Cyclophosphamide-Induced Lipid Peroxidation and Changes in Cholesterol Content: Protective Role of Reduced Glutathione

1. Introduction

         Lipid peroxidation is a degenerative process that affects unsaturated membrane lipids under conditions of oxidative stress [1]. This complex process is believed to contribute to human aging and disease by disrupting the structural conformation, the packing of lipid components and ultimately the function of biological membranes. Oxidative stress may cause lipid peroxidation by damaging the DNA-sugar and oxidizing protein by introducing carbonyl group into the side chains of protein molecules [2, 3]. Reactive oxygen species and other pro-oxidants cause the decomposition of ω₃ and ω₆ polyunsaturated fatty acids of membrane phospholipids leading to the formation of aldehydic end products including malondialdehyde (MDA), 4-hydroxy-2-nonenals and 4-hydroxy-2-alkenals (HAKs) of different chain length. These aldehydic molecules have been considered as ultimate mediators of toxic effects elicited by oxidative stress occurring in biological membrane [4]. Oxidative stress in cells can be initiated by the addition of Fe²⁺ in the presence of dioxygen. This stress will result in lipid peroxidation and subsequent formation of lipid radicals [5, 6]. Free radical mediated oxidative stress results usually from deficient natural antioxidant defenses and act as a main factor in the pathophysiology of various diseases and ageing [7]. Various antioxidants and free radical scavengers have been suggested to be general cytoprotective agents of therapeutic benefit [8]. In case of reduced or impaired defense mechanism and excess generation of free radicals that are not counter balanced by endogenous antioxidant defense exogenously administered antioxidants have been proven useful to overcome oxidative damage [9]. Lipid peroxidation induction capacity of drugs may be related to their toxic potential as exemplified by adriamycin induced cardiotoxicity, which is occurred through free radical mediated process [10]. So the evaluation of antioxidants as suppressor of drug induced lipid peroxidation provides a scope of further investigation for their co-administration with drugs to reduce drug-induced toxicities that are possibly mediated by free radical mechanism.

Figure 1. Effects of reduced glutathione on cyclophosphamide-induced lipid peroxidation: changes in MDA profile in goat liver homogenates (in vitro) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione -treated samples.

      Cyclophosphamide, an alkylating agent widely used in cancer chemotherapy, is an inactive cytostatic, which is metabolized into active metabolites mainly in the liver. During bioactivation reactive oxygen species are also formed, which can modify the components of both healthy and neoplastic cell leading to decreased antioxidative capacity [11]. It has been reported that cyclophosphamide produces genotoxicity and oxidative stress in mice [12]and early lung injury in rats [13]. It also causes fatal cardiotoxicity [14]. Hemorrhagic cystitis is a major dose limiting side effect of cyclophosphamide [15]. It was also found that cyclophosphamide has the ability to produce male germ cell toxicity [16].

Figure 2. Effects of reduced glutathione on cyclophosphamide-induced lipid peroxidation: changes in MDA profile in rabbit blood (n vivo) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione -treated samples.

     Serum cholesterol or its fractions like low density lipoproteins (LDL), high density lipoproteins (HDL) content have been found responsible for many diseases. Cholesterol and lipoprotein levels correlate well with the risk of cardiovascular diseases [17]. Stress in the form of starvation was found to increase lipid peroxidation and alter lipid profile in rabbits [18].

Figure 3. Effects of reduced glutathione on cyclophosphamide-induced lipid peroxidation: changes in 4-HNE profile in goat liver homogenates (in vitro) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione-treated samples

     In view of the above findings and the ongoing search of the present authors for antioxidant that may reduce drug induced lipid peroxidation [19-22], the present work has been carried out in vitro and in vivo to evaluate the antiperoxidative potential of reduced glutathione on cyclophosphamide-induced lipid peroxidation and also to evaluate the effect of reduced glutathione on cyclophosphamide induced changes in cholesterol content in rabbit blood sample.

2. Materials and methods

2.1. Materials

            Thiobarbituric acid (TBA), 2,4-dinitrophenylhydrazine (DNPH), sodium nitrite and trichloroacetic acid (TCA) were purchased from Ranbaxy Fine Chemicals Ltd., New Delhi; 5, 5’ dithiobis-2-nitrobenzoic acid was from SRL Pvt. Ltd., Mumbai; Sulfanilamide was from SD Fine Chem. Ltd., Mumbai; N-naphthylethylenediamine dihydrochloride was from Loba Chemie Pvt. Ltd., Mumbai; 1,1,3,3-tetraethoxypropane, reduced glutathione were from Sigma Chemicals Co. St. Louis, MO, USA; The standard sample of 4-HNE was purchased from ICN Biomedicals INC., Ohio; Cyclophosphamide injection (Oncomide-200) (for in vivo work) was from Khandelwal Laboratories Pvt. Ltd., Mumbai, India and  pure cyclophosphamide (for in vitro work) was form Dabur Research Foundation, Ghaziabud , India. Cholesterol test kit was from Span Diagnostic Ltd., Surat, India. All other reagents were of analytical grade.

Figure 4. Effects of reduced glutathione on cyclophosphamide-induced lipid peroxidation: changes in 4-HNE profile in rabbit blood (in vivo) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione -treated samples.

2.2. Animals

        The in vitro drug-lipid interaction studies were performed using goat liver as lipid source. The liver was collected from Durgapur Municipal Corporation (DMC) approved outlet. Goat liver was selected because of its easy availability and close similarity with human liver in its lipid profile [23]. The in vivo drug-lipid interaction studies were carried out using white New Zealand rabbit (Oryctolagus caniculus) as experimental model. The in vivo animal experiment was carried out in accordance with the protocol of institutional animal ethics committee of Himalayan Pharmacy Institute, Majhitar, East Sikkim, India (sanctioned by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Animal Welfare Division, Govt. of India, Chennai-600041; Registration no of the institute 1028/C/07/CPCSEA). Normal healthy rabbits weighing 1.5-2.0 kg were taken for the study. All the animals were housed in normal ambient temperatures (25-29 °C) and acclimatized in the laboratory for at least 72 h. They were maintained on a standard laboratory diet and water at ad libitum.

 Figure 5. Effects cyclophosphamide on GSH content of goat liver homogenates (in vitro) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione -treated samples.

2.3. Preparation of tissue homogenate for in vitro lipid peroxidation studies

         Goat liver perfused with normal saline through hepatic portal vein was harvested and its lobes were briefly dried between filter papers to remove excess blood and thin cut with a heavy-duty blade. The small pieces were then transferred in a sterile vessel containing phosphate buffer (pH=7.4) solution. After draining the buffer solution as completely as possible, the liver was immediately grinded to make a tissue homogenate (1 g/ml) using freshly prepared phosphate buffer (pH=7.4). The homogenate was divided into four equal parts, which were then treated differently as mentioned below.

Figure 6. Effects cyclophosphamide on GSH level of rabbit blood (in vivo) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione -treated samples.

2.4. Incubation of tissue homogenate with drug and/or antioxidant for in vitro lipid peroxidation studies

         The tissue homogenate was divided into four parts of 50 ml each. The first portion was kept as control (C), while the second portion was treated with cyclophosphamide (D) at a concentration of 0.015 mg/g tissue homogenate. The third portion was treated with cyclophosphamide at a concentration of 0.015 mg/g tissue homogenate and reduced glutathione at a concentration of 0.05 mg/g tissue homogenate (DA) and the fourth one was treated with reduced glutathione alone at a concentration of 0.05 mg/g tissue homogenate (A). After treatment with cyclophosphamide and/or reduced glutathione, the liver homogenates were shaken for 2 h and incubated at 18±2 °C for a period of maximum 24 h for further work.

Figure 7. Effects of cyclophosphamide NO production in goat liver homogenates (in vitro) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione -treated samples.

2.5. Group division of rabbits for in vivo lipid peroxidation and cholesterol profile studies

          Sixty rabbits were divided into five sets. In each set there were twelve animals. Twelve animals were further subdivided into four groups. There were three animals in each group. The first group was control group (C), while the second group (D) was treated with cyclophosphamide intramuscularly at a dose of 15 mg/kg body weight. The third group (DA) was treated both with cyclophosphamide intramuscularly at a dose of 15 mg/kgbody weight and reduced glutathione intramuscularly at a dose of 50 mg/kgbody weight. The final group (A) received only an intramuscular injection of reduced glutathione at a dose of 50 mg/kg body weight.

2.6. Estimation of malondialdehyde (MDA) level from tissue homogenate/rabbit blood

           The extent of lipid peroxidation was measured in terms of malondialdehyde (MDA) content using thiobarbuturic acid (TBA) method [24]. The estimation was done at 2 and 24 h of incubation and repeated for five times. In each case three samples of 2.5 ml of incubation mixture or 2.5 ml of blood withdrawn from ear vein (xylene treated) of rabbits were treated with 2.5 ml of 10% (w/v) trichloroacetic acid (TCA) and centrifuged at room temperature at 3000 rpm for 30 min to precipitate protein. Then 2.5 ml of the supernatant was treated with 5 ml of 0.002 (M) TBA solutions and then volume was made up to 10 ml with distilled water. The mixture was heated on a boiling water bath for 30 min. Then tubes were cooled to a room temperature and the absorbance was measured at 530 nm against a TBA blank (prepared from 5 ml of TBA solution and 5 ml of distilled water) using Shimadju UV-1700 double beam spectrophotometer. The concentrations of MDA were determined from standard curve, which was constructed as follows. Different aliquots from standard 1,1,3,3-tetrahydroxypropane (TEP) solution were taken in graduated stopper test tubes and volume of each solution was made up to 5 ml. To each solution, 5 ml of TBA solution was added and the mixture was heated in a steam bath for 30 min. The solutions were cooled to a room temperature and their absorbances were measured at 530 nm against TBA as blank. By plotting absorbance against concentrations a straight line passing through the origin of grid was obtained. The best-fit equation is A = 0.006502M, where M=nanomoles of MDA, A=absorbance, r=0.9977, SEM=0.0117 and F=2029.42 (df=1, 9).

Figure 8. Effects of cyclophosphamide on NO production in rabbit blood (in vivo) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione -treated samples.

2.7. Estimation of 4-hydroxy-2-nonenal (4-HNE) level from tissue homogenate/rabbit blood

            The estimation was done at 2 and 24 h of incubation and it was repeated for five times. In each case three samples of 2 ml of incubation mixture or 2 ml of blood withdrawn from ear vein (xylene treated) of rabbits were treated with 1.5 ml of 10% (w/v) TCA solution and centrifuged at 3000 rpm for 30 min. Then 2 ml of the filtrate was treated with 1 ml of 2, 4-dinitrophenyl hydrazine (DNPH) (100 mg/100 ml of 0.5 M HCl) and kept for 1 h at room temperature. After that, the samples were extracted with hexane, and the extract was evaporated to dryness under argon at 40 °C. After cooling to a room temperature, 2 ml of methanol was added to each sample and the absorbance was measured at 350 nm against methanol as blank [26]. The values were determined from the standard curve. The standard calibration curve was drawn based on the following procedure. A series of dilutions of 4-HNE in different concentrations of solvent (phosphate buffer) were prepared. From each solution 2 ml of sample pipette out and transferred into stopper glass tube. DNPH solution (1 ml) was added to all of the samples and kept at room temperature for 1 h. Each sample was extracted with 2 ml of hexane for three times. All extracts were collected in stopper test tubes. After that extract was evaporated to dryness under argon at 40 °Cand the residue was reconstituted in 1 ml of methanol. The absorbance was measured at 350 nm using the 0 mM standard as blank. The best-fit equation is: Nanomoles of 4-HNE=(A₃₅₀-0.005603185)/0.003262215, where A₃₅₀=absorbance at 350 nm, r=0.999, SEM=0.007.

Figure 9. Effects of cyclophosphamide on total cholesterol in rabbit blood (in vivo) (n=5); D, DA & A indicate only cyclophosphamide-treated, cyclophosphamide & reduced glutathione -treated and only reduced glutathione -treated samples.

2.8. Estimation of reduced glutathione (GSH) level from tissue homogenate/rabbit blood

           Reduced glutathione (GSH) was measured in accordance with Ellman’s method [25]. The estimation was done at 2 and 24 h of incubation and repeated for five times. In each case, three samples of 1 ml of incubation mixture or 1 ml of blood withdrawn from ear vein (xylene treated) of rabbits were treated with 1 ml of 5% (w/v) TCA in 1 mM EDTA centrifuged at 2000 g for 10 min. After that, 1 ml of the filtrate was mixed with 5 ml of 0.1 M phosphate buffer (pH=8.0) and 0.4 ml of 5,5’-dithiobis-2-nitrobenzoic acid (0.01% in phosphate buffer pH=8.0) (DTNB) was added to it. The absorbances of the solutions were measured at 412 nm against blank (prepared from 6.0 ml of phosphate buffer and 0.4 ml of DTNB). The concentrations of reduced glutathione were determined from standard curve, which was constructed as follows. Different aliquots of standard reduced glutathione stock solution were taken in 10 ml volumetric flasks. To each solution 0.4 ml of DTNB solution was added and volume was adjusted up to the mark with phosphate buffer (pH=8.0). The absorbance of each solution was measured at 412 nm against a blank containing 9.6 ml of phosphate buffer (pH=8.0) and 0.4 ml DTNB solution. By plotting absorbance against concentration a straight line passing through the origin of grid was obtained. The best-fit equation was A=0.000495 M, where M=nanomoles of reduced glutathione, A=absorbance, r=0.9986, SEM=0.0021 and F=4405.78 (df=1, 12).

2.9. Estimation of nitric oxide (NO) level from tissue homogenate/rabbit blood

          The estimation was done at 2 and 24 h of incubation and it was repeated for five times. NO content was determined by reaction with Griess reagent. Griess reagent was prepared by mixing equal volumes of sulphanilamide (1% w/v in 3 N HCl) and (0.1% w/v N-naphthylethylenediamine dihydrochloride) [27]. In each case three samples of 4 ml of tissue homogenate or 4 ml of blood withdrawn from ear vein (xylene treated) of rabbits were treated with 2.5 ml of 10% (w/v) TCA solution and centrifuged at 3000 rpm for 30 min. Then 5 ml of the filtrate were treated with 0.5 ml Griess reagent. After 10 min the absorbances of the solutions were measured at 540 nm against blank (prepared from 5.0 ml of distilled water and 0.5 ml of Griess reagent). The values were calculated from standard curve, which was constructed as follows. Different aliquots from standard sodium nitrite solution were taken in 5 ml volumetric flasks. To each solution 0.5 ml of Griess reagent was added and volume was adjusted up to the mark with phosphate buffer. The absorbances of each solution were noted at 540 nm against a blank containing the buffer and Griess reagent. By plotting absorbance against concentration a straight line passing through the origin was obtained. The best-fit equation is A=0.015846 M, where M=nanomoles of NO, A=absorbance, r=0.9973, SEM=0.0033 and F=1960.35 (df=1, 9).

2.10. Estimation of total cholesterol and HDL-cholesterol from rabbit blood

The estimation of cholesterol content was determined in one step method [28] with the help of cholesterol test kit. The kit contain three reagents such as, reagent 1 (cholesterol reagent), reagent 2 (working cholesterol standard, 200%) and reagent 3 (precipitating reagent). The estimation was done at 2 and 24 h of incubation and it was repeated for five times. In each case there were three samples. After the specified h of incubation, 2 ml of blood was withdrawn from the ear vein of (xylene treated) rabbits. The blood samples were centrifuged at 2000 rpm for 15 min and the supernatant (plasma) was separated out. After that the following procedures were performed for the estimation of total cholesterol and high density lipoprotein cholesterol of the rabbit blood.

2.10.1. Total cholesterol

          The standard solution was prepared by mixing 3 ml of reagent 1 and 0.015 ml of reagent 2 and the test sample was prepared by mixing 3 ml of reagent 1 and 0.015 ml of supernatant in cleaned glass test tubes. Then both the tubes were shaken well and the tubes were immediately kept in the boiling water bath exactly for 90 s. Then tubes were cooled immediately at room temperature under running tap water. The optical density (O.D.) of Standard (S) and Test (T) were measured on the spectrophotometer at 560 nm against reagent 1 as blank. The Total Cholesterol (TC) was calculated by using the following formula

Total Cholesterol (mg/dl) = (O.D. of Test/O.D. of Standard) x 200

2.10.2. HDL cholesterol

Step-I 

            HDL- cholesterol separation: 0.2 ml of the supernatant was transferred into a centrifuge tube and to it 0.2 ml of reagent 3 was added. Then it was shaken well to mix properly and the tubes were kept at room temperature for 10 min. It was centrifuged at 2000 rpm for 15 min to obtain a clear supernatant.

Step-II

            HDL-cholesterol estimation: the test sample was prepared by mixing 3 ml of reagent 1 with 0.12 ml of the supernatant obtained from the step-I. The centrifuge tubes were shaken well and the tubes were kept in the boiling water bath exactly for 90 seconds. The tubes were cooled immediately at room temperature under running tap water. The O.D. of Standard (S) and Test (T) were measured at 560 nm against reagent 1 as blank. The content of HDL- Cholesterol was calculated by using the following formula:

HDL-Cholesterol (mg/dl) = (O.D. of Test/O.D. of Standard) x 50                       

2.11. Statistical analysis

      Both in vitro and in vivo model of experiments, interpretation of the result is supported by analysis of variance (ANOVA) and multiple comparison analysis using least significant different procedure [29, 30] was also performed on the percent changes data of various groups with respect to control group of corresponding time.

3. Results

       The percent changes in MDA, 4-HNE, GSH, NO, total cholesterol and HDL-cholesterol content of different samples at different time of incubation were calculated with respect to the control of the corresponding time of incubation and was considered as indicator of the extent of lipid and cholesterol peroxidation. Average of percent changes of five sets is shown in figures along with corresponding standard error of mean. The results of the studies on cyclophosphamide-induced lipid peroxidation both in vitro and in vivo model and its inhibition with reduced glutathione are shown in Figures 1 to 8. The results of the studies on cyclophosphamide-induced changes in cholesterol content (i.e. changes in Total Cholesterol and HDL-Cholesterol) and the effects of reduced glutathione on these changes are also shown in Figures 9 and 10.

         The data generated from both in vitro and in vivo models indicate that incubation of the liver homogenates or rabbit blood with cyclophosphamide resulted an increase in MDA and 4-HNE content (Figures 1 to 4) in comparison to control to a significant extent after incubation for varying period of time. These observations suggest that cyclophosphamide has the ability to induce lipid peroxidation process. So the lipid peroxidation induction capacity of the drug may be related to its toxic potential. But the MDA and 4-HNE contents were significantly reduced with respect to cyclophosphamide-treated group when the liver tissue homogenates or rabbit blood were treated with cyclophosphamide in combination with reduced glutathione. This implies that the reduced glutathione had the ability to suppress cyclophosphamide-induced lipid peroxidation. This may be due to the protective effects against free radicals that may have been generated within the system due to presence of cyclophosphamide. Again the liver tissue homogenates or rabbit blood were treated only with the reduced glutathione then the MDA and 4-HNE level were reduced in comparison to the control and the cyclophosphamide-treated group. This decrease may be due to the free radical scavenging property of the antioxidants.

          Figures 5 to 8 indicate that incubation of the liver tissue homogenates or rabbit blood with cyclophosphamide result in a decrease in GSH and NO content with respect control to a significant extent. The decrease in GSH and NO content was associated with an increase in lipid peroxidation. These observations also suggest lipid peroxidation induction potential of the cyclophosphamide. When the liver tissue homogenates or rabbit blood were treated with cyclophosphamide along with reduced glutathione then the GSH and NO levels were increased in comparison to cyclophosphamide-treated group of the corresponding time point. Again, when the liver tissue homogenates or rabbit blood were treated only with reduced glutathione the GSH and NO contents were also increased in comparison to the control samples. The increase in GSH and NO level suggest the antiperoxidative potential of reduced glutathione.

        It was observed from Figures 9 and 10 that rabbit treated with cyclophosphamide caused an increase in total cholesterol content with respect to corresponding control. But the HDL-cholesterol level was reduced in comparison to control group. These observations suggest that cyclophosphamide can change the cholesterol profile. It was further found that incubation of blood sample with cyclophosphamide and reduced glutathione produce a decrease/increase in total cholesterol / HDL-cholesterol content respectively in comparison to cyclophosphamide-treated group. Incubation of blood samples only with reduced glutathione also shows a tendency of decrease/increase in total cholesterol/HDL-cholesterol content, respectively in comparison to control and cyclophosphamide-treated group. These results suggest that reduced glutathione could inhibit cyclophosphamide-induced changes in cholesterol profile.

        To compare means of more than two samples, multiple comparison analysis along with analysis of variance was performed on the percent changes data with respect to corresponding h. It is seen that there are significant differences among various groups (F1) such as cyclophosphamide-treated, cyclophosphamide and reduced glutathione-treated and only reduced glutathione-treated. But within a particular group, differences (F2) are insignificant which shows that there is no statistical difference in animals in a particular group. It was found that the content of MDA/4-HNE in cyclophosphamide-treated group is statistically significantly different from the cyclophosphamide and reduced glutathione-treated group and only reduced glutathione-treated group in both in vitro and in vivo models. In case of GSH/NO content of in vitro model suggest that cyclophosphamide-treated group as well as cyclophosphamide and reduced glutathione-treated groups were statistically different from only reduced glutathione-treated group. But the GSH/NO content of in vivo model indicate that the cyclophosphamide-treated group is statistically significantly different from the cyclophosphamide and reduced glutathione-treated group as well as only reduced glutathione- treated group.

        The content of total cholesterol/HDL-cholesterol in cyclophosphamide-treated group, cyclophosphamide and reduced glutathione-treated group and only reduced glutathione-treated groups are statistically significantly different from each other with the exception of total cholesterol content (2 h) in cyclophosphamide-treated group which is statistically significantly different from the cyclophosphamide and reduced glutathione-treated group and only reduced glutathione- treated group.

4. Discussion

        The results from both in vitro and in vivo models presented in this work clearly demonstrate the lipid peroxidation induction potential of cyclophosphamide, which may be related to its toxic potential. This is an analogy to cardiotoxicity of doxorubicin [10] and indomethacin-induced gastric mucosal injury [31]. MDA is a highly reactive three-carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation and arachidonic acid metabolism [32]. Increase in the accumulation of MDA in cells can result into cellular degradation, some biochemical changes and even cell death [33]. 4-Hydroxy-2-nonenal (4-HNE), a lipid aldehydes that form due to lipid peroxidation occurring during episodes of oxidant stress, readily forms adducts with cellular proteins; these adducts can be assessed as a marker of oxidant stress in the form of lipid peroxidation [34]. 4-HNE can be produced from arachidonic acid, linolenic acid or their hydroperoxide in concentration of 1 mM to 5 nM in response to oxidative stress [35]. It can diffuse within or even escape from the cell and attack targets far from the site of the original free radical event [36]. 4-hydroxy-2-nonenal (HNE) can also modify and inactivate proteins and responsible for age related muscular degeneration [37]. So the decrease in MDA and 4-HNE content of liver tissue homogenates/rabbit blood, when treated with cyclophosphamide and reduced glutathione as well as only with reduced glutathione implies the free radical scavenging property of reduced glutathione. Glutathione is a small protein composed of three amino acids, such as cysteine, glutamic acid and glycine [38]. It is an important antioxidant and plays a very important role in the defence mechanism for tissue against the reactive oxygen species [39]. The depletion of GSH is associated with increase in lipid peroxidation. The decrease in GSH level may be a consequence of enhanced utilization of this compound by the antioxidant enzymes, glutathione peroxidase and glutathione-S-transferase. NO plays a very important role in host defense [40]. Nitric oxide has versatile role in biology because it can be a signaling molecule in vasodilatation [41-44], a toxin [44], a pro-oxidant [45] and a potential antioxidant [46-50]. So the increase in GSH and NO content of liver tissue homogenates or rabbit blood, when treated with drug and antioxidant as well as only with antioxidant implies the free radical scavenging activity of the antioxidant. It has been proposed that NO causes chain termination reactions during lipid peroxidation as observed in low-density lipoprotein oxidation as well as in chemical systems [48-50].

       Increase in total cholesterol level/decrease in HDL-cholesterol level in cyclophosphamide-treated group indicates that cyclophosphamide has the ability to change cholesterol profile may be by inducing oxidation of cholesterol. An increase in the lipid peroxidation level was observed in hyperlipidemic and hypercholesterolemic patients [51, 52]. But the increase in HDL-cholesterol level in cyclophosphamide along with reduced glutathione-treated group as well as only reduced glutathione-treated group implies that reduced glutathione and HDL-cholesterol has protective effect against lipid peroxidation. It was also reported that antioxidant like vitamin C prevents oxidation of LDL-cholesterol, decreases total and LDL-cholesterol and triglyceride and also raises HDL-cholesterol level [53-55].

        To explore possibility of any mathematical relationship between MDA/4-HNE /GSH/NO with total cholesterol/HDL-cholesterol and to interpret the data in a better way, regression analysis have been performed between % changes of MDA/4-HNE with % changes of total cholesterol and % changes of GSH/NO with % changes of HDL-cholesterol content of rabbit blood. The results (Table 1 in supplementary material section) indicates that for cyclophosphamide-treated group MDA/4-HNE with total cholesterol have a correlation coefficient 0.89/0.769 at 2 h of incubation and 0.778/0.308 at 24 h of incubation. So it can be said that MDA/4-HNE and total cholesterol have a direct correlation. It was also observed that MDA/4-HNE and total cholesterol have detrimental activity towards living system. Several studies also show that lipid peroxidation increases the total cholesterol level. In rabbits, on cholesterol feeding, increase in serum and blood MDA levels was noticed [56, 57]. The correlation coefficients between GSH and HDL-cholesterol for antioxidant treated group are 0.898 and 0.628 at 2 and 24 h of incubation, respectively. For NO and HDL-cholesterol, the correlation coefficients for drug-treated group are 0.842 and 0.576 at 2 and 24 h of incubation, where as for only antioxidant treated group the correlation coefficients are 0.968 and 0.218, respectively. There are also similar links between GSH/NO with HDL-cholesterol and they are capable of inhibiting cyclophosphamide-induced changes in lipid and cholesterol profile. The quality of some of the equations is not in the acceptable range, such is unavoidable while working directly with raw biological data.

5. Conclusion

        These findings from both in vitro as well as in vivo models indicate the lipid peroxidation induction potential of cyclophosphamide which may be related to its toxic potential. The results also suggest the antiperoxidative effects of reduced glutathione and demonstrate its potential to reduce cyclophosphamide-induced lipid peroxidation and thus to increase therapeutic index of the drug by way of reducing toxicity that may be mediated through free radical mechanisms. It is also observed that cyclophosphamide also has the ability to change the cholesterol profile and reduced glutathione has a protective effect on these changes. However a detailed study of total lipid profile is required in this regard.

Acknowledgements

         This work was supported by All India Council for Technical Education (AICTE), New Delhi, for a financial assistance to one of us (SR) under research promotion scheme (RPS, F.No.: 8023/BOR/RPS-176/2006-07). The authors also wish to thank Dabur Research Foundation, Ghaziabad (U.P), for the free gift sample of cyclophosphamide.