Renal Failure
ISSN: 0886-022X (Print) 1525-6049 (Online) Journal homepage: http://www.tandfonline.com/loi/irnf20
Curcumin protects against gallic acid-induced oxidative stress, suppression of glutathione antioxidant defenses, hepatic and renal damage in rats Sunny O. Abarikwu, Mojisola Durojaiye, Adenike Alabi, Bede Asonye & Oghenetega Akiri To cite this article: Sunny O. Abarikwu, Mojisola Durojaiye, Adenike Alabi, Bede Asonye & Oghenetega Akiri (2015): Curcumin protects against gallic acid-induced oxidative stress, suppression of glutathione antioxidant defenses, hepatic and renal damage in rats, Renal Failure, DOI: 10.3109/0886022X.2015.1127743 To link to this article: http://dx.doi.org/10.3109/0886022X.2015.1127743
Published online: 27 Dec 2015.
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Date: 04 January 2016, At: 07:33
RENAL FAILURE, 2015 http://dx.doi.org/10.3109/0886022X.2015.1127743
LABORATORY STUDY
Curcumin protects against gallic acid-induced oxidative stress, suppression of glutathione antioxidant defenses, hepatic and renal damage in rats Sunny O. Abarikwua, Mojisola Durojaiyeb, Adenike Alabib, Bede Asonyea and Oghenetega Akirib
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a Department of Biochemistry, University of Port Harcourt, East-West Road, Choba, Nigeria; bDepartment of Chemical Sciences, Redeemer’s University, Redemption City, Ogun State, Nigeria
ABSTRACT
ARTICLE HISTORY
Curcumin (Cur) and gallic acid (Gal) are major food additives. Cur has well-known antioxidant properties, whereas Gal has both antioxidant and pro-oxidant effects. The present study investigated the effects of oral administration of Gal with or without Cur on antioxidant enzymes activities, glutathione (GSH) and the enzymes in its metabolism in rat liver in vivo and markers of tissue damage in the serum. Results showed that the increase in serum creatinine level, alkaline phosphatase and lactate dehydrogenase activities by Gal treatment were inhibited by combined administration of Gal and Cur. The decrease in GSH-peroxidase, GSH-S-transferase, superoxide dismutase and GSH-reductase activities by Gal treatment were inhibited when both Gal and Cur were administered together. The malondialdehyde concentration and catalase activity were significantly increased following administration of Gal but not when the administration of Gal was combined with Cur. Finally, the increase in GSH level was seen following administration of Cur alone or in combination with Gal but not with Gal alone. These results suggest that Gal might induce oxidative stress in the rat liver and affect renal function that can be inhibited by the combined administration of Gal and Cur.
Received 28 September 2015 Revised 7 November 2015 Accepted 22 November 2015 Published online 24 December 2015
Introduction Phytochemicals with antioxidant and pro-oxidant properties are both present in human diets and are consumed in large quantities.1 Curcumin and gallic acids (Figure 1) are both food additives with well-known pharmacological properties. Curcumin, the principal component of the curry spice turmeric (Curcuma longa) used in many parts of the world as a spice and medical agent possesses anti-oxidant properties and has no toxic effects.2–5 Gal acids are added to foods to prevent oxygen-induced lipid peroxidation as a result of its antioxidant properties.6,7 However, the potential of Gal to function as antioxidant may be dependent on the dose and their molecular polarities, which could account for the controversial function of Gal as an antioxidant and pro-oxidant molecule.8 Thus, Gal is cytotoxic to human blood lymphocytes by mechanism involving paradoxical generation of reactive oxygen species.7,9 We also recently reported decreased activities of antioxidant enzymes and GSH level without the induction of lipid peroxidation in the testis of rats as a result of Gal treatment for 30 days.5 The potential of Gal to act as prooxidant also explain its toxicity in the liver, kidney and
Curcumin; gallic acid; glutathione metabolism; antioxidant enzymes; malondialdehyde
blood of rats.10 Many phytochemicals, for example, quercetin, hesperetin naringenin, gossypol, myricetin and morin have both antioxidant and pro-oxidant properties depending on dosage, structural characteristics and experimental design.11–13 Apart the antioxidative and pro-oxidant properties, Gal possess anti-inflammatory, anti-bacterial and anticarcinogenic abilities.5,14 Similarly, Cur possesses antiinflammatory effect and is a potential chemopreventive agent with multiple beneficial functions.2 It is also possible that flavonoid pro-oxidant function could be a toll of their other beneficial functions. For example, epigallocatechin gallate that possess gallate moiety promotes apoptosis and has bactericidal activity, which is attributed to its ability to reduce oxygen to yield hydrogen peroxide.15,16 The biological system is well endowed with antioxidants such as GSH, GSH-Px, GR, CAT and SOD, which act as radical scavengers and protect cells and tissues from oxidative damage, and are associated with good health status.5 The adverse effects of polyphenolic molecules on GSH antioxidant system have been reported and pro-oxidant effects could deplete the antioxidative molecules of cell and tissues and lead to oxidative damage.17,18
CONTACT Sunny O. Abarikwu [email protected], [email protected] West Road Choba, PMB 5323, Port Harcourt, Rivers State, Nigeria ß 2015 Taylor & Francis
KEYWORDS
Department of Biochemistry, University of Port Harcourt, East-
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S. O. ABARIKWU ET AL.
Figure 1. Chemical structure of (A) Curcumin (1,7-Bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) and (B) Gallic acid (3,4,5-Trihydroxybenzoic acid.
Despite the long-time use of Cur and Gal as dietary spices and food additives, research efforts towards understanding their combined effects on the GSH antioxidant defence system of experimental models are limited. Therefore, this study was performed to evaluate the combined effects of two well-known dietary spices on the antioxidant enzymes systems and GSH-defensive system in liver and on renal function in vivo.
Materials and methods Chemicals and reagents Curcumin and gallic acid were purchased from SigmaAldrich, (Chemie, GmbH, Taufkirchen, Germany). All other used reagents were of analytical grade and purchased from Sigma unless otherwise stated.
Animal treatment Twenty male Wistar rats (average weight of 128.25 g) were obtained from the animal house of the College of Natural Sciences, Redeemer’s, Redemption City, Ogun State and were allowed to acclimatize for 1 week prior to the start of study. The animals were housed in metal cages under a well-ventilated condition, maintained under a 12-h light/dark cycle and provided with standard commercial pelleted feed and water ad libitum. After acclimatization, the animals were assigned randomly into four groups of five animals, including a control group and three experimental groups. The three treatment groups of rats received 100 mg/kg/day of Cur or Gal, or the combination of Cur and Gal (100 mg/kg/ day + 100 mg/kg/day). Control rats were administered equivalent volumes of corn oil only. Cur and Gal were
diluted with the vehicle (corn oil) and administered to all animals (0.3 mL/kg) by gavage four times a week for 30 days. Experimental protocols were in accordance with the principles and procedures of the National Institute of Health Guidelines for Animal Care and Use of Laboratory Animals. At the end of 30 days, the animals were fasted overnight, weighed and killed by cervical dislocation. Blood samples were collected for biochemical analysis. The liver and kidney were dissected out quickly and washed in 1.15% KCl (ice-cold) and pat-dried. One side of the kidney and a portion of the liver were preserved in neutral buffered formalin. Tissues were sectioned and stained routinely with hematoxylin and eosin (H&E) for microscopy. The dose of Gal (100 mg/kg) was chosen because it is less than the no-observed-adverse-effect level (NOAEL) for Gal in rats.10 The dose of Cur was chosen based on our previous studies and those of others which reported protective function on testicular tissues under various pathological conditions.5,19,20
Estimation of the activities of serum markers enzymes, lipid and hematological profiles Creatinine, acid phosphatase (ACP), g-glutamyl transferase (GGT), alanine amino transferase (ALT), aspartate amino transferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), urea, cholesterol (CHOL), triglycerides (TRIGS), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were estimated in the serum using Randox commercial kits. Heparin-added whole-blood samples, immediately after collection were used for hematological analyses. Hb, PCV, RBC, WBC, platelet, NEUT, leucocytes were analyzed by means of a COBAS MICROS (Roche, Palo Alto, CA) analyzer. Hematological analyses were systematically checked by means of standard human blood CBC-3D Hematology control (R & D System Inc., Minneapolis, MN). A day-today precision of RBC, and PCV measurements (n ¼ 5) in blood were 4.2 and 4.5%, respectively.
Preparation of liver homogenate and assessment of oxidative stress The tissues were homogenized in ice-cold 0.1 M Tris–HCl buffer (pH7.4) to produce 10% homogenate. The homogenate was centrifuged at 10,000 g, 4 C for 30 min and the supernatant was separated to measure the biochemical parameters of oxidative stress described below. Malondialdehyde (MDA) and glutathione (GSH) and the enzymes of its metabolism (glutathione peroxidase, glutathione-S-transferase, glutathione reductase), defensive antioxidant enzymes (superoxide dismutase and catalase).
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Table 1. Hepatic malondialdehyde and reduced glutathione levels, glutathione peroxidase, glutathione-S-transferase and glutathione reductase activities in liver of rats after administration of Cur and Gal alone or in combination for 4 weeks. Variable MDA (mmol/mg) GSH (mg/mg) GSH-Px (Unit/mg) GST (Unit/mg) GR (U/L)
Control
Gal
Cur
Gal + Cur
1.46 ± 0.76 10.36 ± 0.42 1.17 ± 0.03 4.98 ± 1.09 145 ± 10.81
3.26 ± 0.97* 10.29 ± 0.49 0.40 ± 0.02* 11.22 ± 1.12* 87 ± 7.64*
1.58 ± 0.46 11.74 ± 0.94* 1.21 ± 0.05 4.74 ± 1.51 151 ± 12.73
1.79 ± 0.85** 11.79 ± 1.64* 1.13 ± 0.05** 4.05 ± 1.77** 148 ± 12.76**
Cur: curcumin; Gal: gallic acid. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group. **Significantly different from Gal treated group (p50.05).
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Measurement of lipid peroxidation Lipid peroxidation was quantified spectrophotometrically as MDA using thiobarbituric acid for colour development.21 The absorbance of the pink-coloured solution was measured at 532 nm using 1,1,3,3-tetraethoxypropane as a standard. The result was expressed as micromoles per milligram of protein. Protein concentration was determined by the method of Lowry et al.22
Measurements of reduced glutathione and enzymes of its metabolism The reduced GSH level was determined with a spectrophotometer (412 nm) using 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) for colour development.23 The results were expressed as mg/mg protein. The activity of glutathione peroxidase (GSH-Px) was estimated with a spectrophotometer (412 nm) by measuring the residual GSH content after the reaction: H2O2 + 2GSH!2H2O + GSSG (oxidized glutathione) as previously described.24 The enzyme activity was expressed as units/mg protein. The activity of glutathione reductase (GR) was determined in a spectrophotometer (340 nm) by monitoring the oxidation of NADPH using the extinction coefficient 6220 M 1 cm1 for NADPH.25 One unit of NADPH causes the oxidation of 1.0 mmol NADPH at 25 C at pH 7.4. The activity of glutathione S-transferase (GST) was measured in a spectrophotometer (340 nm) using with 1-chloro2,4-dinitrobenzene as a substrate.26
Measurements of defensive antioxidant enzymes The activity of catalase (CAT) was measured spectrophotometrically at 240 nm by calculating the rate of degradation of H2O2, the substrate of the enzyme.27 Activities were calculated using the molar extinction coefficient of H2O2 at 240 nm, 43.59 M 1 cm1. One unit of CAT activity equals the amount of protein that converts 1 mmol H2O2/min. Activity was expressed as micromole of H2O2 consumed per milligram of protein.
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The activity of superoxide dismutase (SOD) was determined spectrophotometrically at 480 nm using freshly prepared epinephrine (0.01%) as substrate.28 One unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline to adrenochrome during one minute. The results were expressed as unit per milligram of protein.
Statistical analyses All values are expressed as mean ± SD. Statistical comparison between different treatments was done by either Student’s t-test or one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test using Graph Pad Prism 5.0 software (Graph Pad Software, Inc., San Diego, CA). Differences between values were considered statistically significant at the p50.05 level.
Results Concentration of glutathione Gallic acid treatment did not change the concentration of reduced GSH compared with basal GSH concentration (10.36 ± 0.42 and 10.29 ± 0.49 mg/mg protein, respectively, p40.05), while the level of GSH of rats treated with Cur (11.74 ± 0.94 mg/mg protein) was significantly increased from that of the basal value (Table 1). This increase was maintained when both Gal and Cur were combined in rats treated with the combination of Gal and Cur compared with Gal alone or control (11.79 ± 1.64, p50.05).
Activities of glutathione peroxidase, reductase and S-transferase Curcumin alone did not influence the basal activity of GSH-Px, GR and GST in liver (1.17 ± 0.03, 145 ± 10.81, 4.98 ± 1.09, respectively). Gal treatment alone decreased the GSH-Px and GR activities (0.4 ± 0.02 Unit/mg and 87 ± 7.64 U/L, respectively) and increased GST activity (11.22 ± 1.12 Unit/mg) compared with basal GSH-Px, GR and GST activities (1.17 ± 0.03 Unit/mg, 145 ± 10.81 and 4.98 ± 1.09, respectively). These decreases in GSH-Px and GR activities and increase in GST activity caused by Gal treatment were prevented by the combined administration of Gal and Cur (1.13 ± 0.05 Unit/mg, 148 ± 12.76 U/L and 4.05 ± 1.77 Unit/mg, respectively, p50.05 compared with Gal alone, Table 1).
Concentration of malondialdehyde The basal MDA concentration in liver was 1.46 ± 0.76 mmol/mg protein and was significantly
S. O. ABARIKWU ET AL.
(A)
3 *
*
Catalase (Unit/mg)
2.5 2 **
1.5 1 0.5 0 Control
Gal
Cur
(B) 10 9 8 7 6 5 4 3 2 1 0 Superoxide dismutase (Unit/mg)
4
Gal+Cur
*
**
*
Control
Gal
Cur
Gal+Cur
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Figure 2. Activities of catalase (A, unit/mg) and superoxide dismutase (B, unit/mg) activities in rat liver after administration of Cur and Gal alone or in combination for 4 weeks. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group, **Significantly different from Gal treated group (p50.05).
increased 123.29-fold following treatment with Gal alone, while Cur alone showed no such effect (Table 1). This increase with Gal alone was prevented by the combined administration of Gal and Cur (1.79 ± 0.85 mmol/mg protein, p50.05 compared with Gal alone).
Activities of superoxide dismutase and catalase The basal SOD and CAT activities in the liver were 3.06 ± 1.32 Unit/mg and 1.59 ± 0.21 Unit/mg, respectively (Figure 2), and SOD was significantly decreased while CAT was significantly increased following treatment with Gal. The changes caused by Gal alone on the activities of SOD and CAT were prevented by the combined administration of Gal and Cur (2.63 ± 0.22 Unit/mg protein and 1.45 ± 0.11 Unit/mg protein, p50.05 compared with Gal alone). Cur treatment alone also significantly increased the activities of SOD and CAT (7.81 ± 0.79 Unit/mg and 2.42 ± 0.13 Unit/mg protein, p50.05 compared with basal values).
Table 2. Serum hepatic and renal markers in rats after treatment with cur and gal alone or in combination for 4 weeks. Variable ACP (Unit/mg) GGT ALT (U/L) AST (U/L) Urea (mmol/L) Total protein (g/L)
Control
Gal
Cur
Gal + Cur
0.92 ± 0.11 29.71 ± 0.76 21.57 ± 2.76 37 ± 8.47 2.36 ± 0.84 75.43 ± 4.86
0.89 ± 0.13 29.5 ± 0.55 22.14 ± 3.8 31 ± 7.57 2.14 ± 0.87 80.57 ± 4.99*
0.91 ± 0.28 29.71 ± 0.76 21 ± 2.31 38.57 ± 8.77 2.21 ± 0.73 77.71 ± 4.82
0.94 ± 0.19 30.29 ± 0.49 20.29 ± 4.57 31.57 ± 6.99 2.69 ± 0.6 74 ± 2.31**
Cur: curcumin; Gal: gallic acid. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group. **Significantly different from Gal treated group (p50.05).
Table 3. Lipid profiles of rats serum after treatment with cur and gal alone or in combination for 4 weeks. Variable CHOL (mmol/L) TRIGS (mmol/L) HDL (mmol/L) LDL (mmol/L)
Control
Gal
Cur
Gal + Cur
2.33 ± 0.48 0.91 ± 0.18 1.31 ± 0.43 1.18 ± 0.37
2.39 ± 0.43 0.93 ± 0.23 1.79 ± 0.47 0.32 ± 0.13*
2.4 ± 0.56 1.11 ± 0.25 1.39 ± 0.23 0.98 ± 0.13
2.09 ± 0.14 1.03 ± 0.28 1.56 ± 0.44 0.34 ± 0.15*
Cur: curcumin; Gal: gallic acid. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group (p50.05).
Blood biochemistry and hematology Gallic acid or Cur alone or in combination did not influence the basal activities of ACP, GGT, ALT, AST, urea, TRIGS, HDL and CHOL (Tables 2 and 3). Treatment with Gal alone significantly decreased LDL level (0.32 ± 0.13 mmol/L, p50.05 compared with basal values (1.18 mmol/L), while Cur alone showed no such effect. This decrease with Gal treatment was not prevented by the combined administration of Gal and Cur (0.34 ± 0.15 mmol/L compared with Gal alone or basal values). The total protein levels, basal creatinine (CREA) level, lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in the serum were 75.43 ± 4.86 g/L, 145.67 ± 17.14 mmol/L, 448 ± 46.62 U/L and 42.14 ± 3.58 U/L, respectively, and were significantly
increased by 6.81%, 136.3%, 29% and 68.81%, respectively, following treatment with Gal alone compared with basal values, while Cur alone showed no such effect (Figure 3). This increase with Gal alone was prevented by the combined administration of Gal and Cur (74 ± 2.31 g/L, 184 ± 42.59 mmol/L, 414.29 ± 25.23 U/L, 45 ± 4.36 U/L, respectively, p50.05 compared with Gal alone). Gallic acid or Cur alone or in combination did not influence basal Hb, PCV, RBC, WBC, neutrophils and leucocytes counts (Table 4). Treatment with Gal alone significantly increased platelet count (283.3 ± 20.7 109/L, p50.05 compared with basal values, 176.7 ± 30.11 109/ L), while Cur alone showed no such effect. However, this increase with Gal treatment was not prevented by the
RENAL FAILURE
(A) 450
(B) 700
Creatinine (µmol/L)
Lactate dehydrogenase (U/L)
*
400 350 300
**
250 200 150 100 50 0
*
600 500
**
400 300 200 100 0
Control
Gal
Cur
Gal+Cur
Alkaline phosphatase (U/L)
(C) 90
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Control
Gal
Cur
Gal+Cur
*
80 70 60 **
50 40 30 20 10 0 Control
Gal
Cur
Gal+Cur
Figure 3. Activities of creatinine, (A, mmol/L), lactate dehydrogenase (B, U/L) and alkaline phosphatase (C, U/L) activities in rat serum after administration of Cur and Gal alone or in combination for 4 weeks. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group, **Significantly different from Gal treated group (p50.05).
Discussion Table 4. Hematology data for rats after treatment with cur and gal alone or in combination for 4 weeks. Variable Hb (g/L) PCV (L/L) RBC (1012/L) WBC ( 109/L) Platelet ( 109/L Neutrophils Leucocytes
Control
Gal
Cur
Gal + Cur
12.43 ± 0.64 37.29 ± 1.98 6.69 ± 0.25 10.11 ± 2.9 176.7 ± 30.11 40 ± 6.16 59.29 ± 5.77
12.76 ± 0.49 38.29 ± 1.5 6.8 ± 0.34 12.1 ± 3.03 283.3 ± 20.66* 41.86 ± 4.14 58.14 ± 4.14
12.51 ± 0.85 37.57 ± 2.51 6.77 ± 0.4 11.79 ± 1.47 196.7 ± 32.66 40.71 ± 3.64 58.43 ± 4.58
12.370.88 37.14 ± 2.61 6.67 ± 0.44 11.43 ± 3.87 318.3 ± 47.5* 39.5 ± 5.96 59.17 ± 6.77
Cur: curcumin; Gal: gallic acid. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group (p50.05).
combined administration of Gal and Cur (318.3 ± 47.5 109/L compared with Gal alone or basal values).
Histopathology of rat kidney and liver There were no visible lesions in the control, Cur and Gal + Cur-treated animals. Gallic acid treatment-related effects were noted in the liver and kidney of rats (Figures 4 and 5). In the liver of Gal-treated rats, there is a very mild periportal cellular infiltration by mononuclear cells. The kidney of control, Cur-and Gal + Cur-treated animals showed normal glomerulus and renal tubules. The kidneys of Gal-treated rats showed inflammatory cells and partially obliterated bowman capsule.
Although both Gal and Cur have been in use as food additive for many years, their combined effects on the liver and kidney of experimental models, for example, rats have not been evaluated in vivo. In this study, their combine effects on the GSH-defensive system and enzymes of its metabolism and on renal function were evaluated. The present results showed that Gal at 100 mg/kg/day decreased GSH-Px, GR and SOD activities and increased lipid peroxidation, GST and CAT activities in rat’s liver. In our previous report, Gal at same dose (100 mg/kg/day) used in the present study may downregulate GSH metabolism in the testes of rats.5 Moreover, Gal at a dose similar than was used in the present experiment induced toxicity to both the kidney and liver of male and female rats.10 These support the toxicity of Gal in the present experimental model and suggest that Gal might have pro-oxidant effects in rat liver. In studies where Gal exhibited antioxidant effects, the doses used were usually smaller than was used in the present study.29,30 Thus, the anti- or prooxidant effect of Gal could be dependent on the dosage of treatment. However, the dose of Gal used in the present study was based on the no-observed-adverseeffect level (NOAEL) which is about 119 mg/kg in male rats10 and has been reported to be biologically relevant dose.5
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Control
Gal
Cur
Cur + Gal
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Figure 4. Representative photomicrograph of hematoxylin and eosin-stained sections of liver of control and experimental rats (A) Control, (B) Cur, 100 mg/kg, (C) Gal, 100 mg/kg, (D) Cur + Gal. (arrows: mild periportal cellular infiltration by mononuclear cells ( 400). Cur: curcumin; Gal: gallic acid.
Control
Gal
Cur
Cur+Gal
Figure 5. Representative photomicrograph of hematoxylin and eosin-stained sections of kidney of control and experimental rats (A) Control, (B) Cur, 100 mg/kg, (C) Gal, 100 mg/kg, (D) Cur + Gal (short arrows: inflammatory cells; long arrows: partially obliterated bowman’s capsule ( 400). Cur: curcumin; Gal: gallic acid.
The mechanism by which Gal induces lipid peroxidation could be that Gal altered the activities of the antioxidant enzymes, SOD and CAT and the GSHmetabolizing enzymes. However, GSH levels remain unchanged in the rat liver. This was unlike in our previous report in the rat testis.5 These two organs might behave differently to Gal effect on GSH level. Several reports indicated that the liver could be more vulnerable to oxidative stress because it plays a central role in the metabolism of foreign compounds and drugs.5 Additionally, some tissues are more vulnerable than others to the induction of oxidative stress.31 This could contribute in the present study to the difference of Gal effect on GSH and MDA as MDA formation was not induced in the testis as reported previously5 but was induced in the liver in the present study. Glutathione plays a central role in antioxidant defense system and a high intracellular concentration of GSH is associated with good health status, due to their protective effects against oxidative stress.1 Thus, the changes in the levels of the antioxidant enzymes, including SOD and CAT and on the activities of GSH-metabolizing enzymes were sufficient for Gal to induce oxidative stress in the rat liver without changing GSH level. These parameters are therefore more vulnerable to the pro-oxidant effect of Gal in the rat liver than GSH itself. One possibility on the mechanism how Gal induces oxidative stress is that the three aromatic phenoxyl
groups of Gal (Figure 1) are prone to oxidation with the formation of H2O2, superoxide anions, quinones and semiquinones.32–34 These metabolites (e.g. superoxides) could be converted to H2O2 by SOD and then the H2O2 converted to water by CAT. The H2O2 is also a substrate for GSH-Px which uses H2O2 to oxidize GSH to GSSG and then the GSSG is reconverted to GSH by GR which then becomes a substrate for GST. The increased GST activity might have compensated for the decreased GR and GSHPx activities resulting to the unchanged GSH level. These observations are in agreement with the results of in vivo studies.35 The roles of oxygen-dependent mechanism in the inhibition of GR and GSH-Px by flavonoids have been reported.1,36,37 Furthermore, it was reported that SOD and CAT activities are reciprocally regulated. For instance, an increase in SOD activity inhibited CAT activity, whereas increase in CAT activity inhibited SOD activity.31,38,39 There are also studies where an increase in SOD activity was not accompanied by change in CAT activity.31,35,39 In these studies, oxidative stress was induced, suggesting that adjustments in SOD and CAT activities in response to a pro-oxidant were accompanied by oxidative damage. Overall these antioxidant systems might have responded in an adaptive manner to keep GSH level constant.39 Moreover, oxidative-stressinduced by some flavonoids such as quercetin, kaemferol and myricetin have been reported to inhibit some GSH-metabolizing enzymes.40 Our present data do not
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allow us to contribute further to the mechanism of Galinduced oxidative stress in rat liver. Interestingly, we observed that the lowered GSH-Px, GR, SOD and elevated CAT and GST activities by four weeks of treatment of Gal appeared fully recovered by combined administration of both Gal and Cur. In addition, GSH, SOD and CAT activities were significantly increased by Cur alone. This increase in GSH concentration by Cur was still maintained by the combined administration of both Cur and Gal, an indication of the antioxidant property of Cur. Thus, Cur might be exerting its antioxidant effect by increasing the level of GSH in rat liver. The toxic effects of Gal included an increase in platelet counts and decrease in LDL which were not prevented by combined treatment of Gal and Cur. Most of the hematological (Hb, PCV, RBC, WBC, NEUT, LEU) and lipid profiles endpoints (CHOL, TRIGS, HDL) investigated in this study were not influenced by Gal or Cur treatment. Other studies have reported hematological toxicity including anemia in Gal treated rats10 which are contrary to the observation of this present study. This difference is probably due to variation in the biokinetics including absorption and hydrolysis of Gal and its derivatives,10 because we administered Gal orally by gavage for four weeks while those of Niho et al.10 administered Gal as dietary mixture for 13 weeks. This difference could have contributed to the changes observed in these two studies. It therefore seems that lipid profiles and hematological data are not relevant parameters in Gal toxicity for the treatment schedule and regimen employed in our study. Furthermore, serum markers, including ACP, GGT, ALT, AST and urea, were not significantly influence in the serum of Gal treated. This observation has also been made previously in rats administered dietary Gal.10 This could be that in the present study, the histopathological liver and renal changes were mild, and thus, the damage was not sufficient to allow the leakage of these marker molecules from the tissues in to the blood.41,42 However, total proteins, LDH, ALP and CREA were significantly increased in the serum of Gal-treated rats. These allow us to conclude that in mild histopathological changes of the liver and kidney, ALP, CREA and LDH are important markers underlying the toxicity of pro-oxidants, such as Gal. Interestingly, the combination of Gal and Cur prevented tissue damage and the leakage of these molecules except that of LDL. These are very interesting findings because Gal is found in a variety of plants, and the gallate moiety is a key component of many foods and drinks, for example, there are two gallate moieties in the important polyphenol, (-)-epi-gallocatechin-3-gallate (EGCG); this and related polyphenols are some of the most
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widely consumed beverages in the world, such as green tea.43 Furthermore, the biosafety of long-term usage of food additives have been the focus of few researchers,7,44 apart that Gal and Cur are commonly used food spices that are consumed in large quantities in most parts of the world. Thus, combined administration of Gal and Cur can abolish Gal-induced inhibition of GSH-related antioxidant system, antioxidant enzymes, renal and hepatic damage. However, the lipid and hematological endpoints investigated in this study are less vulnerable to Gal toxicity than the GSH-related antioxidant enzyme system and renal markers. Taken together, these results suggest that Gal but not Cur induce an adaptive response of the GSH-related enzyme system and antioxidant enzymes of the liver and induce mild renal and hepatic damage in rats treated with long-term biologically significant dose. This change could be abolished by the combined administration of Gal and Cur. Thus, Gal should be used with caution in relation to hepatic and renal health.
Acknowledgements The authors thank Drs Chikwuogwo W. Paul and Oluwasanmi O. Aina of the Department of Anatomy, College of Health Sciences, University of Port Harcourt, Choba, and Department of Veterinary Anatomy, Faculty of Veterinary Medicine, University of Ibadan respectively for their help in performing the histopathological examination.
Declaration of interest The authors report no financial conflicts of interest. The authors alone are responsible for the content and writing of this paper.
References 1. Choi EJ, Lee BH, Lee K, et al. Long-term combined administration of quercetin and daidzein inhibits quercetin-induced suppression of glutathione antioxidant defences. Food Chem Toxicol. 2005;43:793–798. 2. Inano H, Onoda M, Inafuku N, et al. Potent preventive action of curcumin on radiation-induced initiation of mammary tumorigenesis in rats. Carcinogenesis. 2000;21: 1835–1841. 3. Ammon HPT, Wahl MA. Pharmacology of curcuma longa. Planta Med. 1991;57:1–7. 4. Farombi EO, Abarikwu SO, Adedara IA, et al. Kolaviron & curcumin ameliorate di-n-Butylphthalate-induced testicular toxicity in rats. Basic Clin Pharmacol Toxicol. 2007; 100:43–48. 5. Abarikwu SO, Oghenetega FA, Durojaiye MA, et al. Combined administration of curcumin and gallic acid inhibits gallic acid-induced suppression of steroidogenesis, sperm output, antioxidant defences and inflammatory responsive genes. J Steroid Biochem Mol Biol. 2014; 143:49–60.
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6. Taitzoglou IA, Tsantarliotou M, Zervos I, et al. Inhibition of human and ovine acrosomal enzymes by tannic acid in vitro. Reprod 2001;121:131–137. 7. Park W, Chang MS, Kim H, et al. Cytotoxic effect of gallic acid on testicular cell lines with increasing H2O2 level in GC-1 spg cells. Toxicol in Vitro 2008;22: 159–163. 8. Lu Z, Nie G, Belton PS, et al. Structure activity relationship analysis of antioxidant ability and neuroprotective effect of gallic acid derivatives. Neurochem Int. 2006;48: 263–274. 9. Inoue M, Suzuki R, Koide T, et al. Antioxidant, gallic acid, induces apoptosis in HL-60RG cells. Biochem Biophys Res Commun. 1994;204:898–904. 10. Niho N, Shibutani T, Tamura T, et al. Subchronic toxicity study of gallic acid by oral administration in F344 rats. Food Chem Toxicol. 2001;39:1063–1070. 11. Laughton MJ, Halliwell B, Evans PJ, et al. Antioxidant and pro-oxidant actions of the plant phenolics quercetin, gossypol and myricetin: Effects of lipid peroxidation, hydroxyl radical generation and bleomycindependent damage to DNA. Biochem Pharmacol. 1989;38: 2859–2864. 12. Galati G, Moridani MY, Chan TS, et al. Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: Glutathione oxidation and conjugation. Free Radic Biol Med. 2001;30:370–382. 13. Heim KE, Tagliaferro AR, Bobilya DJ. Flavonoid antioxidants: Chemistry, metabolism and structure-activity relationships. J Nutr Biochem. 2002;13:572–584. 14. Li D, Liu Z, Zhao W, et al. A straightforward method to determine the cytocidal and cytopathic effects of the functional groups of gallic acid. Proc Biochem. 2011;46: 2210–2214. 15. Procha´zkova´ D, Bousˇova´ I, Wilhelmova´ N. Antioxidant and prooxidant properties of flavonoids. Fitoterapia. 2011;82: 513–523. 16. Maeta K, Nomur W, Takashume Y, et al. Green tea polyphenols function as prooxidants to active oxidativestress-responsive transcription factors in yeasts. Appl Environ Microbiol. 2007;73:572–580. 17. Choi EJ, Chee KM, Choi MY. The anti-and pro-oxidative effects of orally administered flavonoids in normal rats. Nutr Sci. 2004;7:133–137. 18. Choi EJ, Chee KM, Lee BH. Anti-and pro-oxidative effects of chronic quercetin administration in rats. Eur J Pharmacol. 2003;482:281–285. 19. Sharma P, Singh R. Protective role of curcumin on lindane induced reproductive toxicity in male Wistar rats. Bull Environ Contam Toxicol. 2010;84:378–384. 20. Noorafshan A, Karbalay-Doust S, Valizadeh A, et al. Ameliorative effects of curcumin on the seminiferous epithelium in metronidazole-treated mice: A stereological study. Toxicol Pathol. 2010;38:366–371. 21. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95:351–358. 22. Lowry OH, Rosebrough NJ, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951; 193:265–275. 23. Sedlak J, Lindsay RH. Estimation of total protein-bound and non-protein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem. 1968;25:192–205.
24. Rotruck JT, Pope AL, Ganther HE, et al. Selenium: Biochemical role as a component of glutathione peroxidase. Science. 1973;179:588–590. 25. Staal GE, Visser J, Veeger C. Purification and properties of glutathione reductase of human erythrocytes. Biochim Biophys Acta 1969;185:39–48. 26. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. J Biol Chem. 1974;249:7130–7139. 27. Clairborne A. Handbook of methods for oxygen radical research. Boca Raton (FL): CRC Press; 1995. 28. Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem. 1972;247: 3170–3175. 29. Prasad L, Khan TH, Jahangir T, et al. Effect of gallic acid on renal biochemical alterations in male Wistar rats induced by ferric nitriloacetic acid. Hum Exp Toxicol. 2006;25: 523–529. 30. Vijaya PV, Sowmya P, Arun FT, et al. Protective effect of gallic acid against lindane induced toxicity in experimental rats. Food Chem Toxicol. 2011;49:991–998. 31. Abarikwu SO. Protective effect of quercetin on atrazineinduced oxidative stress in the liver, kidney, brain, and heart of adult wistar rats. Toxicol Int. 2014;21:148–155. 32. Wong SK, Sytnyk W, Wan JKS. Electron spin resonance study of the self disproportionation of some semiquinone radicals in solution. Can J Chem. 1972;50: 3052–3057. 33. Eslami AC, Pasanphan W, Wagner BA, et al. Free radicals produced by the oxidation of gallic acid: An electron paramagnetic resonance study. Chem Cent J. 2010;4:15 34. Gil-Longo J, Gonza´lez-Va´zquez C. Vascular pro-oxidant effects secondary to the autoxidation of gallic acid in rat aorta. J Nutr Biochem. 2010;4:304–309. 35. Abarikwu SO, Adesiyan AC, Oyeloja TO, et al. Changes in sperm characteristics and induction of oxidative stress in the testis and epididymis of experimental rats by a herbicide, atrazine. Arch Environ Contam Toxicol. 2010;58: 874–882. 36. Elliott AJ, Scheiber SA, Thomas C, et al. Inhibition of glutathione reductase by flavonoids. A structure-activity study. Biochem Pharmacol. 1992;44:1603–1608. 37. Yen GC, Duh PD, Tsai HL, et al. Pro-oxidative properties of flavonoids in human lymphocytes. Biosci Biotechnol Biochem. 2003;67:1215–1222. 38. Pillai A, Gupta S. Antioxidant enzyme activity and lipid peroxidation in liver of female rats co-exposed to lead and cadmium: Effects of vitamin E and Mn2+. Free Radic Res. 2005;39:707–712. 39. Adesiyan AC, Oyeloja TO, Abarikwu SO, et al. Selenium provides protection to the liver but not the reproductive organs in an atrazine-model of experimental toxicity. Exp Toxicol Pathol. 2011;63:201–207. 40. Zhang K, Yang EB, Tang WY, et al. Inhibition of glutathione reductase by plant polyphenols. Biochem Pharmacol. 1997;54:1047–1053. 41. Rajesh MG, Latha MS. Preliminary evaluation of the antihepatotoxic activity of Kamilari, a polyherbal formulation. J Ethnopharmacol. 2004;91:99–104. 42. Olaleye MT, Amobonye AE, Komolafe K. Protective effects of Parinari curatellifolia flavonoids against
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acetaminophen-induced hepatic necrosis in rats. Saudi J Biol Sci. 2014;21:486–492. 43. Tachibana H. Molecular basis for cancer chemoprevention by green tea polyphenol EGCG. Forum Nutr. 2009;61: 156–169.
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44. Kang KS, Che JH, Ryu DY, et al. Decreased sperm number and motile activity on the F1 offspring maternally exposed to butyl p-hydroxybenzoic acid (butyl paraben). J Vet Med Sci. 2002;64:227–235.
ISSN: 0886-022X (Print) 1525-6049 (Online) Journal homepage: http://www.tandfonline.com/loi/irnf20
Curcumin protects against gallic acid-induced oxidative stress, suppression of glutathione antioxidant defenses, hepatic and renal damage in rats Sunny O. Abarikwu, Mojisola Durojaiye, Adenike Alabi, Bede Asonye & Oghenetega Akiri To cite this article: Sunny O. Abarikwu, Mojisola Durojaiye, Adenike Alabi, Bede Asonye & Oghenetega Akiri (2015): Curcumin protects against gallic acid-induced oxidative stress, suppression of glutathione antioxidant defenses, hepatic and renal damage in rats, Renal Failure, DOI: 10.3109/0886022X.2015.1127743 To link to this article: http://dx.doi.org/10.3109/0886022X.2015.1127743
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Date: 04 January 2016, At: 07:33
RENAL FAILURE, 2015 http://dx.doi.org/10.3109/0886022X.2015.1127743
LABORATORY STUDY
Curcumin protects against gallic acid-induced oxidative stress, suppression of glutathione antioxidant defenses, hepatic and renal damage in rats Sunny O. Abarikwua, Mojisola Durojaiyeb, Adenike Alabib, Bede Asonyea and Oghenetega Akirib
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a Department of Biochemistry, University of Port Harcourt, East-West Road, Choba, Nigeria; bDepartment of Chemical Sciences, Redeemer’s University, Redemption City, Ogun State, Nigeria
ABSTRACT
ARTICLE HISTORY
Curcumin (Cur) and gallic acid (Gal) are major food additives. Cur has well-known antioxidant properties, whereas Gal has both antioxidant and pro-oxidant effects. The present study investigated the effects of oral administration of Gal with or without Cur on antioxidant enzymes activities, glutathione (GSH) and the enzymes in its metabolism in rat liver in vivo and markers of tissue damage in the serum. Results showed that the increase in serum creatinine level, alkaline phosphatase and lactate dehydrogenase activities by Gal treatment were inhibited by combined administration of Gal and Cur. The decrease in GSH-peroxidase, GSH-S-transferase, superoxide dismutase and GSH-reductase activities by Gal treatment were inhibited when both Gal and Cur were administered together. The malondialdehyde concentration and catalase activity were significantly increased following administration of Gal but not when the administration of Gal was combined with Cur. Finally, the increase in GSH level was seen following administration of Cur alone or in combination with Gal but not with Gal alone. These results suggest that Gal might induce oxidative stress in the rat liver and affect renal function that can be inhibited by the combined administration of Gal and Cur.
Received 28 September 2015 Revised 7 November 2015 Accepted 22 November 2015 Published online 24 December 2015
Introduction Phytochemicals with antioxidant and pro-oxidant properties are both present in human diets and are consumed in large quantities.1 Curcumin and gallic acids (Figure 1) are both food additives with well-known pharmacological properties. Curcumin, the principal component of the curry spice turmeric (Curcuma longa) used in many parts of the world as a spice and medical agent possesses anti-oxidant properties and has no toxic effects.2–5 Gal acids are added to foods to prevent oxygen-induced lipid peroxidation as a result of its antioxidant properties.6,7 However, the potential of Gal to function as antioxidant may be dependent on the dose and their molecular polarities, which could account for the controversial function of Gal as an antioxidant and pro-oxidant molecule.8 Thus, Gal is cytotoxic to human blood lymphocytes by mechanism involving paradoxical generation of reactive oxygen species.7,9 We also recently reported decreased activities of antioxidant enzymes and GSH level without the induction of lipid peroxidation in the testis of rats as a result of Gal treatment for 30 days.5 The potential of Gal to act as prooxidant also explain its toxicity in the liver, kidney and
Curcumin; gallic acid; glutathione metabolism; antioxidant enzymes; malondialdehyde
blood of rats.10 Many phytochemicals, for example, quercetin, hesperetin naringenin, gossypol, myricetin and morin have both antioxidant and pro-oxidant properties depending on dosage, structural characteristics and experimental design.11–13 Apart the antioxidative and pro-oxidant properties, Gal possess anti-inflammatory, anti-bacterial and anticarcinogenic abilities.5,14 Similarly, Cur possesses antiinflammatory effect and is a potential chemopreventive agent with multiple beneficial functions.2 It is also possible that flavonoid pro-oxidant function could be a toll of their other beneficial functions. For example, epigallocatechin gallate that possess gallate moiety promotes apoptosis and has bactericidal activity, which is attributed to its ability to reduce oxygen to yield hydrogen peroxide.15,16 The biological system is well endowed with antioxidants such as GSH, GSH-Px, GR, CAT and SOD, which act as radical scavengers and protect cells and tissues from oxidative damage, and are associated with good health status.5 The adverse effects of polyphenolic molecules on GSH antioxidant system have been reported and pro-oxidant effects could deplete the antioxidative molecules of cell and tissues and lead to oxidative damage.17,18
CONTACT Sunny O. Abarikwu [email protected], [email protected] West Road Choba, PMB 5323, Port Harcourt, Rivers State, Nigeria ß 2015 Taylor & Francis
KEYWORDS
Department of Biochemistry, University of Port Harcourt, East-
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Figure 1. Chemical structure of (A) Curcumin (1,7-Bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) and (B) Gallic acid (3,4,5-Trihydroxybenzoic acid.
Despite the long-time use of Cur and Gal as dietary spices and food additives, research efforts towards understanding their combined effects on the GSH antioxidant defence system of experimental models are limited. Therefore, this study was performed to evaluate the combined effects of two well-known dietary spices on the antioxidant enzymes systems and GSH-defensive system in liver and on renal function in vivo.
Materials and methods Chemicals and reagents Curcumin and gallic acid were purchased from SigmaAldrich, (Chemie, GmbH, Taufkirchen, Germany). All other used reagents were of analytical grade and purchased from Sigma unless otherwise stated.
Animal treatment Twenty male Wistar rats (average weight of 128.25 g) were obtained from the animal house of the College of Natural Sciences, Redeemer’s, Redemption City, Ogun State and were allowed to acclimatize for 1 week prior to the start of study. The animals were housed in metal cages under a well-ventilated condition, maintained under a 12-h light/dark cycle and provided with standard commercial pelleted feed and water ad libitum. After acclimatization, the animals were assigned randomly into four groups of five animals, including a control group and three experimental groups. The three treatment groups of rats received 100 mg/kg/day of Cur or Gal, or the combination of Cur and Gal (100 mg/kg/ day + 100 mg/kg/day). Control rats were administered equivalent volumes of corn oil only. Cur and Gal were
diluted with the vehicle (corn oil) and administered to all animals (0.3 mL/kg) by gavage four times a week for 30 days. Experimental protocols were in accordance with the principles and procedures of the National Institute of Health Guidelines for Animal Care and Use of Laboratory Animals. At the end of 30 days, the animals were fasted overnight, weighed and killed by cervical dislocation. Blood samples were collected for biochemical analysis. The liver and kidney were dissected out quickly and washed in 1.15% KCl (ice-cold) and pat-dried. One side of the kidney and a portion of the liver were preserved in neutral buffered formalin. Tissues were sectioned and stained routinely with hematoxylin and eosin (H&E) for microscopy. The dose of Gal (100 mg/kg) was chosen because it is less than the no-observed-adverse-effect level (NOAEL) for Gal in rats.10 The dose of Cur was chosen based on our previous studies and those of others which reported protective function on testicular tissues under various pathological conditions.5,19,20
Estimation of the activities of serum markers enzymes, lipid and hematological profiles Creatinine, acid phosphatase (ACP), g-glutamyl transferase (GGT), alanine amino transferase (ALT), aspartate amino transferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), urea, cholesterol (CHOL), triglycerides (TRIGS), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were estimated in the serum using Randox commercial kits. Heparin-added whole-blood samples, immediately after collection were used for hematological analyses. Hb, PCV, RBC, WBC, platelet, NEUT, leucocytes were analyzed by means of a COBAS MICROS (Roche, Palo Alto, CA) analyzer. Hematological analyses were systematically checked by means of standard human blood CBC-3D Hematology control (R & D System Inc., Minneapolis, MN). A day-today precision of RBC, and PCV measurements (n ¼ 5) in blood were 4.2 and 4.5%, respectively.
Preparation of liver homogenate and assessment of oxidative stress The tissues were homogenized in ice-cold 0.1 M Tris–HCl buffer (pH7.4) to produce 10% homogenate. The homogenate was centrifuged at 10,000 g, 4 C for 30 min and the supernatant was separated to measure the biochemical parameters of oxidative stress described below. Malondialdehyde (MDA) and glutathione (GSH) and the enzymes of its metabolism (glutathione peroxidase, glutathione-S-transferase, glutathione reductase), defensive antioxidant enzymes (superoxide dismutase and catalase).
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Table 1. Hepatic malondialdehyde and reduced glutathione levels, glutathione peroxidase, glutathione-S-transferase and glutathione reductase activities in liver of rats after administration of Cur and Gal alone or in combination for 4 weeks. Variable MDA (mmol/mg) GSH (mg/mg) GSH-Px (Unit/mg) GST (Unit/mg) GR (U/L)
Control
Gal
Cur
Gal + Cur
1.46 ± 0.76 10.36 ± 0.42 1.17 ± 0.03 4.98 ± 1.09 145 ± 10.81
3.26 ± 0.97* 10.29 ± 0.49 0.40 ± 0.02* 11.22 ± 1.12* 87 ± 7.64*
1.58 ± 0.46 11.74 ± 0.94* 1.21 ± 0.05 4.74 ± 1.51 151 ± 12.73
1.79 ± 0.85** 11.79 ± 1.64* 1.13 ± 0.05** 4.05 ± 1.77** 148 ± 12.76**
Cur: curcumin; Gal: gallic acid. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group. **Significantly different from Gal treated group (p50.05).
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Measurement of lipid peroxidation Lipid peroxidation was quantified spectrophotometrically as MDA using thiobarbituric acid for colour development.21 The absorbance of the pink-coloured solution was measured at 532 nm using 1,1,3,3-tetraethoxypropane as a standard. The result was expressed as micromoles per milligram of protein. Protein concentration was determined by the method of Lowry et al.22
Measurements of reduced glutathione and enzymes of its metabolism The reduced GSH level was determined with a spectrophotometer (412 nm) using 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) for colour development.23 The results were expressed as mg/mg protein. The activity of glutathione peroxidase (GSH-Px) was estimated with a spectrophotometer (412 nm) by measuring the residual GSH content after the reaction: H2O2 + 2GSH!2H2O + GSSG (oxidized glutathione) as previously described.24 The enzyme activity was expressed as units/mg protein. The activity of glutathione reductase (GR) was determined in a spectrophotometer (340 nm) by monitoring the oxidation of NADPH using the extinction coefficient 6220 M 1 cm1 for NADPH.25 One unit of NADPH causes the oxidation of 1.0 mmol NADPH at 25 C at pH 7.4. The activity of glutathione S-transferase (GST) was measured in a spectrophotometer (340 nm) using with 1-chloro2,4-dinitrobenzene as a substrate.26
Measurements of defensive antioxidant enzymes The activity of catalase (CAT) was measured spectrophotometrically at 240 nm by calculating the rate of degradation of H2O2, the substrate of the enzyme.27 Activities were calculated using the molar extinction coefficient of H2O2 at 240 nm, 43.59 M 1 cm1. One unit of CAT activity equals the amount of protein that converts 1 mmol H2O2/min. Activity was expressed as micromole of H2O2 consumed per milligram of protein.
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The activity of superoxide dismutase (SOD) was determined spectrophotometrically at 480 nm using freshly prepared epinephrine (0.01%) as substrate.28 One unit of SOD activity was given as the amount of SOD necessary to cause 50% inhibition of the oxidation of adrenaline to adrenochrome during one minute. The results were expressed as unit per milligram of protein.
Statistical analyses All values are expressed as mean ± SD. Statistical comparison between different treatments was done by either Student’s t-test or one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test using Graph Pad Prism 5.0 software (Graph Pad Software, Inc., San Diego, CA). Differences between values were considered statistically significant at the p50.05 level.
Results Concentration of glutathione Gallic acid treatment did not change the concentration of reduced GSH compared with basal GSH concentration (10.36 ± 0.42 and 10.29 ± 0.49 mg/mg protein, respectively, p40.05), while the level of GSH of rats treated with Cur (11.74 ± 0.94 mg/mg protein) was significantly increased from that of the basal value (Table 1). This increase was maintained when both Gal and Cur were combined in rats treated with the combination of Gal and Cur compared with Gal alone or control (11.79 ± 1.64, p50.05).
Activities of glutathione peroxidase, reductase and S-transferase Curcumin alone did not influence the basal activity of GSH-Px, GR and GST in liver (1.17 ± 0.03, 145 ± 10.81, 4.98 ± 1.09, respectively). Gal treatment alone decreased the GSH-Px and GR activities (0.4 ± 0.02 Unit/mg and 87 ± 7.64 U/L, respectively) and increased GST activity (11.22 ± 1.12 Unit/mg) compared with basal GSH-Px, GR and GST activities (1.17 ± 0.03 Unit/mg, 145 ± 10.81 and 4.98 ± 1.09, respectively). These decreases in GSH-Px and GR activities and increase in GST activity caused by Gal treatment were prevented by the combined administration of Gal and Cur (1.13 ± 0.05 Unit/mg, 148 ± 12.76 U/L and 4.05 ± 1.77 Unit/mg, respectively, p50.05 compared with Gal alone, Table 1).
Concentration of malondialdehyde The basal MDA concentration in liver was 1.46 ± 0.76 mmol/mg protein and was significantly
S. O. ABARIKWU ET AL.
(A)
3 *
*
Catalase (Unit/mg)
2.5 2 **
1.5 1 0.5 0 Control
Gal
Cur
(B) 10 9 8 7 6 5 4 3 2 1 0 Superoxide dismutase (Unit/mg)
4
Gal+Cur
*
**
*
Control
Gal
Cur
Gal+Cur
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Figure 2. Activities of catalase (A, unit/mg) and superoxide dismutase (B, unit/mg) activities in rat liver after administration of Cur and Gal alone or in combination for 4 weeks. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group, **Significantly different from Gal treated group (p50.05).
increased 123.29-fold following treatment with Gal alone, while Cur alone showed no such effect (Table 1). This increase with Gal alone was prevented by the combined administration of Gal and Cur (1.79 ± 0.85 mmol/mg protein, p50.05 compared with Gal alone).
Activities of superoxide dismutase and catalase The basal SOD and CAT activities in the liver were 3.06 ± 1.32 Unit/mg and 1.59 ± 0.21 Unit/mg, respectively (Figure 2), and SOD was significantly decreased while CAT was significantly increased following treatment with Gal. The changes caused by Gal alone on the activities of SOD and CAT were prevented by the combined administration of Gal and Cur (2.63 ± 0.22 Unit/mg protein and 1.45 ± 0.11 Unit/mg protein, p50.05 compared with Gal alone). Cur treatment alone also significantly increased the activities of SOD and CAT (7.81 ± 0.79 Unit/mg and 2.42 ± 0.13 Unit/mg protein, p50.05 compared with basal values).
Table 2. Serum hepatic and renal markers in rats after treatment with cur and gal alone or in combination for 4 weeks. Variable ACP (Unit/mg) GGT ALT (U/L) AST (U/L) Urea (mmol/L) Total protein (g/L)
Control
Gal
Cur
Gal + Cur
0.92 ± 0.11 29.71 ± 0.76 21.57 ± 2.76 37 ± 8.47 2.36 ± 0.84 75.43 ± 4.86
0.89 ± 0.13 29.5 ± 0.55 22.14 ± 3.8 31 ± 7.57 2.14 ± 0.87 80.57 ± 4.99*
0.91 ± 0.28 29.71 ± 0.76 21 ± 2.31 38.57 ± 8.77 2.21 ± 0.73 77.71 ± 4.82
0.94 ± 0.19 30.29 ± 0.49 20.29 ± 4.57 31.57 ± 6.99 2.69 ± 0.6 74 ± 2.31**
Cur: curcumin; Gal: gallic acid. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group. **Significantly different from Gal treated group (p50.05).
Table 3. Lipid profiles of rats serum after treatment with cur and gal alone or in combination for 4 weeks. Variable CHOL (mmol/L) TRIGS (mmol/L) HDL (mmol/L) LDL (mmol/L)
Control
Gal
Cur
Gal + Cur
2.33 ± 0.48 0.91 ± 0.18 1.31 ± 0.43 1.18 ± 0.37
2.39 ± 0.43 0.93 ± 0.23 1.79 ± 0.47 0.32 ± 0.13*
2.4 ± 0.56 1.11 ± 0.25 1.39 ± 0.23 0.98 ± 0.13
2.09 ± 0.14 1.03 ± 0.28 1.56 ± 0.44 0.34 ± 0.15*
Cur: curcumin; Gal: gallic acid. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group (p50.05).
Blood biochemistry and hematology Gallic acid or Cur alone or in combination did not influence the basal activities of ACP, GGT, ALT, AST, urea, TRIGS, HDL and CHOL (Tables 2 and 3). Treatment with Gal alone significantly decreased LDL level (0.32 ± 0.13 mmol/L, p50.05 compared with basal values (1.18 mmol/L), while Cur alone showed no such effect. This decrease with Gal treatment was not prevented by the combined administration of Gal and Cur (0.34 ± 0.15 mmol/L compared with Gal alone or basal values). The total protein levels, basal creatinine (CREA) level, lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in the serum were 75.43 ± 4.86 g/L, 145.67 ± 17.14 mmol/L, 448 ± 46.62 U/L and 42.14 ± 3.58 U/L, respectively, and were significantly
increased by 6.81%, 136.3%, 29% and 68.81%, respectively, following treatment with Gal alone compared with basal values, while Cur alone showed no such effect (Figure 3). This increase with Gal alone was prevented by the combined administration of Gal and Cur (74 ± 2.31 g/L, 184 ± 42.59 mmol/L, 414.29 ± 25.23 U/L, 45 ± 4.36 U/L, respectively, p50.05 compared with Gal alone). Gallic acid or Cur alone or in combination did not influence basal Hb, PCV, RBC, WBC, neutrophils and leucocytes counts (Table 4). Treatment with Gal alone significantly increased platelet count (283.3 ± 20.7 109/L, p50.05 compared with basal values, 176.7 ± 30.11 109/ L), while Cur alone showed no such effect. However, this increase with Gal treatment was not prevented by the
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(A) 450
(B) 700
Creatinine (µmol/L)
Lactate dehydrogenase (U/L)
*
400 350 300
**
250 200 150 100 50 0
*
600 500
**
400 300 200 100 0
Control
Gal
Cur
Gal+Cur
Alkaline phosphatase (U/L)
(C) 90
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5
Control
Gal
Cur
Gal+Cur
*
80 70 60 **
50 40 30 20 10 0 Control
Gal
Cur
Gal+Cur
Figure 3. Activities of creatinine, (A, mmol/L), lactate dehydrogenase (B, U/L) and alkaline phosphatase (C, U/L) activities in rat serum after administration of Cur and Gal alone or in combination for 4 weeks. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group, **Significantly different from Gal treated group (p50.05).
Discussion Table 4. Hematology data for rats after treatment with cur and gal alone or in combination for 4 weeks. Variable Hb (g/L) PCV (L/L) RBC (1012/L) WBC ( 109/L) Platelet ( 109/L Neutrophils Leucocytes
Control
Gal
Cur
Gal + Cur
12.43 ± 0.64 37.29 ± 1.98 6.69 ± 0.25 10.11 ± 2.9 176.7 ± 30.11 40 ± 6.16 59.29 ± 5.77
12.76 ± 0.49 38.29 ± 1.5 6.8 ± 0.34 12.1 ± 3.03 283.3 ± 20.66* 41.86 ± 4.14 58.14 ± 4.14
12.51 ± 0.85 37.57 ± 2.51 6.77 ± 0.4 11.79 ± 1.47 196.7 ± 32.66 40.71 ± 3.64 58.43 ± 4.58
12.370.88 37.14 ± 2.61 6.67 ± 0.44 11.43 ± 3.87 318.3 ± 47.5* 39.5 ± 5.96 59.17 ± 6.77
Cur: curcumin; Gal: gallic acid. Data are presented as the mean ± SD (n ¼ 5). *Significantly different from the control group (p50.05).
combined administration of Gal and Cur (318.3 ± 47.5 109/L compared with Gal alone or basal values).
Histopathology of rat kidney and liver There were no visible lesions in the control, Cur and Gal + Cur-treated animals. Gallic acid treatment-related effects were noted in the liver and kidney of rats (Figures 4 and 5). In the liver of Gal-treated rats, there is a very mild periportal cellular infiltration by mononuclear cells. The kidney of control, Cur-and Gal + Cur-treated animals showed normal glomerulus and renal tubules. The kidneys of Gal-treated rats showed inflammatory cells and partially obliterated bowman capsule.
Although both Gal and Cur have been in use as food additive for many years, their combined effects on the liver and kidney of experimental models, for example, rats have not been evaluated in vivo. In this study, their combine effects on the GSH-defensive system and enzymes of its metabolism and on renal function were evaluated. The present results showed that Gal at 100 mg/kg/day decreased GSH-Px, GR and SOD activities and increased lipid peroxidation, GST and CAT activities in rat’s liver. In our previous report, Gal at same dose (100 mg/kg/day) used in the present study may downregulate GSH metabolism in the testes of rats.5 Moreover, Gal at a dose similar than was used in the present experiment induced toxicity to both the kidney and liver of male and female rats.10 These support the toxicity of Gal in the present experimental model and suggest that Gal might have pro-oxidant effects in rat liver. In studies where Gal exhibited antioxidant effects, the doses used were usually smaller than was used in the present study.29,30 Thus, the anti- or prooxidant effect of Gal could be dependent on the dosage of treatment. However, the dose of Gal used in the present study was based on the no-observed-adverseeffect level (NOAEL) which is about 119 mg/kg in male rats10 and has been reported to be biologically relevant dose.5
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Control
Gal
Cur
Cur + Gal
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Figure 4. Representative photomicrograph of hematoxylin and eosin-stained sections of liver of control and experimental rats (A) Control, (B) Cur, 100 mg/kg, (C) Gal, 100 mg/kg, (D) Cur + Gal. (arrows: mild periportal cellular infiltration by mononuclear cells ( 400). Cur: curcumin; Gal: gallic acid.
Control
Gal
Cur
Cur+Gal
Figure 5. Representative photomicrograph of hematoxylin and eosin-stained sections of kidney of control and experimental rats (A) Control, (B) Cur, 100 mg/kg, (C) Gal, 100 mg/kg, (D) Cur + Gal (short arrows: inflammatory cells; long arrows: partially obliterated bowman’s capsule ( 400). Cur: curcumin; Gal: gallic acid.
The mechanism by which Gal induces lipid peroxidation could be that Gal altered the activities of the antioxidant enzymes, SOD and CAT and the GSHmetabolizing enzymes. However, GSH levels remain unchanged in the rat liver. This was unlike in our previous report in the rat testis.5 These two organs might behave differently to Gal effect on GSH level. Several reports indicated that the liver could be more vulnerable to oxidative stress because it plays a central role in the metabolism of foreign compounds and drugs.5 Additionally, some tissues are more vulnerable than others to the induction of oxidative stress.31 This could contribute in the present study to the difference of Gal effect on GSH and MDA as MDA formation was not induced in the testis as reported previously5 but was induced in the liver in the present study. Glutathione plays a central role in antioxidant defense system and a high intracellular concentration of GSH is associated with good health status, due to their protective effects against oxidative stress.1 Thus, the changes in the levels of the antioxidant enzymes, including SOD and CAT and on the activities of GSH-metabolizing enzymes were sufficient for Gal to induce oxidative stress in the rat liver without changing GSH level. These parameters are therefore more vulnerable to the pro-oxidant effect of Gal in the rat liver than GSH itself. One possibility on the mechanism how Gal induces oxidative stress is that the three aromatic phenoxyl
groups of Gal (Figure 1) are prone to oxidation with the formation of H2O2, superoxide anions, quinones and semiquinones.32–34 These metabolites (e.g. superoxides) could be converted to H2O2 by SOD and then the H2O2 converted to water by CAT. The H2O2 is also a substrate for GSH-Px which uses H2O2 to oxidize GSH to GSSG and then the GSSG is reconverted to GSH by GR which then becomes a substrate for GST. The increased GST activity might have compensated for the decreased GR and GSHPx activities resulting to the unchanged GSH level. These observations are in agreement with the results of in vivo studies.35 The roles of oxygen-dependent mechanism in the inhibition of GR and GSH-Px by flavonoids have been reported.1,36,37 Furthermore, it was reported that SOD and CAT activities are reciprocally regulated. For instance, an increase in SOD activity inhibited CAT activity, whereas increase in CAT activity inhibited SOD activity.31,38,39 There are also studies where an increase in SOD activity was not accompanied by change in CAT activity.31,35,39 In these studies, oxidative stress was induced, suggesting that adjustments in SOD and CAT activities in response to a pro-oxidant were accompanied by oxidative damage. Overall these antioxidant systems might have responded in an adaptive manner to keep GSH level constant.39 Moreover, oxidative-stressinduced by some flavonoids such as quercetin, kaemferol and myricetin have been reported to inhibit some GSH-metabolizing enzymes.40 Our present data do not
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allow us to contribute further to the mechanism of Galinduced oxidative stress in rat liver. Interestingly, we observed that the lowered GSH-Px, GR, SOD and elevated CAT and GST activities by four weeks of treatment of Gal appeared fully recovered by combined administration of both Gal and Cur. In addition, GSH, SOD and CAT activities were significantly increased by Cur alone. This increase in GSH concentration by Cur was still maintained by the combined administration of both Cur and Gal, an indication of the antioxidant property of Cur. Thus, Cur might be exerting its antioxidant effect by increasing the level of GSH in rat liver. The toxic effects of Gal included an increase in platelet counts and decrease in LDL which were not prevented by combined treatment of Gal and Cur. Most of the hematological (Hb, PCV, RBC, WBC, NEUT, LEU) and lipid profiles endpoints (CHOL, TRIGS, HDL) investigated in this study were not influenced by Gal or Cur treatment. Other studies have reported hematological toxicity including anemia in Gal treated rats10 which are contrary to the observation of this present study. This difference is probably due to variation in the biokinetics including absorption and hydrolysis of Gal and its derivatives,10 because we administered Gal orally by gavage for four weeks while those of Niho et al.10 administered Gal as dietary mixture for 13 weeks. This difference could have contributed to the changes observed in these two studies. It therefore seems that lipid profiles and hematological data are not relevant parameters in Gal toxicity for the treatment schedule and regimen employed in our study. Furthermore, serum markers, including ACP, GGT, ALT, AST and urea, were not significantly influence in the serum of Gal treated. This observation has also been made previously in rats administered dietary Gal.10 This could be that in the present study, the histopathological liver and renal changes were mild, and thus, the damage was not sufficient to allow the leakage of these marker molecules from the tissues in to the blood.41,42 However, total proteins, LDH, ALP and CREA were significantly increased in the serum of Gal-treated rats. These allow us to conclude that in mild histopathological changes of the liver and kidney, ALP, CREA and LDH are important markers underlying the toxicity of pro-oxidants, such as Gal. Interestingly, the combination of Gal and Cur prevented tissue damage and the leakage of these molecules except that of LDL. These are very interesting findings because Gal is found in a variety of plants, and the gallate moiety is a key component of many foods and drinks, for example, there are two gallate moieties in the important polyphenol, (-)-epi-gallocatechin-3-gallate (EGCG); this and related polyphenols are some of the most
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widely consumed beverages in the world, such as green tea.43 Furthermore, the biosafety of long-term usage of food additives have been the focus of few researchers,7,44 apart that Gal and Cur are commonly used food spices that are consumed in large quantities in most parts of the world. Thus, combined administration of Gal and Cur can abolish Gal-induced inhibition of GSH-related antioxidant system, antioxidant enzymes, renal and hepatic damage. However, the lipid and hematological endpoints investigated in this study are less vulnerable to Gal toxicity than the GSH-related antioxidant enzyme system and renal markers. Taken together, these results suggest that Gal but not Cur induce an adaptive response of the GSH-related enzyme system and antioxidant enzymes of the liver and induce mild renal and hepatic damage in rats treated with long-term biologically significant dose. This change could be abolished by the combined administration of Gal and Cur. Thus, Gal should be used with caution in relation to hepatic and renal health.
Acknowledgements The authors thank Drs Chikwuogwo W. Paul and Oluwasanmi O. Aina of the Department of Anatomy, College of Health Sciences, University of Port Harcourt, Choba, and Department of Veterinary Anatomy, Faculty of Veterinary Medicine, University of Ibadan respectively for their help in performing the histopathological examination.
Declaration of interest The authors report no financial conflicts of interest. The authors alone are responsible for the content and writing of this paper.
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