Spirulina protects against cadmium-induced hepatotoxicity in rats

Cadmium (Cd) is an environmental and industrial cumulative pollutant that most affects many organs specially the liver. The protective effect of Spirulina, filamentous blue green algae, on Cd – induced oxidative stress and hepatotoxicity was evaluated in male rats. Oral administration of water extract of Spirulina for seven days followed by an acute toxic dose of Cd (3.5 mg kg b.wt. ), reduced the hepatotoxicity and attenuated the Cd-induced oxidative stress. Spirulina treatment showed marked reduction in serum aminotrasferase activities and elevation in lipid peroxidation and recovery of the endogenous levels of antioxidants following Cd-intoxication. The Cd-induced hepatic histopathological changes were also minimized with the tested extract. These results suggest that Spirulina algae might play a role in reducing the toxic effect of Cd and its antioxidant properties seems to mediate such a protective effect.


INTRODUCTION
Although cadmium (Cd) is an industrial and environmental pollutant and is toxic to several tissues such as the liver, kidney and testis, the basis for its toxicity is not yet fully understood. Cadmium initially accumulates in the liver and therefore acute exposure to toxic doses of Cd produces apotosis and necrosis in the liver [1][2][3] . Overproduction of reactive oxygen species (ROS) has been considered the primary mechanism for Cd toxicity [1,4] . More beneficial effects were documented for combined treatment with chelating agent and antioxidant against Cd-induced oxidative stress in rat livers than if they were applied separately for the same purpose [5] .
Spirulina, filamentous blue-green algae (Spirulina platensis, Spirulina maxima, Spirulina fusiformis), has been consumed by man since ancient times. In Mexico and central Africa Spirulina is used as a primary food source and it is currently grown at large scale in many countries for commercial purposes [6] . Spirulina is considered as a valuable additional food source of some macro-and micronutrients including amino acids, chlorophyll, gamma-linolenic acid, carotenoids, vitamins B1 and B2 and trace elements such as iron, iodine, selenium and zinc [7] . Spirulina is currently recieving more attention as a nutraceutical and as a potential source of pharmaceutical targets [8,9] . Spirulina has been found to be active against several viruses including HIV (AIDS virus) and has also been reported to possess immuno-modulatory properties [9] . Anticarcinogenic and antioxidant effects have also been documented in Spirulina species. These properties were largely related to the Spirulina's phycobiliprotein phycocyanin [8,10] . In addition, Spirulina fusiformis has been shown to protect against the cisplatin-and urethane-induced genotoxicity [11] and against mercury intoxication [12] in mice. Spirulina was also found to have biosorption capacity for Cd and lead which enhance the Spirulina's effectiveness to remove Cd and lead from wastewater [13,14] .
The present investigation was set to evaluate the protective effects of Spirulina species against Cdinduced acute hepatotoxicity in male rats and to study the mechanisms underlying these effects. Hepatic toxicity was assessed histopatologically and by determining serum levels of alanine and aspartate transaminases (ALT & AST). Levels of liver antioxidants such as reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and of malondialdehyde (MDA; an index of lipid peroxidation) were measured and were shown to be significantly ameliorated with spirulina. Treatment regime: Cd solution was freshly prepared in a saline solution. Spirulina was gavaged at a dose of one gm kg 1 body weight and dissolved in 5ml distilled water. Rats were divided randomly into four groups (n=6) and were treated as follows. Animals of the Cdtreated group were given a single intraperitoneal dose of Cd (3.5 mg kg b.wt. 1 ). This dose was selected as it has been previously reported to induce liver toxicity in male Wister rats [2] . Animals of the protected group were fed Spirulina solution for seven days prior Cdinjection. Then, Cd-induced liver toxicity was initiated in these protected animals via a single dose injection of Cd (3.5 mg kg b.wt. 1 ). One group of rats was administered with Spirulina alone for 7 days. Animals of the control group were fed with distilled water (5 ml kg b.wt. 1 ) for 7 days and were then injected with saline (vehicle) after 10 days of water administration. Twenty four hours post Cd and/or vehicle injection, blood samples and liver tissues were collected and properly stored for further analyses.

Chemicals
Biochemical assays and histopathology: Blood was collected from the retro-orbital plexus from diethyl ether-anesthetized animals. To obtain serum, blood was collected in centrifuge tubes and centrifuged in a refrigerated centrifuge (4°C) at 3000 r.p.m. for 20 minutes.
Liver tissues were dissected out and homogenized separately in ice-cold KCl (150 mM). Supernatants were collected and processed for lipid peroxidation, SOD, CAT and GSH were done. Protein was estimated by the Lowry's method as modified by Peterson [15] . The methods have been standardized in our laboratory and have been detailed elsewhere [16][17][18][19][20] .
For the histological examinations, pieces of liver tissues were fixed in 10% neutral phosphate-buffered formalin and hydrated tissue sections, 5 m in thickness, were stained with Hematoxylin and Eosin. The sections were examined under a Leica DMRB/E light microscope.
Statistical analysis: Data were expressed as mean ± SE. Statistical significance between the various groups was determined using unpaired t-test and ANOVA (19).

RESULTS
Effects on liver functions: Treatment with Cd significantly (P<0.001) increased the activities of serum ALT and AST (357 and 126 % respectively) compared to the control (Fig. 1). Administration of Spirulina alone did not show any significant changes in the serum levels of these liver enzymes. Pre-treatment with Spirulina attenuated the Cd-induced increase of serum ALT and AST activities (62 and 32% respectively) compared to their levels in Cd-treated group (Fig. 1).

Effects on oxidative stress markers:
In Cd-intoxicated rats, hepatic MDA level (Fig. 2a) was significantly (P<0.01) increased by about 73% compared to control group. Rats of this group have shown a significant (P<0.05) depletion in liver GSH level by about 14% (Fig. 2b). However, rats pre-treated with Spirulina rescued the levels of these oxidative stress markers to almost control levels.
Effects on antioxidant enzymes: A significant change in the activities of antioxidant enzymes was determined following Cd-treatment. While CAT activity was elevated (P<0.05), SOD activity declined (P<0.01) (Fig.  3). Levels of these oxidative stress markers were back to control values in rats pre-treated with Spirulina before Cd-intoxication. Interestingly, treatment with Spirulina prevented the Cd-induced necrotic lesions and the histopathological alterations. The liver appears normal except for few scattered degenerated cells (Fig. 4).

DISCUSSION
The present study evaluates the protective effect of Spirulina against liver damage induced by Cd in male Wister rats. Cd injection at dosage 3.5 mg showed severe hepatic damage associated with marked increase in the serum activity of aminotransferases. These characteristic features of Cd-induced liver toxicity were similar to those previously reported by other investigators [1,2] . Several mechanisms have been suggested for the induction of Cd-associated hepatotoxicity [1] . In this work, Cd-treated rats showed not only a significant increase in MDA level but also a significant decrease in the hepatic levels of GSH and SOD. These results correlate well with other reports, where Cd has been shown to up regulate oxidative stress marker such as MDA and decrease the activity of antioxidants such as GSH and SOD [22][23][24] . Therefore, it is possible that the Cd-induced liver toxicity can be mediated by the upregulation of the reactive oxygen species (ROS; hydroxyl groups, superoxides and hydrogen peroxides) generation which in turn leads to an oxidative damage to lipid contents of membranes. Cd caused CAT activity to increase in liver. CAT is known to catalyze the removal of hydrogen peroxide and therefore its upregulation may provide a compensatory or adaptive response against elevation in hydrogen peroxide [17,23] . Over-production of ROS normally induces oxidative stress unless it was scavenged with endogenous antioxidants. Thus, overproduction of ROS could be attributed to the depletion of antioxidants or to the direct action of Cd on peroxidation reaction and iron-mediated peroxidation [22] .
In the present study, administration of Spirulina alone did not significantly alter the normal level of aminotransferase enzymes, antioxidants and oxidativestress marker. Pretreatment with Spirulina prior Cd intoxication attenuated the levels of ALT & AST as markers for liver function and the MDA as marker for lipid peroxidation. It also normalized the levels of both enzymatic and non-enzymatic antioxidants (GSH, SOD and CAT) in liver. This Spirulina-induced oxidative stress protection may be well correlated with previous studies, where Spirulina has been shown to restored the level of antioxidant enzymes and oxidative stress markers against cyclophosphamide and mitomycin C [25] and cisplatin and urethane [11] in mice. The protective effect of Spirulina against Cd-induced oxidative stress in this study could also be attributed to its antioxidant and chelating effects. It has been reported that Spirulina possess strong antioxidant and free radical scavenging properties [8,10] in addition to its strong chelating [13,14] effect. These characteristics can be attributed to the high levels of antioxidants such as vitamin B1 and B2, carotenoids and phyocyanin reported in Spirulina [7,8] .