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Effect of Dietary n-3 and n-6 Oils with and without Food Restriction on Activity of Antioxidant Enzymes and Lipid Peroxidation in Livers of Cyclophosphamide Treated Autoimmune-Prone NZB/W Female Mice

Department of Medicine, Division of Clinical Immunology, University of Texas Health Science Center, San Antonio, Texas

Address reprint requests to: Gabriel Fernandes, Ph.D., Division of Clinical Immunology, mail code 7874, Department of Medicine, The University of Texas Health Science Center 7703 Floyd Curl Drive, San Antonio, TX 78229-3900. E-mail: fernandes{at}uthscsa.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Objective: Cyclophosphamide (CTX), an alkylating agent, is extensively used in the treatment of lupus nephritis, but its administration has been associated with free radical mediated oxidative stress. The present study was designed to investigate the effect of dietary corn oil (CO), fish oil (FO) and food restriction (FR) on the activities of hepatic antioxidant enzymes, fatty acid composition and lipid peroxidation following CTX administration in autoimmune-prone NZB/W female mice.

Methods: Autoimmune-prone NZB/W female mice were fed either ad libitum (AL) or food restricted (60% of AL intake), semipurified diets containing 5% CO or 5% FO supplemented with equal levels of antioxidants and injected with either phosphate buffered saline (PBS), or CTX (50 mg/kg body weight) every 10 days. Proteinuria was measured biweekly. The treatment was stopped at 10 months and diets were continued until the mice were killed at 12 months. Fatty acid composition, activity of antioxidant enzymes and lipid peroxidation were analyzed in liver homogenates, and anti-DNA antibodies were analyzed in the serum.

Results: Mice in the FO/AL dietary group exhibited significantly higher liver catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities compared to the CO/AL dietary group. CTX significantly decreased SOD and GSH-Px activity in the FO/AL group and CAT and GSH-Px in the CO/AL group. In AL fed mice given CTX, activities of CAT, GSH-Px and GST were significantly higher in mice fed FO diets than in mice fed CO diets. FR increased the activity of enzymes in both the CO and FO diet groups. In FR mice, CTX decreased CAT and GSH-Px activity in both the CO and FO dietary groups, but glutathione S-transferase (GST) only in the CO group. The decrease in SOD activity was not significant in either of the restricted groups. CTX significantly increased generation of thiobarbituric acid reactive substances (TBARS) in both AL groups. FR significantly decreased lipid peroxidation in both the CO and FO groups, with or without CTX. CTX decreased serum anti-DNA antibody levels in both the CO and FO dietary groups. FR also decreased antibody titer in both the CO and FO dietary groups, and it was decreased further with CTX treatment. FO fed animals had higher levels of n-3 fatty acids, whereas CO fed animals had high levels of n-6 fatty acids. CTX significantly increased 20:4 and decreased 18:1 in CO/AL fed animals, whereas it increased 18:1 and decreased 22:6 in FO/AL fed animals.

Conclusions: Results obtained in the present study suggests that FO and, more significantly, FO combined with FR can have a beneficial effect in hepatic tissues subjected to CTX induced oxidative stress by regulating the activity of antioxidant enzymes. In addition, the study also indicates that n-3 and n-6 dietary lipids are susceptible to lipid peroxidation, particularly in the presence of a prooxidant like CTX, and that FR is beneficial in decreasing lipid peroxidation. The study also suggests that FO and CTX can have additive effects in preventing kidney disease in NZB/W mice.

Key words: cyclophosphamide, antioxidant enzymes, lipid peroxidation, fish oil, fatty acid composition, NZB/W mice, liver, anti-DNA antibodies

Abbreviations: AL = ad libitum • ANOVA = analysis of variance • B/W = NZB x NZW F1 • BCA = bicinchoninic acid • CAT = catalase • CDNB = 1-chloro-2,4-dinitrobenzene • CO = corn oil • CTX = cyclophosphamide • DHA = docosahexaenoic acid • EDTA = ethylenediamine tetra acetic acid • ELISA = enzyme linked immunosorbent assay • EPA = eicosapentaenoic acid • FID = flame ionization detector • FO = fish oil • FR = food restricted • GC = gas chromatography • GSH = glutathione • GSH-Px = glutathione peroxidase • GST = glutathione S-transferase • LPO = lipid peroxidation • MUFA = mono-unsaturated fatty acid • NADPH = nicotinamide adenine dinucleotide phosphate (reduced form) • PBS = phosphate buffered saline • PBS-T = PBS-Tween 20 • PUFA = polyunsaturated fatty acid • RA = rheumatoid arthritis • SEM = standard error of the mean • SLE = systemic lupus erythematosus • SOD = superoxide dismutase • SS = Sjogren’s syndrome • TBA = thiobarbituric acid • TBARS = TBA reactive substances

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Autoimmune-prone (NZB X NZW) F₁ (B/W) female mice develop an autoimmune disease, which resembles human systemic lupus erythematosus (SLE). MRL-lpr, BXSB and NZB/W F₁ mice serve as excellent models to study autoimmune diseases like rheumatoid arthritis (RA), SLE and Sjogren’s syndrome (SS). SLE is characterized by the presence of a heterogeneous group of autoantibodies cross reactive with DNA, phospholipids, cardiolipin, surface antigens, erythrocytes and ribonucleoproteins [1]. In particular, the NZB/W F₁ mouse model of SLE [2] has been used to study the effects of diet and immunosuppressive drugs on autoimmunity [3]. These mice begin to develop nephritis at five months of age and usually die at 6 to 12 months of age due to glomerular disease and renal failure.

Lupus nephritis is a major cause of mortality and morbidity among patients with SLE and also in (NZB X NZW) F₁ (B/W) lupus-prone mice [4]. Cyclophosphamide (CTX) is an alkylating agent and has been primarily used as an antineoplastic agent for the treatment of various forms of cancer, and as an immunosuppressive drug for certain non-neoplastic conditions, before organ transplantation [5,6] and also commonly in the treatment of lupus nephritis [7–16]. Earlier studies on murine models showed that administration of CTX retards the progression of kidney disease [4]. It is used extensively in patients with renal lupus for diffuse proliferative glomerulonephritis [4]. Although CTX has been shown to be beneficial in murine lupus, there has been concern about its side effects and degree of immunosuppression [4,17]. Besides its toxic effects, studies also indicate that CTX has a pro-oxidant character and its administration leads to generation of oxidative stress in liver, lungs and serum of mice and rats with a resulting decrease in the activities of antioxidant enzymes and increase in lipid peroxidation in these tissues [18–23]. These deleterious effects are significant side effects of CTX when used as a chemoprotective agent [24–28]. It is quite possible that free radical generation is one of the mechanisms by which CTX and its derivatives exert their toxic effects in different tissues.

Dietary fish oil decreases the risk of cardiovascular diseases, malignancy and autoimmune diseases [29–33]. Fish oil (FO) contains considerable amounts of long-chain polyunsaturated (n-3) fatty acids (in particular, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) whereas corn oil (CO) does not contain EPA or DHA, but is rich in (n-6) polyunsaturated fatty acids. Experiments carried out on NZB/W F1 female mice have indicated the beneficial effects of FO when substituted for CO in delaying the development of anti-DNA antibodies and extending life span in these autoimmune disease prone mice [34–36]. FO has also been shown to be particularly effective in ameliorating autoimmune diseases like RA [37], SLE [38,39] and SS [40]. Similar results have also been obtained using calorie restriction, prostaglandin E1 administration and hormonal therapy, and feeding diets deficient in the essential fatty acids, linoleic acid (18:26), linolenic acid (18:33) and arachidonic acid (20:46) [41]. Dietary alterations like calorie restriction and/or food restriction have proved effective in extending life span, lowering oxidative stress (which has been recognized as one of the major causes for aging), delaying/preventing disease development (in particular, autoimmune diseases) and reducing drug toxicity [42,43].

It has been well established that different dietary lipids in combination with food restriction (FR) have differential effects on activities of antioxidant enzymes, lipid peroxidation and tissue lipid profiles [44–50]. FR is known to prevent the decrease of antioxidant activity, especially during the aging process [49]. Studies published from this laboratory have shown that FO augments the activity of antioxidant enzymes and that these changes are not tissue specific [51,52]. In a recent study from our laboratory, FO/FR was shown to prolong life span and augment the activities of antioxidant enzymes in kidney tissues of autoimmune disease prone NZB/W female young and old mice [44]. One of our previous studies also indicated that FO increases the activities and mRNA levels of catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in liver tissues of autoimmune-prone mice [46].

Since CTX is extensively used for the treatment of lupus nephritis [53], we investigated whether treatment with CTX in autoimmune-prone B/W mice is associated with increased oxidative stress and whether dietary oils or dietary oils in combination with food restriction can prevent this phenomenon as well as further delay the onset of kidney disease. To explore this aspect, we studied the effect of corn oil or fish oil in combination with cyclophosphamide and food restriction on certain biochemical parameters, which are indicative of oxidative stress such as antioxidant enzymes and TBARS generation in liver tissues of NZB/W mice. We also studied the fatty acid composition of liver tissues from CTX treated and untreated mice.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Animals and Experimental Diets
Female NZB/W mice (age, two months) were purchased from Jackson Laboratories, Bar Harbor, ME. They were immediately divided into four dietary groups of 9–10 each fed semipurified AIN-76 diets containing either 5% CO—ad libitum (CO/AL) or food restricted (CO/FR) or 5% FO—ad libitum (FO/AL) or food restricted (FO/FR). In the FO diet groups, 1% CO was added to 4% FO to prevent essential fatty acid deficiency. The AL mice received 3 gm diet/day initially and increased to 4 gm/day, whereas the FR mice received 2 gm diet/day initially and increased to 2.4 gm/day (40% food restriction) at three months. The composition of the semipurified diet is provided in Table 1. Both the diets were supplemented with equal amounts of vitamin E to prevent peroxidative damage during storage [44]. The fatty acid composition of corn oil and fish oil is presented in Table 2. Fresh diet was prepared and provided daily and the animals were maintained on a 12/12 hour light/dark cycle. NIH guidelines provided under "The guide for the care and use of laboratory animals" were strictly followed and all studies were approved by the Institutional Laboratory Animal Care and Use Committee.

Collection of Blood and Tissues
The animals were killed by cervical dislocation; blood was collected and centrifuged at 10,000 g for 10 minutes at 4°C. Serum was stored at -70°C until analysis of anti-DNA antibodies. Livers were collected and immediately frozen in liquid nitrogen then stored at -80°C until the time of study.

Chemicals
Xanthine, xanthine oxidase, ferricytochrome C, ß-nicotinamide adenine dinucleotide phosphate (reduced form) (ß-NADPH), reduced glutathione, 30% hydrogen peroxide, glutathione reductase, 1-chloro-2,4-dinitrobenzene, cumene hydroperoxide, 1,1,3,3-tetraethoxypropane and thiobarbituric acid were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fatty acid methyl ester standards were purchased from Nu-Chek (Elysian, MN). All other chemicals were of analytical grade.

Preparation of Liver Homogenates
The livers were rinsed in ice-cold physiological saline and minced with scissors. 10% homogenates were prepared in 0.01 M Tris-HCl buffer (pH 7.4), centrifuged at 10,000 x g and the supernatant was used for antioxidant enzyme assays. For the determination of lipid peroxidation, liver tissues were homogenized in 1.15% KCl solution to obtain a 10% (w/v) homogenate. The protein content of liver homogenates was determined using the bicinchoninic acid (BCA) protein assay reagent (Pierce Chemical Company, Rockford, IL).

Fatty Acid Extraction and Analysis
Total lipid extraction of tissues was performed by the method of Bligh and Dyer [54] using chloroform-methanol (1:2) and chloroform-methanol-water (1:2:0.8). Extractions were performed in an atmosphere of nitrogen and butylated hydroxy toluene (BHT) was added to prevent oxidation during processing. The organic phase containing the total lipid extracts was evaporated under a stream of nitrogen and the residue methylated according to the method of Kates [55]. Fatty acid methyl esters were separated and quantified by gas-liquid chromatography using a Hewlett-Packard 5890A series II gas chromatograph, equipped with a capillary column (Supelco, Bellafonte, PA) and a flame ionization detector (FID). The injector and detector port temperatures were 225°C and 250°C, respectively. The oven temperature was maintained at 170°C for one minute and then increased to 215°C at the rate of 5°C/minute. Helium was used as the carrier gas. The running time of each sample was approximately 36 minutes. The fatty acid methyl esters were identified by comparison of retention times with fatty acid methyl ester standard (68A) from Nu Chek, Elysian, MN. Quantification was done by an integrator (Hewlett-Packard 3396 series II) attached to the GC machine.

Determination of Lipid Peroxidation (LPO) in Liver Tissues
Thiobarbituric acid reactive substances (TBARS) were measured by a modification of the method of Ohkawa et al. [56]. For each sample to be assayed, four tubes were set up containing 100, 150, 200 and 250 µL of tissue homogenate, 100 µL of 8.1% SDS, 750 µL of 20% acetic acid, and 750 µL of 0.8% aqueous solution of TBA. The volume was made up to 4 mL with distilled water, mixed thoroughly and heated at 95°C for 60 minutes. After cooling, 4 mL of n-butanol was added to each tube and the contents mixed thoroughly then centrifuged at 3000 rpm for 10 minutes. The absorption of the clear upper (n-butanol) layer was read at 532 nm. 1, 1, 3, 3 tetraethoxy propane (97%) was used as the external standard. Results are expressed as nmoles TBARS/mg tissue.

Catalase Activity (CAT)
The activity of CAT was measured using its peroxidatic function according to the method of Johansson and Borg [57]. Briefly, 50 µL potassium phosphate buffer (250 mM, pH 7.0) was incubated with 50 µL methanol and 10 µL hydrogen peroxide (0.27%). The reaction was initiated by addition of 100 µL sample with continuous shaking at room temperature (20°C). After 20 minutes, reaction was terminated by addition of 50 µL 7.8 M potassium hydroxide. Immediately 100 µL of purpald (4-Amino-3-hydrazino-5-mercapto-1,2,4-triazole, 34.2 mM in 480 mM HCl) was added and the mixture was again incubated for 10 minutes at 20°C with continuous shaking. Potassium periodate (50 µL 65.2 mM) was added to obtain a colored compound. The absorbance was read at 550 nm in a spectrophotometer. Results are expressed as micromoles of formaldehyde produced/mg protein.

Superoxide Dismutase Activity (SOD)
SOD activity was determined by the inhibition of cytochrome C reduction by the method of Flohe and Otting [58]. The reduction of cytochrome C was mediated by superoxide anions generated by xanthine/xanthine oxidase system and monitored at 550 nm. One unit of SOD was defined as the amount of enzyme required to inhibit the rate of cytochrome C reduction by 50%. Results are expressed as units/mg protein.

Glutathione Peroxidase Activity (GSH-Px)
GSH-Px activity was measured by NADPH oxidation using a coupled reaction system consisting of glutathione, glutathione reductase, and cumene hydroperoxide [59]. Briefly, 100 µl of enzyme sample was incubated for five minutes with 1.55 ml stock solution (prepared in 50 mM Tris buffer, pH 7.6 with 0.1 mM EDTA) containing 0.25 mM GSH, 0.12 mM NADPH and 1 unit glutathione reductase. The reaction was initiated by adding 50 µL of cumene hydroperoxide (1 mg/mL) and the rate of disappearance of NADPH with time was determined by monitoring absorbance at 340 nm. One unit of enzyme activity is defined as the amount of enzyme that transforms 1 µmol of NADPH to NADP per minute. Results are expressed in units/mg protein.

Glutathione-S-transferase Activity (GST)
GST activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate [60]. To 100 µL of 0.5 M phosphate buffer, 100 µL homogenate, 1.7 mL of water and 100 µL of 30 mM (CDNB) were added and incubated at 37°C for 15 minutes. After incubation, 100 µL of 30 mM reduced glutathione was added. The increase in absorbance with time was recorded at 340 nm. The enzyme activity is expressed as micromoles of CDNB-GSH conjugate formed/min/mg protein.

Measurement of Serum Anti-dsDNA Antibodies
Serum anti-ds DNA antibodies were determined by ELISA as previously described [61]. Costar brand 96 well microtiter plates were coated with calf thymus DNA (sigma), sealed and incubated overnight at 4°C to improve anti-ds DNA antigen binding. The next day, plates were washed three times with PBS-T (phosphate-buffered saline (PBS-0.01 M, pH 7.3)-0.05% Tween-20) wash buffer and blotted dry. Non-specific sites on the plate were blocked with PBS-1% BSA by incubating for two hours at 37°C. Plates were again washed four times with PBS-T wash buffer and blotted dry. Appropriately diluted standard and samples at different concentrations were added in duplicate to the wells (final volume 100 µL) and incubated at 37°C for two hours. Plates were again washed with PBS-T wash buffer and blotted dry. Next, alkaline phosphatase labeled secondary antibody (100 µL) was added to each well and incubated overnight at 4°C. Plates were again washed with PBS-T wash buffer and blotted dry. Phosphatase substrate (Sigma diagnostic) (100 µL) was added to each well. Color developed in 5 to 10 minutes at room temperature, which was read at 410 nm using a microplate reader. Absorbance of samples and standards were noted and corrected for by subtracting background from mean values of samples and standards.

Proteinuria
Proteinuria levels in urine were monitored biweekly using chemstrips (Roche Diagnostic Corporation, Indianapolis, IN) till the time of killing at 12 months.

Statistical Analysis
Results are expressed as mean ± SEM. Data were statistically analyzed using one-way ANOVA and p < 0.05 for F ratio was considered statistically significant. Newman-Keuls multiple comparison test was used to test the differences between groups for significance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Survival and Proteinuria Levels in Urine
Proteinuria levels for the different dietary groups have been summarized in Table 3. The mice fed 5% CO/AL developed proteinuria much earlier than mice fed 5% FO/AL. However, both CO/FR and FO/FR dietary groups started developing proteinuria by 10 months of age. There was 100% survival in FO/AL + CTX, CO/FR, FO/FR and FO/FR + CTX dietary groups. FO and CTX seemed to have a synergistic effect on proteinuria.

The effects of dietary lipids, FR and CTX on total saturates, MUFAs, PUFAs, polyunsaturates/saturates (P/S) ratio, n-6/n-3 ratio and peroxidizibility index in rat liver total lipids are presented in Table 5. Analyzing using ANOVA for main effects indicated that MUFAs, P/S, n-6, n-6/n-3 were significantly higher and saturates, n-3, and peroxidation index were significantly lower in CO fed mice compared to FO fed mice. Dietary corn oil or fish oil had no overall effect on PUFA levels. All the variables except MUFAs and n-6/n-3 were significantly higher in food-restricted mice when compared to ad libitum mice. CTX had no effect on any of the variables studied although the interaction of FR/CTX was significant for PUFA and P/S levels.

View larger version (35K): [in this window] [in a new window]   Fig. 1. Effect of dietary lipids and food restriction on liver catalase activity in cyclophosphamide treated and untreated mice. Values (mean ± SEM, 3–5 mice/group) with different superscripts are significantly different at p < 0.05. CO = corn oil, FO = fish oil, CTX = cyclophosphamide, AL = ad libitum, FR = food restriction.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Effect of dietary lipids and food restriction on liver superoxide dismutase activity in cyclophosphamide treated and untreated mice. Values (mean ± SEM, 3–5 mice/group) with different superscripts are significantly different at p < 0.05. CO = corn oil, FO = fish oil, CTX = cyclophosphamide; AL = ad libitum, FR = food restriction.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Effect of dietary lipids and food restriction on liver glutathione peroxidase activity in cyclophosphamide treated and untreated mice. Values (mean ± SEM, 3–5 mice/group) with different superscripts are significantly different at p < 0.05. CO = corn oil, FO = fish oil, CTX = cyclophosphamide, AL = ad libitum, FR = food restriction.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Effect of dietary lipids and food restriction on liver glutathione transferase activity in cyclophosphamide treated and untreated mice. Values (mean ± SEM, 3–5 mice/group) with different superscripts are significantly different at p < 0.05. CO = corn oil, FO = fish oil, CTX = cyclophosphamide, AL = ad libitum, FR = food restriction.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Effect of dietary lipids and food restriction on liver TBARS generation in cyclophosphamide treated and untreated mice. Values (mean ± SEM, 3–5 mice/group) with different superscripts are significantly different at p < 0.05. CO = corn oil, FO = fish oil, CTX = cyclophosphamide, AL = ad libitum, FR = food restriction.

View larger version (23K): [in this window] [in a new window]   Fig. 6. Effect of dietary lipids and food restriction on serum anti-dsDNA antibody levels in cyclophosphamide treated and untreated mice. Values (mean ± SEM, 3–5 mice/group) with different superscripts are significantly different at p < 0.05. CO = corn oil, FO = fish oil, CTX = cyclophosphamide, AL = ad libitum, FR = food restriction.

	DISCUSSION

Antioxidants form an important part of a cells defense against free radical damage. Antioxidant enzymes, in particular, constitute a major part of this defense. SOD detoxifies the superoxide radicals giving rise to hydrogen peroxide (H₂O₂) and is the only known enzyme that uses free radicals as a substrate. However, H₂O₂ is itself a potent free radical generator and can generate toxic hydroxyl radicals by reacting with ferrous ions, which can induce lipid peroxidation of cell membranes. Cellular CAT and GSH-Px detoxify H₂O₂. It is important that an enhanced SOD activity be followed up by increased activities of CAT and GSH-Px to prevent accumulation of toxic H₂O₂ [67]. In addition to these enzymes, the glutathione transferases are a group of isoenzymes capable of detoxifying exogenous and endogenous substances in conjugation with glutathione including organic hydroperoxides.

Lipid peroxidation is widely used as an indicator to reflect oxidative stress and cell membrane damage [68]. Free radicals like superoxide anion and hydroxyl radical exert their toxic effect by acting on DNA, membrane proteins and lipids. Anticancer drugs like cyclophosphamide are known to exert their cytotoxic effects by a free radical mediated mechanism [69]. While such reactions might prove beneficial in chemotherapy, they will result in the build up of free radicals in normal animals and lead to membrane damage and inactivation or alteration of membrane bound enzymes. Feeding dietary oils rich in unsaturated fatty acids makes target tissues such as kidney more vulnerable to peroxidative damage [70]. This is reflected in higher LPO production in livers of animals fed these diets. However, in this study the TBARS levels were similar in both CO/AL and FO/AL groups. The high level of dietary vitamin E used in this study may have protected the liver tissues from peroxidative damage [70]. However, LPO significantly increased in the FO/AL + CTX group in comparison to the CO/AL + CTX group. This finding probably reflects the increased susceptibility of FO in the presence of a pro-oxidant like CTX because of its high polyunsaturated fatty acid content. A decrease in polyunsaturated fatty acids in hepatic tissues (in particular, 22:63) in the FO/AL + CTX group possibly reflects the above observation. FR decreased level of TBARS in FO fed group as compared to CO fed group. FR significantly decreased LPO in both the CTX treated dietary groups. This indicates the beneficial effect of FR in preventing free radical induced peroxidative damage in both the treatment groups. However, FO/FR and FO/FR + CTX had much lower TBARS values compared to corresponding groups in CO indicating enhanced effect of FR in the FO dietary groups. In this study, activity of GSH-Px was higher in the FO treated groups. GSH-Px works in conjugation with reduced glutathione and is known to scavenge H₂O₂ and lipid peroxides [71]. Although reduced glutathione activity was not measured in the present study, an enhanced GSH-Px activity is likely to counter increased LPO in the FO/AL + CTX group.

In the present study, we attempted to examine the combined effects of CO or FO with FR and CTX therapy on the activities of antioxidant enzymes, the extent of lipid peroxidation and changes in fatty acid composition in liver in an animal model of lupus nephritis. CTX is extensively used for the treatment of lupus nephritis. However, studies in animal models other than lupus nephritis show that CTX administration may be toxic and is associated with considerable lipid peroxidation in serum and hepatic tissues and induces lung injury [22–30]. Moreover, there is decrease in the activities of hepatic antioxidant enzymes following its administration. This can eventually lead to a build up of oxidative stress and could cause tissue damage. In one such study, a one-time administration of cyclophosphamide (40 mg/kg, i.p.) induced significant oxidative stress and decreased hepatic levels of SOD, CAT, GSH-Px, reduced glutathione and GST enzymes and increased LPO in liver of mice [23]. Other studies involving CTX administration over a period of time have cited similar results. However, reports of the effect of CTX on GST and glutathione reductase (GR) have varied between studies with one study indicating no change [25] and others indicating significant decrease with treatment [18,23]. The total period of drug administration and the dosage itself seem to regulate the activity of antioxidant enzymes. In the present study, we found CTX to be associated with increased lipid peroxidation in both the dietary groups. However, the intensity of lipid peroxidation seems to be determined by the type of dietary fat and the duration of CTX administration.

CTX is not cytotoxic by itself but undergoes activation by cytochrome P450 in the liver [69] to phosphoramide mustard (also an alkylating agent) and acrolein. Molecules such as proteins, membrane lipids and RNA may interact with either of these metabolites resulting in production of unstable reactive oxygen species (ROS). This raises the possibility that effects of food restriction on CTX activity may be attributed to changes in cytochrome P450 levels in food restricted animals. Food restriction was reported to have no effect on cytochrome P450 levels in mice [72] in one study, and others have reported that cytochrome P450 patterns change with age but are maintained closer to the young profile in FR mice [73].

Fish oil combined with food restriction has been shown previously to enhance the activity of antioxidant enzymes. An earlier study from this laboratory showed that FO supplementation significantly increases the activities and mRNA expression of CAT, SOD and GSH-Px enzymes in autoimmune-prone NZB/W mice [46]. In that study, it was proposed that one of the mechanisms by which n-3 lipids delay onset of autoimmune diseases in this autoimmune-prone strain of mice may be the maintenance of higher activity and expression of hepatic antioxidant enzymes. Omega-3 fatty acids at hypotriglyceridemic doses are known to enhance the activities of the hepatic antioxidant enzymes SOD, CAT, GSH-Px and GST [74]. Fish oil n-3 fatty acids have been shown to enhance antioxidant activity in other tissues as well indicating that their effects are not tissue specific [51,52]. In a more recent study from this laboratory, FO and FO/FR were shown to significantly increase the activities of SOD, CAT and GSH-Px but not GST, compared to CO and CO/FR in kidney tissues of four-month and eight-month-old lupus prone B/W mice [44].

CTX did not reverse proteinuria in CO/AL fed B/W mice but FO and CTX seemed to have additive effects in preventing proteinuria. This finding supports a previous study where CTX failed to reverse proteinuria but reduced anti-ds DNA antibody titer in B/W mice [4]. Maintenance of low proteinuria levels in FO/FR + CTX and CO/FR + CTX groups seems to be more an effect of FR rather than CTX. Anti-ds DNA antibody titers serve as markers to evaluate the onset and severity of renal disease. FO and CTX are both known to lower serum levels of anti-ds DNA antibody levels in B/W mice. In this study, FO/AL dietary group had higher antibody titer than CO/AL treatment group. CTX treatment significantly decreased the antibody titer in both the dietary groups. However, the difference between the two groups was not statistically significant. In the case of the food-restricted diets, CO/FR and FO/FR had lower anti-dsDNA antibody titers than the corresponding AL groups. Anti-dsDNA antibody titer decreased further with CTX in both the groups, although the results were again not statistically significant.

It is firmly established that fatty acid compositions of different tissues reflect the fatty acid composition of dietary lipids [70]. Corn oil fed mice exhibited higher hepatic levels of n-6 fatty acids, whereas fish oil fed mice had higher n-3 fatty acids. This can be attributed to presence of high levels of 18:2n-6 in corn oil and high levels of n-3 fatty acids in fish oil. These findings are in accordance with previously published reports [70]. CO/AL had higher levels of 18:26 and 20:46, and low level of 22:63 compared to FO/AL in liver tissues. The percentages of 20:46 and 22:63 increased in CO/AL + CTX group but decreased in the FO/AL + CTX group possibly due to elevated LPO in the fish oil group. Lower levels of 18:2n-6 and 20:4n-6 in FO/AL and FO/AL + CTX groups can result in lower prostaglandin E₂ levels, which can alleviate the pro-inflammatory immune response [45]. Furthermore, the 20:5n-3 level was unaffected by CTX administration and can contribute towards lowering of inflammatory mediators during the autoimmune process [45]. Elevated levels of 20:46 and 22:63 were observed in both CO/FR and FO/FR groups, which was indicative of diminished susceptibility to oxidative stress. There was a significant increase in EPA and DHA levels in the FO/FR + CTX group compared to rest of the FO groups indicating that FR significantly reduced LPO associated with FO + CTX intake. This was clearly established by a decreased production of TBARS in this group of mice.

	CONCLUSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
Based on the results obtained, we can conclude that although CTX prevents renal disease in B/W mice, its administration is associated with oxidative stress in hepatic tissues in the presence of n-6 fatty acids. However, FO/AL and more significantly, FO/FR can prevent this effect by regulating the activities of hepatic antioxidant enzymes. Furthermore, it can be concluded that the type of dietary oil, whether or not food intake is restricted and whether or not CTX is given, can differentially affect the fatty acid composition of hepatic tissues. Also, FO, FR and CTX may have additive effects in delaying the onset of kidney disease in these mice. This might be of paramount importance in patients being treated for lupus nephritis, where treatment with CTX is associated with increased production of free radicals and accumulation of oxidative stress. We propose that agents, which can augment tissue antioxidant status and minimize these effects, such as n-3 fatty acids, when given along with CTX or other immunosuppressive drugs in these patients, may be quite useful in preventing side effects. Furthermore, studies are urgently needed to confirm these new findings in order to apply these favorable results in the prevention of early morbidity and mortality in SLE patients.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 
This work was supported in part by NIH grants AG14541 and AG20239.

Received December 9, 2003. Accepted May 13, 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS CONCLUSION ACKNOWLEDGMENTS REFERENCES

 

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