Effect of the Carotenoid-Producing Alga,Dunaliella bardawil,on CCl 4 -Induced Toxicity in Rats

Abstract

Dunaliella bardawil is a carotenoid-producing alga that is being considered for use in nutraceuticals. To evaluate potential protective effects of consumption of this alga, rats were treated with two different doses of D. bardawil (2.5 and 5.0 g kg^–1 body weight [bw]) as a biomass suspension daily for 14 days. Animals were tested against Carbon tetrachloride (CCl₄; 2 ml kg^–1)–induced liver toxicity as measured by various biochemical marker enzymes in liver and blood. All measurements were taken 6 h following the single dose of CCl₄. The results of this study show that there was a slight, but statistically significant mean serum enzyme values, with D. bardawil treatment, compared to higher mean values in animals receiving CCl₄ alone. Lipid peroxidation is measured by thiobarbituric acid–reactive substance (TBARS) activity was likewise slightly less elevated with algae treatment. The results also demonstrated protection against DNA strand breaks in hepatocytes, as measured by single cell gel electrophoresis. Liver histopathology was less severe with D. bardawil treatment, supporting the apparent protective action of 14-day treatment on hepatic oxidative injury.

Carotenoids have been demonstrated to be capable of providing health benefits, including their protective role in diseases such as cancer of various organs like skin, lungs, stomach, cervix, pancreas, colon, rectum, breast, prostate, and ovary (Bast et al. 1996; Ames 1983). The biological functions of carotenoids are attributed to their ability to scavenge free radicals, physically quench the singlet oxygen. Among the carotenoids, ß-carotene is a potent antioxidant and also a well-known precursor of vitamin A (Burton and Ingold 1984).

Carbon tetrachloride (CCl₄) is an industrial toxicant, known to cause not only the hepatic necrosis but also free radical generation in kidney, heart, lung, testis, brain, and blood (Ahmad, Cowan, and Sun 1987; Ohta et al. 1997; Ozturk et al. 2003). Reactive oxygen species (ROS) can destroy cellular membranes, cellular proteins, and nucleic acids. The toxic effects of CCl₄ are the consequences of production of free radicals, which initiate cell damage (Slater 1984; Kadiiskaa et al. 2005).

In recent years algae are gaining importance in diet supplements, as they possess nutraceutical properties. Dunaliella is a eukaryotic photosynthetic microalgae belonging to the Chlorophyceae, which can produce ß-carotene in large quantities under high stress of solar radiation (Ben Amotz, Kartz, and Avron 1983). The alga lacks a rigid cell wall, which makes it easily digestible and accessible by most animal species (Ben Amotz et al. 1989). Previous studies reported that Dunaliella has no toxic effects in rats and can be utilized as a potential source of food supplement (Kuroiwa et al. 2006; Mokady, Abramovici, and Cogau 1989). Dunaliella has been known to possess various nutraceutical properties. Nagasawa et al. (1989) showed that the mammary tumourigenesis was significantly inhibited, when D. bardawil was supplemented in the diet as a spray dried powder and oily solution of D. salina. D. bardawil is known to promote the growth of normal mammary gland cells, but inhibits proliferation of neoplastic cells (Fujii et al. 1991, 1993). Dry algal powder of Dunaliella bardawil can be used as a retinol supplement in chick and rat diet (Ben Amotz, Edelstein, and Avron 1986; Ben Amotz et al. 1986).

Published data on biological activity of whole cell biomass or carotenoids of D. bardawil in protecting against CCl₄-induced hepatotoxicity, nephrotoxicity, and genotoxicity are scanty. In this connection the present study was aimed to evaluate the possible protective effects of Dunaliella bardawil biomass against CCl₄-induced toxicity by monitoring changes in serum enzyme levels, serum bilirubin, creatinine content, and lipid peroxidation in hepatic and renal tissues. We have also studied the DNA damage induced by free radicals in hepatocytes and histopathology of liver tissues to understand the role of D. bardawil biomass in protection against CCl₄-induced toxicity.

MATERIALS AND METHODS

Source of D. bardawil and Culture Condition

An authenticated indigenous strain of D. bardawil was isolated from the Sambar lake of Rajasthan, India. The culture was maintained in AS-100 medium, (Vonshak 1986) with modification. Composition of the medium was MgSO₄·7H₂0 2.5 mM; MgCl₃ 2.5 mM; CaCl₂·2H₂O 0.3 mM; KH₂PO₄ 0.2 mM; KNO₃ 5.0 mM; NaCl 1.5 mM; NaHCO₃ 5.0 mM; H₃BO₃ 0.5 mM; CoCl₂·6H₂O 1.0 mM; MnCl₂·4H₂O 7 mM; ZnCl₂ 1.0 mM; FeCl₃ 1.5 mM; (NH₄)₆ MO₇O₂·4H₂O 1.0 mM (trace metal mixture); along with EDTA-chelated iron solution. The 14-day-old culture was subjected to carotenogenesis under high-irradiance stress. After 4 days, cells were harvested using online centrifuge at 8000 rpm (M/s Sharples, UK), the wet biomass was lyophilized and used for feeding the experimental animals.

Estimation of Carotenoid Content from Dunaliella

The carotenoids from the biomass were extracted using ethyl acetate, concentrated and redissolved in known amount of mobile phase. Estimation of carotenoids from Dunaliella was carried out by high-performance liquid chromatography on a Bondapak C18 column (5μ × 250 mm) with methanol:acetonitrile:chloroform (47:47:6) mobile phase at a flow rate of 1 ml min^–1. Parameters were controlled by a Shimadzu LC 10-AS liquid chromatograph equipped with a dual pump and a photodiode array detector (Model SPD-10A) set at 450 nm. The recorder Shimadzu C-R7A chromatopac was set at a chart speed of 2.5 cm min^-1. Injection volume was 10 μl, injected with Rheodyne 7125 injector. Peak identification was achieved by comparing with the retention time of standards (Sigma, USA) and confirmed by spiking the standards with individual samples.

Experimental Design

Albino rats of Wister strain (120 to 150 g body weight) bred in the Animal House of Central Food Technological Research Institute were used for the study. Animals were grouped into following groups each consisting six rats (n = 6) (three males and three females, maintained separately). The carotenoid rich biomass of D. bardawil was fed at two different doses, i.e., 2.5 and 5.0 g/kg body weight (approximately equivalent to 50 and 100 mg of ß–carotene kg^–1 body weight) and synthetic ß-carotene at 50 mg kg^–1 body weight was fed for 14 days. Group 1 served as normal (receiving normal basal diet without toxin treatment); group 2, control (receiving normal basal diet with toxin treatment); group 3, D. bardawil biomass was fed at 5 g kg^–1 body weight; group 4, D. bardawil biomass was fed at 2.5 g kg^–1 body weight along with CCl₄; group 5, D. bardawil biomass fed at 5 g kg^–1 body weight with CCl₄; and group 6, treated with synthetic ß-carotene at 50 mg kg^–1 body weight along with CCl₄. The dosage of CCl₄ was decided based on our earlier reports (Chidambara Murthy, Singh, and Jayaprakasha 2002). The animals of all the groups except groups 1 and 3 were given single dose of CCl₄ (2 ml kg^–1 body weight [bw]), on the 14th day, dissolved in olive oil (1:1). Animals of groups 1 and 3 were given same dose of olive oil as vehicle.

The animals were housed in a room with a barrier system, and maintained under the following conditions: temperature 24°C ± 1°C, relative humidity 55% ± 5%, and a 12-h light/dark cycle. The animals were housed in polypropylene cages (3 rats/cage) on soft sawdust bedding. Throughout the experiment, rats were given commercial basal diet and water ad libitum. All the experiments were carried out under the regulation of Institute Animal Ethical Committee.

Test Compound and Administration Dose Levels

D. bardawil biomass was ground with minimal amount of water to form uniform slurry. The animals in groups 1 and 2 received the commercial pellet diet (basal diet; Lipton India). Experimental groups were fed with Dunaliella biomass once in a day for 14 days by forced feeding using a oral gavage, followed by a single administration of 2 ml kg^–1 bw CCl₄ (Merck, Mumbai, India) in olive oil. On the 14th day the animals were sacrificed 6 h after the CCl₄ dosage.

Biochemical Screening

Clinical signs and general appearances were checked daily and body weights were measured once in a week. Before the day of necropsy, the animals were deprived of food overnight and sacrificed by anaesthetizing the animals with ether. Blood was collected from the animals and the serum obtained was analyzed. The liver and kidney were transferred to ice-cold containers for various estimations. Livers of all the animals were stored in 10% formalin solution and subjected for histopathological examination using hematoxylin and eosin (H& E) staining. The slides were evaluated with bright field microscope (Olympus, BX40) and documented.

Estimation of Serum Enzymes

Serum alanine and aspartate aminotransferases (ALT and AST) were measured by the DNPH (2,4-Dinitrophenyl hydrazine) method (King 1965). Serum alkaline phosphatase (ALP) activity was assayed by the method of King and Armstrong (1988) using the commercially available kits (M/s SPAN diagnostic reagent kits, Mumbai, India).

Lipid Peroxidation in Hepatic and Renal Tissues

Liver and kidney were homogenized in 0.1 mol/L Trisbuffer (pH 7.4) and centrifuged. The particle-free homogenate was used for the biochemical analysis. Extent of lipid peroxidation was measured by quantifying the malondialdehyde formed in terms of thiobarbituric acid–reactive substances (TBARS) and expressed in terms of nmol/mg protein (Buege and Aust 1978).

Estimation of Protein, Serum Creatinine, and Bilirubin Content

Protein content was determined using the method of Lowry et al. (1951). Serum creatinin and bilirubin content was estimated using diagnostic kits from SPAN Diagnostics, India.

Measurement of DNA Strand Breaks (Comet Assay)

The isolation of hepatocytes and comet assay was performed under alkaline condition (Singh et al. 1988). Briefly, 200 mg liver tissue was mixed with 3 ml of hypotonic buffer (75 mM NaCl, 24 mM EDTA, pH 7.5), minced, and resuspended in hypotonic buffer. The isolated liver cells were embedded in low-melting agarose and deposited over 1% agarose layer. After solidification one more agarose layer was deposited. The slides were dipped in lysis buffer (2.5 M NaCl, 0.15 M NaOH, 100 mM EDTA, 10 mM Tris along with1% Triton X-100 and 10% DMSO pH 10) for 2 h at 4°C in dark. After lysis, only the isolated nucleus remained in the agarose. The slides were electrophoresed (10 N NaOH, 200 mM EDTA at pH >13) at 24 V and approximately 300 mA for 30 min. The slides were washed with neutralization buffer (0.4 M Tris at pH 7.5), stained with ethidium bromide, washed with water, and scored using an Olympus microscope equipped with fluorescence filters (Olympus, BX40; emission wavelength 450 to 480 nm, excitation wavelength 510 to 550 nm). Tail lengths were measured from the trailing edge of the comet tail. One hundred cells per treatment from three independent experiments were analyzed for DNA migration and categorized into different groups (Collins 2004).

Statistical Analysis

Mean and standard deviation values were determined for all the parameters studied. In all the analysis, the groups pretreated with normal diet supplemented with synthetic ß-carotene and Dunaliella biomass followed by single dose of CCl₄ are compared with groups fed with normal diet with single dose of CCl₄. Results were statistically analyzed by analysis of variance (ANOVA) using MS-Excel and statistical analysis software AGRES. Statistical significance was considered at p < .01 and p < .05.

RESULTS

Estimation of Carotenoid Content from Dunaliella

HPLC estimation revealed total 3.0% carotenoids in D. bardawil, among which, ß-carotene constituting around 75% and other being lutein (20%), α-carotene (3%), and lycopene (1%), and traces of chlorophyll were also seen.

Effect of Algal Feeding on Body Weight and Organ Weight

Administrations of Dunaliella did not show significant variation in body weight when compared to controls with normal diet (data not shown). No clinical signs of any toxicity as well as notable behavioral changes were observed among rats fed with both the concentration of algae. Further, no mortality occurred during the experimental period. No significant variation in the relative weight of vital organs (Table 1) were observed; whereas, increase in relative weight of liver has been observed in the groups treated with CCl₄, indicating the symptoms of fatty liver.

Histological Observation

In case of normal group (without CCl₄ treatment), hepatocytes with normal architecture was noticed (Figure 1a). Similar characteristics were noticed in groups fed with D. bardawil (without CCl₄ treatment; Figure 1c). Histopathological analysis of CCl₄-treated animals (Figure 1b) showed fatty liver symptoms, total loss of hepatic architecture, acute liver injury, centrilobular necrosis with hemorrhages, and degenerated hepatic cords and hepatocytes. Experimental groups fed with D. bardawil followed by CCl₄ treatment has retained better hepatic architecture compared to control. The level of protection against liver injury and necrosis was higher in case of groups fed with Dunaliella whole cells (Figure 1d and e) when compared to synthetic ß-carotene treatment (Figure 1f). No significant variations were noticed among male and females in terms of hepatic architecture in different groups.

Changes in Liver Marker Enzymes

Feeding of D. bardawil (5 g kg^–1 bw) alone did not alter the activity of liver marker enzymes. Rats treated with a single dose of CCl₄ produced a marked increase in the activities of AST, ALT, and ALP, indicating the hepatic damage (Table 2). Administration of Dunaliella biomass (2.5 and 5.0 g kg^–1 bw) and synthetic ß-carotene (50 mg kg^–1 bw) decreased the deleterious effect of CCl₄, as evident by the decreased level of liver marker enzymes (Table 2).

Effect on Serum Bilirubin

The serum bilirubin content was increased in CCl₄-treated group, indicating injury to liver. Both the doses of D. bardawil and synthetic ß-carotene attenuated the CCl₄-induced elevated levels of serum bilirubin (Figure 2).

Effect on the Hepatic and Renal TBARS Levels

Lipid peroxidation was measured in terms of TBARS activity and expressed as nmol/mg protein. CCl₄ challenge caused a marked lipid peroxidation in both liver and kidney. As with the liver serum enzymes discussed above, there was slight restoration of lipid peroxidation was noticed after 14 days of D. bardawil treatment after 14 days of D. bardawil treatement (Figure 3).

Effect on Serum Creatinine

The serum creatinine content were increased in the CCl₄-treated group, indicating injury to kidney (Figure 4). Decreased level of serum creatinine was observed in groups fed with D. bardawil biomass.

Measurement of DNA Strand Breaks in Isolated Hepatocytes

The tail momentum in single-cell gel electrophoresis was considered as 100% in rats fed with normal diet without CCl₄ treatment and percentage difference in tail momentum in comparison with the control group was presented in Figure 2. CCl₄ induced severe DNA strand breaks and it was evident from an increase of tail momentum. The results demonstrated three- to fourfold increase in tail momentum in groups treated with CCl₄. Decrease in tail momentum was observed in groups treated with Dunaliella biomass and synthetic ß-carotene, respectively. The visual scoring of comets revealed up to 30% comets under scoring category 0 (no damage) in CCl₄-treated group; up to 30% each under scoring category 1 (low damage) and scoring category 2 (mild damage); 12% under scoring category 3 (moderate damage); and 5% under scoring category 4 (severe damage). Pretreatment with Dunaliella demonstrated the protection against DNA strand breaks and 70% were under scoring category 0 (no damage) (Figure 5). The results indicated that feeding of D. bardawil biomass provided protection against DNA strand breaks induced by CCl₄.

DISCUSSION

Algae are known as good nutritional supplements. In this article we report hepatoprotection and protection against DNA damage in rat models fed with diet supplemented with algae, D. bardawil. In earlier studies we have reported the high antioxidant potency of Dunaliella salina compared to synthetic ß-carotene (Chidambara Murthy et al. 2005).

The hepatotoxicity of CCl₄ is widely known (Slater 1987; Recknagel et al. 1989). CCl₄ is also known to cause DNA damage due to the formation of free radicals (Kadiiskaa et al. 2005).

Many different natural compounds and their extracts have been shown to prevent the toxicity of CCl₄ by quenching the free radicals, and inhibition of lipid peroxidation, generated from CCl₄ metabolism (Biasi et al. 1991; Chidambara Murthy, Singh, and Jayaprakasha 2002). Because the changes associated with CCl₄-induced liver damage are similar to that of acute viral hepatitis (Rubinstein 1962), CCl₄-mediated hepatotoxicity was taken here as an experimental model for liver damage. Our results demonstrated effects of CCl₄-induced oxidative stress in the rat plasma and liver as evident by the increased levels of serum enzymes and lipid peroxidation after CCl₄ treatment. This elevation could be explained on the basis that CCl₄ causes necrosis of liver cells. As a result of necrosis of the hepatic cells caused by acute infection or chronic liver disease, the marker enzymes are known to be released into the circulation with consequent rise in the serum levels (Wroblewski and La Due 1955) and a persistent rise in serum transaminase level is presumably an indication of liver cell damage. The elevation of the serum transaminases, especially AST and ALP activitives, is due to the hepatic damage caused by CCl₄. Treatment with CCl₄ also causes increase in liver weight, mainly due to fatty liver symptoms. This is mainly due to a fall in the concentration of plasma triglycerides and the accumulation of triglycerides in the liver. The development of fatty liver in CCl₄-treated rats is due to a block in the release of hepatic triglycerides to the plasma. The liver rapidly converts the free fatty acids to triglycerides, but fails to release it into the plasma. As a consequence, triglycerides accumulate within the liver and decrease in the plasma (Lombard and Ugazio 1965).

The present study reveals that rats pretreated with D. bardawil biomass at two different levels for duration of 14 days restored the increased levels of liver marker enzymes. Many of the antioxidants present in plants are known for their hepatoprotective activity against CCl₄-induced hepatic damage (Chidambara Murthy, Singh, and Jayaprakasha 2002). Hepatic damage also increases the serum bilirubin levels due to the leakage of cellular content from hepatocytes to the serum (Recknagel et al. 1989).

The enhanced level of lipid peroxidation (LP) in the CCl₄-treated group expressed in terms of thiobarbituric acid–reactive substances (TBARS), indicating liver and renal damage. CCl₄ is known to increase lipid peroxidation (Glei et al. 2002; Recknagel et al. 1989). In our study D. bardawil biomass as well as synthetic ß-carotene showed restoration of the elevated levels of AST, ALT, ALP, bilirubin, and creatinine content that are coupled with marked hepatic and renal oxidative stress. Natural antioxidants in fruits and vegetables, viz. vitamins C and E, carotenoids, flavonoids, etc., are found to scavenge the reactive oxygen free radical, before they cause damage to any cellular organ (Volkovova, Dusinska, and Collins 2006).

The hepatoprotective activity of Dunaliella may be attributed to the presence of different carotenoids, mainly ß-carotene. Carotenoids are known to possess high radical-scavenging activity and thereby provide adequate protection against free radical–induced liver damage. We have also compared the protective effect of synthetic ß-carotene and carotenoids from D. bardawil. The results clearly revealed the protection of carotenoids against free radical–induced hepatic damage. This could be further attributed to the fact that D. bardawil is a rich source of a diverse array of carotenoids. Our results demonstrate that mixture of carotenoids from natural source possess high degree of free radical–scavenging activity compared to synthetic ß-carotene.

Among the parameters studied in the kidney, marked restoration of renal TBARS and serum creatinine was observed. This suggests that upon CCl₄ administration there is damage to the renal tissues too. Fadhel and Amran (2002) had also reported increased levels of renal TBARS in rats after CCl₄ exposure, which could be restored by black tea extract.

CCl₄ is known to cause renal functional impairment (Ogeturk et al. 2005; Ozturk et al. 2003). Ozturk et al. (2003) detected glomerular necrosis and histologic alterations in proximal and distal tubules. Tubular epithelial cell alterations, including vacuolization, atrophy, and, finally, detachment of the epithelial cells, indicate tubular necrosis. In our results the increase in serum creatinine levels along with increased TBARS activity indicate possible involvement of renal functional impairment caused due to CCl₄ intoxication. Fadhel and Amran (2002) demonstrated that the protective effects of black tea against CCl₄-induced lipid peroxidation in liver, kidneys, and testes are due at least partly to its antioxidant properties, scavenging CCl₄-associated free radicals. Our results are in accordance with the above reports and carotenoids from Dunaliella are found to protect against CCl₄-induced toxicity in renal and hepatic systems.

CCl₄ is also known to cause hepatocellular DNA damage in rats (Zhou et al. 1996). We sought to study whether the carotenoid-rich Dunaliella diet protects the hepatic DNA against free radical–induced genotoxicity. Earlier reports suggest that synthetic carotenoids may protect DNA against oxidative damage (Collins 2001). Glei et al. (2002) observed that ß-carotene enters the cell and prevents the damages caused by bleomycin in human lymphocytes. Of the several different techniques that can be used to evaluate oxidative DNA damage, alkaline single-cell gel electrophoresis (SCGE), or the Comet assay, is a sensitive method for detecting double- and single-strand DNA breaks caused by multiple toxicants (Singh et al. 1988). Our studies with single-cell electrophoresis revealed a high degree of protection conferred by Dunaliella carotenoids against CCl₄-induced DNA damage. However, the protective effect of D. bardawil carotenoids on chronic hepatotoxicity and genotoxicity will be of great importance for future studies.

CONCLUSION

The results of this study indicate that repeated daily administration of D. bardawil slightly ameliorates CCl₄-induced liver toxicity in rats. Because algae contain a good amount of protein and carotenoids of biological significance, it can be utilized as a food supplement for the prevention of radical-mediated hepatic damages that might be caused by various drugs and natural products. This also supports the use of D. bardawil biomass as nutraceuticals claiming antioxidant properties.