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Abstract

In this study, the protective effects of vanillin were evaluated against carbon tetrachloride (CCl₄)-induced kidney damages in Wistar albino rats. CCl₄ (1 ml/kg, intraperitoneally [i.p.]) caused a significant induction of renal disorder, oxidative damage and DNA fragmentation as evidenced by increased plasma creatinine, urea and uric acid levels, increased lipid peroxidation (malondialdehyde [MDA]) and protein carbonyl. Furthermore, glutathione levels, catalase, superoxide dismutase, glutathione transferase and glutathione peroxidase activities were significantly decreased. A smear without ladder formation on agarose gel was also shown, indicating random DNA degradation. Pretreatment of rats with vanillin (150 mg/kg/day, i.p.), for 3 consecutive days before CCl₄ injection, protected kidney against the increase of MDA and degradation of membrane proteins compared to CCl₄-treated rats and exhibited marked prevention against CCl₄-induced nephropathology, oxidative stress and DNA damage. Kidney histological sections showed glomerular hypertrophy and tubular dilatation in CCl₄-treated rats, however, in vanillin pretreated rats, these histopathological changes were less important and present a similar structure to that of control rats. These data indicated the protective role of vanillin against CCl₄-induced nephrotoxicity and suggested its significant contribution of these beneficial effects.

Keywords

carbon tetrachloride vanillin antioxidant system nephropathy

Introduction

Carbon tetrachloride (CCl₄) is a manufactured chemical and most of it is used in the production of chlorofluorocarbons (CFCs) and other chlorinated hydrocarbons.

CCl₄ intoxication in animals is an experimental model that mimics oxidative stress in many physiological situations.¹ The initial step in the tissue injury induced by CCl₄ is its cytochrome P450-mediated transfer of a single electron to the C–Cl bond, giving a radical anion as a transient intermediate that eliminates chlorine to form a carbon-centered radical, the trichloromethyl radical (•CCl₃) and chloride.^2,3 The trichloromethyl radical can dismutate to chloroform, bind to macromolecules or attack polyenoic fatty acids in cellular membranes. The double allylic hydrogen atoms in these acids are particularly susceptible to abstraction by free radicals, giving chloroform and secondary lipid radicals which react rapidly with molecular oxygen to form lipid peroxyradicals. The trichloromethyl radical can also react with oxygen to form the peroxytrichloromethyl free radical (•CCl₃O₂), which is more reactive than the 3,2 trichloromethyl radical and produces similar kinds of damage. The eventual decomposition of peroxidized fatty acids gives rise to a number of stable end products, such as carbonyls including malondialdehyde (MDA), ethane and pentane.⁴ With respect to its molecular characteristics, CCl₄ is considered a hepatotoxic and nephrotoxic chemical. Radicals generated by CCl₄ induce damages and dysfunctions of DNA and proteins in addition to lipid peroxidation. The renal alterations caused by CCl₄ are possibly mediated by oxidative stress. The key enzyme involving the CCl₄-induced nephrotoxicity is P₄₅₀ cytochrome, which is localized in the cortical tubule cells,⁵ and then increased lipid peroxidation is evident in the renal brush border.⁶ CCl₄ treatment also affects renal mitochondrial function, including calcium flux across mitochondrial membranes.⁶

In order to cope with the excess of free radicals produced upon oxidative stress, organisms have developed enzymatic and nonenzymatic antioxidant systems to scavenge or detoxify reactive oxygen species (ROS), block their production or sequester transition metals which are the source of free radicals.⁷ The generation of small amounts of free radicals appears to have an important biological function, but oxidative stress is caused by the excessive production of ROS, which are implicated in the pathogenesis of several diseases. Excessive production of free radicals and oxidative stress can be induced by a variety of factors such as ionizing radiation or exposure to drugs and xenobiotics (e.g. CCl₄). Various investigations have established that CCl₄ causes renal injuries in rats^8,9 in addition to hepatic toxicity through the generation of free radicals.¹⁰ Alteration of antioxidant status with CCl₄ may potentially cause nephropathies in rats.

The administration of antioxidants could conceivably protect tissues from the effects of free radicals, ROS and lipid peroxidation and thereby retard the progress of many chronic diseases.^11,12 A number of plants have been shown to possess protective properties by improving the antioxidant and anti-inflammatory status, for example flax, pumpkin, sesame and peanut.^13–15 However, there is still a lack of scientific evidence to authenticate the protective properties of some natural products which are traditionally used to treat disorders. Vanillin, for example, is a chemical compound that confers the smell and flavor of vanilla, an important flavoring agent used in various confectionaries, sleep prevention agent, and aphrodisiac.¹⁶ Currently, a lot of new functional uses of vanillin have been discovered. It exhibits chemopreventive effects on multiorgan carcinogenesis models in rats¹⁷ and suppresses the invasion and migration of cancer cells^18–20 and could be used to treat sickle cell anemia.²¹ Recently, vanillin has been shown to inhibit lipopolysaccharide-stimulated nuclear factor kappa B (NF-κB) activation and cyclooxygenase 2 gene expression in murine macrophages.²² In addition, according to Sasaki et al.,²³ vanillin is reported to be effective in reducing substance-induced mutations. As an antimutagen product, it is also able to prevent and treat cancer, a disease closely related to mutations.²⁴

Figure 1.

Structure of 4-hydroxy-3-methoxybenzaldehyde (vanillin).

Despite the beneficial biological properties of vanillin, its protective effect against CCl₄ nephrotoxicity has not so far been explored. In the present study, we investigated the antioxidant effects of vanillin against CCl₄-induced nephrotoxicity in rats on relevant oxidative stress parameters, including thiobarbituric acid reactive substances (TBARS), protein carbonyl (PCO), glutathione (GSH) levels, antioxidant enzyme (catalase [CAT], superoxide dismutase [SOD], glutathione peroxidase [GPx] and glutathione transferase [GST]) activities, renal injury biomarkers (creatinine, uric acid and urea), genotoxicity (DNA damage) and renal histopathology.

Materials and methods

CCl₄ intoxication, vanillin pretreatment and samples collection

The investigation was conducted in accordance with the international principles for laboratory animals use and care as found in the guidelines.²⁵ The investigation was approved by the ethics committee of the faculty of science of Sfax, Tunisia.

A total of 32 male Wistar rats (weighing between 180 and 200 g) were housed in cages at 22 ± 2°C on a 12-h day/night regimen with access to a diet and water ad libitum. They were divided into 4 groups of 8 rats each: group 1 (control group [C]); group 2 (CCl₄ group [CCl₄]); group 3 (vanillin group [Va]) and group 4 (vanillin + CCl₄ group [Va + CCl₄]).

The rats in groups 2 and 4 were i.p. injected with 1.0 ml/kg body weight of CCl₄ dissolved in olive oil, v/v. Rat of the group 1 received an equal volume of olive oil. The administered dose was according to the reference dose for chronic oral exposure (RfD) as recommended for CCl₄ (CASRN 56-23-5).²⁶ Groups 3 and 4 were i.p. injected daily with vanillin (150 mg/kg body weight), during 3 days before CCl₄ injection. Twenty-four hours after the CCl₄ intoxication, all rats of each group were killed. Kidney tissue was quickly removed, cleaned and washed in ice-cold saline solution. It was finely minced and homogenized in phosphate buffer (0.1 M; pH 7.4) and centrifuged at 8000g for 20 min at 4°C. The supernatant was used to assay biochemical parameters.

Determination of protein carbonyl content in kidney

PCOs were measured using the method of Reznick and Packer.²⁷ The absorbance of the sample was measured at 370 nm. The carbonyl content was calculated based on the molar extinction coefficient of 2,4-dinitrophénylhydrazine ([DNPH] ∊ = 2.2 × 10⁴ cm⁻¹ M⁻¹) and expressed as nmol/mg protein.

Measurement of malondialdehyde in kidney

Concentrations of MDA in kidney, an index of lipid peroxidation, were determined spectrophotometrically according to Draper and Hadley.²⁸ The absorbance of TBA-MDA complex was determined at 532 nm. Lipid peroxidation was expressed as nmol of TBARS, using 1,1,3,3-tetraethoxypropane as standard.

Antioxidant enzymes and glutathione assays in kidney

Total superoxide dismutase activity

SOD activity was estimated according to Beauchamp and Fridovich.²⁹ The blue color developed in the reaction was measured at 560 nm. Units of SOD activity were expressed as the amount of enzyme required to inhibit the reduction of nitroblue tetrazolium by 50% and the activity was expressed as units per mg of protein.

Catalase activity

CAT activity was assayed by the method of Aebi.³⁰ Changes in absorbance were recorded at 240 nm. CAT activity was calculated in terms of nmol H₂O₂ consumed/min/mg of protein.

Glutathione peroxidase

GPx activity was measured according to Flohe and Gunzler.³¹ The enzyme activity was expressed as nmoles of GSH oxidized/min/mg protein.

Glutathione transferase

GST activity was determined following the procedure of Habig et al.³² The change in color was monitored by recording the absorbance (340 nm) at 30 s intervals for 3 min. The enzyme activity was calculated as nmoles 1-chloro-2,4-dinitrobenzene (C-DNB) conjugate formed/min/mg protein.

Glutathione levels

GSH in tissues was determined by the method of Jollow et al.³³ based on the development of a yellow color when 5,5-dithiobis-2 nitro benzoic acid was added to compounds containing sulfhydryl groups. The absorbance was measured at 412 nm after 10 min. Total GSH content was expressed as µg/mg of tissue.

Estimation of urea, uric acid and creatinine

The levels of urea, uric acid and creatinine in plasma were estimated spectrophotometrically using commercial diagnostic kits, respectively (Refs. 20151, 20143, and 20091), purchased from Biomagreb (Ariana, Tunisia).

DNA fragmentation analysis

The extent of DNA fragmentation in the kidney tissue was determined by the method described by Kanno et al.³⁴ Briefly, kidney tissue was homogenized in lysis buffer. The lysate was incubated for 3 h with proteinase K at 56°C. DNA was purified with an equal volume of Tris-saturated phenol/chloroform/isoamyl alcohol (25:24:1), incubated in ice and centrifuged at 10,000g at 4°C. The clear supernatant containing DNA was transferred to another tube and mixed and saturated sodium acetate was added, with the same volume of ice-cold isopropanol. The precipitated DNA was separated by centrifugation; it was washed twice with ethyl alcohol (70%) and finally dissolved in 100 µl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8). The suspended DNA was incubated with RNase A for 60 min at 37°C in a water bath. DNA samples (10 µg of DNA/lane) were kept at 80 V for 1 h on 0.8% agarose gel in Tris-acetate-EDTA buffer, containing 0.5 μg/ml ethidium bromide. The gel was observed under an ultraviolet lamp and a photograph has been taken.

Histological examination

Kidney samples, intended for histological examination by light microscopy, were removed and immediately fixed in formalin solution, embedded in paraffin, serially sectioned at 5 μm and stained with hematoxylin–eosin.

Statistical analysis

The data were analyzed using the statistical package program stat view 5 software for windows (SAS Institute, Berkley, CA, USA). Statistical analysis between all groups was performed with one-way analysis of variance followed by Student’s t test. All data were expressed as means ± SD. The results were considered significant if p ≤ 0.05.

Results

Table 1 shows the effects of vanillin on the plasma levels of urea, uric acid and creatinine in rats. Nephrotoxicity was evidenced by a significant alteration in these plasma parameters in the CCl₄ group when compared with those of controls. Pretreatment with vanillin significantly ameliorated the concentrations of urea, uric acid and creatinine in the plasma of Va-CCl₄ rats when compared with CCl₄-treated rats. The Va group showed no significant variation in these biomarkers as compared to the control group.

Table 1.

Plasma levels of creatinine, urea and uric acid of C-, Va-, CCl₄- and Va + CCl₄-treated rats^a

Parameters and treatments	C	Va	CCl₄	Va + CCl₄
Creatinine (μmol/L)	102.24 ± 7.34	110.46 ± 7.78	190.2 ± 5.95^b	137.85 ± 7.54^c
Uric acid (μmol/L)	337.5 ± 12.24	312.2 ± 11.23	302.63 ± 8.74^b	374.2 ± 13.06^c
Urea (mmol/L)	9.78 ± 1.62	10.03 ± 2.22	10.54 ± 1.52^b	15.12 ± 1.95^e

C: control, CCl₄: carbon tetrachloride, Va: vanillin.

^aValues are expressed as mean ± SD (n = 8).

^bSignificant differences between the CCl₄ and C groups, p < 0.001.

^cSignificant differences between the Va + CCl₄ and CCl₄ groups, p < 0.001.

^dSignificant differences between the CCl₄ and C groups, p < 0.05.

^eSignificant differences between the Va + CCl₄ and CCl₄ groups, p < 0.01.

A significant increase in PCO and MDA content (44% and 59%, respectively), an end product of protein and lipid oxidation, respectively, in the kidney of CCl₄ rats was observed when compared with the control group. Pretreatment with vanillin of CCl₄ group significantly reduced the PCO and MDA content in the kidney (27% and 29%, respectively), when compared with that of rats administered CCl₄ only, thus restoring it back to the range in the controls (Table 2).

Table 2.

Effect of vanillin pretreatment on the lipid peroxidation (MDA) and protein carbonyls levels (PCO) in the kidneys of rats^a

Parameters and treatments	C	Va	CCl₄	Va + CCl₄
MDA (nmol/100 mg)	5.32 ± 1.15	5.54 ± 0.23	8.45 ± 4.05^b	6.01 ± 1.21^c
PCO (nmol/mg protein)	12.54 ± 2.35	11.82 ± 1.84	18.04 ± 1.32^d	13.11 ± 1.43^e

C: control, CCl₄: carbon tetrachloride, MDA: malondialdehyde, PCO: protein carbonyl, Va: vanillin.

^aValues are expressed as mean ± SD (n = 8).

^bSignificant differences between the CCl₄ and C groups, p < 0.001.

^cSignificant differences between the Va + CCl₄ and CCl₄ groups, p < 0.01.

^dSignificant differences between the CCl₄ and C groups, p < 0.01.

^eSignificant differences between the Va + CCl₄ and CCl₄ groups, p < 0.05.

Antioxidant enzyme activities (CAT, SOD, GPx and GST) and GSH level in kidney of control and tested groups are shown in Table 3. In CCl₄ group, significant decreases in enzymes activities (CAT, SOD, GPx and GST by 46%, 45%, 64% and 63%, respectively) and GSH level (53%) were observed in kidney, as compared to the control group. Pretreatment with vanillin improved, in Va + CCl₄ rats, GSH levels by 109%, CAT, SOD, GPx and GST activities by 34%, 49%, 135% and 99%, respectively, in kidney. The Va group showed no significant variations in these parameters as compared to control group.

Table 3.

Glutathione levels and enzymes activities in the kidneys of C-, Va-, CCl₄- and Va + CCl₄-treated rats^a

Parameters and treatments	C	Va	CCl₄	Va + CCl₄
SOD (Units/mg protein)	23.23 ± 2.54	21.13 ± 2.83	12.84 ± 2.85^b	19.18 ± 2.34^c
CAT (µmoles H₂O₂ degraded/min/mg protein)	16.64 ± 2.17	14.89 ± 1.58	8.95 ± 1.72^d	11.97 ± 1.21^e
GST (nmole C-DNB conjugate formed/min/mg protein)	17.41 ± 2.21	16.39 ± 3.10	6.49 ± 2.01^d	12.97 ± 2.15^e
GPx (nmole GSH/min/mg protein)	74.14 ± 6.34	73.32 ± 5.11	26.72 ± 7.62^d	62.89 ± 3.07^e
GSH (mg/ml in plasma and mg/mg tissue)	10.12 ± 1.11	11.22 ± 1.49	4.78 ± 1.15^b	9.97 ± 1.15^e

C: control, CAT: catalase, CCl₄: carbon tetrachloride, GPx: glutathione peroxidase, GSH: glutathione, GST: glutathione transferase, SOD: superoxide dismutase, Va: vanillin.

^aValues are expressed as mean ± SD (n = 8).

^bSignificant differences between the CCl₄ and C groups, p < 0.01.

^cSignificant differences between the Va + CCl₄ and CCl₄ groups, p < 0.01.

^dSignificant differences between the CCl₄ and C groups, p < 0.001.

^eSignificant differences between the Va + CCl₄ and CCl₄ groups, p < 0.001.

As shown in Figure 2, a smear (hallmark of necrosis) without ladder formation on agarose gel, indicating random DNA degradation, was observed in the kidney tissue of the CCl₄-treated rats. Vanillin pretreatment exerted a protective effect against CCl₄ by reducing the smear formation. No difference could be observed in DNA damage among the control and the vanillin groups.

Figure 2.

Agarose gel electrophoresis of DNA fragmentation. M: marker (1 kb DNA ladder); lane 1: C group; lane 2: Va + CCl₄ group; lane 3: CCl₄ group; lane 4: Va group.

Histopathological studies of the kidney of control, Va and Va + CCl₄ animals showed normal histology. In CCl₄-treated rats, significant kidney injury was observed confirmed by damage in renal structure, showing marked glomeruli and tubular damages (Figure 3).

Figure 3.

Kidney histological sections of C, Va, CCl₄ and Va + CCl₄ rats (hematoxylin and eosin, ×400).

Discussion

The present in vivo study demonstrated the protective potential of vanillin by reversing CCl₄-induced nephrotoxicity. CCl₄ has been commonly used in rat experimental models to investigate the oxidative stress induced in various organs. In recent years, there has been considerable interest in free radical-mediated damage in biological systems due to pesticide exposure^35,36 and the search for herbal and natural drugs with antioxidant activity has gained importance as the dietary intake of antioxidants obtained from natural sources is considered to be relatively safe and involves no side effects.³⁷

CCl₄ intoxication generates free radicals that trigger a cascade of events resulting in nephrotoxicity in rat. The pathogenesis of kidneys is a major public health problem. It is well known that the kidneys play a pivotal role in the regulation of various chemicals. Administration of CCl₄ causes nephrotoxicity as indicated by the modification in the plasma levels of urea, creatinine and uric acid. These pathological changes signify a potential damage in liver and/or kidneys induced with CCl₄ treatment.^38,39 The increased plasma level of creatinine can be attributed to the damaged structural integrity of nephron. The present study revealed that pretreatment with vanillin of CCl₄-treated rats ameliorated the toxic effects of CCl₄ by restoring the markers mentioned above to normal levels.

Toxicity was also evidenced by a significant increase in kidney PCO and MDA content after the administration of CCl₄ only. These parameters are also used according to Tokyay et al.⁴⁰ to determine early liver oxidative stress. They are used to investigate the oxidative damage of proteins and lipid peroxidation of the membrane and lipoproteins as a possible pathogenic mechanism for tissues injury.⁴¹ Lipid peroxidation, an autocatalytic process and a common consequence of cell death, may cause peroxidative tissue damage in inflammation, cancer and aging.⁴² It has been reported that lipid peroxidation is one of the major causes of CCl₄-induced nephrotoxicity mediated by the production of free radical derivatives of CCl₄. In the present study, the administration of CCl₄ resulted in a significant elevation in renal PCO and MDA levels, indicating increased protein and lipid oxidation as indicated by the severe injuries in the histopathology of kidneys. Interestingly, preadministration of vanillin markedly reduced the extent of protein and lipid oxidation by decreasing the PCO and MDA levels, which confirms the nephroprotective effect of vanillin against the renal lipid peroxidation induced by CCl₄.

Generally, antioxidant enzymes such as CAT and SOD are easily inactivated by lipid peroxides or ROS, which results in a decrease in their activities as reported by our results in CCl₄-treated rats. These enzymes are also extremely effective antioxidant enzymes responsible for catalytic dismutation of highly reactive toxic superoxide radicals to H₂O₂ and for the catalytic decomposition of H₂O₂ to oxygen and water, respectively.⁴³ Thus, antioxidant enzymes play an important role in the detoxification of xenobiotics, catalyzing their conjugation with the reduced GSH. The ability of vanillin to modulate the activity of renal antioxidant enzymes such as SOD and CAT has been demonstrated in our study, indicating the antioxidative action of this molecule.

The GSH system includes reduced glutathione, GPx and glutathione-S-transferase. The status of these antioxidant enzymes is an appropriate indirect way to assess the prooxidant–antioxidant status in tissues. These enzymes are lowered due to enhanced lipid peroxidation and are also accompanied by a decrease in GSH level. GSH concentration is known to influence the redox status of cells. Therefore, GSH-dependent enzymes will be affected when its level is depleted in the cells. GSH acts as a nonenzymatic antioxidant both intracellularly and extracellularly in conjunction with various enzymatic processes, which reduces hydrogen peroxides and hydroperoxides by redox and detoxification reactions.⁴⁴ We obtained a significant depletion of GPx, glutathione-S-transferase and GSH in renal tissues of CCl₄-treated rats. These results support the view that renal injury induced by CCl₄ is the best characterized system of xenobiotic-induced nephrotoxicity, as it causes extensive oxidative stress in renal tissues. Administration of vanillin attenuated the CCl₄ toxicity, thereby increasing the level of GSH and activities of GPx and glutathione-S-transferase compared to the CCl₄ group.

Agarose gel electrophoresis showed undetectable DNA laddering (DNA fragmentation) in the kidney of the control rats. The DNA intact band appears to be condensed near the application point with no DNA smearing, suggesting no DNA fragmentation. On the other hand, in our study, CCl₄-treatment resulted in massive DNA fragmentations with a subsequent formation of a DNA smear on agarose gel, a hallmark feature of necrosis⁴⁵ without ladder formation, suggesting CCl₄-induced renal cell damage. Vanillin pretreatment was found to be effective in preventing this CCl₄-induced smear formation.

The biochemical parameters were correlated with the renal histological studies. In fact, we revealed that CCl₄ caused a significant damage to renal structure, showing marked glomeruli and tubular damages, probably due to the generation of reactive radicals and to subsequent lipid peroxidation induced by its generated metabolites. So, hydroperoxides accumulated in kidney could cause cytotoxicity associated with membrane phospholipids peroxidation, the basis for renal cellular damage and necrotic renal cells. Pretreatment of rats with vanillin improved the histological alterations induced by CCl₄, which could be attributed to its antiradical/antioxidant activities.

The mechanism by which vanillin prevented generation of free radicals against CCl₄-induced oxidation could be explained by (i) a decrease in the metabolic activation of CCl₄ or (ii) acting as a chain-breaking antioxidant for scavenging free radicals or (iii) a combination of these effects. Recently, Tai et al.⁴⁶ demonstrate that vanillin shows a stronger antioxidant activity than ascorbic acid and Trolox, and it reacts with radicals via adduct formation or self-dimerization mechanism, which contribute to the high reaction against ABTS•⁺ and AAPH-derived radicals.

In conclusion, our results demonstrate that vanillin present a protective effect on kidney toxicity induced by CCl₄. The mechanisms of protection include the inhibition of protein and lipid oxidation processes, the increase in antioxidant enzymes activities and the inhibition of DNA damage, which results in the recovery of biological parameters and the integrity of kidney histological aspects.

Footnotes

M Makni and Y Chtourou contributed equally to this work.

Acknowledgements

The authors thank the skilful technical assistance of Miss Kchaou Dalinda (Histopathology Laboratory of CHU Habib Bourguiba, Sfax, Tunisia). We also extend our thanks to Mr Bejaoui Hafed, teacher of English at the Sfax Faculty of Science, who helped proofread and edit this manuscript.

Funding

The present work was supported by the grants of DGRST (Appui à la Recherche Universitaire de base, ARUB 99/UR/08-73), Tunisia.

References

Ivor

Schneider

. Learning from failure: congestive heart failure in the postgenomic age. review series introduction. J Clin Invest 2005; 115: 495–499.

Slater

. Free radical mechanisms in tissue injury. Biochem J 1984; 222: 1–15.

Halliwell

Gutteridge

. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984; 219: 1–14.

Rechnagel

Glende

Dolak

Waller

. Mechanisms of carbon tetrachloride toxicity. J Pharmacol Exp Ther 1989; 43: 139–154.

Ronis

MJJ

Huang

Longo

Tindberg

Ingelman- Sundberg

Badger

. Expression and distribution of cytochrome P450 enzymes in male rat kidney: effects of ethanol, acetone and dietary conditions. Biochem Pharmacol 1998; 55: 123–129.

Natarajan

Basivireddy

Ramachandran

Thomas

Ramamoorthy

Pulimood

. Renal damage in experimentally-induced cirrhosis in rats: role of oxygen free radicals. Hepatology 2006; 43: 1248–1256.

Chaudiere

Ferrari-Iliou

. Intracellular antioxidants: from chemical to biochemical mechanisms. Food Chem Toxicol 1999; 37: 949–962.

Khan

Rizvi

Khan

Shaheen

. Carbon tetrachloride induced nephrotoxicity in rat: protective role of Digera muricata . J Ethnopharmacol 2009; 122: 91–99.

Khan

Sahreen

. Evaluation of Launaea procumbens use in renal disorders: a rat model. J Ethnopharmacol 2010; 128: 452–461.

10.

Lin

Tseng

Wang

Lin

Chou

. Hepatoprotective effects of Solanum nigrum Linn. extract against CCl₄-induced oxidative damage in rats. Chem Biol Interact 2008; 171: 283–293.

11.

Lai

Chou

Chao

. Studies on the antioxidative activities of Hsian-tsao (Mesona procumbens Hemsl) leaf gum. J Agric Food Chem 2001; 49: 963–968.

12.

Gulcin

Oktay

Kirecci

Aslam

. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem 2003; 83: 371–382.

13.

Makni

Fetoui

Gargouri

Garoui

Jaber

Makni

. Hypolipidemic and hepatoprotective effects of flax and pumpkin seed mixture rich in ω-3 and ω-6 fatty acids in hypercholesterolemic rats. Food Chem Toxicol 2008; 46: 3714–3720.

14.

Makni

Fetoui

Gargouri

Garoui

Jaber

Makni

. Hypolipidemic and hepatoprotective seeds mixture diet rich in ω-3 and ω-6 fatty acids. Food Chem Toxicol 2010; 48: 2239–2246.

15.

Makni

Fetoui

Gargouri

Garoui

Jaber

Makni

. Flax and pumpkin seeds mixture ameliorates diabetic nephropathy in rats. Food Chem Toxicol 2010; 48: 2407–2412.

16.

Bythrow

. Vanillin as a medical plant. Semin Integr Med 2005; 3: 129–131.

17.

Akagi

Hirose

Hoshiya

Mizoguchi

Ito

Shirai

. Modulating effects of elagic acid, vanillin and quercetin in a rat medium term multi-organ carcinogenesis model. Cancer Lett 1995; 94: 113–121.

18.

Lirdprapamongkol

Kramb

Suthiphongchai

Surarit

Srisomsap

Dannhardt

. Vanillin suppresses metastatic potential of human cancer cells through PI3K inhibition and decreases angiogenesis in vivo. J Agric Food Chem 2009; 57: 3055–3063.

19.

Cheng

Hsiang

Lai

. Relationship between San-Huang-Xie-Xin- Tang and its herbal components on the gene expression profiles in HepG2 cells. Am J Chin Med 2008; 36: 783–797.

20.

Liang

Hsiang

. Vanillin inhibits matrix metalloproteinase-9 expression through down-regulation of nuclear factor-κB signaling pathway in human hepatocellular carcinoma cells. Mol Pharmacol 2009; 75: 151–157.

21.

Zhang

Lian

Chen

Abdulmalik

Vassilev

. Antisickling effect of MX-1520, a prodrug of vanillin: an in vivo study using rodents. Br J Haematol 2004; 125: 788–795.

22.

Murakami

Hirata

Ito

Shoji

Tanaka

Yasui

. Re-evaluation of cyclooxygenase-2-inhibiting activity of vanillin and guaiacol in macrophages stimulated with lipopolysaccharide. Anticancer Res 2007; 27: 801–807.

23.

Sasaki

Ohta

Imanishi

Watanabe

Matsumoto

Kato

. Suppressing effects of vanillin, cinnamaldehyde, and anisaldehyde on chromosome aberrations induced by X-rays in mice. Mutat Res 1990; 243: 299–302.

24.

Latifah

Norsharina

Maznah

. Toxicology study of vanillin on rats via oral and intra-peritoneal administration. Food Chem Toxicol 2011; 49: 25–30.

25.

Council of European Communities. Council instructions about the protection of living animals used in scientific investigations. Official Journal of the European Communities (JO 86/609/CEE) 1986; L358: 1–18.

26.

Bruckner

MacKenzie

Muralidhara

Luthra

Kyle

Acosta

. Oral toxicity of carbon tetrachloride: acute, subacute and subchronic studies in rats. Fundam Appl Toxicol 1986; 6: 16–34.

27.

Reznick

Packer

. Methods in enzymology. New York: Academic Press, 1994, p.357.

28.

Draper

Hadley

. Malondialdehyde determination as index of lipid peroxidation. Method Enzymol 1990; 86: 421–431.

29.

Beauchamp

Fridovich

. Superoxide dismutase: improved assays and an assay applicable to acrylamide gel. Anal Biochem 1971; 44: 276–287.

30.

Aebi

. Catalase in vitro. Method Enzymol 1984; 105: 121–126.

31.

Flohe

Gunzler

. Assays of glutathione peroxidase. Method Enzymol 1984; 105: 114–121.

32.

Habig

Pabst

Jokoby

. Glutathione-S-transferase: the first enzymatic step in mercapturic acid formation. J Biol Chem 1974; 249: 7130–7139.

33.

Jollow

Mitchell

Zamppaglione

Gillette

. Bromobenzene induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolites. Pharmacology 1974; 11: 1 51–157.

34.

Kanno

Shouji

Hirata

Asou

Ishikawa

. Effects of naringin on cytosine arabinoside (Ara-C)-induced cytotoxicity and apoptosis in P388 cells. Life Sci 2004; 75: 353–365.

35.

Banerjee

Seth

Bhattacharya

. Biochemical effects of some pesticides on lipid peroxidation and free radical scavengers. Toxicol Lett 1999; 107: 33–47.

36.

Banerjee

Seth

Ahmed

. Pesticides induced oxidative stress: perspectiveness and trends. Rev Environ Health 2001; 16: 1–40.

37.

Xavier

Rekha

Bairy

. Health perspective of pesticide exposure and dietary management. Malays J Nutr 2004; 10: 39–51.

38.

Ozturk

Ucar

Ozturk

Vardi

Batcioglu

. Carbon tetrachloride induced nephrotoxicity and protective effect of betaine in Sprague–Dawley rats. Urology 2003; 62: 353–356.

39.

Ogeturk

Kus

Colakoglu

Zararsiz

Ilhan

Sarsilmaz

. Caffeic acid phenyl ester protects kidney against carbon tetrachloride toxicity in rats. J Ethnopharmacol 2005; 97: 273–280.

40.

Tokyay

Kaya

Gur

Tuncel

Ozbek

Oztuk

. Prostaglandin synthetase inhibition reduces peritonitis induced early liver oxidant stress. Surg Today 1999; 29: 42–46.

41.

Kocic

Viahovic

Pavlovic

Kocic

Cvetkovict

Stojanovic

. The possible importance of the cation binding site for the oxidative modification of liver 5-nucleotidase. Arch Physiol Biochem 1998; 106: 91–99.

42.

Bhadauria

Nirala

. Reversal of acetaminophen induced subchronic hepatorenal injury by propolis extract in rats. Environ Toxicol Pharmacol 2009; 27: 17–25.

43.

Reiter

Tan

Osuna

Gitto

. Actions of melatonin in the reduction of oxidative stress. J Biomed Sci 2000; 7: 444–458.

44.

Kadiska

Gladen

Baird

Dikalov

Sohal

Hatch

. Biomarkers of oxidative stress study: are plasma antioxidants markers of CCl₄ poisoning? Free Radic Biol Med 2000; 28: 838–845.

45.

Ueda

Shah

. Tubular cell damage in acute renal failure-apoptosis, necrosis or both. Nephrol Dial Transplant 2000; 15: 318–323.

46.

Tai

Sawano

Yazama

Ito

. Evaluation of antioxidant activity of vanillin by using multiple antioxidant assays. Biochim Biophys Acta 2011; 1810: 170–177.

Carbon tetrachloride-induced nephrotoxicity and DNA damage in rats

Abstract

Keywords

Introduction

Materials and methods

CCl4 intoxication, vanillin pretreatment and samples collection

Determination of protein carbonyl content in kidney

Measurement of malondialdehyde in kidney

Antioxidant enzymes and glutathione assays in kidney

Total superoxide dismutase activity

Catalase activity

Glutathione peroxidase

Glutathione transferase

Glutathione levels

Estimation of urea, uric acid and creatinine

DNA fragmentation analysis

Histological examination

Statistical analysis

Results

Discussion

Footnotes

Acknowledgements

Funding

References

CCl₄ intoxication, vanillin pretreatment and samples collection