Efficient amelioration of carbon tetrachloride induced toxicity in isolated rat hepatocytes by Syzygium cumini Skeels extract

Toxicology in Vitro 22 (2008) 1440–1446

Contents lists available at ScienceDirect

Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit

Efficient amelioration of carbon tetrachloride induced toxicity in isolated rat hepatocytes by Syzygium cumini Skeels extract Jyothi M. Veigas, Richa Shrivasthava, Bhagyalakshmi Neelwarne * Plant Cell Biotechnology Department, Central Food Technological Research Institute, Mysore 570020, India

a r t i c l e

i n f o

Article history: Received 12 July 2007 Accepted 15 April 2008 Available online 29 April 2008 Keywords: Anthocyanin Antioxidant Black Java plum Antioxidant enzymes Hepatoprotective

a b s t r a c t Syzygium cumini, Indian black plum or Java plum, is a rich source for anthocyanins (230 mg/100 g DW) showing high antioxidant activity in vitro. In the following study it is further demonstrated that S. cumini peel extract rich in anthocyanins (SCA) offers considerable protection against carbon tetrachloride (CCl4)induced damage in rat hepatocytes. SCA itself being non-toxic to primary rat hepatocytes at concentrations ranging from 50 to 500 ppm, was found to suppress CCl4-induced LDH leakage by 54% at 50 ppm, thereby improving the cell viability by 39%. The SCA significantly reversed the CCl4 induced changes in cellular glutathione (GSH) level, lipid peroxidation and activity of the antioxidant enzyme glutathione peroxidase. Exposure of hepatocytes to SCA after CCl4 treatment was found to elevate GSH and GPx activities by 2-folds, whereas the activities of catalase and superoxide dismutase were not significantly affected. The fruit pulp extract (SPE) was less effective in offering protection to rat hepatocytes, particularly in terms of total GSH content and a consequent increase in lipid peroxidation although the higher GPx activity suggests the probable involvement of GSH as a substrate for GPx. These observations suggest that the fruit peel extract of S. cumini, is largely responsible for the reversal of CCl4-induced oxidative damage in rat hepatocytes. Both peel and pulp extract appear to offer protection to rat hepatocytes through GPx along with other biological pathways independent of catalase and superoxide dismutase. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction The liver is the major organ in the body since it performs an astonishingly large number of tasks of regulating several functions in higher animals and humans. The major functions are metabolism of carbohydrates, lipids, proteins, drugs and xenobiotics that are ingested by the body. Thus the liver is prone to injury due to the chronic exposure to drugs, environmental toxicants and other xenobiotics. The major consequence of this is an indirect effect on virtually all other organs. Carbon tetrachloride, a by-product of sewage and drinking water chlorination, is one of the major hepatotoxins which is metabolized in the liver by the cytochrome P450 oxidase system resulting in the reactive trichloromethyl rad ical ðCCl3 Þ and subsequently resulting in an even more reactive form, the trichloromethyl-peroxyl (OOCCl3) radical (Ruch et al., 1986). In addition, CCl4 generates free radicals which fuel a series of reactions ultimately resulting in the initiation of membrane lipid peroxidation and a consequent liver injury (Slater, 1984). For these reasons, CCl4 is often used in experiments associated with toxicity of the liver. Inhibition of free radical generation or scavenging the free radicals generated by other biochemical reactions is an important fac* Corresponding author. Tel.: +91 821 2516501; fax: +91 821 2517233. E-mail address: [email protected] (B. Neelwarne). 0887-2333/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2008.04.015

tor in the prevention of hepatic damage. Several natural products have been proven to be effective against hepatic damage either by acting as antioxidants or as positive modulators of other cellular machineries. Fruits of Syzygium cumini Skeels (Syn. Eugenia jambolana Lam.; E. cumini Druce; Fam; Myrtaceae) (Fig. 1) – commonly known as Java plum or Jambolan in English are known to possess anti-hyperglycemic and anti-oxidant effects (Banerjee et al., 2005; Sharma et al., 2006). In addition, our earlier study showed the presence of three major anthocyanins–glucoglucosides of delphinidin, petunidin and malvidin where the ethanol extract of the fruit peel was partially purified and demonstrated to possess high anti-oxidative potential in vitro (Veigas et al., 2007). In addition to their colorful characteristics, anthocyanins are known to possess excellent antioxidant properties (Kong et al., 2003). Although a recent study reported that these pigments are hydrolysed by the intestinal microflora and the anti-oxidative effects are due to the byproducts such as phenolic acids (Keppler and Humpf, 2005) an earlier clinical study involving elderly women has clearly established that these pigments are in fact found as glycosides in plasma and urine (Cao et al., 2001). Probably for these reasons, anthocyanins from different sources have been reported to inhibit lipid peroxidation and platelet aggregation (Ghiselli et al., 1998). Cyanidin has been found to form co-pigmentation complex with DNA thus conferring a mutual protection against oxidative stress. This phenomenon might be a possible defense mechanism against

J.M. Veigas et al. / Toxicology in Vitro 22 (2008) 1440–1446

1441

Fig. 1. Photograph depicting part of S. cumini plant with fruits, flowers and leaves. Inset shows the fully ripe berries.

oxidative damage of DNA having physiological implications as an antioxidant tool (Sarma and Sharma, 1999). Thus the knowledge on antioxidative effects of flavonoid glycosides is not new. Nevertheless, it is important to establish the antioxidant properties of a new compound using in vivo models in order to hypothesize a role for human beings especially when there are specific studies showing hepato-toxic effects of phenolics (Galati et al., 2006). Therefore in the present study, besides providing empirical evidence for the hepatoprotection rendered by a new source of anthocyanin, two additional questions have been addressed: (1) Do S. cumini anthocyanins (SCAs) also protect the key enzymes that normally protect a cell from oxidative damage? (2) Do SCAs elevate the activity levels of key radical scavenging enzymes? Therefore, the present study addresses whether the anthocyanin rich extract of S. cumini imparts protection against CCl4 intoxication in primary rat hepatocytes, and if so, the mode through which it acts. This is the first report on the S. cumini fruit extracts demonstrating modulation of antioxidant enzymes in rat primary hepatocytes. 2. Materials and methods 2.1. Chemicals XAD-7 (Amberlite polymeric adsorbent of 20–50 mesh) and pyrogallol were purchased from Fluka (Germany). Ascorbic acid, DTNB (5,5-dithiobis-2-nitrobenzoic acid), EDTA (ethylene diaminotetraacetic acid), NADPH (reduced nicotinamide adenine dinucleotide phosphate), NADH (nicotinamide adenine dinucleotide), BSA (bovine serum albumin), collagenase (Type IV), HEPES, Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), antibiotic–antimycotic solution, Tris, reduced glutathione (GSH), epigallo-catechin-3-gallate (EGCG), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) and glutathione reductase were procured from Sigma-Aldrich (Steinheim, Germany). Insulin, dexamethasone were from Novartis, India. All other reagents and chemicals were of analytical grade and purchased

from Merck (Darmstadt, Germany). The stock solutions were prepared fresh prior to use. 2.2. Plant material The peels of fully ripe berries obtained from the local market were manually separated and immediately transferred to solvent for pigment extraction. 2.3. Preparation of S. cumini anthocyanins (SCA) Anthocyanins were extracted with 0.1% HCl in 100% ethanol (Francis, 1986) by way of soaking the fruit peel in 10-fold volume of the solvent for 3 h on an orbital shaker set at 100 rpm (25 °C ± 1). The extractives were collected by filtration and the residue was repeatedly extracted until the filtrate obtained was nearly colorless. The extracts were pooled and concentrated in a Buchi Rotavapour (Flawil, Switzerland) under vacuum at 30 °C ± 1, partitioned against ethyl acetate before application on an Amberlite XAD-7 column (Veigas et al., 2007; Andersen et al, 1995). The column eluate was lyophilized and stored at 20 °C for further use. 2.4. Preparation of S. cumini pulp extract (SPE) The pulp obtained after the removal of peel was, mashed and boiled in distilled water for 5 min, filtered using a Buchner’s funnel and concentrated in a Buchi Rotavapour (Flawil, Switzerland) under vacuum at 40 °C ± 1. The concentrate was lyophilized and stored at 20 °C until further use. 2.5. Preparation of rat hepatocytes and treatment Rat primary hepatocytes were isolated from male Wistar rat (150–250 g) by two-step collagenase perfusion of the liver (YingJie et al., 1998). Cell viability, determined by trypan blue exclusion,

J.M. Veigas et al. / Toxicology in Vitro 22 (2008) 1440–1446

was over 85%. The isolated rat hepatocytes were cultured in DMEM supplemented with 10% FBS, 1% antibiotic–antimycotic solution, 0.1 U/ml insulin and 0.01 lM dexamethasone for 3 h at 37 °C in a humidified atmosphere of 95% O2 and 5% CO2. Cells (2  105) were plated in 96 well plates for cell viability assessment and in 12 well plates for other assays. The cells were treated with 7 mM of CCl4 for 1 h at the end of which the medium was replaced with fresh medium containing different concentrations (0–500 ppm for cell viability; 0, 50 and 100 ppm for the other assays) of the extracts. The cultures were subsequently incubated for further 3 h at 37 °C in a humidified atmosphere of 95% relative humidity with 95% O2 and 5% CO2. At the end of the treatment period, the cells were assessed for cell viability and other biochemical indices such as membrane damage (LDH leakage), GSH content, level of lipid peroxidation and activities of antioxidant enzymes such as catalase, superoxide dismutase and glutathione peroxidase. 2.6. Assessment of cell viability and integrity Cell viability was assessed by MTT assay and lactate dehydrogenase (LDH) activity. LDH activity in the culture medium was measured as an index of plasma membrane integrity. The assay was carried out in 96 well plates. At the end of the treatment period, 50 ll of the medium from each treatment was removed for LDH leakage determination and treated with freshly prepared b-NADH solution (4.58 mg pyruvate, 5.32 mg NADH, and 7.49 NaHCO3 in 10 ml 0.05 M potassium phosphate buffer, pH 7.4). The plates were incubated in dark for 20 min, after which the absorbance of the reaction mixture was measured at 340 nm using an ELISA microtitre plate reader. Hepatocyte viability was expressed as the quantum fraction of LDH released into the culture medium relative to the total cellular activity observed after cell lysis with 0.1% v/v Triton-X 100 (Haidara et al., 1999). To the remaining medium, MTT (15 ll of a 1 mg/ml solution) was added and incubated for 4 h at 37 °C. The medium was removed and the formazan crystals formed were dissolved in 200 ll of DMSO and the absorbance was measured at 570 nm using an ELISA microtitre plate reader (Neuman et al., 1993). The data are expressed as percentage of control. 2.7. Preparation of cell lysate After the treatment period, the cells were centrifuged at 5000g for 5 min and the pellet was washed with ice-cold PBS, pH 7.4. The cell pellet was resuspended in PBS and sonicated with an ultrasonicator (Bandelin Sonopuls HD 2200, Berlin) for 3 cycles at 30 sec/ cycle. The cell extract was centrifuged at 12,000g for 20 min at 4 °C and the supernatant was used for enzyme assays and GSH content determination. Total protein was estimated using Lowry’s method (Lowry et al., 1951). 2.8. Glutathione assay Reduction in glutathione level was determined in the cell lysate by the method of Popat et al. (2002) with slight modification. A 50 ll of the cell lysate was treated with 150 lL of DTNB reagent (12 mM NADPH, 0.1 mM DTNB, 50 U/ml GSH reductase in 0.1 mM sodium phosphate buffer with 1 mM EDTA, pH 7.5) and absorbance was read at 415 nm using an ELISA microtitre plate reader. The GSH content was expressed as nM/mg protein using a calibration curve prepared using known concentrations of reduced glutathione.

2.0 ml ice-cold mixture of 0.25 N HCl containing 15% trichloroacetic acid (TCA), 0.3% thiobarbituric acid (TBA) and 0.05% butylated hydroxyl toluene (BHT) and was heated at 90C for 60 min. The samples were cooled and centrifuged at 7000g for 5 min. The absorbance of the supernatant was measured spectrophotometrically at 535 nm and the results were expressed as percentage lipid peroxidation (Buege and Aust, 1978). 2.10. Antioxidant enzyme assays Superoxide dismutase (SOD) activity in the cell lysate was measured by the inhibition of pyrogallol auto-oxidation (Kamendulis et al., 1999). Briefly, 50 ll of the cell lysate was mixed with 100 ll of assay buffer (50 mM Tris HCl, 1 mM EDTA pH 8.2) and 50 ll of 0.2 mM pyrogallol. The absorbance was monitored at 420 nm for 3 min. One unit of SOD activity is defined as the amount of enzyme required to produce a 50% inhibition of pyrogallol auto-oxidation. Catalase activity was determined by spectrophotometry by measuring reduction in H2O2 absorbance at 240 nm (Aebi, 1984). The reaction mixture contained 500 ll of 30 mM H2O2, 400 ll of phosphate buffered saline (pH 7.4) and 100 ll of cell lysate. One unit of catalase activity is defined as the amount of enzyme that decomposes 1 mM H2O2 per minute. Glutathione peroxidase activity of the cell lysate was determined spectrophotometrically at 340 nm. Assay mixture was composed of 0.05 M phosphate buffer (pH 7.0), 1 mM EDTA, 3 U glutathione reductase, 1 mM GSH, 0.2 mM NADPH, 12 mM tertiary butyl hydroperoxide and cell lysate. Oxidation of NADPH was recorded at 340 nm at 15 s intervals for 3 min. Oxidation of NADPH at 37 °C was determined spectrophotometrically at 340 nm. One unit of activity was defined as the amount of GPx required to oxidize 1 nM of NADPH per min per mg protein (Flohe and Gunzler, 1984). 2.11. Data analysis All experiments were done in triplicates and the data presented are the averages of mean of three independent experiments with standard deviation. The data were analyzed by one-way analysis of variance (ANOVA) using Microsoft Excel XP (Microsoft Corp., Redmond, WA), and post-hoc mean separations were performed by Duncan’s multiple-range test at p < 0.05 (Harter, 1960). 3. Result 3.1. Cell viability and integrity The initial screening by MTT assay showed that the extracts of S. cumini were non-toxic to rat hepatocytes at the tested concentra-

MTT reduction (% of control)

1442

120.00 100.00 80.00 60.00 40.00 20.00 0.00 SCA 50ppm

SPE 100ppm

250ppm

500ppm

2.9. Lipid peroxidation The cell lysate (0.5 ml) obtained after sonication was incubated with 1.0 ml of KCl (0.15 M) and 250 ll of 0.2 mM ferric chloride solution at 37 °C for 30 min. The reaction was stopped by adding

Fig. 2. Effect of the extracts on the viability of rat hepatocytes. Rat hepatocytes were isolated and incubated for 3 h with DMEM containing different concentrations of SCA and SPE. At the end of the incubation period, the viability was determined by MTT reduction assay and expressed as percentage of control. Data are expressed as average ± SD of three independent experiments.

1443

tions (Fig. 2) and the viability increased in a dose dependent manner. Different concentrations (0–500 ppm) of the extracts were tested against CCl4 induced cytotoxicity (data not shown) and only those concentrations enhancing the cell viability to above 95% were selected for detailed study. Upon CCl4 treatment, a 60% reduction in viability was observed compared to the untreated control cells. The viability was considerably alleviated (to an extent of more than 95%) in the presence of the extracts after CCl4 treatment (Fig. 3). Similar effects were found even with EGCG when used at levels 0.1, 1.0, 5.0 and 10.0 lmol/l. Intracellular LDH leakage, as a result of the plasma membrane breakdown and a concomitant alteration in its permeability, was evaluated as a marker of cell membrane integrity and viability. While the control cells (untreated) showed less than 5% leakage of LDH, the CCl4-exposed cells showed a substantially high value of 45% leakage of LDH (Fig. 4). Exposure of the CCl4-treated cells to extracts significantly brought down the levels of LDH leakage indicating the reversal of the damage induced by CCl4 in primary rat hepatocytes (Fig. 4). Treatment of the rat hepatocytes with S. cumini extracts post-CCl4 exposure showed an increase in the viability of the same. This was further substantiated by a consequent suppression of LDH leakage by SCA and SPE at concentrations ranging from 50 to 500 ppm suggesting protection against CCl4 toxicity. The protection offered was slightly higher with SCA (>90%) as compared to that with SPE (<90%) at 50 ppm. The higher concentrations of EGCG increases LDH leakage but at a level significantly lower than that of CCl4 (p < 0.001). Though the LDH leakage in SPE treated cells was higher than that of SCA treated ones, the levels were still significantly less than that in CCl4 treated cells (p < 0.001). Based on the results of LDH and MTT assay, 50 and 100 ppm of the extracts and 0.1 and 1.0 lmol/l EGCG were chosen for further study since these concentrations provided sufficient protection (>95%) against CCl4 induced cell death. Fig. 5 summarises the effect of SCA, SPE and EGCG on intracellular GSH content. There was a significant (13%) fall in the intracellular GSH levels in CCl4 treated cells as compared to the control cells. Treatment of such cells with the SCA substantially increased the GSH content, more at 50 ppm than 100 ppm. The increase was nearly 2-fold (6.46 nM/mg protein) at 50 ppm as compared to the GSH levels in cells treated with CCl4 (3.67 nM/mg protein). However further depletion of intracellular GSH levels by SPE was observed. The cells showed a higher

LDH leakage (%)

J.M. Veigas et al. / Toxicology in Vitro 22 (2008) 1440–1446

50 45 40 35 30 25 20 15 10 5 0

*

* *

**

CCl 4

50ppm

EGCG 100ppm

*

**

*

*

* Control

* SCA 250ppm

SPE 500ppm

Fig. 4. Effect of the extracts and EGCG  on the LDH leakage in rat hepatocytes. Rat hepatocytes were isolated and incubated with 7 mmol/l CCl4 for 1 h after which the medium was replaced by fresh medium containing either SCA or SPE each ranging in concentration from 50 to 500 ppm and cultures incubated for further 3 h. Untreated (labeled as ‘‘control”) and CCl4 treated controls were treated with fresh medium alone. Viability was expressed percentage LDH released into the culture medium relative to the total cellular activity determined after cell lysis with 0.1% v/ v Triton-X 100. Data are expressed as mean ± SD of three independent experiments: * Indicates statistically very high significant difference compared to CCl4 treated group p < 0.001; **The concentrations of EGCG were 0.1, 1.0, 5.0 and 10.0 lmol/l.

recovery response to SCA than EGCG in terms of GSH content (Fig. 5). Results of lipid peroxidation assay showed a substantial protection offered by the SCA compared to the others. Carbon tetrachloride induced a 46% peroxidation of lipid membranes as against a 28% in untreated, control cells. SCA (50 and 100 ppm) and EGCG (1 lM) significantly reduced the CCl4-induced lipid peroxidation in rat hepatocytes to near-normal levels (Fig. 6). However, SPE did not significantly alter the level of lipid peroxidation initiated by CCl4. Table 1 shows the effect of different extracts on antioxidant enzymes activities in CCl4 intoxicated rat hepatocytes indicating a significant reduction in the activity of SOD (8.89 U/mg protein) as compared to the control cells (11.34 U/mg protein). However, the extracts (100 ppm) as well as EGCG brought about a negligible 2% increase in the SOD activity brought down by CCl4 but was inefficient in restoring the enzyme activity to the normal level. A marginal increase in CAT activity (25.39 U/mg protein) was achieved at 100 ppm of SCA in CCl4 intoxicated rat hepatocytes

140

†

120

MTT reduction (% of control)

*

‡

‡† ‡ ‡‡

100

5 ppm 10 ppm 20 ppm

80

25 ppm

*

50 ppm

60

100 ppm 40

250 ppm 500 ppm

20 0

CCl4

EGCG

S CA

SPE

Fig. 3. Effect of the extracts and EGCG§ on the viability of rat hepatocytes. Rat hepatocytes were isolated and incubated with 7 mmol/l CCl4 for 1 h after which the medium was replaced by fresh medium containing either SCA or SPE each ranging in concentration from 50 to 500 ppm and cultures incubated for further 3 h. Untreated (labeled as ‘‘control”) and CCl4 treated controls were treated with fresh medium alone. At the end of the incubation period, the viability was determined by MTT reduction assay and expressed as percentage of control. Data are expressed as mean ± SD of three independent experiments: *Indicates a very high statistically significant difference compared to control at p < 0.001;  Indicates a statistically significant difference compared to CCl4 treated group at p < 0.05; àIndicates a statistically significant difference compared to CCl4 treated group at p < 0.01; §The concentrations of EGCG were 0.01, 0.025, 0.05, 0.075, 0.1, 1.0, 5.0 and 10.0 lmol/l.

1444

J.M. Veigas et al. / Toxicology in Vitro 22 (2008) 1440–1446

7.00 6.00

nmol/mg protein

mg protein). Supplementing the cells with anthocyanin extract significantly enhanced the GPx activity in a dose dependent manner. At 100 ppm, SCA and SPE showed an activity of 13.59 and 17.77 U/ mg protein, respectively. EGCG evoked a significant 4-fold increase in GPx activity in the isolated rat hepatocytes.

*‡ ‡

5.00

† †

4.00 3.00

*‡ *‡

2.00 1.00 0.00

Control

CCl4 50ppm

EGCG

SCA 100ppm

SPE

Lipid peroxidation (%)

Fig. 5. Effect of the extracts and EGCG§ on the total intracellular GSH content in rat hepatocytes. Rat hepatocytes were isolated and incubated with 7 mmol/l CCl4 for 1 h after medium was replaced by fresh medium containing either SCA or SPE each ranging in concentration from 50 to 500 ppm and cultures incubated for further 3 h. Untreated (labeled as ‘‘control”) and CCl4 treated controls were treated with fresh medium alone. Data are expressed as average ± SD of three independent experiments: *Indicates a statistically significant difference compared to control (p < 0.001);  Indicates a statistically significant difference compared to control (p < 0.05); à Indicates a statistically significant difference compared to CCl4 treated group (p < 0.001); §The concentrations of EGCG were 0.1 and 1.0 lmol/l.

60 50 40 30

*

*

*

*

20 10 0 CCl4

Control

EGCG 50ppm

SCA

SPE

100ppm

Fig. 6. Effect of the extracts and EGCG** on the lipid peroxidation status in rat hepatocytes. Rat hepatocytes were isolated and incubated with 7 mmol/l CCl4 for 1 h after medium was replaced by fresh medium containing either SCA or SPE each ranging in concentration from 50 to 500 ppm and cultures incubated for further 3 h. Untreated (labeled as ‘‘control”) and CCl4 treated controls were treated with fresh medium alone. Lipid peroxidation is expressed as percentage with respect to blank. Data are expressed as average ± SD of three independent experiments: *Indicates a statistically significant difference compared to CCl4 treated group (p < 0.001); **The concentrations of EGCG were 0.1 and 1.0 lmol/l.

as compared to the treatment with CCl4 alone (23.3 U/mg protein). EGCG and SPE further reduced the CAT activity to 15.3 and 18.9 U/ mg protein, respectively, at 50 ppm. Carbon tetrachloride showed about 28% reduction in GPx activity (5.22 U/mg protein) as compared to the untreated cells (7.32 U/

4. Discussion Anthocyanins are naturally occurring polyphenols abundant in several edible fruits and vegetables and beverages such as red wine and other fruit juices. There is considerable anecdotal and epidemiological evidence that dietary anthocyanins and polyphenols confer preventive and therapeutic roles in a number of human diseases. The dried flower extract of Hibiscus sabdariffa (a rich source of anthocyanins) has been reported to inhibit CCl4-induced liver fibrosis in rats (Liu et al., 2006). Therefore, identifying new sources of such compounds having potent antioxidant and hepatoprotective activity is considered an important milestone. After establishing the antioxidant potential of the anthocyanin rich extract of S. cumini peel through our previous study (Veigas et al., 2007), the present study was conducted to further understand the mechanisms through which the extracts of S. cumini might render protection against CCl4-induced oxidative insult in rat primary hepatocytes. To the best of our knowledge the literature does not provide any information on this subject. Specific concentrations of a green tea polyphenol – Epigallo catechin gallate (EGCG) is known to show excellent antioxidant and cytoprotective activity (Lin et al., 2000; Potapovich and Kostyuk, 2003). An earlier study has recorded inhibition of lipid oxidation at physiological levels (0.1–1.0 lM) of EGCG (Intra and Kuo, 2007). Due to these reasons, EGCG was used to know how far our test materials (i.e., the extracts of S. cumini) compare with such a compound in our experimental model. Carbon tetrachloride is metabolized by the cytochrome P450 system to produce toxic trichloromethyl radicals, which act as free radical cascade initiators (Johnston and Kroening, 1998). The rapid loss of cytochrome P450 system during the development of the culture renders cultured hepatocytes unsuitable for CCl4 toxicity studies (Adzet et al., 1987). For this and for various other reasons stated in previous studies (Moldeus et al., 1978), freshly isolated cells were used in the present study. The biochemical mechanisms involved in the hepatoprotective activity of the S. cumini extract against CCl4-induced injury were studied by measuring the levels of LDH, GSH, lipid peroxidation and by screening for the activities of primary antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase. The MTT and LDH assays are well established and widely used methods to assess the mitochondrial competence and cell membrane integrity respectively (Ljubuncic et al., 2005). These assays

Table 1 Effect of the extracts and EGCG on the activities of the antioxidant enzymes CAT, GPx and SOD in rat hepatocytesa Sample

CAT (U/mg protein) 50 ppm

Control CCl4 SCA SPE

29.60 ± 0.32a 23.30 ± 0.19cd 24.20 ± 0.30c 18.97 ± 0.95de 0.1 lmol

EGCG

15.30 ± 0.43e

100 ppm 29.60 ± 0.32bc 23.30 ± 0.19c 25.39 ± 0.61bc 11.61 ± 0.49de 1.0 lmol. 6.57 ± 0.24e

GPx (nmol NADPH oxidised/min/mg protein)

SOD (U/mg protein)

50 ppm

50 ppm

7.32 ± 0.17e 5.22 ± 0.06e 12.09 ± 0.42b 9.41 ± 0.25bc 0.1 lmol 9.41 ± 0.31bc

100 ppm 7.32 ± 0.17e 5.22 ± 0.06e 13.59 ± 0.25d 17.77 ± 1.11c 1.0 lmol. 29.27 ± 0.62a

11.34 ± 0.53b 8.89 ± 0.26c 8.93 ± 0.07bc 3.54 ± 0.11e 0.1 lmol 4.52 ± 0.05de

100 ppm 11.34 ± 0.53a 8.89 ± 0.26e 9.08 ± 0.06e 9.03 ± 0.02e 1.0 lmol. 9.53 ± 0.19de

Data are expressed as average ± SD of three independent experiments: Data followed by different letters within each column are significantly different according to Duncan’s multiple-range test at (p < 0.05). a Rat hepatocytes were isolated and incubated with 7 mmol/l CCl4 for 1 h after which the medium was replaced by fresh medium containing either SCA or SPE each ranging in concentration from 50 to 500 ppm and cultures incubated for further 3 h. Untreated and CCl4 treated controls were treated with fresh medium alone.

J.M. Veigas et al. / Toxicology in Vitro 22 (2008) 1440–1446

showed that both the SCA and SPE significantly reduced the LDH release, confirming the cytoprotective effects rendered by S. cumini. In addition, the results of MTT assay showed a dose dependent increase in the cell viability which was highest in treatment with 500 ppm of SCA (Fig. 3). Thus, the suppression of the LDH leakage as well as the alleviation of cell viability by the extract clearly establishes its role as a cytoprotective agent in post CCl4-treated cells. GSH, a low molecular weight endogenous antioxidant thiol present in abundance in the liver acts either by directly scavenging the free radicals or by acting as a substrate to GPx and glutathione transferase during the detoxification of hydrogen peroxides, lipid peroxides and electrophiles as well as by preventing oxidation of –SH groups of proteins (Galati et al., 2006; Masella et al., 2005; Lu, 1999). Treatment with CCl4 reduced the levels of intracellular GSH with a reciprocal decrease in GPx activity which was significantly reversed by SCA, on the contrary, SPE reduced the GSH level. Since there was no reduction in the viability of cells treated with SPE, the latter fraction appears to offer protection through pathways other than glutathione redox pathway. Similarly, a high GPx activity in this treatment suggests the utilization of GSH as a substrate for the activity of the said enzyme. Another practical and more meaningful explanation that can be offered here is the conjugation of the GSH by the phenolic acids (Galati et al., 2006) present in the pulp extract (Anonymous, 1976), thus making the free GSH unavailable for the assay. A similar observation was made with buthionine sulfoximine, which reduced GSH level in cultured rat hepatocyte without reducing the cell viability (Lee and Farrell, 2001). The high GSH content may also be responsible for a direct scavenging of free radicals generated in vivo in addition to acting as a substrate to the enzyme GPx. This hypothesis needs a thorough investigation where an assessment at transcriptional levels of these enzymes under the influence of free radical generator and scavengers will give insights into the mode of action of the extracts. Catalase and SOD play a major role as antioxidant enzymes in the mammalian system, by detoxifying hydrogen peroxide generated by various oxidases and other enzymatic and non-enzymatic auto oxidation of compounds (Eaton, 1989). Glutathione peroxidase counteracts the free radicals using GSH as a substrate. As a result, GSH is oxidized to GSSG (oxidized glutathione), which in turn is reduced to GSH by glutathione reductase at the expense of NADPH, forming a redox cycle. Our results show that CCl4 significantly reduced the activities of CAT and SOD. However, the extracts failed to revive the activities of these two antioxidant enzymes. Earlier studies have established that the depletion of GSH by CCl4 is followed by peroxidation of cellular lipids resulting in death of the hepatocytes (Miccadei et al., 1988). This was further supported in our study where the CCl4-exposed hepatocytes clearly exhibited a depletion of GSH content and an increase in lipid peroxidation (Figs. 5 and 6) while the reverse was observed when the cells were treated with SCA and EGCG post CCl4 exposure. This suggests that the anthocyanins present in the extract may play an important role in preventing initiation and propagation of the lipid peroxidation process by scavenging the free radicals via the GSH (Martin-Aragon et al., 2001). SCA at 50 ppm level diminishes lipid peroxidation. Probably this being an ideal situation, higher level of extract containing un-reacted anthocyanins having an absorption maximum of 535 may contribute for erroneous results. This has been indicated as one of the reasons for high lipid peroxidation value obtained at high concentrations of SCA and SPE, although this argument needs to be confirmed by detailed analytical studies. Contrarily, SPE showed a reduction in GSH content, not offering any protection against lipid peroxidation despite the high viability of cells (GSH content is generally regarded as an index of cell death). Intracellular LDH is released as a result of membrane

1445

breakdown and concomitant increase in membrane permeability. Therefore, the extracts might be offering protection against the lipid peroxidation although complete reversal of CCl4-damage was not imparted. Since MTT and LDH assays clearly show cytoprotection by SPE, there may be other mechanisms involved in cell protection rather than the involvement of GSH alone. The involvement of other enzymes such as glutathione reductase and respective transferase (not followed in this study) cannot be ignored. The overall results show that the extracts, especially SCA, mainly act via the glutathione redox system rather than through CAT and SOD enzyme system. The failure of SCA and SPE in restoring the SOD and CAT activities suggest that the two extracts reverse cell damage through biological pathways independent of these antioxidant enzymes. Health benefits from the use of natural products have evoked interest in finding new and novel sources of such compounds for application in nutraceutical, pharmaceutical and cosmetic industry. The present study gives an insight into the cytoprotective effects of S. cumini anthocyanins, particularly in reversing CCl4induced cell damage indicating its application in ameliorating toxic effects of various other chemicals and xenobiotics. Acknowledgements Authors are grateful to Dr. V. Prakash, Director, CFTRI, for his encouragement for the work and Dr. S.P. Muthukumar, scientist, Biochemistry and Nutrition Department for providing the rat liver. JMV and RS are grateful to Council for Scientific and Industrial Research, Government of India, for the Grant of fellowship. References Adzet, T., Camarasa, J., Laguna, J.C., 1987. Hepatoprotective activity of polyphenolic compounds in isolated rat hepatocytes from Cynara scolymus against CCl4 toxicity. Journal of Natural Products 50 (4), 612–617. Aebi, H.E., 1984. Catalase in vitro. Methods Enzymology 105, 121–126. Andersen, O.M., Viksund, R.I., Pedersen, A.T., 1995. Malvidin 3-(6-acetylglucoside)5-glucoside and other anthocyanins from flowers of Geranium sylvaticum. Phytochemistry 38 (6), 1513–1517. Anonymous, 1976. Syzygium cumini. In: Chadha, Y.R. (Ed.), The Wealth of India, Raw materials – A Dictionary of Indian Raw Materials and Industrial Products, Publication and Information Directorate, Council for Scientific and Industrial Research, New Delhi, India, vol. X, p. 100. Banerjee, A., Dasgupta, N., De, B., 2005. In vitro study of antioxidant activity of Syzygium cumini fruit. Food Chemistry 90 (4), 727–733. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymology 52, 302–310. Cao, G., Muccitelli, H.U., Sanchez-Moreno, C., Prior, R.L., 2001. Anthocyanins are absorbed in glycated forms in elderly women: a pharmacokinetic study. American Journal of Clinical Nutrition 73 (5), 920–926. Eaton, J.W., 1989. Actalasemia. In: Scriver, C.R., Beaudet, A.L., Sly, W.S. (Eds.), The Metabolic Basis of Inherited Diseases. McGraw-Hill, New York, pp. 1551–1561. Flohe, A., Gunzler, W.A., 1984. Assays of glutathione peroxidase. Methods Enzymology 105, 114–120. Francis, F.J., 1986. Analysis of anthocyanins. In: Markakis, P. (Ed.), Anthocyanins as Food Colors. Academic press, New York, pp. 181–207. Galati, G., Lin, A., Sultan, A., O’Brien, P.J., 2006. Cellular and in vivo hepatotoxicity caused by green tea phenolic acids and catechins. Free Radical Biology and Medicine 40 (4), 570–580. Ghiselli, A., Nardini, M., Baldi, A., Scaccini, C., 1998. Antioxidant activity of different phenolics fractions separated from Italian red wine. Journal of Agricultural and Food Chemistry 46 (2), 361–367. Haidara, K., Moffat, P., Denizeau, F., 1999. Metallothionein induction attenuates the effects of glutathione depletors in rat hepatocytes. Toxicological Sciences 49 (2), 297–305. Harter, L.N., 1960. Critical values for Duncan’s new multiple range test. Biometrics 16 (4), 671–685. Intra, J., Kuo, S., 2007. Physiological levels of tea catechins increase cellular lipid antioxidant activity of vitamin C and vitamin E in human intestinal Caco-2 cells. Chemico-Biological Interactions 169 (2), 91–99. Johnston, D.E., Kroening, C., 1998. Mechanism of early carbon tetrachloride toxicity in cultured rat hepatocytes. Pharmacology and Toxicology 83 (6), 231–239. Kamendulis, L.M., Jiang, J., Xu, Y., Klaunig, J.E., 1999. Induction of oxidative stress and oxidative damage in rat glial cells by the acrylonitrile. Carcinogenesis 20 (8), 1555–1560.

1446

J.M. Veigas et al. / Toxicology in Vitro 22 (2008) 1440–1446

Keppler, K., Humpf, H., 2005. Metabolism of anthocyanins and their phenolic degradation products by the intestinal microflora. Bioorganic and Medicinal Chemistry 13 (17), 5195–5205. Kong, J., Chia, L., Goh, N., Chia, T., Brouillard, R., 2003. Analysis and biological activities of anthocyanins. Phytochemistry 64 (5), 923–933. Lee, A.U., Farrell, G.C., 2001. Mechanism of azathioprine-induced injury to rat hepatocytes: roles of glutathione depletion and mitochondrial injury. Journal of Hepatology 35, 756–764. Lin, J.K., Chen, P.C., Ho, C.T., Lin-Shiau, S.Y., 2000. Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3, 3-digallate, (-)-epigallocatechin-3-gallate and propyl gallate. Journal of Agricultural and Food Chemistry 48 (7), 2736–2743. Liu, J., Chen, C., Wang, W., Hsu, J., Yang, M., Wang, C., 2006. The protective effects of Hibiscus sabdariffa extract on CCl4-induced liver fibrosis in rats. Food and Chemical Toxicology 44 (3), 336–343. Ljubuncic, P., Portnaya, I., Cogan, U., Azaizeh, H., Bomzon, A., 2005. Antioxidant activity of Crataegus aronia extract used in traditional Arab medicine in Israel. Journal of Ethnopharmacology 101 (1–3), 153–161. Lowry, O.H., Rosenberg, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin–phenol reagent. Journal of Biological Chemistry 193, 265–267. Lu, S.C., 1999. Regulation of hepatic glutathione synthesis: current concepts and controversies. FASEB Journal 13 (10), 1169–1183. Martin-Aragon, S., De Las Heras, B., Sanchez-Reus, M.I., Benedi, J., 2001. Pharmacological modification of endogenous antioxidant enzymes by ursolic acid on tetrachloride-induced liver damage in rats and primary cultures of rat hepatocytes. Experimental Toxicology and Pathology 53, 199–206. Masella, R., Di Benedetto, R., Vari, R., Filesi, C., Giovannini, C., 2005. Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. Journal of Nutritional Biochemistry 16 (10), 577–586.

Miccadei, S., Kyle, M.E., Gilfor, D., Farber, J.L., 1988. Toxic consequence of the abrupt depletion of glutathione in cultured rat hepatocytes. Archives of Biochemistry and Biophysics 265 (2), 311–320. Moldeus, P., Hogberg, J., Orrenius, S., 1978. Isolation and use of liver cells. Methods Enzymology 52, 60–71. Neuman, M.G., Koren, G., Tiribelli, C., 1993. In vitro assessment of the ethanol induced hepatotoxicity on HepG2 cell line. Biochemical and Biophysical Research Communications 197 (2), 932–941. Popat, A., Shear, N.H., Malkiewicz, I., Thomson, S., Neuman, M.G., 2002. Mechanism of Impila (Callilepsis laureola)-induced cytotoxicity in HepG2 cells. Clinical Biochemistry 35 (1), 57–64. Potapovich, A.I., Kostyuk, V.A., 2003. Comparative study of antioxidant properties and cytoprotective activity of flavonoids. Biochemistry 68 (5), 514–519. Ruch, R.J., Klaunig, J.E., Schultz, N.E., Askari, A.B., Lacher, D.A., Pereira, M.A., Goldblatt, P.J., 1986. Mechanisms of chloroform and carbon tetrachloride toxicity in primary cultured mouse hepatocytes. Environmental Health Perspectives 69, 301–305. Sarma, A.D., Sharma, R., 1999. Anthocyanin–DNA copigmentation: mutual protection against oxidative damage. Phytochemistry 52 (7), 1313–1318. Sharma, S.B., Nasir, A., Prabhu, K.M., Murthy, P.S., 2006. Antihyperglycemic effect of the fruit-pulp of Eugenia jambolana in experimental diabetes mellitus. Journal of Ethnopharmacology 104 (3), 367–373. Slater, T.F., 1984. Free-radical mechanisms in tissue injury. Biochemical Journal 222 (1), 1–15. Veigas, J.M., Narayan, M.S., Laxman, P.M., Neelwarne, B., 2007. Chemical nature, stability and bioefficacies of anthocyanins from fruit peel of Syzygium cumini Skeels. Food Chemistry 105 (2), 619–627. Ying-Jie, W., Meng-Dong, L., Yu-Min, W., Jian, D., Qing-He, N., 1998. Simplified isolation and spheroidal aggregate culture of rat hepatocytes. World Journal of Gastroenterology 4 (1), 74–76.