Protective effect of cactus cladode extract against cisplatin induced oxidative stress, genotoxicity and apoptosis in balb/c mice: combination with phytochemical composition
© Brahmi et al.; licensee BioMed Central Ltd. 2012
Received: 18 August 2011
Accepted: 21 June 2012
Published: 31 July 2012
Cis-Platinum (II) (cis-diammine dichloroplatinum; CDDP) is a potent antitumor compound widely used for the treatment of many malignancies. An important side-effect of CDDP is nephrotoxicity. The cytotoxic action of this drug is often thought to induce oxidative stress and be associated with its ability to bind DNA to form CDDP–DNA adducts and apoptosis in kidney cells. In this study, the protective effect of cactus cladode extract (CCE) against CDDP-induced oxidative stress and genotoxicity were investigated in mice. We also looked for levels of malondialdehyde (MDA), catalase activity, superoxide dismutase (SOD) activity, chromosome aberrations (CA) test, SOS Chromotest, expressions of p53, bax and bcl2 in kidney and we also analyzed several parameters of renal function markers toxicity such as serum biochemical analysis.
Adult, healthy balb/c (20–25 g) male mice aged of 4–5 weeks were pre-treated by intraperitonial administration of CCE (50 mg/Kg.b.w) for 2 weeks. Control animals were treated 3 days a week for 4 weeks by intraperitonial administration of 100 μg/Kg.b.w CDDP. Animals which treated by CDDP and CCE were divided into two groups: the first group was administrated CCE 2 hours before each treatment with CDDP 3 days a week for 4 weeks. The second group was administrated without pre-treatment with CCE but this extract was administrated 24 hours after each treatment with CDDP 3 days a week for 4 weeks.
Our results showed that CDDP induced significant alterations in all tested oxidative stress markers. In addition it induced CA in bone morrow cells, increased the expression of pro-apoptotic proteins p53 and bax and decreased the expression of anti-apoptotic protein bcl2 in kidney. On the other hand, CDDP significantly increased the levels of urea and creatinine and decreased the levels of albumin and total protein.The treatment of CCE before or after treatment with CDDP showed, (i) a total reduction of CDDP induced oxidative damage for all tested markers, (ii) an anti-genotoxic effect resulting in an efficient prevention of chromosomal aberrations compared to the group treated with CDDP alone (iii) restriction of the effect of CDDP by differential modulation of the expression of p53 which is decreased as well as its associated genes such as bax and bcl2, (iiii) restriction of serums levels of creatinine, urea, albumin and total protein resuming its values towards near normal levels of control.
We concluded that CCE is beneficial in CDDP-induced kidney dysfunction in mice via its anti-oxidant anti-genotoxic and anti-apoptotic properties against CDDP.
KeywordsCactus CDDP Genotoxicity Antioxidant Protective effect
CDDP (cis-dichlorodiammineplatinum (II), CDDP) is a synthetic anticancer drug extensively used clinically for the treatment of several human malignancies such as ovarian, testicular, bladder, head and neck, and uterine cervix carcinomas [1–3]. Various data indicate that CDDP induces oxidative stress, lipid peroxides  and DNA damage [5, 6]. Also, there is evidence suggesting that the generation of free radicals causes nephrotoxic effects by CDDP [7, 8]. There is a continuous search for agents that provide nephroprotection against CDDP and other platinum drugs; these include antioxidants, modulators of nitric oxide, diuretics, and cytoprotective and apoptotic agents . However, none of these were found to be suitable/safe for clinical use in protecting against CDDP-induced nephrotoxicity.
In the past few years, much interest has been centered on the role of naturally occurring dietary substances for the control and management of various chronic diseases [10, 11] such as cactus Opuntia ficus indica which grows all over the semiarid countries and is mainly cultivated for its fruit (cactus pear) and cladode which are rich in nutritional compounds . In Chinese medicine cactus pear is used against inflammation and snakebite . Different parts of Opuntia ficus-indica are used in the traditional medicine in several countries: the cladodes are utilized to reduce serum cholesterol level and blood pressure, for treatment of ulcers, rheumatic pain and kidney conditions . The fruits have shown antiulcerogenic  and neuroprotective activity .
But a few studies have examined the cytoprotective effect of cladodes that is why we chose CCE against toxicity of CDDP.
Taking into consideration the potential clinical use of CDDP and the numerous health benefits of CCE. The aim of the present study was to find out the eventual protective effect of CCE against CDDP-induced oxidative stress and genotoxicity and nephrotoxicity in vivo using balb/c mice. We evaluated the antioxidant and antigenotoxic potential CCE against CDDP. To this end we also measured (i) levels of MDA, level of catalase and SOD activity, evaluated (ii) chromosome aberrations, p53, bax and bcl2 protein expressions we also analyzed several parameters of renal function markers toxicity. It is also of interest to find whether there is any correlation between total phenolic and total flavonoid contents of plant extract and the different activities.
CDDP salt (cis-diamineplatinum (II) dichloride, CAS no. 15663-27-1) was purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). It was dissolved in water. Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate disodium salt (BCIP) were from Sigma Aldrich, France. Mouse monoclonal anti-p53, anti-bax and anti-bcl2 and the secondary antibody (phosphatase-conjugated) were from Invitrogen. All other chemicals used were of the highest grade available from commercial sources.
Extract of cactus cladodes
Young cactus cladodes of Opuntia ficus-indica (2–3 weeks of age) collected from the local area were washed with water chopped into small pieces and then pressed using a hand-press, homogenized with 10 mM Tris–HCl, pH 7.4 at 4°C and centrifuged 30 min at 3500 g at 4°C. The supernatant was collected and lyophilized. Prior to use, the lyophilized extract was dissolved in water.
Determination of total polyphenol and flavonoid contents
Determination of tannin content
where ε: molar extinction coefficient (l g-1 cm-1) of tannic acid (3.27 L g-1 cm-1).
DPPH radical scavenging assay
Radical scavenging activity (RSA) of the CCE was measured using the free radical α, α-diphenyl-b-picrylhydrazyl (DPPH) . According to the method, 0.1 g of the sample was extracted in 2.9 ml of methanol by centrifuging at 5000 rpm for 15 min. The content was filtered through Whatman No.1 filter paper. Methanolic DPPH (0.5 ml, 500 μM) was added to the tubes containing this supernatant and shaken vigorously. The tubes were incubated at room temperature for 45 min in the darkness. Vitamin E was used as a reference compound in the same concentration range as the test compounds.
When DPPH reacts with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in colour (from deep—violet to light—yellow) were measured at 515 nm on a UV/visible light spectrophotometer (Spectronic Genesys).
Animals and treatments
Adult, healthy balb/c (20–25 g) male mice aged of 4–5 weeks provided from an animal breeding centre (SEXAL St. Doulchard, France following the agreement of the Ethics Committee named National committee of Medical ethics CNEM, BP 74 - Pasteur Institute Tunis 1002 TUNISIA) were used. The animals were kept for acclimatization 1 week under constant conditions of temperature and a light/dark cycle of 12 h: 12 h. Animals had free access to standard granulated chow and drinking water.
All animals were divided in 8 groups of 6 animals per group and treated as follows:
Group 1: Mice given H2O (100 μl) by intraperitonial route (ip)
Group 2: Mice given CCE 50 mg/Kg b.w (ip) for 45 days.
Group 3: Mice given CDDP 100 μg/Kg b.w for15 days treatment.
Group 4: Mice are pre-treated with only CCE for 15 days and they given CDDP 100 μg/Kg b.w + CCE 50 mg/Kg b.w for other 15 days (second treatment with CCE is before 2 hours injection with CDDP).
Group 5: Mice are pre-treated with only CCE for 15 days and they given CDDP 100 μg/Kg b.w + CCE 50 mg/Kg b.w for other 15 days (second treatment with CCE is after 24 hours injection with CDDP). NB: Groups 6, 7 and 8 are not pre-treated with CCE
Group 6: Mice given CDDP 100 μg/Kg b.w for 30 days treatment
Group 7: Mice given CDDP 100 μg/Kg b.w + CCE 50 mg/Kg b.w (before 2 hours injection with CDDP for 30 days treatment)
Group 8: Mice given CDDP 100 μg/Kg b.w + CCE 50 mg/Kg b.w (after 24 hours injection with CDDP for 30 days treatment).
At the end of the experiment, animals were sacrificed under light ether anesthesia by decapitation and the kidneys were immediately removed.
Evaluation of lipid peroxidation status
Lipid peroxidation was determined indirectly by measuring the production of MDA in the renal extracts following the method of Aust et al. (1985) . Briefly, 200 μl of kidney extracts were mixed with 150 μl of TBS (Tris 50 mM and NaCl 150 mM, pH 7.4) and 250 μl TCA–BHT (20% TCA and BHT 1%). The mixture was vigorously vortexed and centrifuged at 1500 g for 10 min. 400 μl of the supernatant were added with HCl 0.6 N and 320 μl Tris-TBA (Tris 26 mM and TBA 120 mM), the content was mixed and incubated 10 min at 80°C. The absorbance was measured at 535 nm. The optic density corresponding to the complex formed with the TBA–MDA is proportional to the concentration of MDA and to the lipid peroxide. The concentration of μmol of MDA/mg of proteins is calculated from the absorbance at 530 nm using the molar extinction coefficient of MDA 1.56 × 105 M-1 cm-1.
Determination of catalase activity
Catalase activity was measured in the kidney extracts at 240 nm, according to Clairbone (1985) . Briefly, 20 μl of the extracts were added in the quartz cuvette contain 780 μl phosphate buffer and 200 μl of H2O2 0.5 M. The activity of catalase was calculated using the molar extinction coefficient (0.04 Mm-1 cm-1). The results were expressed as μmol of H2O2/min/mg of proteins.
Determination of SOD activity
Kidney tissue was homogenized with 10 volumes of ice-cold 1.15% KCl buffer containing 0.4 Mm PMSF and was centrifuged at 2000 rpm for 10 min (4°C). Total (Cu–Zn and Mn) SOD activity was determined according to Sun et al. (1988) . The method is based on the inhibition of nitro blue tetrazolium (NBT) reduction by the xanthine-xanthine oxidase system as a superoxide generator. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. SOD activity was also expressed as units per milligram protein (U/mg protein).
Chromosome aberration assay
24 hours before sacrifice, animals were given a suspension of yeast powder (100 mg/500 μl) to accelerate mitosis of bone-marrow cells. Vinblastine (200 μl; 250 μg/ml) was injected into the animals 45 min before sacrifice in order to block dividing cells in metaphasis. Bone-marrow cells from femurs and tibias were collected, subjected to hypotonic shock (KCl 0.075 M) and fixed three times using methanol-acetic acid . The cells were spread on glass slides that were blazed on a flame for 5s, then air-dried for conservation at room temperature and finally stained by 4% dilution of Giemsa reagent in water for 15 min. After coding of the slides, the chromosomes of 100 cells in metaphase were examined for abnormalities at a magnification of 1000× using an optical microscope (Carl Zeiss, Germany). This was done for each one of three replicates (300 metaphases per dose level) for negative controls, positive controls and treated groups. Chromosome aberrations were identified according to criteria described by Savage (1975) . Metaphases with chromosome breaks, gaps, rings and centric fusions (robertsonian translocation) were recorded and expressed as percentage of total metaphases per group.
The S9 microsome fraction was prepared from the liver of rats treated with Aroclor 1254 . The composition of the activation mixture is the following per 10 ml of S9 mix: salt solution (1.65 M KCl + 0.4 M MgCl2 6H2O) 0.2 ml; G6P (1 M) 0.05 ml; NADP (0.1 M) 0.15 ml; Tris buffer (0.4 M pH7.4) 2.5 ml; Luria broth medium 6.1 ml; S9 fraction 1 ml.
where IF1 is the induction factor in the presence of the test compound and the genotoxin, IF2 the induction factor in the absence of the test compound and in the presence of the genotoxin, and IF0 is the induction factor of the negative control. Data were collected as a mean ± S.D. of experiments.
Protein extraction and Western blot analysis
Equal amounts of proteins (20 μg) were separated by 12% SDS-polyacrylamide gel electrophoresis. Separated proteins were electro-blotted on nitrocellulose membrane in the transfer buffer (10 ml Tris-base, pH 8.3, 96 mM glycine and 10% methanol). The membrane was then blocked in TBS (20 mM Tris–HCl, Ph 7.5, 500 mM sodium chloride) containing 5% of BSA, washed in TBS (TBS containing 0.3% Tween 20) and probed with an antibody for p53 or bax or bcl2 at a 1:1000 dilution for 6 h at room temperature. The membrane was then washed and incubated with goat anti-mouse alkaline phosphate conjugated at a 1:3000 dilution for 1 h. Next, the membrane was washed and the chromogenic substrate BCIP/NBT was added to localize antibody binding. P53, bax and bcl2 levels were then determined by computer-assisted densitometric analysis (Densitometer, GS-800, BioRad Quantity One).
Creatinine, urea were performed spectrophotometrically using an autoanalyzer (Opera, Techicon, Bayer, USA). Total protein was determined in plasma samples by the Biuret method according to Gornall et al. (1949) .
Albumin concentration was determined by the method of Doumas et al. (1977) .
Experimental values are expressed as mean ± SD. Comparison of mean values between groups was performed by one way-analysis of variance (oneway-ANOVA) followed by post- hoc Tukey test. Expression of p53, bax and bcl2 were determined by Kruskal–Wallis Test. The level of significance was accepted with P<0.05 was used for statistical analysis.
Preliminary phytochemical analysis
Determination of total polyphenol, flavonoid and tannin contents
Quantitative polyphenols, flavonoids and tannins of cactus cladode extract
Cactus cladode extract
Total polyphenols μg (Gallic acid equivalents)
600,18 ± 6,1
Flavonoids μg (Quercetin equivalents)
223,04 ± 2,2
Tannins μg (Tannic acid equivalent)
53,33 ± 0,5
The radical scavenging activity was evaluated by the DPPH assay. DPPH is a molecule containing a stable free radical; In the presence of an antioxidant that can donate an electron to DPPH, the purple color typical of the free DPPH radical decays, a change that can be followed spectrophotometrically at 517 nm. This simple test can provide information on the ability of a compound to donate an electron, the number of electrons a given molecule can donate, and on the mechanism of antioxidant action.
Effect of CCE on oxidative stress induced by CDDP
Evaluation of lipid peroxidation status
Determination of catalase activity
Determination of SOD activity
Effect of CCE on DNA damage induced by CDDP
Eventual prevention of CDDP-induced chromosome aberrations by CCE
The SOS chromotest assay
Genotoxic activity of cactus cladode extract and CDDP by the SOS chromotest in the presence of E.coli PQ37
0,65 ± 0,001
1,9 ± 0,001
9,21 ± 0,005
2,5 ± 0,003
1,98 ± 0,004
2,5 ± 0,002
1,1 ± 0,002
1,7 ± 0,001
CDDP + CCE
1,56 ± 0,002
1,25 ± 0,002
Effect of CCE on apoptosis status
Determination of p53 expression
Determination of bax expression
Determination of bcl2 expression
Effect of CCE on CDDP-induced nephrotoxicity parameters in serum
Effect of CCE on several serum parameters with/without CDDP treatment
CDDP 15 days
CDDP + CCE (pre-treatment)
CDDP + CCE (post-treatment)
CDDP 30 days
CDDP + CCE (pre-treatment)
CDDP + CCE (post-treatment)
0,8 ± 0,14
0,75 ± 0,1
1,79 ± 0,0045*
0,56 ± 0,005*
0,88 ± 0,0048*
2,13 ± 0,005*
0,87 ± 0,0044
0,95 ± 0,0052
52,34 ± 0,12
51,54 ± 0,17
79,13 ± 0,12*
58,5 ± 0,14*
60,13 ± 0,19*
94,2 ± 0,12*
60,12 ± 0,18
68,5 ± 0,15
10,44 ± 0,14
10,72 ± 0,15
8,01 ± 0,17*
9,8 ± 0,14*
8,48 ± 0,18*
7,12 ± 0,16*
9,91 ± 0,12
9,24 ± 0,16
Total protein (g/dl)
9,95 ± 0,19
9,78 ± 0,21
7,12 ± 0,25*
8,67 ± 0,19*
8,03 ± 0,26*
6,2 ± 0,193*
8,7 ± 0,23
8,12 ± 0,26
CDDP is an extensively used anti-cancer agent for the management of germ cell tumors, head and neck cancers, bladder cancer, cervical cancer and as a salvage treatment for other solid tumors . Although higher doses of CDDP are more efficacious for the suppression of cancer but high dose therapy manifests irreversible renal dysfunction  and damage to non-tumor cells. The concept of cancer and chronic kidney diseases prevention using naturally occurring substances that can be included in the diet consumed by the human population is gaining increasing attention. In this line, different types of fruits and vegetables have been re-evaluated and recognized as valuable sources of nutraceuticals. Polyphenolic compounds are abundant in foods of plant origin. The application of such bioactive plant components may increase the stability of foods and, at the same time, improve their health properties associated with anti-cancer, antiallergic and anti-inflammatory activities of polyphenols in the human body [34–36].
The total polyphenol content of the cladode extracts from Opuntia ficus indica was expressed as gallic acid equivalents. The total flavonoids contents of the CCE is determined by using the method of Zhishen et al. (1999)  and expressed as quercetin equivalents [37, 38]. Significantly high total polyphenols and flavonoids content of the CCE may be corroborated with the observed antioxidant and antigenotoxic activities (Table 1).
The present study was performed to test the hypothesis that CCE would ameliorate CDDP induced oxidative stress and genotoxicity causes of nephrotoxic effect allowing the clinical use of CDDP in the treatment of various malignancies and minimizing its side effects. To this end, we evaluated the effect of pre or post-treatment by CCE 50 mg/kg b.w in balb/c mice. The intraperitonial route for administration of the CCE in this dose was chosen based on reports in our studies which have shown that after testing several doses of cactus this dose is appropriate to induce a good prevention against oxidative stress induced by mycotoxine zearalenone in balb/c mice [39, 40].
To evaluate the oxidative status, we looked for an eventual lipid peroxidation which constitutes one of the most common indices used to assess oxidative stress. MDA is the end product of lipoperoxydation, considered as a late biomarker of oxidative stress and cellular damage [41, 42]. In the present study, exposure to CDDP induces a marked increase in MDA formation in kidney but administration of CCE significantly reduced MDA level which dropped to the control level (Figure 2). Yuce et al. (2007) and Al-Majed et al. (2006) [43, 44] have also reported an increase in MDA and a decrease in the activities of antioxidant enzyme upon similar CDDP treatment of rats.
Also we looked for measured level of antioxidant enzymes catalase activity and SOD activity, were significantly (p<0.005) decreased compared to control (Figures 3 and 4); Yuce et al. (2007)  reported a similar decrease of antioxidant enzymes catalase and SOD by CDDP treatment in vivo. Also several investigators have demonstrated that CDDP induces ROS in renal epithelial cells primarily by decreasing the activity of antioxidant enzymes and by depleting intracellular concentrations of GSH, catalase and SOD activities [45, 46]. The presence of CCE with CDDP by pre or post treatment normalized the levels of the antioxidant enzyme catalase and SOD to nearly the normal values of control. CCE ability to prevent and protect against oxidative damage is certainly associated to the presence of several antioxidants such as ascorbic acid, flavonoids and phenolic acids actually detected in cladodes (Table 1) and in fruit [47–50].
Percentage of different type of chromosomal damage induced by CDDP and reversed with cactus cladodes extract before or after treatment with CDDP
1,5 ± 1,25
1,00 ± 0,43
3,28 ± 0,25
0 ± 0,00
3,78 ± 1,5
1,67 ± 0,75
1 ± 1,33
0 ± 0,00
1 ± 0,67
3,67 ± 1,78
CDDP 15 days
2,13 ± 0,28*
1 ± 0,17
10 ± 2,02*
2,13 ± 1,5*
15,26 ± 1,75*
CDDP + CCE Pre-treatment
1,66 ± 0,55*
0 ± 0,17
6 ± 1,68*
1 ± 0,24
8,66 ± 1,53*
CDDP + CCE Post-treatment
2,03 ± 1,5*
1 ± 0,33
7,3 ± 1,45*
0 ± 0,00
10,33 ± 2,06*
CDDP 30 days
5 ± 1,77*
2,66 ± 2,67*
20 ± 1,57*
5 ± 1,25*
32,66 ± 2,14*
CDDP + CCE Pre-treatment
3 ± 1,48*
1,33 ± 0,58*
5 ± 2,01*
1 ± 1,00*
10,33 ± 1,45*
CDDP + CCE Post-treatment
2 ± 1,78*
1 ± 0,25*
6 ± 0,05*
3,58 ± 1,45*
12,58 ± 1,67*
The absence of genotoxicity is not a characteristic of all natural products in use, since other medicinal plants, assayed with the SOS chromotest have resulted positively in genotoxicity . These tests showed that CDDP present a genotoxic effect and that the treatment with CCE is able to remove this genotoxicity (Table 2).
The preliminary chemical study of CCE of Opuntia ficus indica, revealed the presence of important quantities of polyphenol compounds flavonoids and tannins in aqueous extracts. These results could be correlated to the antigenotoxic activity detected in this extract. In fact the CCE showed significant anti-genotoxicity towards CDDP. This suggests that CCE inhibit microsomal activation or that they directly protect DNA strands from the electrophilic metabolite of the mutagen. They may inhibit several metabolic intermediates and reactive oxygen species (ROS) formed during the process of microsomal enzyme activation which are capable of breaking DNA strands. Anti-genotoxic activity of CCE may be ascribed to flavonoids  and tannins [56, 57] which are detected in our extract.
We cannot however, exclude the possibility that other compounds with anti-genotoxic properties, participate in the anti-genotoxic effect of CCE. On the other hand, CCE exhibited a significant antioxidant activity towards the free radical DPPH. These results were correlated with the chemical composition of these extract. In fact, the chemical study of CCE, revealed the presence of important quantities of flavonoids. We believe that flavonoids are the most likely candidates among the compounds known to be present CCE for preventing oxidative lesions and providing antigenotoxic effect [58, 59]. These compounds may inhibit free radicals and reactive oxygen species produced by CDDP.
The cytotoxicity of CDDP is believed to be due to the formation of DNA adducts, which include DNA-protein cross-links, DNA monoadducts, and interstrand and intrastrand DNA cross-links [60, 61]. Further studies demonstrated that the cytotoxicity of CDDP is probably due to a combination of insults, including mitochondrial dysfunction , inhibition of protein synthesis  and DNA injury . It has recently reported that DNA damage induced by CDDP leads to a rapid activation of ataxia telangiec- tasia and Rad3-related (ATR) which phosphorylates Chk2, a checkpoint kinase. ATR/Chk2 signaling is largely responsible for p53 phosphorylation and activation during CDDP treatment and the p53 protein binds DNA .
Many studies have now documented the rapid activation and nuclear translocation of p53 in response to CDDP both in kidneys  and in cultured renal proximal tubular cells . It is well known that both CDDP-induced DNA damage and CDDP-induced oxidant stress are potent activators of p53 [68, 69], and that p53 can in turn activate bax [70, 71]. It is therefore likely that this regulatory mechanism may play a crucial role in CDDP-induced apoptosis.
In the present study, the modulatory effect of CCE on CDDP toxicity was suggested to carry out through alterations in cell death pathway, p53 and the ratio of bax/bcl2 plays an important role in determining whether cells will undergo apoptosis. Our results showed that treatment by CDDP for 15 and 30 days induced high expressions of p53 and bax, an apoptotic marker in kidney tissues of CDDP treated mice than controls and down-regulation of antiapoptotic protein bcl2 (Figures 6a, 6b, 7a, 7b, 8a, 8b). Our study showed that the CCE treatment after or before CDDP treatment has been shown to induce an anti-apoptotic effect via inhibition of p53 and bax expression (Figure 6a, 6b, 7a, 7b and 8a, 8b). This indicates that CCE modulates the p53 dependent apoptotic pathway to restrict the CDDP toxicity in kidneys.
Kidneys represent the major control system maintaining body homeostasis. The plasma concentrations of urea and creatinine determine renal function and are thus biomarkers for kidney disease . Mice treated with CCE alone showed no significant change in the levels of urea and creatinine compared to control. While, CDDP treatment caused significant increase (p<0.05) in levels of both parameters accompanied by significant decrease in blood levels of albumin and total protein. The serum albumin concentration may be directly altered, as results to loss albumin through damaged glomeruli in case of renal failure . Consequently, in the present study, the significant decrease in albumin may be evidence on CDDP-induced nephrotoxicity. Mice exposed to CCE before or after CDDP exhibited a significant (p<0.05) decrease in the levels of urea and creatinine and increase levels of albumin and total protein; in fact, CCE seemed to restore serums levels of creatinine, urea, albumin and total protein resuming its values towards near normal levels of control (Table 3). The increase in both urea and creatinine levels due to CDDP treatment has been previously reported by Shemida et al. (2005), Iseri et al. (2007) and Mansour et al. (2002) [73–75]. Nephrotoxic damage by CDDP is indicated by increase in blood urea and creatinine levels. Excretion of CDDP is predominantly renal, and the kidney is considered to be the primary target organ for CDDP toxicity. Consequently, the impairment of kidney function by CDDP is recognized as the main side effect and the dose limiting factor associated with its use, occurring either acutely or after repeated treatment .
Total protein concentration is likely to be decreased if there is inhibition of protein synthesis or if degradation of protein is promoting . CDDP diminishes DNA, RNA and protein synthesis. Ribosomal DNA accumulates CDDP-induced DNA adducts. This is consistent with the CDDP- induced injury. Moreover, CDDP-induced transcription highjacking is another reason for the inhibition of protein synthesis associated with CDDP. Transcription highjacking refers to the consequences of the ability of certain transcription factors to bind to DNA adducts caused by organoplatinum compounds. This leads to the sequestration of these transcription factors from their usual promoter binding sites .
In agents in the treatment of cancer. In spite of its clinical usefulness, there are many occasions in which it is difficult to continue the administration of the drug due to its nephrotoxicity. In the present study, it is clear that CDDP exposure resulted oxidative stress, genomic DNA damage, apoptotic cell death in kidney, increased serum creatinine and blood urea, and decreased levels of albumin and total protein biomarkers for kidney disease. But CCE exposure prior and post to CDDP provided near complete protection in terms of generation of oxidative stress, genomic DNA integrity and modulate apoptosis status. According to our results we noticed a similar effect between the pre and post treatment for the antioxidant and antigenotoxic effects of CCE. Our results indicate that antioxidant of CCE would support biological resistance to free radicals, suggesting the capacity of this extract to play a role in antigenotoxic, anti-apoptotic and anti-nephrotoxic effects of CCE. The protective effect of CCE makes them promising candidates for further studies designed to obtain more evidence on their components with potential chemo-preventive activity.
This research was funded by the Tunisian Ministry of Scientific Research and Technology through the Laboratory for Research on Biologically Compatible Compounds (LRSBC), Faculty of Dentistry of Monastir and the Research Unit of Macromolecular Biochemistry and Genetics (BMG), Faculty of Sciences of Gafsa.
- Thigpen T, Vance R, Puneky L, Khansurt T: Chemotherapy in advanced ovarian carcinoma: current standard of care based on randomized trials. Gynecological Oncology. 1994, 55: 597-607.View ArticleGoogle Scholar
- Tikoo K, Bhatt DK, Gaikawad AB, Sharma V, Kabra DG: Differential effects of tannic acid on cisplatin induced nephrotoxicity in rats. FEBS Lett. 2007, 581: 2027-2035. 10.1016/j.febslet.2007.04.036.View ArticlePubMedGoogle Scholar
- Ajith TA, Usha S, Nivitha V: Ascorbic acid and a-tocopherol protect anticancer drug cisplatin induced nephrotoxicity in mice. A comparative study. Clin Chim Acta. 2007, 375: 82-86. 10.1016/j.cca.2006.06.011.View ArticlePubMedGoogle Scholar
- Matsushima H, Yonemura K, Ohishi K, Hishida A: The role of oxygen free radicals in cisplatin-induced acute renal failure in rats. J Lab Clin Med. 1998, 131: 518-526. 10.1016/S0022-2143(98)90060-9.View ArticlePubMedGoogle Scholar
- Lieberthal W, Triaca V, Levine J: Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis. AJP. 1996, 270: F700-F708.Google Scholar
- Badary OA, Abdel-Maksoud S, Ahmed WA, Owieda GH: Naringenin attenuates cisplatin nephrotoxicity in rats. Life Sci. 2005, 76: 2125-2135. 10.1016/j.lfs.2004.11.005.View ArticlePubMedGoogle Scholar
- Ishikawa M, Takayanagi Y, Sasaki K: Enhancement of cisplatin toxicity by buthionine sulfoximine, a glutathionedepleting agent, in mice. Res Comm Chem Pathol Pharmacol. 1990, 67: 131-141.Google Scholar
- Uslu R, Bonavida B: Involvement of the mitochondrion respiratory chain in the synergy achieved by treatment of human ovarian carcinoma cell lines with both tumor necrosis factor-alpha and cis diamminedichloroplatinum. Cancer. 1996, 77: 725-732. 10.1002/(SICI)1097-0142(19960215)77:4<725::AID-CNCR19>3.0.CO;2-2.View ArticlePubMedGoogle Scholar
- Ali BH, Al Moundhri SM: Agents ameliorating or augmenting the nephrotoxicity of cisplatin and other platinum compounds: a review of some recent research. Food Chem Toxicol. 2006, 44: 1173-1183. 10.1016/j.fct.2006.01.013.View ArticlePubMedGoogle Scholar
- Alschuler L: Green tea: healing tonic. Am J Nat Med. 1998, 5: 28-31.Google Scholar
- Connor WE: Importance of n-3 fatty acids in health and disease. Am J Clin Nutr. 2000, 71: 171S-175S.PubMedGoogle Scholar
- Stintzing FC, Herbach KM, Mosshammer MR, Carle R, Yi WG, Sellappan S, Akoh R, Bunch CC, Felke P: Color, betalain pattern, and antioxidant properties of cactus pear (Opuntia spp.) clones. J Agric Food Chem. 2005, 53: 442-451. 10.1021/jf048751y.View ArticlePubMedGoogle Scholar
- Zou DM, Brewer M, Garcia F, Feugang JM, Wang J, Zang R, Liu H, Zou C: Cactus pear: natural product in cancer chemoprevention. Nutr J. 2005, 4: 25-36. 10.1186/1475-2891-4-25.View ArticlePubMedPubMed CentralGoogle Scholar
- Agozzino P, Avellone G, Caraulo L, Ferrugia M, Flizzola F: Volatile profile of Sicilian prickly pear (Opuntia ficus-indica) by SPME-GC/MS analysis. Ital J Food Sci. 2005, 17: 341-348.Google Scholar
- Galati EM, Mondello MR, Giuffrida D, Dugo G, Miceli N, Pergolizzi S, Taviano MF: Chemical characterization and biological effects of Sicilian Opuntia ficus indica (L.) Mill. fruit juice: antioxidant and antiulcerogenic activity. J Agric Food Chem. 2003, 51: 4903-4908. 10.1021/jf030123d.View ArticlePubMedGoogle Scholar
- Dok-Go H, Lee KH, Kim HJ, Lee EH, Lee J, Song YS, Lee Y-H, Jin C, Lee YS, Cho J: Neuroprotective effects of antioxidative flavonoids, quercetin, (+)- dihydroquercetin and quercetin 3-methyl ether, isolated from Opuntia ficus indica var.saboten. Brain Res. 2003, 965: 130-136. 10.1016/S0006-8993(02)04150-1.View ArticlePubMedGoogle Scholar
- Yuan VY, Bone DE, Carrington F: Antioxidant activity of dulse (Palmaria palmata) extract evaluated in vitro. Food Chem. 2005, 91: 485-494. 10.1016/j.foodchem.2004.04.039.View ArticleGoogle Scholar
- Kumar A, Chattopadhyay S: DNA damage protecting activity and antioxidant potential of pudina extract. Food Chem. 2006, 100: 1377-1384.View ArticleGoogle Scholar
- Zhishen J, Mengcheng T, Jianming W: The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999, 64: 555-559. 10.1016/S0308-8146(98)00102-2.View ArticleGoogle Scholar
- Pearson D: The Chemical Analysis of Foods. 1976, London: Churchill Livingstone, 572-7Google Scholar
- Nwabueze TU: Effect of process variables on trypsin inhibitor activity (TIA) phytic acid and tannin content of extruded African breadfruit-corn-soy mixtures: a response surface analysis. Lebensm Wiss Technol. 2007, 40: 21-29. 10.1016/j.lwt.2005.10.004.View ArticleGoogle Scholar
- Blois MS: Antioxidant determination by the use of a stable free radical. Nature. 1958, 181: 1199-1200. 10.1038/1811199a0.View ArticleGoogle Scholar
- Aust SD, Morehouse LA, Thomas CE: Role of metals in oxygen radical reactions. J Free Radic Biol Med. 1985, 1: 3-25. 10.1016/0748-5514(85)90025-X.View ArticlePubMedGoogle Scholar
- Claiborne A: Catalase activity. Handbook of methods for oxygen research. Edited by: Greenwald RA. 1985, Boca Raton, Fla: CRC Press, 283-284.Google Scholar
- Sun Y, Oberley LW, Li Y: A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988, 34: 497-500.PubMedGoogle Scholar
- Evans EP, Breckon G, Ford CE: An air drying method for meiotic preparation from mammalian tests. Cytogenet. 1960, 3: 613-616.Google Scholar
- Savage JRK: Classification and relationships of induced chromosomal structural changes. J Med Genet. 1975, 12: 103-122.Google Scholar
- Maron DM, Ames BN: Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983, 113: 173-215. 10.1016/0165-1161(83)90010-9.View ArticlePubMedGoogle Scholar
- Quillardet P, Hofnung M: The SOS Chromotest, a colorimetric bacterial assay for genotoxins: procedures. Mutat Res. 1985, 147: 65-78. 10.1016/0165-1161(85)90020-2.View ArticlePubMedGoogle Scholar
- Gornall AG, Bardwill CS, David MM: Determination of serum proteins by means of biuret reaction. J Biol Chem. 1949, 177: 751-766.PubMedGoogle Scholar
- Doumas B, Tn Watson WA, Biggs HG: Albumin standards and measurement of serum albumin with bromocresol green. Clinica Chimica Acta. 1977, 31: 87-96.View ArticleGoogle Scholar
- Chester JD, Hall GD, Forster M, Protheroe AS: Systemic chemotherapy for patients with bladder cancer-current controversies and future directions. Cancer Treat Rev. 2004, 30: 343-358. 10.1016/j.ctrv.2003.12.005.View ArticlePubMedGoogle Scholar
- Taguchi T, Nazneen A, Abid MR, Razzaque MS: Cisplatin associated nephrotoxicity and pathological events. Contrib Nephrol. 2005, 148: 107-121.View ArticlePubMedGoogle Scholar
- Moure A, Jose M, Cruz D, Franco M, Domínguez J, Sineiro J, Domínguez H, Núñez MJ, Parajó JC: Natural antioxidants from residual sources. Food Chem. 2001, 72: 145-171. 10.1016/S0308-8146(00)00223-5.View ArticleGoogle Scholar
- Rice-Evans CA, Miller NJ, Paganga G: Structure antioxidant activity relationships of flavonoids and phenolic acids. Free Radic Biol Med. 1996, 20: 933-956. 10.1016/0891-5849(95)02227-9.View ArticlePubMedGoogle Scholar
- Capecka E, Mareczek A, Leja M: Antioxidant activity of fresh and dry herbs of some Lamiaceae species. Food Chem. 2005, 93: 223-226. 10.1016/j.foodchem.2004.09.020.View ArticleGoogle Scholar
- Luximon-Ramma A, Bahorun T, Soobrattee MA, Aruoma O: Antioxidant activities of phenolic, proanthocyanidin, and flavonoid components in extract of Cassia fistula. J Agric Food Chem. 2002, 50 (18): 5042-5047. 10.1021/jf0201172.View ArticlePubMedGoogle Scholar
- Eberhardt MV, Lee CY, Liu RH: Antioxidant activity of fresh apples. Nature. 2000, 405: 903-904.PubMedGoogle Scholar
- Zourgui L, Ayed-Boussema I, Ayed Y, Bacha H, Hassen W: The antigenotoxic activities of Cactus (Opuntia ficus indica) cladodes against the mycotoxin zearalenone in Balb/c mice Prevention of micronuclei, chromosome aberrations and DNA fragmentation. Food Chem Toxicol. 2009, 47: 662-667. 10.1016/j.fct.2008.12.031.View ArticleGoogle Scholar
- Zourgui L, Golli EE, Bouaziz C, Bacha H, Hassen W: Cactus (Opuntia ficus indica) Cladodes prevent oxidative damage induced by the mycotoxin zearalenone in Balb/c mice. Food Chem Toxicol. 2008, 46: 1817-1824. 10.1016/j.fct.2008.01.023.View ArticlePubMedGoogle Scholar
- Kim HS, Kwack SJ, Lee BM: Lipid peroxidation, antioxidant enzymes, and benzo[a]pyrene-quinones in the blood of rats treated with benzo[a]pyrene. Chem Biol Interact. 2000, 127: 139-150. 10.1016/S0009-2797(00)00177-0.View ArticlePubMedGoogle Scholar
- Dotan Y, Lichtenberg D, Pinchuk I: Lipid peroxidation cannot be used as a universal criterion of oxidative stress. Prog Lipid Res. 2004, 43: 200-227. 10.1016/j.plipres.2003.10.001.View ArticlePubMedGoogle Scholar
- Yuce A, Ates s ahin A, Ceribas AO, Aksakal M: Ellagic acid prevents cisplatin-induced Oxidative stress in liver and heart tissue of rats. Basic Clin Pharmacol Toxicol. 2007, 101: 345-349. 10.1111/j.1742-7843.2007.00129.x.View ArticlePubMedGoogle Scholar
- Al-Majed AA, Sayed-Ahmed AA, Al-Yahya AA, Aleisa AM, Al-Rejaie SS, Al Shabanah OA: Propionyl-l-carnitine prevents the progression of cisplatin induced cardiomyopathy in a carnitine-depleted rat model. Pharmacol Res. 2006, 3: 278-286.View ArticleGoogle Scholar
- Husain K, Morris C, Whitworth C, Trammell GL, Rybak LP: Somani SM Protection by ebselen against cisplatin-induced nephrotoxicity: antioxidant system. Mol Cell Biochem. 1998, 178: 127-133. 10.1023/A:1006889427520.View ArticlePubMedGoogle Scholar
- Huang Q, Dunn RT, Jayadev S, DiSorbo O, Pack FD, Farr SB, Stoll RE, Blanchard KT: Assessment of cisplatin-induced nephrotoxocity by microarray technology. Toxicol Sci. 2001, 63: 196-207. 10.1093/toxsci/63.2.196.View ArticlePubMedGoogle Scholar
- Kuti JO: Antioxidant compounds from four Opuntia cactus pear fruit varieties. Food Chem. 2004, 85: 527-533. 10.1016/S0308-8146(03)00184-5.View ArticleGoogle Scholar
- Tesoriere L, Fazzari M, Allegra M, Livrea MA: Biothiols, taurine, and lipid-soluble antioxidants in the edible pulp of Sicilian cactus pear (Opuntia ficus-indica) fruits and changes of bioactive juice components upon industrial processing. J Agric Food Chem. 2005, 20: 7851-7855.View ArticleGoogle Scholar
- Panico AM, Cardile V, Garufi F, Puglia C, Bonina F, Ronsisvalle G: Protective effects of Capparis spinosa on chondrocytes. Life Sci. 2005, 77: 2479-2488. 10.1016/j.lfs.2004.12.051.View ArticlePubMedGoogle Scholar
- Shim HC, Hwang HJ, Kang KJ, Lee BH: An antioxidative and anti inflammatory Agent for potential treatment of osteoarthritis from Ecklonia cava. Arch Pharm Res. 2006, 29: 165-171. 10.1007/BF02974279.View ArticleGoogle Scholar
- Albertini RJ, Ardell SK, Judice SA, Jacobson S, Allegretta M: Hypoxanthine–guanine phosphoribosyltransferase reporter gene mutation for analysis of in vivo clonal amplification in patients with HTLV type 1-associated Myelopathy/Tropical spastic paraparesis. AIDS Res Hum Retroviruses. 2000, 16: 1747-1752. 10.1089/08892220050193254.View ArticlePubMedGoogle Scholar
- Jamieson ER: Lippard SJ Structure, recognition, and proces- sing of cisplatin–DNA adducts. Chem Rev. 1999, 99: 2467-2498. 10.1021/cr980421n.View ArticlePubMedGoogle Scholar
- Goldstein RS, Mayor GH: The nephrotoxocity of cisplatin. Life Sci. 1983, 32: 685-690. 10.1016/0024-3205(83)90299-0.View ArticlePubMedGoogle Scholar
- De Carvalho MCRD, Barca FNTV, Agnez-Lima LF, de Medeiros SRB: Evaluation of mutagenic activity in an extract of pepper tree stem barks (Schinus terebinthifolius Raddi). Environ Mol Mutagen. 2003, 42: 185-191. 10.1002/em.10183.View ArticlePubMedGoogle Scholar
- Calomme M, Pieters L, Vlietink A, Berghe DV: Inhibition of bacterial mutagenesis flavonoids. Planta Med. 1996, 92: 222-226.View ArticleGoogle Scholar
- Lee KT, Sohn IC, Park HJ, Kim DW, Jung GO, Park KY: Essential moiety of antimutagenic and cytotoxic activity of hederagenin monodesmosides and bidesmosides isolated from the stem bark of Kalapanox pictus. Planta Med. 2000, 66: 329-332. 10.1055/s-2000-8539.View ArticlePubMedGoogle Scholar
- Baratto MC, Tattini M, Galardi C, Pinelli P, Romani A, Visioli F, Basosi R, Pongim R: Antioxidant activity of galloyl quinic derivatives isolated from P.lentiscus leaves. Free Radical Res. 2003, 37: 405-412. 10.1080/1071576031000068618.View ArticleGoogle Scholar
- Edenharder R, Grunhage D: Free radical scavenging abilities of flavonoids as mechanism of protection against mutagenicity induced by tertbutyl hydroperoxide or cumene hydroperoxide in Salmonella typhimurium TA102. Mutat Res. 2003, 540: 1-18. 10.1016/S1383-5718(03)00114-1.View ArticlePubMedGoogle Scholar
- Park KY, Jung GO, Lee KT, Choi J, Choi MY, Kim GT, Jung HJ, Park HJ: Antimutagenic activity of flavonoids from the heartwood of Rhus verniciflua. J Ethnopharmacol. 2004, 90: 73-79. 10.1016/j.jep.2003.09.043.View ArticlePubMedGoogle Scholar
- Eastman A: Reevaluation of interaction of cis-dichloro(ethylenediamine) platinum(II) with DNA. Biochemistry. 1986, 25: 3912-3915. 10.1021/bi00361a026.View ArticlePubMedGoogle Scholar
- Ingber DE: Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J Cell Sci. 1993, 104: 613-627.PubMedGoogle Scholar
- Brady HR, Kone BC, Stromski ME, Zeidel ML, Giebisch G, Gullans SR: Mitochondrial injury: an early event in cisplatin toxicity to renal proximal tubules. Am J Physiol. 1990, 258: F1181-F1187.PubMedGoogle Scholar
- Leibbrandt MEI, Wolfgang GHI, Metz AL, Ozobia AA, Haskins JR: Critical subcellular targets of cisplatin and related platinum analogs in rat renal proximal tubule cells. Kidney Int. 1995, 48: 761-770. 10.1038/ki.1995.348.View ArticlePubMedGoogle Scholar
- Kharbanda S, Ren R, Pandey P, Shafman TD, Feller SM, Weichselbaum RR, Kufe DW: Activation of the c-Abl tyrosine kinase in the stress response to DNA-damaging agents. Nature. 1995, 376: 785-788. 10.1038/376785a0.View ArticlePubMedGoogle Scholar
- Pabla N, Huang S, Mi QS, Daniel R, Dong Z: ATR-Chk2 signaling in p53 activation and DNAdamage response during cisplatin-induced apoptosis. J Biol Chem. 2008, 283: 6572-6583. 10.1074/jbc.M707568200. 6572View ArticlePubMedGoogle Scholar
- Miyaji T, Kato A, Yasuda H, Fujigaki Y, Hishida A: Role of the increase in p21 in cisplatin-induced acute renal failure in rats. J Am Soc Nephrol. 2001, 12: 900-908.PubMedGoogle Scholar
- Cummings BS, Schnellmann RG: Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways. J Pharmacol Exp Ther. 2002, 302: 8-17. 10.1124/jpet.302.1.8.View ArticlePubMedGoogle Scholar
- Muller M, Wilder S, Bannasch D, Israeli D, Lehlbach K, Li-Weber M, Friedman SL, Galle PR, Stremmel W, Oren M, Krammer PH: P53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. J Exp Med. 1998, 188: 2033-2045. 10.1084/jem.188.11.2033.View ArticlePubMedPubMed CentralGoogle Scholar
- Chandel NS, Vander Heiden MG, Thompson CB, Schumaker PT: Redox regulation of p53 during hypoxia. Oncogene. 2000, 19: 3840-3848. 10.1038/sj.onc.1203727.View ArticlePubMedGoogle Scholar
- Miyashita H, Nitta Y, Mori S, Kanzaki A, Nakayama K, Terada K, Sugiyama T, Kawamura H, Sato A, Morikawa H, Motegi K, Takebayashi Y: Expression of copper-transporting P-type adenosine triphosphatase (ATP7B) as a chemoresistance marker in human oral squamous cell carcinoma treated with cisplatin. Oral Oncol. 2003, 39: 157-162. 10.1016/S1368-8375(02)00038-6.View ArticlePubMedGoogle Scholar
- Burns TF, El-Deiry WS: The p53 pathway and apoptosis. J Cell Physiol. 1999, 181: 231-239. 10.1002/(SICI)1097-4652(199911)181:2<231::AID-JCP5>3.0.CO;2-L.View ArticlePubMedGoogle Scholar
- Venkatesan N, Punithavathi D, Arumugam V: Curcumin prevents adriamycin nephrotoxicity in rats Brit. J Pharmacol. 2000, 129: 231-234.Google Scholar
- Shemida Y, Hirotani Y, Akimoto YS, Ahindou K, Ijiri Y, Nishihori T, Tanaka K: Protective effects of capsaicin against cisplatin-induced nephrotoxicity in rats. Biol Pharm Bull. 2005, 28: 1635-1638. 10.1248/bpb.28.1635.View ArticleGoogle Scholar
- Iseri S, Ercan F, Gedik N, Yuksel M, Alican I: Simvastatin attenuates cisplatin-induced kidney and liver damage in rats. Toxicology. 2007, 230: 256-264. 10.1016/j.tox.2006.11.073.View ArticlePubMedGoogle Scholar
- Mansour MA, Mostafa AM, Nagi MN, Khattab MM, Al-Shabanah OA: Protective effect of aminoguanidine against nephrotoxicity induced by cisplatin in normal rats. Comp Biochem Physiol Toxicol Pharmacol. 2002, 2: 123-128.View ArticleGoogle Scholar
- Saad SY, Al-Rikabi AC: Protection effects of taurine supplementation against cisplatin-induced nephrotoxicity in rats. Chemotherapy. 2002, 48: 42-48. 10.1159/000048587.View ArticlePubMedGoogle Scholar
- Heidenreich O, Neininger A, Schratt G, Zinck R, Cahill MA, Engel K: MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo. J Biol Chem. 1999, 274: 14434-14443. 10.1074/jbc.274.20.14434.View ArticlePubMedGoogle Scholar
- Tarloff J, Lash L: Toxicology of the Kidney. 2004, Boca Raton: CRC Press, 3Google Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/111/prepub
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