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

Abstract

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.

• cyclophosphamide
• antioxidant enzymes
• lipid peroxidation
• fish oil
• fatty acid composition
• NZB/W mice
• liver
• anti-DNA antibodies

• AL = ad libitum
• ANOVA = analysis of variance
• B/W = NZB × 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

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