Free Radical Biology & Medicine, Vol. 29, No. 1, pp. 1–7, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter
PII S0891-5849(00)00284-5
Original Contribution VITAMIN A DEFICIENCY CAUSES OXIDATIVE DAMAGE TO LIVER MITOCHONDRIA IN RATS TERESA BARBER, ELISA BORRA´ S, LUIS TORRES, CONCHA GARC´ıA, FRANCISCO CABEZUELO, ANA LLORET, FEDERICO V. PALLARDO´ , and JUAN R.VIN˜ A Departamentos de Bioquı´mica y Biologı´a Molecular y Fisiologı´a, Facultades de Medicina y Farmacia, Universidad de Valencia, Valencia, Spain (Received 30 November 1999; Revised 30 March 2000; Accepted 6 April 2000)
Abstract—Mitochondrial damage in rat liver induced by chronic vitamin A-deficiency was studied using three different groups of rats: (i) control rats, (ii) rats fed a vitamin A-free diet until 50 d after birth and (iii) vitamin A-deficient rats re-fed a control diet for 30 d. No statistical difference in body weight and food intake was found between control and vitamin A-deficient rats. Liver GSH concentration was similar in both groups. However, in vitamin A-deficient rats, the mitochondrial GSH/GSSG ratio was significantly lower and the levels of malondialdehyde (MDA) and 8-oxo-7,8dihydro-2⬘-deoxyguanosine (oxo8dG) were higher when compared to control rats. These values were partially restored in re-fed rats. The mitochondrial membrane potential of vitamin A-deficient rats was significantly lower than in control rats and returned to normal levels in restored vitamin A rats. Two populations of mitochondria were found in vitamin A-deficient rats according to the composition of membrane lipids. One population showed a similar pattern to the control mitochondria and the second population had a higher membrane lipid content. This report emphasizes the protective role of vitamin A in liver mitochondria under physiological circumstances. © 2000 Elsevier Science Inc. Keywords—Vitamin A, Liver, Mitochondria, mtDNA, Oxidative stress, GSH, Free radicals
INTRODUCTION
(11-cis-retinal) and reproduction [1]. Recently, using murine 3T3 cells, vitamin A (retinol) has been identified as the serum survival factor for fibroblasts. The physiological retinol derivate 14-hydroxy-4,14-retro-retinol, but not retinoic acid, can replace retinol in rescuing or activating 3T3 cells [2]. The normal concentration of retinol in plasma is 1–2 M and this is directly related to the liver concentration of this vitamin. In liver from vitamin A-deficient rats there is a down-regulation of CYP2C11 [3] and also a loss of liver membrane-bound low-affinity glucocorticoid binding site activity [4]. This can be prevented with either retinyl esters or all-trans-retinoic acid, and it has also been shown that the effect of vitamin A deficiency on CYP2C11 in male rat liver is mediated by androgen deficiency [3,5]. Our experimental setting is a good model to study the protective role of vitamin A at physiological levels on oxidative liver damage because we tested three groups: the first was the control group with an intake of vitamin A as recommended by AIN; the second was the vitamin A-deficient group; and the third was a group of vitamin A-deficient rats that were re-fed a control diet for 30 d.
Vitamin A (retinol) is an essential nutrient and its major dietary forms are retinyl esters and -carotene, which can be cleavaged in vivo to give retinal, which can be reduced to retinol or oxidized to retinoic acid. The absorption of dietary vitamin A requires hydrolysis, and the free retinol in the intestine is re-esterified with palmitic or stearic acid. Dietary retinol reaches the liver as chylomicron remnants and includes movement from parenchymal cells into stellate cells. Retinol is released from the liver when vitamin A-dependent tissues requiered it, and binds to a serum retinol-binding protein and to transthyretin. Retinol is metabolized in mammalian cells by oxidative reactions to retinal and retinoic acid, which shares only some of the activities of retinol because it can support growth and differentation in a variety of systems but it is unable to support processes such as vision Address correspondence to: Juan R.Vin˜a, Departamento de Bioquı´mica y Biologı´a Molecular, Facultad de Medicina y Odontologı´a, Av. Blasco Iban˜ez 17, Valencia 46010, Spain; Tel: ⫹34 963864187; Fax: ⫹34 963864173; E-Mail:
[email protected]. 1
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The antioxidant property of vitamin A was shown for the first time when small concentrations of vitamin A inhibited the oxygen uptake of linoleic acid for hours [6]. This antioxidant activity is achieved by the hydrophobic chain of polyene units, which can quench singlet oxygen, neutralize thiyl radicals, and stabilize and combine with peroxyl radicals [7,8]. The antioxidant activity of vitamin A against lipid peroxidation induced by doxorubicin in heart and brain membrane lipids has been shown in vivo [9] and vitamin A supplementation inhibits chemiluminescence and lipid peroxidation in isolated rat liver microsomes and mitochondria [10]. For the first time we have been able to show how vitamin A deficiency causes oxidative damage to liver mtDNA, which is related to the mitochondrial GSH/GSSG ratio. These effects are accompanied by changes in the lipid composition of mitochondria membranes and also by a drop in mitochondria membrane potential (MMP). These changes are partially restored when the vitamin A-deficient rats are re-fed a normal diet (with vitamin A) for 30 d. MATERIALS AND METHODS
Rats Rats were made deficient in vitamin A by being fed a solid diet devoid of this vitamin [11]. Pregnant rats were housed in individual cages in a room maintained at 22°C with a 12-h light-dark cycle. One day after the pup birth, dams were fed either a control diet or a vitamin A-deficient diet. Milk production was evaluated during lactation in both groups. After weaning, the rats were fed the same corresponding diet until 50 d old. Over this period of time, food intake and rat weights were similar in both groups (Table 1). However, after this period (when experiments were performed), vitamin A-deficient rats started eating less food, their body weight reached a nadir, and they died prematurely compared to controls. A third group was formed with vitamin A-deficient rats that were re-fed with a control diet over a period of 30 d.
Retinol determination Serum vitamin A concentration was determined using a spectrofluorimetric method taking retinol palmitate as standard [12]. Isolation of mitochondria Mitochondria were isolated by using a standard differential centrifugation procedure [13]. Glutathione determination Reduced glutathione was measured using the glutathione-S-transferase assay [14]. Oxidized glutathione was measured using an HPLC method with UV-V detection, which was developed to measure GSSG in the presence of a large excess of GSH [15]. Purification of mitochondrial DNA The method of Latorre et al. [16] was used with minor modifications. The purity of mtDNA was determined using gel electrophoresis and the spectrophotometric method, which measures the absorbance ratio at 260/280 nm; the value found was around 1.8. Digestion of mtDNA to nucleosides After isolation, mtDNA was digested to nucleosides using nuclease P1, then alkaline phosphatase was added in order to liberate the nucleosides from phosphate residues. The resultant solution was incubated at 37°C for 60 min; 8-oxo-dG from Sigma Chemical Co. (St. Louis, MO, USA) was used as standard. Malondialdehyde measurement Lipid peroxides were determined as MDA according to Wong et al. [17].
Table 1. Average Body Weight and Diet Ingested by Control and Vitamin A-Deficient Rats Body weight (g) Week 4 5 6 7
(days (days (days (days
22–28) 29–35) 36–42) 43–49)
Intake (g/d)
Control
Vitamin A-deficient
Control
Vitamin A-deficient
83.2 ⫾ 10.4 141.4 ⫾ 9.2 171.3 ⫾ 6.8 194.8 ⫾ 12.5
85.4 ⫾ 10.6 137.3 ⫾ 5.4 160.7 ⫾ 7.7 184.1 ⫾ 9.6
16.4 ⫾ 1.0 17.0 ⫾ 0.7 19.8 ⫾ 1.4 21.3 ⫾ 0.7
15.4 ⫾ 0.6 17.8 ⫾ 0.6 20.7 ⫾ 1.5 18.7 ⫾ 0.7
The data are means ⫾ SEM from 15 control rats and 17 vitamin A-deficient rats. There are no significant differences between groups.
Vitamin A and oxidative stress
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Measurement of 8-oxo-7,8-dihydro-2⬘-deoxyguanosine (oxo8dG) The oxo8dG present in mtDNA hydrolysates was separated isocratically on a 20 ⫻ 0.46 cm ODS-2 Spherisob column, with a mobile phase of 2% acetonitrile in 25 mM potassium phosphate buffer, pH 5.5. The flow rate was 1 ml/min. Electrochemical detection of oxo8dG was performed on an ESA Coulochem II model 5200 equipped with a 5011 analytical cell and a 5021 guard cell. The settings for the dual coulometric detector were 100mV for detector 1 and 400mV for detector 2. Flow cytometry Flow cytometry was performed using an EPICS PROFILE II flow cytometer (Coulter Electronics, Hialeah, FL, USA). Fluorochromes were excited with an argon laser tuned at 488 nm. Forward-angle light scatter and side-angle light scatter (90°) were measured and fluorescence was detected through a 488 nm blocking filter, a 550 nm long pass dichroic, a 525 nm band pass, or a 575 nm long pass; 10,000 mitochondria were counted for each sample. The composition of polar and apolar lipids in the mitochondrial membrane was assessed by fluorescence emission of nile red (5 l of a 100- mol/l solution per milliliter of sample). Mitochondrial membrane potential from liver was measured using rhodamine 123 (RH123). Mean values for each experiment were calculated by the flow cytometry parameters using the histogram display analysis program (EPICS PROFILE II). Statistics Values are presented as mean ⫾ SEM for the number of observations indicated. Statistical analyses were performed by the least-significant difference test, which consists of two steps. First an ANOVA was performed. The null hypothesis was accepted for all the values of these sets in which the F value was nonsignificant at P ⬎ 0.05. The data for which the F value was significant were examined by the Student’s t test. RESULTS
Physiological parameters and retinol concentration in plasma After delivery, dams were fed either a control diet or a vitamin A-deficient diet. Milk production was evaluated at the peak of lactation in both groups, and no difference was found. After weaning, the pups either ate the solid control diet or the vitamin A-deficient diet; no statistical difference in body weight or food intake was found between the groups (Table 1). The concentration
Fig. 1. Liver mitochondrial membrane potential in control, vitamin A-deficient and vitamin A-restored rats. Panel A: A representative experiment showing an overlay plot of the MMP using mitochondria from control, deficient, and restored rats. Panel B: The percentage of change from control (100%) is plotted. Data are mean ⫾ SEM for control (white, n ⫽ 7), deficient (dotted, n ⫽ 4), and in restored rats (stripped, n ⫽ 3).
of retinol (M) in control rats was 1.62 ⫾ 0.20 (n ⫽ 6), 0.16 ⫾ 0.03* (n ⫽ 6) in vitamin A-deficient rats, and 1.37 ⫾ 0.12 (n ⫽ 3) in vitamin A-restored rats. Mitochondrial membrane potential (MMP) This was determined in isolated liver mitochondria using rhodamine 123 (Rh 123) as fluorescent dye. The MMP is the driving force for Rh 123 uptake. The MMP of vitamin A-deficient rats was significantly lower than in isolated liver mitochondria of control rats. This change was reversed in vitamin A-restored rats (Fig. 1). Membrane lipid composition The size and shape of the isolated liver mitochondria were similar in the three groups, i.e., the control group, the vitamin A-deficient group, and the vitamin A-re-
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Fig. 2. Liver mitochondrial lipid membrane composition: A representative experiment of a flow cytometry analysis. (A) control, (B) deficient, and (C) restored. Green fluorescence measures apolar lipids and red fluorescence measures polar lipids, orange fluorescence is a mixture of both lipids. FS ⫽ forward side, SS ⫽ scattered side.
stored group (Fig. 2). The membrane lipid composition showed a homogenous population for both polar and apolar lipids in mitochondria from control rats. However, two populations of mitochondria were found in vitamin A-deficient rats according to the composition of membrane lipids. One population showed a similar fluorescence intensity to that of the control mitochondria, and the second population had a higher lipid content than the other population. This occurred in both apolar (green) (222 ⫾ 82%, n ⫽ 4 from control) and polar lipids (red) (224 ⫾ 79%, n ⫽ 4 from control). Results in restored animals tended to show a decrease in the relative proportion of mitochondria with high lipid content when compared to the vitamin A-deficient group.
and in vitamin A-restored rats it was 5.8 ⫾ 0.6 (5). There were no significant differences between the groups.
Vitamin A and GSH values in total liver
Malondialdehyde (MDA) concentration
In control liver the GSH concentration (mol/g) was 5.9 ⫾ 0.3 (9), in vitamin A-deficient rats was 6.2 ⫾ 0.6 (8)
Mitochondrial lipid peroxidation measured as malondialdehyde increased significantly in vitamin A-deficient
Vitamin A status and mitochondrial glutathione oxidation Vitamin A deficiency caused a significant increase in GSSG levels concurrent with a decrease in GSH levels in isolated mitochondria. In the restored rats, although GSH values returned to normal, the GSSG values did not. The GSH/GSSG ratio decreased significantly in the vitamin A-deficient rats when compared to control rats, and in the restored rats this ratio was significantly higher than the value from the vitamin A-deficient group, but it did not reach the control value (Table 2).
Vitamin A and oxidative stress
rats. Results in restored animals were similar to the values found in the vitamin A-deficient group (Table 2). Production of 8-oxo-dG by isolated mitochondria Oxygen free radicals attack guanine residues converting deoxyguanosine into 8-oxo-dG, which is one of the best indicators of oxidative damage to DNA. In vitamin A-deficient rats the production of 8-oxo-dG was significantly higher than in controls. The values returned to control values in the vitamin A-restored group (Table 2). DISCUSSION
Vitamin A malnutrition has increased dramatically in this decade because more than 250 million people have a subclinical deficit of this vitamin [18]. Over the years, supplementation of -carotene in excess of physiological needs produced neither benefit nor harm in terms of incidence of malignant neoplasms, cardiovascular disease, or death from all these causes [19]. After an average of 4 years of supplementation, the combination of -carotene and vitamin A had no benefit and may have had an adverse effect on the incidence of lung cancer and on the risk of death from lung cancer, cardiovascular disease, and any cause in smokers and workers exposed to asbestos [20]. Thus, to study the antioxidant properties of this vitamin at physiological levels we have used the experimental model of three different groups of rats: (i) control rats, (ii) vitamin A-deficient rats, and (iii) vitamin A-restored rats. Mitochondria, besides functioning as organelles responding to changes in ATP demand, have emerged as active signaling organelles as shown in experiments performed in cells depleted of mitochondrial DNA (mtDNA) [21]. Mitochondrial defects occur in several degenerative diseases, aging, and cancer; and three of the more important aspects of mitochondrial oxidative phosphorylation for disease pathogenesis are (i) energy production, (ii) generation of reactive oxygen species, and (iii) regulation of programmed cell death [22]. Moreover, oxidative stress is involved in different pathological con-
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ditions, such as AIDS, Alzheimer’s disease, Parkinson’s disease and in ischemia-reperfusion injuries, as well as normal aging. mtDNA appears to be a common target for oxygen radical damage and accumulates 10 times more damage than nuclear DNA [23]. These observations have been attributed to the proximity of mtDNA to metabolic oxidation generation [24]. The importance of mtDNA as a specific target of age-associated free radical attack has been proposed [25]. Using the production of 8-oxo-7,8dihydro-2⬘-deoxyguanosine (oxo8dG), oxidation of mtDNA was shown to be very high in aging [26]. These changes found in aging are in agreement with the results found using high-density oligonucleotide arrays, which have revealed a lower expression of genes involved in energy metabolism (which includes the ERV1 gene) [27]. mtDNA, which encodes only 13 polypeptide genes (including some essential components of oxidative phosphorylation), has a high mutation rate and, as the percentage of mutant mtDNA increases, the cellular capacity diminishes until it falls below the bioenergetic threshold. The oxidation of mitochondrial GSH has been proposed as an index of oxidative stress and it has been described as a direct relationship between the mitochondrial GSH/GSSG and the production of oxo8dG [26]. A similar relationship occurs in this study because a decrease in the GSH/GSSG ratio in vitamin A deficiency is followed by an increase in the production of 8-oxo-dG, and these changes are reversed in the vitamin A-restored rats. Recently, it has been found that mitochondrial GSH/ GSSG ratio in old rodents is lower, and the production of oxo8dG from different tissues is significantly higher, than in the young rats. Treatment with antioxidants protects against oxidation of the mitochondrial glutathione pool and the increase of mtDNA oxidation [26]. Similar results were found in the liver of mice treated with zidovudine (3⬘-azido-2⬘,3⬘-dideoxythymidine [AZT]), which is a drug that inhibits human immunodeficiency virus replication and delays the progression of AIDS. The production of oxo8dG is significantly higher when compared to controls due to the increased production of peroxides by liver mitochondria from AZT-treated mice.
Table 2. Glutathione Status, Malondialdehyde Concentration and Levels of Oxo8dG in Mitochondria from Liver of Control Rats, Vitamin A-Deficient Rats and Vitamin A-Restored Rats
GSH (nmol/mg protein) GSSG (nmol/mg protein) GSH/GSSG MDA (nmol/mg protein) Oxo8dG (pmol/g mt DNA)
Control
Vitamin A-deficient
Vitamin A-restored
9.3 ⫾ 1.3 (6) 0.12 ⫾ 0.01 (7) 80 ⫾ 10 (6) 1.7 ⫾ 0.2 (6) 0.22 ⫾ 0.05 (7)
4.0 ⫾ 0.7 (5)* 0.27 ⫾ 0.05 (5)* 18 ⫾ 6 (5)* 2.9 ⫾ 0.6 (5)* 0.35 ⫾ 0.05 (8)*
10.8 ⫾ 1.1 (6) 0.23 ⫾ 0.02 (7)* 51 ⫾ 8 (6)* 3.3 ⫾ 0.8 (3)* 0.25 ⫾ 0.05 (5)
Results are means ⫾ SEM with the numbers of animals indicated in parentheses. Values significantly different from that of controls are shown by * P ⬍ .05.
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These changes were prevented by dietary administration of vitamins C and E [28]. Mitochondrial changes in the heart of vitamin A-deficient rats have been reported previously [29] and are due to a lower NADH coenzyme Q oxidoreductase activity (respiratory complex I of mitochondria), which transfers electrons from NADH to coenzyme Q. This was accompanied by a high-CoQ content to maintain the mitochondrial electron transfer rate. The antioxidant properties of vitamin A have been shown in vitro and in vivo [6 –10]; however, in the present work we studied the antioxidant role of this vitamin in mitochondria and we found a mitochondria GSH depletion, an increase in the production of 8-oxodG, and a decrease of MMP in vitamin A-deficient rats. This was accompanied by changes in lipid composition of liver mitochondria, probably due to a decrease in the membrane turnover [30]. These changes are partially restored when rats are re-fed a diet containing vitamin A. Recently, a “death sequence” similar to our findings has been proposed after treatment of leukemic blasts with 1-beta-D-arabinofuranosylcytosine, which includes GSH depletion, loss of MMP, and phosphatidylserine exposure, which preceded a state of high reactive oxygen generation [31]. In short, the deficit of dietary vitamin A produced an increase in oxidized glutathione, MDA, 8-oxo-dG, an 80% decrease in the GSH/GSSG ratio, and a drop in MMP. This emphasizes the relevance of mitochondria as targets for free radical damage associated with vitamin A deficiency and this was partially restored when rats were re-fed with vitamin A. Moreover, by keeping mitochondria functioning, vitamin A not only attenuates oxidative damage but may also modulate the expression of signal transduction. Acknowledgements — This work was supported by the Fondo Investigacio´n Sanitaria. FIS 98/1461, FIS 99/1157 and by the Generalitat Valenciana GV-D-VS-20-152-96. Elisa Borra´s holds a predoctoral fellowship from the Danone Institute, SPAIN. We thank Marilyn R. Noyes for supervising the manuscript.
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