0.03a 7.17 ± 0.10a 71.50 ± 0.82a 5.37 ± 0.11a 7.08 ± 0.48a F Cadmium + Liv.52 0.75 ± 0.06a 6.17 ±0.37d 66.26 ± 1.64d 4.79 ± 0.25d 5.84 ± 0.45d G Spirulina + Liv.52 0.63 ± 0.03a 7.23 ± 0.09a 71.62 ± 1.45a 5.46 ± 0.12a 7.19 ± 0.38a

H Cadmium + spirulina + Liv.52 0.73 ± 0.05a 6.43 ± 0.20d 67.14 ± 0.28d 4.92 ± 0.31d 6.08 ± 0.56a,d

*=Units/mg protein (amount of enzyme required to inhibit 50% reduction of NBT) #=Units/mg protein (µm of H2O2 decomposed/mg protein/min) $=Units/mg protein (µg of GSH consumed/min/mg protein) Values with same superscript did not differ significantly (p<0.01). Hasan et al.46 indicated that adverse effect of heavy metals (cadmium, lead, arsenic, mercury) would be antagonized by the administration of zinc, copper and selenium. Spirulina contains superoxide dismutase (SOD) that can prevent the cell damage by free radicals47. The enzymes SOD and CAT constitute the first lien of defense against free radical induced damage and the restoration of these enzyme activity by Liv.52 and spirulina may account for their protective effect.

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Fig. 1 – Liver section of rats (a) control showing normal arrangement of hepatocytes, central vein and portal triad. No inflammation is observed (H&E x 100); (b) cadmium treated rats with periportal inflammation (PI) (H&E x 100); (c) cadmium treated rats showing Microvesicular steatosis (MS), balloon degeneration (BD) (H&E x 450); (d) cadmium and spirulina treated rats showing normal architecture with mild residual microvesicular steatosis MRMS) (H&E x 450); (e) cadmium and Liv-52 treated rats showing normal hepatocytes with no significant damage. (H&E x 100); (f) cadmium + spirulina + Liv-52 treated rats showing normal hepatocytes (NH) with reversal of cadmium induced hepatic damage (H&E x 450).

Table 4: Effect of spirulina and Liv.52 against cadmium induced toxicity in kidney

(Values are mean ± SD of 6 rats)

Gro

ups

Treatment TBARS (nm/mg protein)

SOD* (Units/mg protein)

CAT# (Units/mg protein)

GSH (µg/mg protein)

GSx$ (Units/mg protein)

A Normal control 1.14 ± 0.03a 5.43 ± 0.21a 61.38 ± 1.62a 4.62 ± 0.11a 5.23 ± 0.65a B Cadmium treated 2.79 ± 0.04b 2.41 ± 0.45b 29.58 ± 2.42b 2.45 ± 0.08b 1.52 ± 0.19b C Spirulina alone 1.12 ± 0.03a 5.56 ± 0.18a 61.96 ± 0.94a 4.70 ± 0.07a 5.39 ± 0.52a D Cadmium + Spirulina 1.68 ± 0.02c 4.23 ± 0.13c 52.47 ± 1.36c 3.86 ± 0.08c 3.62 ± 0.41c E Liv.52 alone 1.11 ± 0.07a 5.60 ± 0.10a 62.27 ± 0.57a 4.68 ± 0.13a 5.48 ± 0.79a F Cadmium + Liv.52 1.29 ± 0.08d 4.58 ± 0.10d 59.99 ± 1.38a,d 4.24 ±0.09d 4.33 ±0.48a,c G Spirulina + Liv.52 1.10 ± 0.04a 5.63 ± 0.06a 62.86 ± 0.68a 4.72 ± 0.10a 5.77 ± 0.57a

H Cadmium + spirulina + Liv.52 1.25 ± 0.17a,d 4.77 ± 0.77d 58.53 ± 2.82d 4.34 ± 0.10d 4.52 ± 0.37a,c

*=Units/mg protein (amount of enzyme required to inhibit 50% reduction of NBT) #=Units/mg protein (µm of H2O2 decomposed/mg protein/min) $=Units/mg protein (µg of GSH consumed/min/mg protein) Values with same superscript did not differ significantly (p<0.01).

In rats treated with cadmium (Gr. B) there was a significant decrease (p<0.01) in the level GSH and GPx when compared to normal control (Gr.A) (Tables 3 and 4). This could be probably due to either increased utilisation of GSH by the cells to act as scavengers of free radicals caused by toxic chemical agents, or enhanced utilization of GSH by GPx48, or decreased availability of selenium which leads to inefficient disposal of peroxides and results in elevated lipid peroxidation49. Simultaneous administration of either spirulina (Gr.D) or Liv.52 (Gr.F) or both Liv.52 and spirulina (Gr.H), along with cadmium, significantly increased (p<0.01) the levels of GSH and GPx when compared to rats treated with cadmium along (Gr.B). This may be due to either increased supply of GSH or selenium by these drugs for the activation of GPx. Selenium is an integral component of GPx which can capable of reducing peroxides and hydrogen peroxide19. Histopathology of liver section of control rats showed the normal hepatocytes, central vein and portal triad (Fig. 1a). Liver section of

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Fig. 2 – Kidney section of rats (a) control showing normal architecture (H&E x 100); (b) cadmium treated rats with cellular glomeruli (CG) (H&E x 450); (c) cadmium treated rats with tubular necrosis (TN). The interstitium appears normal (H&E x 100); (d) cadmium and spirulina treated rats showing normal architecture with mild residual tubular necrosis (MRTN) (H&E x 100); (e) cadmium and Liv-52 treated rats with normal architecture. (H&E 100); (f) cadmium + Spirulina + Liv-52 treated rats showing normal architecture with reversal of cellular glomeruli and tubular necrosis. (H&E x 100).

cadmium treated rats showed periportal inflammation, fatty change, congestion of sinusoids and microvesicular steatosis (Fig. 1b and c). Liver section of cadmium and spirulina treated rats showed the normal architecture with mild residual microvesicular steatosis (Fig. 1d). Administration of either Liv.52 alone (Fig. 1e) or in combination with spirulina (Fig. 1f) in cadmium intoxicated rats showed the normal hepatocytes with reversal of cadmium induced hepatic damage. Histopathology of kidney section of control rats showed normal glomeruli and renal tubules (Fig. 2a). The kidney section of cadmium treated rats showed cellular glomeruli, congestion of blood vessels, and tubular necrosis (Fig. 2b, c). Kidney section of cadmium and spirulina treated rats showed normal architecture with mild residual tubular necrosis (Fig. 2d). Administration of either Liv.52 along (Fig. 2e) or in combination with spirulina (Fig. 1f) in cadmium intoxicated rats indicated the normal architecture with reversal of cadmium induced renal damage.

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