Liver endogenous antioxidant defenses in mice fed AIN-76A diet and infected with murine AIDS

ELSEVIER

Chemico-Biological Interactions 99 (1996) 17-28

Liver endogenous antioxidant defenses in mice fed AIN-76A diet and infected with murine AIDS Linda H. Chen*a, Side Xib, Donald A. Cohenc aDepartment of Nutrition and Food Science, University of Kentucky, Lexington, KY 40506~tXl54, USA bNutritional Sciences Program, University of Kentucky, Lexington, KY 40.506-0057, USA ‘Department of Microbiology and Immunology University of Kentucky, Lexington, KY 40506-0084. USA

Received 12 May 1995; revision received 24 July 1995; accepted 28 July 1995

Abstract The effects of murine AIDS infection on endogenous antioxidant defenses in mice fed the AIN-76A liquid diet were investigated. C57BW6 female mice were divided into 2 groups: one group was injected interperitoneally with LP-BMS murine retrovirus (MAIDS) stock, and the other group served as the non-infected control. Two weeks after the infection, the mice were killed and livers were excised for biochemical analysis of the antioxidant defenses. Liver reduced glutathione (GSH) levels and activities of both cytosolic superoxide dismutase (SOD) and mitochondrial SOD were significantly depressed by MAIDS infection. Activities of glutathione reductase (GR), selenium (Se)-dependent glutathione peroxidase (GPx), catalase and glutathione-S-transferase (GST) toward I-chloro-2,4-dinitrobenzene (CDNB) were not affected by MAIDS infection. A previous study by this laboratory using the Lieber-DeCarli (L-D) all purpose liquid diet caused a decline in total SOD activity and GPx activity, but not GSH levels. The results suggest that MAIDS infection depresses liver antioxidant defenses; however, MAIDS infection of mice fed the AIN-76A liquid diet depresses different liver antioxidant defense parameters when compared to those of the mice fed the L-D all purpose liquid diet. Keywords:

Murine AIDS; Glutathione;

Superoxide dismutase;

Glutathione

Catalase

* Corresponding author. 0009-2797/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-2797(95)03657-g

peroxidase;

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L. H. Chen er ul. / Chemrco-Biologicd

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1. Introduction

HIV infection affects multiple aspects of the immune systems, including the number and function of CD4+ and CD8+ lymphocytes, B lymphocytes and mononuclear phagocytes. Acquired immunodeficiency syndrome (AIDS) has been identified as a major public health problem in the world because of its epidemic nature, increasing incidence and high mortality. The pathogenic mechanisms underlying HIV infection and AIDS progression are complex and not clearly understood. However, it is becoming apparent that the level of oxidative stress is greater in HIV-infected individuals, as compared to the normal population, and may contribute to the progression of AIDS [ 11. Oxidative stress in cells and tissues refers to increased generation of reactive oxygen species (ROS) (such as superoxide radicals, hydroxyl radicals and HzOz) and/or a decline in antioxidant defenses causing an imbalance of prooxidants and antioxidant defense equilibrium in favor of prooxidants. The cellular antioxidant status of the HIV-infected cells, which is of importance for the regulation of ROS levels, may play an important role in modulating the activity of the immunodeficiency virus, and in determining the latency period of HIV infection [2]. In addition, diminution of antioxidant defenses may lower the ability of an HIV-infected individual to handle ROS generated by secondary opportunistic infections and thus contribute individually to associated tissue damage during these infections. Because of the involvement of ROS in AIDS, it is important to develop an animal model in which to examine changes in antioxidant defenses during progression of AIDS. The objective of this study focused on the effect of murine retroviral infection on endogenous antioxidant defenses using the LP-BMS murine retrovirus model of AIDS (MAIDS) [3]. Murine AIDS infection has been shown to lead to immunodeficiency in both CD4+ and CD8+ T cell subsets, lymphoadenopathy, hypergammaglobulinemia and to increase susceptibility to a variety of opportunistic infections. Whether murine AIDS infection alters cellular nonenzymatic and enzymatic antioxidant defense systems in a manner similar to HIV is not yet known. A number of potential sites in the antioxidant defense systems could be affected by MAIDS. GSH can react directly with ROS as a free radical scavenger. It also protects thiol groups in protein from oxidation and serves as the substrate for several enzymes such as GPx and GST. GSH can also directly modulate proliferation of T cells [4], is involved in the synthesis of proteins and DNA precursors, induces initiation and progression of lymphocyte activation, and is critical for the function of natural killer cells and for lymphocyte-mediated cytotoxicity [5]. Major enzymatic antioxidant defense systems include SOD, catalase and GPx. There are two types of SODS: Cu/ZnSOD (contains Cu and Zn as the prosthetic metals) and MnSOD (contains Mn as the prosthetic metal). SODS, found in cytosol (CuiZnSOD) and mitochondria, catalyze the dismutation of two superoxide anions to form hydrogen peroxide and oxygen. Catalase, found in peroxisomes, catalyzes the reduction of two hydrogen peroxide to two water molecules and oxygen. GPx protects the cell components from damaging effects of organic hydroperoxides and hydrogen peroxide using GSH as the substrate. Because the intracellular concentration of GSH that is required by GPx may vary, catalase is an important enzyme in

L. H. Chen et al. / Chemico-Biological Interactions 99 (I 9%) 17-28

19

the protection against hydrogen peroxide-mediated damage at the physiological levels. In a previous study by this laboratory [6], we compared the effects of MAIDS infection on antioxidant defenses in the livers of mice fed mice chow and the L-D allpurpose liquid diet (Lieber-DeCarli, 1986) [7]. In the present study, we further evaluated the effects of MAIDS infection on antioxidant defenses in mice fed AIN76A liquid diet. AIN-76A liquid diet was chosen for this study, because the L-D all purpose liquid diet contains a large excess of vitamin A and a higher level of vitamin E when compared to the NAS-NRC recommendation for mice and rats (Table l), and may have an influence on the cellular antioxidant defenses. As shown in Table 1, the vitamin A level of the L-D liquid diet is 51 times the recommended level, whereas the vitamin A level of the AIN-76A liquid diet is only 8 times the recommended level. The vitamin E level in the L-D liquid diet is 2.5 times the level in the AIN-76A liquid diet and the recommended level. The levels of Se, Cu, Zn and Mn in the two diets are comparable to the NAS-NRC recommendations. 2. Materials and methods 2.1. Animals and diets Twelve C57BW6 female mice (6-8 weeks old) were obtained from Harlan Sprague Dawley Inc. (Indianapolis, IN) and were caged individually in sterile microisolator cages. Animals were monitored by the Division of Laboratory Animal Resources at the University of Kentucky according to the guidelines of the Animal Welfare Act. The mice were fed AIN-76A liquid diet (Dyets Inc., Bethlehem, PA). All mice were fed diet and sterile water ad libitum. 2.2. Virus infection LP-BMS is a mixture of murine leukemia viruses that contains the disease-causing defective retrovirus of MAIDS. Virus stocks were prepared from chronically infected SC-l cells according to the procedure of Mosier et al. [3] and stored at -80°C. Mice were divided into 2 groups of 6 mice each. One group of mice was injected intraperitoneally with 1 ml of cell-free virus stock 1 week after the mice were placed on the diets. The mice were killed 2 weeks after MAIDS virus infection. Mice were anesthetized by Metophane and exsanguinated by cardiac puncture prior to removal of liver for biochemical assay. Viral infection was verified at the time of sacrifice by the occurrence of splenomegally as we have previously described [6,8]. 2.3. Biochemical assays A 10% liver homogenate in 0.1 M phosphate buffer (pH 7.4) was prepared at 4°C. An aliquot of liver homogenate was immediately used for GSH determination by the method of Owen and Belcher [9]. Another aliquot of liver homogenate for catalase and protein determinations was frozen at -80°C until analyses were performed. The remaining liver homogenate was centrifuged at 1000 x g for 15 min, and the supernatant was further centrifuged at 9000 x g for 10 min to separate mitochondria. After washing the mitochondria once with 0.25 M sucrose in 0.1 M potassium phos-

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L. H. Chen et al. / Chemico-Biological

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phate buffer (pH 7.4) the mitochondria pellet was suspended in the same buffer with a glass homogenizer, and frozen at -80°C for SOD and GPx activity determinations. The 9000 x g supernatant was also frozen at -80°C for the determinations of GR, Cu/ZnSOD, GPx and GST activities, and protein concentrations. Se-dependent GPx activity in the mitochondria suspension and the supernatant was determined by the method of Paglia and Valentine [lo] using hydrogen peroxide as the substrate. Catalase activity in liver homogenate was determined by the method of Beers and Sizer [l11. SOD activity in the mitochondria suspension and the 9000 x g supernatant was determined by the method of McCord and Fridovich [ 121. GST activity in the supernatant was determined by the method of Habig et al. [ 131 using CDNB as the substrate. The units of GSH levels and the enzyme activities were expressed as per mg protein. Protein concentrations in the homogenate and the supernatant were determined by the method of Lowry et al. [14]. 2.4. Statistical analysis of data Data were analyzed by the Student’s sidered significant.

t-test. A probability

of P < 0.05 was con-

3. Results Fig. 1 shows the effect of MAIDS on liver GSH levels 2 weeks after infection. MAIDS infection significantly decreased GSH levels. However, the activity of GR, the enzyme that catalyzes the regeneration of GSH from GSSG, was not affected by MAIDS infection (Fig. 2). Se-dependent GPx, which catalyzes the decomposition of hydrogen peroxide, can be found in both the cytosol and the mitochondria. Fig. 3 shows that neither cytosolic nor mitochondrial Se-dependent GPx activity was affected by MAIDS infection. Similarly, the activity of GST, using CDNB as the substrate, also was not affected by MAIDS infection (Fig. 4).

70 ,

Control Fig. 1. Reduced glutathione (GSH) at P < 0.05.

levels (mean l LE.).

MAIDS ‘Significantly

different from the control group

L. H. Chen et al. / Chemico-Biological

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40

0 Control

MAIDS

Fig. 2. Glutathione reductase (GR) activities.

SODS catalyze the dismutation of superoxide anions in the cytosol and in the mitochondria. Interestingly, the activities of both cytosolic and mitochondrial SODS were significantly depressed by MAIDS infection (Fig. 5). Finally, the activity of catalase, which is located in peroxisomes and catalyzes the decomposition of hydrogen peroxide, was not affected by MAIDS infection (Fig. 6). 4. Discussion Increased blood levels of malonaldehyde (MDA) [ 151, thiobarbituric acid reactants [ 16,171 and hydroperoxides [ 161 (indicators of lipid peroxidation) have been observed in the plasma of patients in different stages of HIV infection when com-

1.25

T

0.00

/

1

Control

Fig. 3. Glutathione-S-transferase as the substrate.

MAIDS

(GST) activities* (mean f S.E.). *I-chloro-2,4-dinitrobenzene

(CDNB)

L.H. Chen et al. / Chemico-Biological Interactions 99 (1996) 17-28

2-

1q

Mito.GP

1-

MAIDS

Control

Fig. 4. Selenium-dependent

glutathione

peroxidase

(GF’x) activities

(mean f S.E.)

pared with the non-infected controls. Increased lipid fluorescence, diene conjugates and increased ethane exhalation (also indicators of lipid peroxidation) have also been observed in MAIDS-infected mice [18]. MAIDS infection has significantly decreased hepatic concentrations of vitamin A, vitamin E and Cu; splenic concentrations of vitamin A, vitamin E, Zn and Cu; and thymic and serum concentrations of vitamin A and vitamin E; while vitamin E supplementation has completely or partially restored concentrations of these nutrients in the tissues [19]. Infection with MAIDS virus for 2 weeks in the present study depressed both cytosolic and mitochondrial SOD activities, and thus supports our previous study [6] in which it was shown that total SOD activity was depressed at 2 weeks and 4 weeks after MAIDS virus infection. A possible explanation of a decreased MnSOD

F ._

”

0

Cu/Zn-SOD

60

q

MmSOD

t

Control

MAIDS

Fig. 5. Superoxide dimutase (SOD) activities (mean f SE.). ‘Significantly ding control group at P < 0.05.

different

from the correspon-

L. H. Chen et al. / Chemico-Biological Interactions 99 (1996)

activities

23

MAIDS

Control Fig. 6. Catalase

17-28

(mean f

S.E.).

activity is the direct suppression of the expression of cellular MnSOD by tat protein of the virus [20,21]. The alteration of gene expression of cytokines in MAIDSinfected mice may also affect SOD [22]. The decrease in Cu/ZnSOD activity may be explained by a decrease in Cu concentration in the livers of mice with MAIDS [ 191. In addition, the decrease in SOD activity may also be associated with an increase in H,Oz production because it is known that H202 is an effective inhibitor of SOD [23]. Bondy [24] suggested that a decreased activity of SOD is a reflection of oxidative denaturation, because SOD is a sulfhydryl enzyme. SOD catalyzes the dismutation of superoxide radicals to form hydrogen peroxide. In the chain reaction leading to the formation of hydroxyl free radical, superoxide anion reduces ferric ions to ferrous ions, and thus is responsible for promoting the following reaction: Fe*+ + H202 - Fe3+ + ’ OH + OHAs a result, hydroxyl radicals, which are very active species that can attack all biological molecules [25], are prevented from forming. However, when the SOD activity in cytosol and mitochondria is depressed, more hydroxyl radicals will be formed in the presence of ferric and/or ferrous ions, and causing more oxidative stress. Thus, the decline in SOD activity may render MAIDS-infected animals significantly more susceptible to free radical mediated damage generated during primary and secondary infections. MAIDS infection in the present study significantly decreased liver GSH levels. This result is consistent with the decline in GSH levels in the plasma [5,26], and lung epithelial lining fluid [5] of the HIV-infected patients. The decrease in GSH levels was not due to alteration in GR activity or GST activity; and may be due to increased production of ROS and decreased SOD activity, because the decreases in the expression and activity of MnSOD caused by tat protein are associated with decreases in GSH levels and the ratio of GSH/GSSG (211. Since GSH is the substrate for GPx,

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a depressed level of GSH may allow hydrogen peroxide to accumulate. If hydrogen peroxide is allowed to accumulate, it not only can inactivate SOD [23], but may also contribute to inhibition of T-lymphocyte activation, cause damage to synthesis of interleukin-2 [27], and cause damage to lymphocyte DNA [28]. Treatment with compounds which can increase intracellular GSH levels, such as cysteine or N-acetyl cysteine (NAC), has been shown to inhibit HIV-l replication 129,301;NAC is a direct ROS scavenger, and can easily enter the cell and deacetylate to form cysteine which supports GSH synthesis [31]. Whether pharmacological elevation of GSH levels in MAIDS-infected mice delays or reverses virus replication or the onset of immunodeliciency is yet to be studied. The results of this study and those of our previous study using the same animal model but fed the L-D all-purpose liquid diet [6] for the same feeding period were compared. Both studies showed no effect of MAIDS on the activities of GR, catalase and GST toward CDNB substrate. The previous study reported that MAIDS infection depressed total SOD activity, and this study further demonstrated that MAIDS infection depressed both cytosolic SOD and mitochondrial SOD activities. However, several differences in results were observed between the two studies using different diets. Liver GSH levels were decreased by MAIDS in the present study in which the mice were fed AIN-76A diet, but were not affected in the previous study in which the mice were fed the L-D diet. This difference may be explained by the higher level of vitamin E in the L-D diet than the AIN-76A diet. It has been shown that vitamin E levels in the liver and the spleen of the MAIDS-infected mice fed AIN-76A diet are significantly decreased when compared to the uninfected control group [ 191,and that vitamin E deficiency decreases liver GSH levels 1321.Moreover, it has been suggested that vitamin E supplementation be used as an adjunct therapy in HIV and AIDS because vitamin E is an antioxidant, free radical scavenger and immunoenhancer [33]. Thus, the normal GSH levels in MAIDS-infected mice on the L-D

Table 1 Selected micronutrient

levels in two liquid diets and NAS-NRC

recommendations”

Nutrient

AIN-76A liquid diet

Lieber-DeCarli liquid diet

NAS-NRC recommendationb for mice

Vitamin A (IU) Vitamin E (II-J)

941 II.8

6ooo 30 0.025 1.5 7.5 13.5

118

Selenium (mg) Copper (mg) Zinc (mg) Manganese (mg)

0.023 I.4 8.2 12.7

Il.8 (0.009)C (1.2)C 11.8 4.7

aPer 1000 kcal diet. bNutritional Requirements of Laboratory Animals, No. IO, Second Revised Edition, emy of Sciences. CRecommendation for rats (recommendation for mice not available).

National

Acad-

L. H. Chen et al. / Chemico-Biological Interactions 99 (19%)

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Table 2 Macronutrient

composition

Diet

AIN-76A

of AIN-76A

and Lieber-DeCarli

Protein

liquid diet

Lieber-DeCarli

liquid diet

liquid diets”

Lipid

Carbohydrate

20

Ilb

69

18

3F

47

“Percent of calorie. bCorn oil. ‘Corn oil, 8.5; olive oil, 28.4; and safllower

oil, 2.7; g/1/1000 kcal

diet may reflect the potential of dietary antioxidants to modulate the AIDS-like disease. Another difference in the results of the two studies is in GPx activity: the enzyme activity was depressed by MAIDS infection when the L-D diet was used; however, the enzyme activity was not affected by MAIDS infection when the AIN-76A diet was used. This difference in GPx activity may be explained by greater oxidative damage to lipids due to the higher level of polyunsaturated fatty acids in the L-D diet than the AIN-76A diet (Table 2). It is known that superoxide anions and hydroperoxides can cause irreversible inactivation of GPx [34]. The levels of saturated fatty acid, monounsaturated fatty acids and polyunsaturated fatty acid in the L-D diet are 5.34, 22.71 and 11.52 g/1000 kcal, respectively; whereas the levels of saturated fatty acid, monounsaturated fatty acid and polyunsaturated fatty acid in the AIN-76A liquid diet are 1.49, 2.89 and 7.37 g/1000 kcal, respectively. Because the lipid composition of the liver is reflected by the lipid composition of the diet, the lipid in the livers of the mice fed the L-D diet would contain higher levels of polyunsaturated fatty acids than those of the mice fed the AIN-76A diet; and a higher level of polyunsaturated fatty acid in the tissue would render the tissue more susceptible to oxidative stress. The vitamin E level in the L-D diet is about 2.5 times that in the AIN-76A diet (Table l), and may have potentially compensated for its higher polyunsaturated fatty acid content. However, the results in infected mice fed the L-D diet suggest that sufficient protection was not achieved with this level of vitamin E. In mice fed L-D diet, GPx activity was depressed by MAIDS infection suggesting that there was not an adequate amount of Se (as sodium selenite) in the diet. Decreases in plasma Se levels and GPx activity have been observed in AIDS patients [16]. In recent reports, the importance of Se as a supportive measure in early and advanced stages of HIV-infected disease has been suggested [35-371. Because Se is a constituent of GPx, Se supplementation can increase GPx activity in latently infected T-lymphocytes; thus, Se supplementation can protect against the cytotoxic effects of hydrogen peroxide and can also decrease NF-,B activation by hydrogen peroxide [35]. In addition, Se inhibits reverse transcriptase activity in RNA-virusinfected subjects; therefore, Se supplementation may prevent the HIV and slow down the development of AIDS in newly HIV-infected individuals [36].

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L.H. Chen et al. / Chemico-Biological Interactions 99 (1996) 17-D

Our results showed that MAIDS infection depressed liver antioxidant defense capability that contributes to oxidative stress. In addition to a decline in antioxidant defense capability, increased production of ROS in MAIDS also contributes to oxidative stress. Increased basal production of superoxide anions by polynuclear cells has been observed in both the asymptomatic [38] and the advanced stages [39]. In addition, HIV infection causes dysregulation of cytokines known to affect the level of oxidative stress. Increased production of the inflammatory cytokines including interleukin-1 (IL-l) and tumor necrosis factor-o (TNF-(Y) from monocytes and macrophages [40] can cause oxidative stress because increased generation of ROS in IL- I and TNF-CY responses in HIV patients have been observed [41,42]. Recent evidence has suggested the involvement of ROS in the activation of gene expression by induction of the transcription factor, nuclear factor-kappaB (NF-,B), which can also trigger the activation and replication of HIV-l provirus [43]. In conclusion, MAIDS infection of mice fed the AIN-76A liquid diet significantly decreased liver antioxidant defenses by causing a decline in GSH levels and activities of both cytosolic and mitochondrial SOD. The results demonstrate that MAIDS infection lowers antioxidant defenses in the livers of mice, suggesting that MAIDSinfected mice are exposed to greater oxidative stress during the AIDS-like disease and may be less able to protect against ROS generated during secondary opportunistic infections. The results suggest that the choice of diet may have a significant impact on the extent to which MAIDS infection suppresses antioxidant defenses. References 111 S. Baruchel and M.A. Weinberg, The role of oxidative stress in disease progression in individuals infected by the human immunodeficiency virus, J. Leuk. Biol.. 52 (1992) I I l-l 14. virus replication, Nutr. 121 D.H. Baker, Cellular antioxidant status and human immunodeficiency Rev., 50 (1992) 15-18. [31 D.E. Mosier, R.A. Yetter and H.C. Morse, Retrovtral induction of acute lymphoproliferative disease and profound immunosuppression in adult C57BLi6 mice. J. Exp. Med.. I61 (1985) 766-784. M.E. Anderson, V.K. Sharma and A. Meister, Glutathione regulates activation[41 M. Suthanthiran, dependent DNA synthesis in highly purified normal human T-lymphocytes stimulated via the CD2 and CD3 antigens, Proc. Natl. Acad. Sci. USA, 87 (1990) 3343-3347. [51 R. Buhl, H.A. Jaffe, K.J. Holroyd, F.B. Wells, A. Mastrangeli, C. Saltini, A.M. Cantin and R.G. Crystal, Systemic glutathione deficiency in symptom-free HIV sero-positive individuals, Lancet. 2 (1989) 1294-1298. 161 C.Y. Huang, L.H. Chen, Y. Osio and D.A. Cohen, Effects of diet composition on liver antioxidant defense and detoxification enzymes in mice with murine AIDS, Nutr. Res.. I4 (1994) 1841-1851. 1989 Update. Alco171C.S. Lieber and L.M. DeCarli, Liquid diet technique of ethanol administration: hol Alcohol., 24 (1989) 197-21 I. [81 E.A. Fitzpatrick, J.S. Bryson, CA. Rhoads. A.M. Kaplan and D.A. Cohen. Deficient transmembrane signalling in CD4+ T cells retroviral-induced immunodeficient mice, J. Immunol., I48 (1992) 3377-3384. micro-method for the determination of 191 C.W.I. Owens and R.V. Belcher, A calorimetric glutathione, Biochem. J., 94 (1965) 705-71 I. of 1101 D.E. Paglia and W.N. Valentine, Studies on the quantitative and qualitative characterization erythrocyte glutathione peroxidase, J. Lab. Clin. Med.. 70 (1967) 158-169. method for measuring the breakdown of hy1111 R.F. Beers Jr. and I.W. Sizer, A spectrophotometric drogen peroxide by catalase. J. Biol. Chem.. I95 (1952) 133-140.

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