Scholarly article on topic 'Protective effect of thymoquinone on sodium fluoride-induced hepatotoxicity and oxidative stress in rats'

Protective effect of thymoquinone on sodium fluoride-induced hepatotoxicity and oxidative stress in rats Academic research paper on "Chemical sciences"

CC BY-NC-ND
0
0
Share paper
OECD Field of science
Keywords
{"Sodium fluoride" / Hepatotoxicity / "Oxidative stress" / Thymoquinone / Rat}

Abstract of research paper on Chemical sciences, author of scientific article — Wessam M. Abdel-Wahab

Abstract Many active ingredients extracted from herbal and medicinal plants are extensively studied for their beneficial effects. Antioxidant activity and free radical scavenging properties of thymoquinone (TQ) have been reported. The present study evaluated the possible protective effects of TQ against the toxicity and oxidative stress of sodium fluoride (NaF) in the liver of rats. Rats were divided into four groups, the first group served as the control group and was administered distilled water whereas the NaF group received NaF orally at a dose of 10mg/kg for 4weeks, TQ group was administered TQ orally at a dose of 10mg/kg for 5weeks, and the NaF-TQ group was first given TQ for 1week and was secondly administered 10mg/kg/day NaF in association with 10mg/kg TQ for 4weeks. Rats intoxicated with NaF showed a significant increase in lipid peroxidation whereas the level of reduced glutathione (GSH) and the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPx) were reduced in hepatic tissues. The proper functioning of the liver was also disrupted as indicated by alterations in the measured liver function indices and biochemical parameters. TQ supplementation counteracted the NaF-induced hepatotoxicity probably due to its strong antioxidant activity. In conclusion, the results obtained clearly indicated the role of oxidative stress in the induction of NaF toxicity and suggested hepatoprotective effects of TQ against the toxicity of fluoride compounds.

Academic research paper on topic "Protective effect of thymoquinone on sodium fluoride-induced hepatotoxicity and oxidative stress in rats"

The Journal of Basic & Applied Zoology (2013) 66, 263-270

The Egyptian German Society for Zoology

The Journal of Basic & Applied Zoology

www.egsz.org

www.sciencedirect.com

Protective effect of thymoquinone on sodium fluoride-induced hepatotoxicity and oxidative stress in rats

Wessam M. Abdel-Wahab *

Department of Zoology, Faculty of Science, Alexandria University, Alexandria, Egypt

Received 12 March 2013; revised 31 March 2013; accepted 12 April 2013 Available online 15 May 2013

Abstract Many active ingredients extracted from herbal and medicinal plants are extensively studied for their beneficial effects. Antioxidant activity and free radical scavenging properties of thymo-quinone (TQ) have been reported. The present study evaluated the possible protective effects of TQ against the toxicity and oxidative stress of sodium fluoride (NaF) in the liver of rats. Rats were divided into four groups, the first group served as the control group and was administered distilled water whereas the NaF group received NaF orally at a dose of 10 mg/kg for 4 weeks, TQ group was administered TQ orally at a dose of 10 mg/kg for 5 weeks, and the NaF-TQ group was first given TQ for 1 week and was secondly administered 10 mg/kg/day NaF in association with 10 mg/kg TQ for 4 weeks. Rats intoxicated with NaF showed a significant increase in lipid peroxidation whereas the level of reduced glutathione (GSH) and the activity of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPx) were reduced in hepatic tissues. The proper functioning of the liver was also disrupted as indicated by alterations in the measured liver function indices and biochemical parameters. TQ supplementation counteracted the NaF-induced hepatotoxicity probably due to its strong antioxidant activity. In conclusion, the results obtained clearly indicated the role of oxidative stress in the induction of NaF toxicity and suggested hepatoprotective effects of TQ against the toxicity of fluoride compounds. © 2013 Production and hosting by Elsevier B.V. on behalf of The Egyptian German Society for Zoology.

KEYWORDS

Sodium fluoride; Hepatotoxicity; Oxidative stress; Thymoquinone; Rat

* Address: Department of Zoology, Faculty of Science, Alexandria University, 21511 Moharram Bey, Alexandria, Egypt. Tel.: +20 122 3526219; fax: +20 3 3911794.

E-mail address: Profwessam@hotmail.com Peer review under responsibility of The Egyptian German Society for

Introduction

Fluoride (F) anions are widely distributed in the environment in different forms and their compounds are extensively used. Fluoride anions are naturally present in water sources and drinking water as they are released from the runoff of F-containing rocks and soils and leach into groundwater (ATSDR, 2003). In some areas drinking water is artificially fluoridated, therefore water consumption is typically the largest contributor to daily F intake. Furthermore, F anions are incorporated in various insecticide formulations, fluoridated foodstuffs, dentifrices, drugs, vapors emitted from industries using fluoride containing compounds (NRC, 2006).

Zoology.

2090-9896 © 2013 Production and hosting by Elsevier B.V. on behalf of The Egyptian German Society for Zoology. http://dx.doi.org/10.1016/jjobaz.2013.04.002

Fluoride is often described as a double edged sword because in small doses, it is an essential trace element with remarkable protective effect in preventing dental caries and osteoporosis. On the other hand, excessive exposure to F exerts harmful effects on the organism. It may directly or indirectly modulate the enzyme activity by forming complexes with the metal part of enzyme molecules (Pawowska-Goral et al., 1998). In this way F interferes with the metabolic processes involving carbohydrates, lipids and proteins (Blaszczyk et al., 2011; Nabavi et al., 2012). Fluoride inhibits enzymes involved in major metabolic pathways for example glycolysis and the Krebs cycle. In addition, F inhibits fatty acid oxidation and reduces the activity of pyruvate dehydrogenase, which reduces the amount of acetyl-CoA in the cells. Sodium fluoride negatively regulates the activity of ATPase - an enzyme important in the polymerization of amino acids, thus inhibiting the process of bonding the amino acids to peptides and blocking DNA synthesis (Hordyjewska and Pasternak, 2004). Long-term exposure to F compounds induce morphological changes in many organs in particular the liver, leading to an impairment of their function (Koodziejczyk et al., 2000; Chinoy, 2003). Pathological changes occur also in the pancreas, lungs, cardiac and skeletal muscles and kidney (Sinha et al., 2008; Stawiarska-Pieta et al., 2009).

Oxidative stress is one of the most important factors that exacerbate damage by certain drugs and environmental chemicals. Free radical generation is known to be one of the most important mechanisms of F toxicity (Nabavi et al., 2012). Fluoride has the ability to initiate respiratory burst and stimulate the generation of free radicals which change the structure and permeability of cell membranes and impair the cell function (Chl-ubek, 2003). Numerous studies indicated an increased oxidative stress in the serum, liver and brain of animals exposed to F (Grucka-Mamczar et al., 2009). Moreover, F affects the activity of enzymes constituting the cell antioxidant system whose role is to protect against free radicals (Chinoy, 2003).

Natural herbal constituents are extensively studied for their ability to protect cells from miscellaneous damages. Currently, the use of phytochemicals as a therapy in diseases related to oxidative stress has gained immense interest for their ability to quench free radicals by electron or proton donation and their capability to protect body tissues against oxidative stress (Nabavi et al., 2012). Nigella sativa has a long history in medicinal use for centuries. Thymoquinone (TQ), the major bioac-tive constituent of N. sativa seed has been reported to exhibit many pharmacological effects including immunomodulation (Ali and Blunden, 2003), anti-inflammatory (Houghton et al., 1995) and antitumor activities against a broad spectrum of cancer cells including colon, ovarian, lung, osteosarcoma and myeloblastic leukemia (Norwood et al., 2006; Wilson-Simpson et al., 2007). TQ has been reported to possess strong antioxidant properties (Houghton et al.,1995). Oral administration of TQ is capable of protecting several organs against oxidative damage induced by free radical-generating agents including doxorubicin-induced cardiotoxicity (Nagi and Mansour, 2000) and carbon tetrachloride-evoked hepatotoxicity (Nagi et al., 1999). TQ acts as scavenger of superoxide, hydroxyl radical and singlet molecular oxygen (Badary et al., 2003).

The role of TQ against NaF-induced toxicity has not so far been studied. Therefore, the present study was carried out to investigate: (1) the alterations in biochemical parameters and antioxidant status of liver induced by NaF in male rats, (2)

the role of TQ in protecting the liver against the induced changes.

Material and methods

Chemicals

Sodium fluoride and thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals used in the present study were of analytical grade. Kits used in the present study were the products of Biodiagnostic Co. (Egypt) and Biosystems Co. (Spain).

Animals and experimental design

This study was performed on thirty two male albino Sprague-Dawley rats of approximately 170-190 g body weight. The animals were obtained from the animal house of the High Institute of Public Health, Alexandria University, Alexandria, Egypt. Rats were housed in stainless steel cages (4 rats/cage). The animals were maintained under controlled conditions of a 12 h light-dark cycle, room temperature of 22-25 0C, relative humidity of 40-50%. Rats were allowed free access to standard rat chow diet and water. After 2 weeks of acclimatization to the laboratory conditions, rats were randomly divided into 4 experimental groups (8 rats in each) as follows: The first group served as normal control group and was administered distilled water. Rats of the second group were intoxicated with 10 mg/kg NaF (Blaszczyk et al., 2011) once daily for 4 weeks. The third group was treated with TQ at a dose of 10 mg/kg/ day (Nagi and Mansour, 2000) for 5 weeks. The fourth group was treated with both NaF and TQ. NaF and TQ were dissolved in distilled water and were administered orally by gavage (oral ingestion is the main route of fluoride intake). TQ treatment started 1 week before NaF and continued throughout the duration of the experiment. The doses of NaF and TQ were calculated according to the animal's body weight before treatment. All rats were handled in accordance with the standard guide for the use and care of laboratory animals.

Blood sampling and preparation of serum

At the end of the experimental duration, rats were fasted overnight with free access to water. Under light anesthesia with diethyl ether, rats were sacrificed by cervical decapitation and the blood was collected into non-heparinized tubes. Serum was collected from blood by centrifugation at 4000 rpm for 15 min and was stored at —20 0C till analysis. The liver tissue was collected and perfused with normal saline to remove blood and used for the preparation of tissue homogenate.

Preparation of liver homogenate

A known weight of the liver tissue was washed in ice-cold isotonic saline containing 1 mM EDTA. The tissue was then homogenized separately in 10 volumes of potassium phosphate buffer (50 mM, pH 7.4) containing 1 mM EDTA using a homogenizer at 4 0C. The crude tissue homogenate was then centrifuged at 8000 rpm for 15 minutes at 4 0C and the superna-

tant was collected and kept at —20 0C for the estimation of mal-ondialdehyde (MDA), reduced glutathione (GSH) as well as the activity of superoxide dismutase (SOD), catalase (CAT), gluta-thione-S-transferase (GST) and glutathione peroxidase (GPx).

Estimation of lipid peroxidation level in liver homogenate

Lipid peroxidation (LPO) in liver tissue was determined by measuring MDA in the supernatant from liver homogenate using the method of Ohkawa et al. (1979). This method measures the absorbance of the pink-colored complex formed by the reaction of MDA with thiobarbituric acid (a LPO end product) in acidic medium at 534 nm. Results were expressed in nmol/g protein.

Determination of enzymatic and non-enzymatic antioxidants in liver homogenate

The activity of SOD was assayed depending on the ability of the enzyme to inhibit the phenazine methosulphate-mediated reduction of nitro blue tetrazolium dye (NBT). Results were expressed in U/mg protein (Nishikimi et al., 1972). Catalase activity was assayed according to the method of Aebi (1984) where the CAT reacts with a known quantity of H2O2. The reaction is stopped exactly after one minute with CAT inhibitor. In the presence of peroxidase, the remaining H2O2 reacts with 3,5-dichloro-2-hydroxybenzene sulfonic acid (DHBS) and 4-aminophenazone (AAP) to form a chromophore with a color intensity that is inversely proportional to the amount of CAT in the original sample. Results were expressed in U/ mg protein. The activity of GST was assayed by monitoring the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione. The conjugation is accompanied by an increase in absorbance which is measured at 340 nm. The rate of increase is directly proportional to the GST activity in the sample (Habig et al., 1974). The activity of GPx was determined spectrophotmetrically as described by Paglia and Valentine (1967) where the GPx catalyzes the oxidation of glutathione. In the presence of glutathione reductase and NADPH the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decrease in the absorbance (A340) is directly proportional to the GPx activity in the sample. Results were expressed in U/mg-protein. Reduced glutathione was determined according to the method of Beutler et al. (1963). The assay is based on the reduction of Elman's reagent (5,5'dithiobis (2-nitrobenzoic acid) "DTNB") with glutathione to produce a yellow complex. The reduced chromogen is directly proportional to glutathione concentration and its absorbance can be measured at 405 nm. Results are expressed in nmol/g protein.

Liver function biomarkers in the serum

Alanine aminotransferase and aspartate aminotransferase were determined colorimetrically by measuring the amount of pyru-vate or oxaloacetate produced by forming 2,4-dinitrophenylhy-drazine, the color of which was measured at 546 nm (Retiman and Frankel, 1957). Alkaline phosphatase was assayed by transforming phenyl phosphate into phenol and phosphate in the presence of the enzyme. The liberated phenol is measured color-imetrically at 520 nm in the presence of 4-aminophenazone and

potassium ferricyanide (Belfield and Goldberg, 1971). Lactate dehydrogenase was determined according to the method of Vassault (1983) which depends on the oxidation of lactate to pyruvate with the simultaneous conversion of the cosubstrate NADH to NAD. The decrease in absorbance (measured at 340 nm) due to this conversion is directly proportional to LDH activity. Total bilirubin was measured according to the method of Walter and Gerade (1970) depending on the reaction between bilirubin in the sample and the diazonium salt of sulph-anilic acid to produce azobilirubin which shows a maximum absorption at 535 nm in an acid medium.

Serum chemistry

Serum biomarkers were determined using the available assay kits and were performed according to the manufacturers' protocols and instructions. Total lipids, total cholesterol and triglycerides concentrations were assayed according to the methods of Zollner and Kirsch (1970), Richmond (1973), Fossati and Principle (1982), respectively. Total protein, albumin and glucose concentrations were assayed according to the methods of Lowry et al. (1951), Doumas et al. (1971), Trinder (1969), respectively. Insulin level was estimated by radioimmu-noassay using double antibody procedure essentially according to Morgan and Lazarow (1963).

Statistical analysis

Data are expressed as mean ± standard error (SE). Data were analyzed using Statistical Package for Social Science (SPSS/ Version 17.0) software. Significance between experimental groups was determined using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test for comparison between two groups. p values less than 0.05 were considered statistically significant.

Results

Effect of NaF and TQ on LPO in the hepatic tissue

Results concerning the effect of NaF and/or TQ on hepatic LPO are shown in Fig. 1. Administration of NaF at a dose of

Figure 1 Level of hepatic malondialdehyde (nmol/g protein) in rats administered sodium fluoride and/or thymoquinone. Data are expressed as mean ± SE for each experimental group (n = 8) a and b represent significance to control and NaF groups, respectively. p < 0.05, p < 0.01.

10 mg/kg for 4 weeks evoked a significant increment (p < 0.01) in LPO as evidenced by increase (by 262.2%) in hepatic MDA level compared to the control group. Thymoquinone administration to NaF-intoxicated rats mitigated the enhanced LPO as evidenced by significant (p < 0.05) decrease in liver MDA level.

Effect of NaF and TQ on antioxidative status of the liver

The antioxidative status of liver from the normal control and different experimental groups is presented in Table 1. According to this table, the activity of the intracellular antioxidant enzymes namely SOD, CAT, GST and GPx in addition to the level of the non-enzymatic antioxidant GSH in liver homogenates was found to be significantly (p < 0.01) decreased in response to F consumption for 4 weeks. The mean values decreased by 49.3%, 51.5%, 32.5%, 56.9%, and 56.17%, respectively compared to those of the control group. Supplementation with TQ at a dose of 10 mg/kg to NaF-intoxicated rats completely restored the suppressed antioxidants and brought their value near to that of the normal control. It is worth to note that administration of TQ alone has no significant effect on in these antiox-idants compared to the control group.

Effect of NaF and TQ on the level of serum biomarkers related to hepatic dysfunction

Data presented in Table 2 show the effect of NaF, TQ and their combination on the serum liver function indices. Expo-

sure to NaF resulted in impairment in liver function as indicated by significant increase in the activity of AST, ALT, ALP, LDH and in the concentration of total bilirubin. The mean values increased by 73.1%, 131.8%, 63.2%, 56.1%, and 310.4%, respectively compared to the control group. Supplementation with TQ alone resulted in non-significant changes in these liver indices when compared to the control group. Administration of TQ at a dose of 10 mg/kg protected the liver against NaF toxicity and improved its functioning as shown by the significant decrease in these liver function bio-markers compared to the NaF group.

Effect on lipid profile, total proteins, albumin, glucose and insulin levels in serum

The lipid profile, protein content, glucose level and insulin level in the serum of rats administered NaF or TQ or both are presented in Table 3. These results demonstrated that the concentration of total lipids, triglycerides and total cholesterol showed significant (p < 0.01) increase (by 47.9%, 74.3%, and 61.2%, respectively) while the concentration of total proteins and albumin significantly (p < 0.05) decreased (by 25.7% and 27.7%, respectively) in the NaF group compared to the control group. Non significant change in these parameters was observed in rats supplemented with TQ alone compared to the control group. Treatment of NaF-intoxicated rats with TQ improved to some extent the observed alterations in lipid profile and protein contents in the serum as evidenced by the

Table 1 Effect of thymoquinone (TQ) on sodium fluoride-induced changes in the activity of hepatic superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx) and the level of glutathione (GSH).

Parameter Experimental groups

Control NaF TQ NaF + TQ

SOD (U/mg protein) CAT (u/mg protein) GST (nmol/mg protein) GPx (U/mg protein) GSH (nmol/g protein) 19.13 ± 0.80 23.4 ± 1.28 282.5 ± 12.45 27.25 ±1.10 32.45 ± 2.40 9.70 ± 0.84a** 11.35 ± 1.54a** 190.73 ± 17.26a** 11.73 ± 0.58a** 14.23 ± 1.42a** 18.39 ± 0.83 17.20 ± 1.10b* 24.41 ± 1.67 19.85 ± 0.92b* 290.25 ± 11.6 264.83 ± 5.27b* 26.19 ± 1.17 24.62 ± 0.81b* 33.11 ± 3.23 28.36 ± 1.86b*

Data are expressed as mean ± respectively. * p < 0.05. ** p < 0.01. SE for each experimental group (n = 8). a and b represent significance compared to control and NaF groups,

Table 2 Liver function biomarkers, aspartate transferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) activities and bilirubin level in the serum of different experimental groups.

Parameter Experimental groups

Control NaF TQ NaF + TQ

AST (U/l) ALT (U/l) ALP (U/l) LDH (U/l) Total bilirubin (mg/dl) 68.45 ± 2.38 34.23 ± 1.61 116.0 ± 3.36 737.30 ± 20.11 0.48 ± 0.02 118.52 ± 3.51a** 79.36 ± 3.21a** 189.3 ± 5.23a** 1123.72 ± 31.47a 1.97 ± 0.12a** 64.10 ± 3.41 31.14 ± 2.23 119.52 ± 3.31 * 740.52 ± 24.72 0.52 ± 0.04 82.18 ± 3.40b** 43.25 ± 2.42b** 127.2 ± 4.24b* 782.82 ± 19.35b** 0.93 ± 0.04b**

Data are expressed as mean ± SE for each experimental group (n = 8). a and b respectively. * p < 0.05. ** p < 0.01. represent significance compared to control and NaF groups

Table 3 Levels of some biochemical parameters in the serum of rats exposed to sodium fluoride and/or thymoquinone.

Parameter Experimental groups

Control NaF TQ NaF + TQ

Total lipids (mg/dl) 282.80 ± 11.20 418.25 ± 19.35a** 273.7 ± 13.40 345.2 ± 16.31ab*

Triglycerides (mg/dl) 78.45 ± 1.47 136.72 ± 4.51a** 80.21 ± 3.24 104.7 ± 3.41ab*

Total cholesterol (mg/dl) 148.0 ± 3.95 238.63 ± 5.82a** 140.84 ± 4.15 182.21 ± 5.43ab*

Total proteins (mg/dl) 7.31 ± 0.21 5.43 ± 0.13a* 6.94 ± 0.18 6.51 ± 0.23ab**

Albumin (mg/dl) 5.01 ± 0.08 3.62 ± 0.14a* 5.21 ± 0.10 4.31 ± 0.12ab**

Glucose level (mg/dl) 86.42 ± 4.47 143.56 ± 9.1a** 82.38 ± 6.15 102.73 ± 8.14b*

Insulin level (lU/l) 16.7 ± 0.62 10.72 ± 0.87a** 15.92 ± 0.69 10.1 ± 0.82a**

Data are expressed as mean ± SE for each experimental group (n = 8). a and b represent significance compared to control and NaF groups

respectively.

* p < 0.05.

** p < 0.01.

decrease in lipid parameters and the increase in protein content parameters although their values did not reach the normal value of the control group. Rats administered NaF for 4 weeks showed a significant (p < 0.01) increase in the blood glucose level (by 66.1%) with a concomitant decrease in the insulin level (by 37%) compared to the control group. TQ-treated group did not show any significant change compared to the control group. TQ supplementation alleviated to a large extent the NaF-induced alteration in blood glucose level as shown by the significant (p < 0.05) decrease in glucose level compared to the NaF group. On the other hand, TQ supplementation failed to improve the decrease in insulin level as indicated by the observed significant (p < 0.01) difference compared to the control group (Table 3).

Discussion

The present study revealed a disturbance in the antioxidative status in rat liver and impairment in its proper functioning as a result of sodium fluoride consumption. The administration of thymoquinone to the intoxicated rats improved the antioxidant status and mitigated the alterations in the analyzed biochemical parameters. Oxidative stress describes a state of uncontrolled overproduction of free radicals beyond a threshold for proper antioxidant neutralization causing damage to macromolecules such as DNA, proteins and lipids (Halliwell and Guttteridge, 2007). Lipid peroxidation (LPO), as the fundamental index of oxidative damage, has been found to be a major contributor in the toxicity of many xenobiotics (Anane and Creppy, 2001). In the present study, administration of NaF for 4 weeks increased LPO as reflected by the increased MDA level (marker of LPO) in the liver. Fluoride consumption is associated with the production of free radicals which can react with polyunsaturated fatty acids to yield lipid hydroperoxides which in turn initiates a lipid-radical chain reaction leading to oxidative damage to cell membrane. The enhanced hepatic LPO observed in this study agreed with previous studies (Blaszczyk et al., 2011; Grucka-Mamczar et al., 2009; Nab-avi et al., 2012). Increased LPO can be counteracted by administrating antioxidant molecules. In the current study, TQ administration reversed the enhanced level of LPO as indicated by the decreased MDA level which could be attributed to the strong antioxidant potential of TQ (Houghton et al., 1995). The antioxidative effect of TQ may be related to the redox properties of the quinone structure of TQ molecule and its

unlimited ability to cross morpho-physiological barriers and in turn its easy access to subcellular compartments, all of which facilitates the radical scavenging effect (Badary et al., 2003).

The body has its own antioxidant defense mechanisms to stabilize oxidative molecules and keep them in balance. Cells are equipped with endogenous antioxidants, either enzymatic or non-enzymatic which are crucial for preventing or at least slowing the incidence and progression of diseases (Jacob, 1995). In the present study, exposure to NaF for 4 weeks was found to be associated with a reduction in GSH content and in the activity of SOD, CAT, GST and GPx in the liver indicating an impaired function of the hepatic antioxidant defense system. This impairment interferes with the elimination of H2O2 and LPO products and causes their accumulation in the cells leading to the damage of cell membranes. These results are in agreement with Sharman and Chinoy (1998), Chinoy (2003), Blaszczyk et al. (2011) who illustrated that F affected the activity of enzymes constituting the cell antioxida-tive system. Due to its chemical nature, F is capable of interrelating with metals and thus can alter the activity of the enzymes that contain a transition metal as part of their cofac-tors or in their active site (Chinoy, 2003; Sharman and Chinoy, 1998). SOD, CAT, GST and GPx which are the main antiox-idant enzymes contain a transition metal as a cofactor. The interaction of NaF with metals of these enzymes may explain the observed inhibition in the activities of these enzymes. Therefore, enhancing endogenous enzymatic and non-enzymatic antioxidant status by administrating exogenous compounds can provide an effective strategy to prevent NaF-induced toxicity. Supplementation of NaF-intoxicated rats with TQ normalized the assayed antioxidants indicating its ability to restore antioxidative homeostasis. Woo et al. (2012) reported that TQ can scavenge free radical and preserve the activity of various antioxidant enzymes such as CAT, GPx and GST. Previous studies have also shown that TQ could upregulate the GST, GPx and CAT genes with the consequent elevation of hepatic GST, GPx and CAT levels to overcome oxidative stress induced during diethylnitrosamine metabolism (Ismail et al., 2010; Nagi and Almakki, 2009).

Aminotransferases (AST and ALT) mediate the catalysis of aminotransfer reactions and are considered to be markers for clinical diagnosis of liver injury. Alkaline phosphatase, another marker for hepatic damage, is a hydrolase enzyme responsible for removing the phosphate group from nucleo-

tides and proteins. Lactate dehydrogenase is a general indicator of acute or chronic hepatic damage. Data obtained in the current study demonstrate impaired liver function in NaF group as reflected by increased serum indices of liver function namely AST, ALT, ALP, LDH, and total bilirubin. It is well known that elevation in these markers indicates hepatocellular damage (Bulle et al., 1990). The observed elevation in these indices could be a secondary event following NaF-induced LPO of hepatocyte membranes with the subsequent increase in the leakage of these biomarkers from the liver tissue. LPO of cell membranes leads to loss of membrane fluidity, changes in membrane potential and an increase in membrane permeability (Nehru and Anand, 2005), all of which lead to leakage of the enzymes from the liver cells. TQ supplementation could alleviate hepatic toxicity induced by NaF as reflected by normalization of the measured liver function markers suggesting a potential protective effect for TQ against NaF-induced liver damage. This protective effect could be due to the ability of TQ to antagonize the enhanced LPO and in turn stabilize the integrity of the cellular membranes leading to preventing or at least decreasing the leakage of liver enzymes. Thymoqui-none as an antioxidative agent has been reported to prevent the membrane LPO in hepatocytes (Mansour et al., 2002).

Hepatotoxicity is manifested by altered lipid metabolism. Findings of the present study showed that oral administration of NaF induced a significant increase in the level of total lipids, triglycerides and total cholesterol. High levels of NaF lead to its accumulation in the liver leading to disturbance of lipid metabolism and in turn to the reported elevation the lipid profile. As mentioned above, administration of NaF resulted in increased LPO and loss of membrane integrity which might be important determinants of altered lipid metabolism and are closely associated with the observed hyperlipidemia. Abnormal enzyme activities seem to be one of the chief factors responsible for the rise in serum triglycerides and cholesterol. It appears that F inhibits lipases, phospholipases, unspecific esterases and pyro-phosphatase (Machoy-Mokrzyriska et al., 1994; Grucka-Mamczar et al., 2004). Fluoride was found to cause hypercholesterolemia which is believed to be due to the lowered levels of insulin (Garcia-Montalvo et al., 2009). Thy-moquinone supplementation alleviated the previously mentioned alteration in the lipid profile. Previous report illustrated that TQ produce significant reduction in the level of total cholesterol, triglycerides, low density lipoproteins and high density lipoproteins in rats (Bamosa et al., 2002). The mechanism underlying these hypolipidemic effects is unclear. It was suggested that the hypolipidimic activity of TQ may be attributed to inhibition of oxidative stress (Sinha et al., 2008).

Abnormal protein metabolism is considered a sign of hep-atotoxicity. In the present study, the administration of NaF resulted in a significant decrease in the concentration of total protein and albumin as compared to the control group. Previous study reported a similar reduction in protein content in NaF-treated animals and related it to inhibition of decarboxyl-ation of branched chain amino acids and simultaneously promoting protein breakdown (Shashi et al., 1992). Fluoride affects cellular protein synthesis mainly due to the impairment of peptide chain initiation (Godehaux and Atwood, 1976). Sodium fluoride-generated free radicals down-regulate the activity of enzymes important in the polymerization of amino acids, thus inhibiting the process of elongation of peptides (Hord-

yjewska and Pasternak, 2004). Free radicals are also a major source for DNA damage, which can cause strand breaks and base alteration in the DNA (Trivedi et al., 2008). Therefore the reduction in protein content observed in the present study may be due to either direct effect of F on protein synthesis or indirectly through DNA and RNA damage. Furthermore, the observed decrease in protein content may be explained in part by the reduction in insulin level since insulin has an anabolic effect on protein metabolism in that it stimulates protein synthesis and retards protein degradation (Murray et al., 1999). Previous reports have shown that protein synthesis is decreased in all tissues due to absolute or relative deficiency of insulin (Chatterjea and Shinde, 1994). Supplementation with TQ improved the reduced levels of total proteins and albumin of NaF-intoxicated rats. This tendency to increase the level of protein contents could be ascribed to suppression of NaF-in-duced oxidative stress and liver damage with the subsequent improvement in liver synthetic function following TQ treatment.

Impaired carbohydrate metabolism is a major indicator for hepatotoxicity. The present study revealed a significant reduction in the insulin level with a concomitant increase in the blood sugar level in rats administered NaF for 4 weeks. Previous studies demonstrated similar results (Menoyo et al., 2005; Rigalli et al., 1995). From the above mentioned results, the observed NaF-induced hyperglycemia may be explained in part by reduction in insulin secretion. Blood glucose level is controlled by the hormone insulin released from b-cells in the pancreas. Administration of F has been shown to inhibit the secretion of insulin in rats and human beings leading to lower plasma levels of the hormone (Rigalli et al., 1995). Fluoride has been reported to affect insulin secretion through altering the intracellular signaling pathway related to the secretion of insulin (Menoyo et al., 2005). High levels of F were found to be associated with b-cell dysfunction (Menoyo et al., 2008). b-cell dysfunction is associated with the secretion of intact and partially processed proinsulin (insulin precursor) (Yoshio-ka et al., 1988). Furthermore, F has been found to decrease the sensitivity of pancreatic tissue toward glucose stimulus (Me-noyo et al., 2008). Hyperglycemia may be implicated in the generation of reactive oxygen species as hydroxyl radicals and superoxide which can cause LPO (Matkovics et al., 1997). This view further supports the observed NaF-induced LPO and its subsequent damage to membrane of hepatocytes and in turn to the reported leakage of liver enzymes. Hypoin-sulinemia results in various pathologic lesions in the liver and alters various metabolic and enzymatic functions of liver (Zafar et al., 2009). Thymoquinone supplementation evoked a significant decrease in the NaF-induced hyperglycemia without improving the insulin level. These results indicated that the hypoglycemic effect of TQ could be mediated through enhancing peripheral glucose oxidation and/or reduction of gluconeo-genesis and not through affecting insulin release. These results are in accordance with Fararh et al. (2005) who recorded a significant reduction in liver glucose output in diabetic hamasters treated with TQ. Furthermore, El-Dakhakhny et al. (2002) mentioned that N. sativa oil mediated its hypoglycemic effect through extrapancreatic action.

In conclusion, oral administration of thymoquinone counteracted the sodium fluoride-induced toxicity and oxidative stress in rats' liver probably by reducing the level of peroxida-tion and/or enhancing the activities of enzymatic and non-

enzymatic antioxidants of the liver. These effects make thymo-quinone a promising prophylactic agent in a variety of pathological conditions in liver where cellular damage is a consequence of oxidative stress. A limitation of this study is that the efficiency of thymoquinone was evaluated with one dose only (10 mg/kg). Therefore, further dose-dependent study is required to find out the optimal curative dose of thymoqui-none in such cases of hepatotoxicity.

References

Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121-126. Agency for Toxic Substances and Disease Registry (ATSDR), 2003. Toxicological Profile for Fluorides, Hydrogen Fluoride, and Fluorine. US Department of Health and Human Services, Atlanta, US. Ali, B.H., Blunden, G., 2003. Pharmacological and toxicological

properties of Nigella sativa. Phytother. Res. 17, 299-305. Anane, R., Creppy, E.E., 2001. Lipid peroxidation as pathway of aluminium cytotoxicity in human skin fibroblast cultures: prevention by superoxide dismutase + catalase and vitamins E and C. Hum. Exp. Toxicol. 20, 477-481. Badary, O.A., Taha, R.A., Gamal el-Din, A.M., Abdel-Wahab, M.H., 2003. Thymoquinone is a potent superoxide anion scavenger. Drug Chem. Toxicol. 26, 87-98. Bamosa, A.O., Ali, B.A., Hawsawi, Z.A., 2002. The effect of thymoquinone on blood lipids in rats. Ind. J. Physiol. Pharmacol. 46, 195-201.

Belfield, A., Goldberg, D.M., 1971. Revised assay for serum phenyl phosphatase activity using 4-aminoantipyrine. Enzyme 12, 561-573. Beutler, E., Duron, O., Kelly, M., 1963. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 61, 882-888. Blaszczyk, I., Birkner, E., Kasperczyk, S., 2011. Influence of methionine on toxicity of fluoride in the liver of rats. Biol. Trace Elem. Res. 139, 325-331. Bulle, F., Mavier, P., Zafrani, E.S., Preaux, A.M., Lescs, M.C., Siegrist, S., 1990. Mechanism of gamma-glutamyl transpeptidase release in serum during intrahepatic and extrahepatic cholestasis in the rat: a histochemical, biochemical and molecular approach. Hepatology 11, 545-550. Chatterjea, M.N., Shinde, R., 1994. Metabolism of carbohydrate. Textbook of Medical Biochemistry. Jaypee Brothers Medical Publishers Pvt Ltd., New Delhi, India, p. 421. Chinoy, N.J., 2003. Fluoride stress on antioxidant defense systems.

Fluoride 36, 138-141. Chlubek, D., 2003. Fluoride and oxidative stress. Fluoride 36 (4), 217228.

Doumas, B.T., Watson, W.A., Biggs, H.G., 1971. Albumin standards and the measurement of serum albumin with bromocresol green. Clin. Chim. Acta 31, 87-96. El-Dakhakhny, M., Mady, N., Lembert, N., Ammon, H.P., 2002. The hypoglycemic effect of Nigella sativa oil is mediated by extrapan-creatic actions. Planta Med. 68 (5), 465-466. Fararh, K.N., Shimizu, Y., Shiina, T., Nikami, H., Ghanem, M.M., Takewaki, T., 2005. Thymoquinone reduces hepatic glucose production in diabetic hamsters. Res. Vet. Sci. 79, 219-223. Fossati, P., Principle, L., 1982. Serum triglycerides determined colorimetrically with an enzyme that produces hydrogen peroxide. Clin. Chem. 28, 2077-2080. Garcia-Montalvo, E.A., Reyes-Perez, H., Del-Razo, L.M., 2009. Fluoride exposure impairs glucose tolerance via decreased insulin expression and oxidative stress. Toxicology 263, 75-83. Godehaux, W., Atwood, K.C., 1976. Structure and function of

initiation complexes. J. Biol. Chem. 251, 292-301. Grucka-Mamczar, E., Birkner, E., Kasperczyk, S., Kasperczyk, A., Chlubek, D., Samujlo, D., 2004. Lipid balance in rats with fluoride induced hyperglycemia. Fluoride 37, 195-200.

Grucka-Mamczar, E., Birkner, E., Baszczyk, I., Kasperczyk, S., Wielkoszyn, T., Swietochowska, E., Stawiarska-Pie, B., 2009. The influence of sodium fluoride on antioxidants and the concentration of malondialdehyde in rat blood plasma. Fluoride 42 (2), 101-104.

Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferases: the first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 7130-7139.

Halliwell, B., Guttteridge, J.M., 2007. Free Radicals in Biology and Medicine. Oxford University Press, Clarendon, Oxford, pp. 221238.

Hordyjewska, A., Pasternak, K., 2004. Influence of fluoride on organism of human. J. Elementol. 9 (4), 883-987.

Houghton, P.J., Zarka, R., Delasheras, B., Hoult, J.R., 1995. Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Med. 61, 33-36.

Ismail, M., Al-Naqeep, G., Chan, K., 2010. Nigella sativa thymoqui-none-rich fraction greatly improves plasma antioxidant capacity and expression of antioxidant genes in hypercholesterolemic rats. Free Radic. Biol. Med. 48, 664-672.

Jacob, R.A., 1995. The integrated antioxidant system. Nutr. Res. 15 (5), 755-766.

Koodziejczyk, L., Put, A., Grzela, P., 2000. Liver morphology and histochemistry in rats resulting from ingestion of sodium selenite and sodium fluoride. Fluoride 33 (1), 6-16.

Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275.

Machoy-Mokrzyriska, A., Put, A., Ceglecka, M., Mysliwiec, Z., 1994. Influence of essential phospholipids on selected biochemical parameters of lipid metabolism in rats chronically exposed to ammonium fluoride vapours. Fluoride 27 (4), 201-204.

Mansour, M.A., Nagi, M.N., El-Khatib, A.S., Al-Bekairi, A.M., 2002. Effects of thymoquinone on antioxidant enzyme activities, lipid peroxidation and DT-diaphorase in different tissues of mice: a possible mechanism of action. Cell Biochem. Funct. 20, 143-151.

Matkovics, S., Kotorman, M., Varga, I.S., 1997. Proantioxidant and filtration changes in blood of type 1 diabetic patient. Acta Physiol. Hung. 85, 99-106.

Menoyo, I., Rigalli, A., Puche, R.C., 2005. Effect of fluoride on the secretion of insulin in the rat. Arzneimittelforschung 55, 455-460.

Menoyo, I., Puche, R.C., Rigalli, A., 2008. Fluoride-induced resistance to insulin in the rat. Fluoride 41 (4), 260-269.

Morgan, C.R., Lazarow, A., 1963. Immunoassay of insulin: two antibody system. Plasma insulin levels in normal, subdiabetic and diabetic rats. Diabetes 12, 115-119.

Murray, R.R., Granner, D.K., Mayes, P.A., Rodwell, V.W., 1999. Harper's Biochemistry, 25th ed. Appleton and Lange, Stamford, Connecticut, pp. 610-617.

Nabavi, S.M., Nabavi, S.F., Eslami, S., Moghaddam, A.H., 2012. In vivo protective effects of quercetin against sodium fluoride-induced oxidative stress in the hepatic tissue. Food Chem. 132, 931-935.

Nagi, M.N., Alam, K., Badary, O.A., Al-Sawaf, H.A., Al-Bekairy, A.M., 1999. Thymoquinone protects against CCl4 hepatotoxicity in mice via antioxidant mechanism. Biochem. Mol. Biol. Int. 47, 153159.

Nagi, M.N., Mansour, M.A., 2000. Protective effect of thymoquinone against doxorubicin-induced cardiotoxicity in rats: a possible mechanism of protection. Pharmacol. Res. 41, 283-289.

Nagi, M.N., Almakki, H.A., 2009. Thymoquinone supplementation induces quinone reductase and glutathione transferase in mice liver: possible role in protection against chemical carcinogenesis and toxicity. Phytother. Res. 23, 1295-1298.

National Research Council (NRC), 2006. Fluoride in Drinking-Water. A scientific review of EPA's standards, Washington.

Nehru, B., Anand, P., 2005. Oxidative damage following chronic aluminum exposure in adult and pup rat brains. J. Trace Elem. Med. Biol. 19, 203-208.

Nishikimi, M., Appaji, N., Yagi, K., 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun. 46, 849-854.

Norwood, A.A., Tan, M., May, M., Tucci, M., Benghuzzi, H., 2006. Comparison of potential chemotherapeutic agents, 5-fluoruracil, green tea, and thymoquinone on colon cancer cells. Biomed. Sci. Inst. 42, 350-356.

Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351-358.

Paglia, D.E., Valentine, W.N., 1967. Determination of Glutathione peroxidase. J. Lab. Clin. Med. 70, 158-169.

Pawowska-Goral, K., Wardas, W., Wardas, M., Kusa, Z., 1998. Influence of fluoride compounds upon the human body. Ann. Acad. Med. Siles. 34-35, 105-115.

Retiman, S., Frankel, S., 1957. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 56-63.

Richmond, W., 1973. Cholesterol enzymatic colorimetric test chop-PAP method of estimation of total cholesterol in serum. Clin. Chem. 191, 1350-1356.

Rigalli, A., Alloatti, R., Menoyo, I., Puche, R.C., 1995. Comparative study of the effect of sodium fluoride and sodium monofluoro-phosphate on glucose homeostasis in the rat. Arzneimittelforschung 45, 289-292.

Sharman, A., Chinoy, N.J., 1998. Role of free radicals in fluoride-induced toxicity in liver and kidney of mice and its reversal. Fluoride 31, S26.

Shashi, A., Singh, J.P., Thapar, S.P., 1992. Protein degradation in skeletal muscle of rabbit during experimental fluorosis. Fluoride 25 (3), 155-158.

Sinha, M., Manna, P., Sil, P.C., 2008. Terminalia arjuna protects mouse hearts against sodium fluoride-induced oxidative stress. J. Med. Food 11 (4), 733-740.

Stawiarska-Pieta, B., Paszczela, A., Grucka-Mamczar, E., Szafarska-Stojko, E., Birkner, E., 2009. The effect of antioxidative vitamins A and E and coenzyme Q on the morphological picture of the lungs and pancreata of rats intoxicated with sodium fluoride. Food Chem. Toxicol. 47, 2544-2550.

Trinder, P., 1969. Determination of blood glucose using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochem. 6, 24-27.

Trivedi, M.H., Verma, R.J., Chinoy, N.J., 2008. Ameliroation by black tea of sodium fluoride-induced effects on DNA, RNA and protein content of liver and kidney on serum transaminase activities in swiss albino mice. Fluoride 41 (1), 61-66.

Vassault, A., 1983. Lactate dehydrogenase. UV-method with pyruvate and NADH. In: Bergmeyer, J., Grabl, M. (Eds.), Methods of Enzymatic Analysis. Verlag-Chemie, Deerfield Beach, Florida, pp. 119-126.

Walter, M., Gerade, H., 1970. Bilirubin assay. Microchem. J. 15, 231236.

Wilson-Simpson, F., Vance, S., Benghuzzi, H., 2007. Physiological responses of ES-2ovarian cell line following administration of epigallocatechin-3-gallate (EGCG), thymoquinone (TQ), and selenium (SE). Biomed. Sci. Inst. 43, 378-383.

Woo, C.C., Kumar, A.P., Sethi, G., Tan, K.H., 2012. Thymoquinone: potential cure for inflammatory disorders and cancer. Biochem. Pharmacol. 83 (4), 443-451.

Yoshioka, N., Kuzuya, T., Matsuda, A., Taniguchi, M., Iwamato, Y., 1988. Serum proinsulin levels of fasting and after oral glucose load in patients with Type 2 diabetes mellitus. Diabetologia 31, 355-360.

Zafar, M., Naqvi, N.S., Ahmed, M., Kaimkhani, Z.A., 2009. Altered liver morphology and enzymes in streptozotocin-induced diabetic rats. Int. J. Morphol. 27 (3), 719-725.

Zollner, N., Kirsch, K., 1970. Serum total lipids determination colorimetrically. Z. Ges. Exp. Meal. 1335, 54.