Original Contribution

VITAMIN A DEFICIENCY CAUSES OXIDATIVE DAMAGE TO LIVERMITOCHONDRIA IN RATS

TERESA BARBER, ELISA BORRAS, LUIS TORRES, CONCHA GARCıA, FRANCISCO CABEZUELO, ANA LLORET,FEDERICO V. PALLARD O, and JUAN R.VINA

Departamentos de Bioquı´mica y Biologıa Molecular y Fisiologı´a, Facultades de Medicina y Farmacia, Universidad de Valencia,Valencia, Spain

(Received30 November1999;Revised30 March 2000;Accepted6 April 2000)

Abstract—Mitochondrial damage in rat liver induced by chronic vitamin A-deficiency was studied using three differentgroups of rats: (i) control rats, (ii) rats fed a vitamin A-free diet until 50 d after birth and (iii) vitamin A-deficient ratsre-fed a control diet for 30 d. No statistical difference in body weight and food intake was found between control andvitamin A-deficient rats. Liver GSH concentration was similar in both groups. However, in vitamin A-deficient rats, themitochondrial GSH/GSSG ratio was significantly lower and the levels of malondialdehyde (MDA) and 8-oxo-7,8-dihydro-29-deoxyguanosine (oxo8dG) were higher when compared to control rats. These values were partially restoredin re-fed rats. The mitochondrial membrane potential of vitamin A-deficient rats was significantly lower than in controlrats and returned to normal levels in restored vitamin A rats. Two populations of mitochondria were found in vitaminA-deficient rats according to the composition of membrane lipids. One population showed a similar pattern to the controlmitochondria and the second population had a higher membrane lipid content. This report emphasizes the protective roleof vitamin A in liver mitochondria under physiological circumstances. © 2000 Elsevier Science Inc.

Keywords—Vitamin A, Liver, Mitochondria, mtDNA, Oxidative stress, GSH, Free radicals

INTRODUCTION

Vitamin A (retinol) is an essential nutrient and its majordietary forms are retinyl esters andb-carotene, whichcan be cleavaged in vivo to give retinal, which can bereduced to retinol or oxidized to retinoic acid. The ab-sorption of dietary vitamin A requires hydrolysis, and thefree retinol in the intestine is re-esterified with palmiticor stearic acid. Dietary retinol reaches the liver as chy-lomicron remnants and includes movement from paren-chymal cells into stellate cells. Retinol is released fromthe liver when vitamin A-dependent tissues requiered it,and binds to a serum retinol-binding protein and totransthyretin. Retinol is metabolized in mammalian cellsby oxidative reactions to retinal and retinoic acid, whichshares only some of the activities of retinol because it cansupport growth and differentation in a variety of systemsbut it is unable to support processes such as vision

(11-cis-retinal) and reproduction [1]. Recently, usingmurine 3T3 cells, vitamin A (retinol) has been identifiedas the serum survival factor for fibroblasts. The physio-logical retinol derivate 14-hydroxy-4,14-retro-retinol,but not retinoic acid, can replace retinol in rescuing oractivating 3T3 cells [2].

The normal concentration of retinol in plasma is 1–2mMand this is directly related to the liver concentration of thisvitamin. In liver from vitamin A-deficient rats there is adown-regulation of CYP2C11 [3] and also a loss of livermembrane-bound low-affinity glucocorticoid binding siteactivity [4]. This can be prevented with either retinyl estersor all-trans-retinoic acid, and it has also been shown that theeffect of vitamin A deficiency on CYP2C11 in male ratliver is mediated by androgen deficiency [3,5]. Our exper-imental setting is a good model to study the protective roleof vitamin A at physiological levels on oxidative liverdamage because we tested three groups: the first was thecontrol group with an intake of vitamin A as recommendedby AIN; the second was the vitamin A-deficient group; andthe third was a group of vitamin A-deficient rats that werere-fed a control diet for 30 d.

Address correspondence to: Juan R.Vin˜a, Departamento de Bio-quımica y Biologıa Molecular, Facultad de Medicina y Odontologı´a,Av. Blasco Iban˜ez 17, Valencia 46010, Spain; Tel:134 963864187;Fax: 134 963864173; E-Mail: Juan.R.Vina@uv.es.

Free Radical Biology & Medicine, Vol. 29, No. 1, pp. 1–7, 2000Copyright © 2000 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/00/$–see front matter

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The antioxidant property of vitamin A was shown forthe first time when small concentrations of vitamin Ainhibited the oxygen uptake of linoleic acid for hours [6].This antioxidant activity is achieved by the hydrophobicchain of polyene units, which can quench singlet oxygen,neutralize thiyl radicals, and stabilize and combine withperoxyl radicals [7,8]. The antioxidant activity of vita-min A against lipid peroxidation induced by doxorubicinin heart and brain membrane lipids has been shown invivo [9] and vitamin A supplementation inhibits chemi-luminescence and lipid peroxidation in isolated rat livermicrosomes and mitochondria [10]. For the first time wehave been able to show how vitamin A deficiency causesoxidative damage to liver mtDNA, which is related to themitochondrial GSH/GSSG ratio. These effects are ac-companied by changes in the lipid composition of mito-chondria membranes and also by a drop in mitochondriamembrane potential (MMP). These changes are partiallyrestored when the vitamin A-deficient rats are re-fed anormal diet (with vitamin A) for 30 d.

MATERIALS AND METHODS

Rats

Rats were made deficient in vitamin A by being fed asolid diet devoid of this vitamin [11]. Pregnant rats werehoused in individual cages in a room maintained at 22°Cwith a 12-h light-dark cycle. One day after the pup birth,dams were fed either a control diet or a vitamin A-defi-cient diet. Milk production was evaluated during lacta-tion in both groups. After weaning, the rats were fed thesame corresponding diet until 50 d old. Over this periodof time, food intake and rat weights were similar in bothgroups (Table 1). However, after this period (when ex-periments were performed), vitamin A-deficient ratsstarted eating less food, their body weight reached anadir, and they died prematurely compared to controls. Athird group was formed with vitamin A-deficient rats thatwere re-fed with a control diet over a period of 30 d.

Retinol determination

Serum vitamin A concentration was determined usinga spectrofluorimetric method taking retinol palmitate asstandard [12].

Isolation of mitochondria

Mitochondria were isolated by using a standard dif-ferential centrifugation procedure [13].

Glutathione determination

Reduced glutathione was measured using the gluta-thione-S-transferase assay [14]. Oxidized glutathionewas measured using an HPLC method with UV-V de-tection, which was developed to measure GSSG in thepresence of a large excess of GSH [15].

Purification of mitochondrial DNA

The method of Latorre et al. [16] was used with minormodifications. The purity of mtDNA was determinedusing gel electrophoresis and the spectrophotometricmethod, which measures the absorbance ratio at 260/280nm; the value found was around 1.8.

Digestion of mtDNA to nucleosides

After isolation, mtDNA was digested to nucleosidesusing nuclease P1, then alkaline phosphatase was addedin order to liberate the nucleosides from phosphate res-idues. The resultant solution was incubated at 37°C for60 min; 8-oxo-dG from Sigma Chemical Co. (St. Louis,MO, USA) was used as standard.

Malondialdehyde measurement

Lipid peroxides were determined as MDA accordingto Wong et al. [17].

Table 1. Average Body Weight and Diet Ingested by Control and Vitamin A-Deficient Rats

Week

Body weight (g) Intake (g/d)

Control Vitamin A-deficient Control Vitamin A-deficient

4 (days 22–28) 83.26 10.4 85.46 10.6 16.46 1.0 15.46 0.65 (days 29–35) 141.46 9.2 137.36 5.4 17.06 0.7 17.86 0.66 (days 36–42) 171.36 6.8 160.76 7.7 19.86 1.4 20.76 1.57 (days 43–49) 194.86 12.5 184.16 9.6 21.36 0.7 18.76 0.7

The data are means6 SEM from 15 control rats and 17 vitamin A-deficient rats. There are no significantdifferences between groups.

2 T. BARBER et al.

Measurement of 8-oxo-7,8-dihydro-29-deoxyguanosine(oxo8dG)

The oxo8dG present in mtDNA hydrolysates wasseparated isocratically on a 203 0.46 cm ODS-2 Spher-isob column, with a mobile phase of 2% acetonitrile in25 mM potassium phosphate buffer, pH 5.5. The flowrate was 1 ml/min. Electrochemical detection of oxo8dGwas performed on an ESA Coulochem II model 5200equipped with a 5011 analytical cell and a 5021 guardcell. The settings for the dual coulometric detector were100mV for detector 1 and 400mV for detector 2.

Flow cytometry

Flow cytometry was performed using an EPICS PRO-FILE II flow cytometer (Coulter Electronics, Hialeah,FL, USA). Fluorochromes were excited with an argonlaser tuned at 488 nm. Forward-angle light scatter andside-angle light scatter (90°) were measured and fluores-cence was detected through a 488 nm blocking filter, a550 nm long pass dichroic, a 525 nm band pass, or a 575nm long pass; 10,000 mitochondria were counted foreach sample. The composition of polar and apolar lipidsin the mitochondrial membrane was assessed by fluores-cence emission of nile red (5ml of a 100-mmol/l solutionper milliliter of sample). Mitochondrial membrane po-tential from liver was measured using rhodamine 123(RH123). Mean values for each experiment were calcu-lated by the flow cytometry parameters using the histo-gram display analysis program (EPICS PROFILE II).

Statistics

Values are presented as mean6 SEM for the numberof observations indicated. Statistical analyses were per-formed by the least-significant difference test, whichconsists of two steps. First an ANOVA was performed.The null hypothesis was accepted for all the values ofthese sets in which theF value was nonsignificant atP .0.05. The data for which theF value was significant wereexamined by the Student’st test.

RESULTS

Physiological parameters and retinol concentration inplasma

After delivery, dams were fed either a control diet ora vitamin A-deficient diet. Milk production was evalu-ated at the peak of lactation in both groups, and nodifference was found. After weaning, the pups either atethe solid control diet or the vitamin A-deficient diet; nostatistical difference in body weight or food intake wasfound between the groups (Table 1). The concentration

of retinol (mM) in control rats was 1.626 0.20 (n 5 6),0.16 6 0.03* (n 5 6) in vitamin A-deficient rats, and1.376 0.12 (n 5 3) in vitamin A-restored rats.

Mitochondrial membrane potential (MMP)

This was determined in isolated liver mitochondriausing rhodamine 123 (Rh 123) as fluorescent dye. TheMMP is the driving force for Rh 123 uptake. The MMPof vitamin A-deficient rats was significantly lower thanin isolated liver mitochondria of control rats. This changewas reversed in vitamin A-restored rats (Fig. 1).

Membrane lipid composition

The size and shape of the isolated liver mitochondriawere similar in the three groups, i.e., the control group,the vitamin A-deficient group, and the vitamin A-re-

Fig. 1. Liver mitochondrial membrane potential in control, vitaminA-deficient and vitamin A-restored rats. Panel A: A representativeexperiment showing an overlay plot of the MMP using mitochondriafrom control, deficient, and restored rats. Panel B: The percentage ofchange from control (100%) is plotted. Data are mean6 SEM forcontrol (white,n 5 7), deficient (dotted,n 5 4), and in restored rats(stripped,n 5 3).

3Vitamin A and oxidative stress

stored group (Fig. 2). The membrane lipid compositionshowed a homogenous population for both polar andapolar lipids in mitochondria from control rats. However,two populations of mitochondria were found in vitaminA-deficient rats according to the composition of mem-brane lipids. One population showed a similar fluores-cence intensity to that of the control mitochondria, andthe second population had a higher lipid content than theother population. This occurred in both apolar (green)(222 6 82%,n 5 4 from control) and polar lipids (red)(224 6 79%, n 5 4 from control). Results in restoredanimals tended to show a decrease in the relative pro-portion of mitochondria with high lipid content whencompared to the vitamin A-deficient group.

Vitamin A and GSH values in total liver

In control liver the GSH concentration (mmol/g) was5.96 0.3 (9), in vitamin A-deficient rats was 6.26 0.6 (8)

and in vitamin A-restored rats it was 5.86 0.6 (5). Therewere no significant differences between the groups.

Vitamin A status and mitochondrial glutathioneoxidation

Vitamin A deficiency caused a significant increase inGSSG levels concurrent with a decrease in GSH levels inisolated mitochondria. In the restored rats, although GSHvalues returned to normal, the GSSG values did not. TheGSH/GSSG ratio decreased significantly in the vitaminA-deficient rats when compared to control rats, and in therestored rats this ratio was significantly higher than thevalue from the vitamin A-deficient group, but it did notreach the control value (Table 2).

Malondialdehyde (MDA) concentration

Mitochondrial lipid peroxidation measured as malon-dialdehyde increased significantly in vitamin A-deficient

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 fluorescenceis a mixture of both lipids. FS5 forward side, SS5 scattered side.

4 T. BARBER et al.

rats. Results in restored animals were similar to thevalues found in the vitamin A-deficient group (Table 2).

Production of 8-oxo-dG by isolated mitochondria

Oxygen free radicals attack guanine residues convert-ing deoxyguanosine into 8-oxo-dG, which is one of thebest indicators of oxidative damage to DNA. In vitaminA-deficient rats the production of 8-oxo-dG was signif-icantly higher than in controls. The values returned tocontrol values in the vitamin A-restored group (Table 2).

DISCUSSION

Vitamin A malnutrition has increased dramatically inthis decade because more than 250 million people have asubclinical deficit of this vitamin [18]. Over the years,supplementation ofb-carotene in excess of physiologicalneeds produced neither benefit nor harm in terms ofincidence of malignant neoplasms, cardiovascular dis-ease, or death from all these causes [19]. After an aver-age of 4 years of supplementation, the combination ofb-carotene and vitamin A had no benefit and may havehad an adverse effect on the incidence of lung cancer andon the risk of death from lung cancer, cardiovasculardisease, and any cause in smokers and workers exposedto asbestos [20]. Thus, to study the antioxidant propertiesof this vitamin at physiological levels we have used theexperimental model of three different groups of rats: (i)control rats, (ii) vitamin A-deficient rats, and (iii) vitaminA-restored rats.

Mitochondria, besides functioning as organelles re-sponding to changes in ATP demand, have emerged asactive signaling organelles as shown in experiments per-formed in cells depleted of mitochondrial DNA(mtDNA) [21]. Mitochondrial defects occur in severaldegenerative diseases, aging, and cancer; and three of themore important aspects of mitochondrial oxidative phos-phorylation for disease pathogenesis are (i) energy pro-duction, (ii) generation of reactive oxygen species, and(iii) regulation of programmed cell death [22]. Moreover,oxidative stress is involved in different pathological con-

ditions, such as AIDS, Alzheimer’s disease, Parkinson’sdisease and in ischemia-reperfusion injuries, as well asnormal aging. mtDNA appears to be a common target foroxygen radical damage and accumulates 10 times moredamage than nuclear DNA [23]. These observations havebeen attributed to the proximity of mtDNA to metabolicoxidation generation [24]. The importance of mtDNA asa specific target of age-associated free radical attack hasbeen proposed [25]. Using the production of 8-oxo-7,8-dihydro-29-deoxyguanosine (oxo8dG), oxidation ofmtDNA was shown to be very high in aging [26]. Thesechanges found in aging are in agreement with the resultsfound using high-density oligonucleotide arrays, whichhave revealed a lower expression of genes involved inenergy metabolism (which includes the ERV1 gene)[27]. mtDNA, which encodes only 13 polypeptide genes(including some essential components of oxidative phos-phorylation), has a high mutation rate and, as the per-centage of mutant mtDNA increases, the cellular capac-ity diminishes until it falls below the bioenergeticthreshold.

The oxidation of mitochondrial GSH has been pro-posed as an index of oxidative stress and it has beendescribed as a direct relationship between the mitochon-drial GSH/GSSG and the production of oxo8dG [26]. Asimilar relationship occurs in this study because a de-crease in the GSH/GSSG ratio in vitamin A deficiency isfollowed by an increase in the production of 8-oxo-dG,and these changes are reversed in the vitamin A-restoredrats. Recently, it has been found that mitochondrial GSH/GSSG ratio in old rodents is lower, and the production ofoxo8dG from different tissues is significantly higher,than in the young rats. Treatment with antioxidants pro-tects against oxidation of the mitochondrial glutathionepool and the increase of mtDNA oxidation [26]. Similarresults were found in the liver of mice treated withzidovudine (39-azido-29,39-dideoxythymidine [AZT]),which is a drug that inhibits human immunodeficiencyvirus replication and delays the progression of AIDS.The production of oxo8dG is significantly higher whencompared to controls due to the increased production ofperoxides by liver mitochondria from AZT-treated mice.

Table 2. Glutathione Status, Malondialdehyde Concentration and Levels of Oxo8dG in Mitochondriafrom Liver of Control Rats, Vitamin A-Deficient Rats and Vitamin A-Restored Rats

Control Vitamin A-deficient Vitamin A-restored

GSH (nmol/mg protein) 9.36 1.3 (6) 4.06 0.7 (5)* 10.86 1.1 (6)GSSG (nmol/mg protein) 0.126 0.01 (7) 0.276 0.05 (5)* 0.236 0.02 (7)*GSH/GSSG 806 10 (6) 186 6 (5)* 516 8 (6)*MDA (nmol/mg protein) 1.76 0.2 (6) 2.96 0.6 (5)* 3.36 0.8 (3)*Oxo8dG (pmol/mg mt DNA) 0.226 0.05 (7) 0.356 0.05 (8)* 0.256 0.05 (5)

Results are means6 SEM with the numbers of animals indicated in parentheses. Values significantlydifferent from that of controls are shown by *P , .05.

5Vitamin A and oxidative stress

These changes were prevented by dietary administrationof vitamins C and E [28].

Mitochondrial changes in the heart of vitamin A-de-ficient rats have been reported previously [29] and aredue to a lower NADH coenzyme Q oxidoreductase ac-tivity (respiratory complex I of mitochondria), whichtransfers electrons from NADH to coenzyme Q. This wasaccompanied by a high-CoQ content to maintain themitochondrial electron transfer rate.

The antioxidant properties of vitamin A have beenshown in vitro and in vivo [6–10]; however, in thepresent work we studied the antioxidant role of thisvitamin in mitochondria and we found a mitochondriaGSH depletion, an increase in the production of 8-oxo-dG, and a decrease of MMP in vitamin A-deficient rats.This was accompanied by changes in lipid compositionof liver mitochondria, probably due to a decrease in themembrane turnover [30]. These changes are partiallyrestored when rats are re-fed a diet containing vitamin A.Recently, a “death sequence” similar to our findings hasbeen proposed after treatment of leukemic blasts with1-beta-D-arabinofuranosylcytosine, which includes GSHdepletion, loss of MMP, and phosphatidylserine expo-sure, which preceded a state of high reactive oxygengeneration [31]. In short, the deficit of dietary vitamin Aproduced an increase in oxidized glutathione, MDA,8-oxo-dG, an 80% decrease in the GSH/GSSG ratio, anda drop in MMP. This emphasizes the relevance of mito-chondria as targets for free radical damage associatedwith vitamin A deficiency and this was partially restoredwhen rats were re-fed with vitamin A. Moreover, bykeeping mitochondria functioning, vitamin A not onlyattenuates oxidative damage but may also modulate theexpression of signal transduction.

Acknowledgements— This work was supported by the Fondo Inves-tigacion Sanitaria. FIS 98/1461, FIS 99/1157 and by the GeneralitatValenciana GV-D-VS-20-152-96. Elisa Borra´s holds a predoctoralfellowship from the Danone Institute, SPAIN. We thank Marilyn R.Noyes for supervising the manuscript.

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7Vitamin A and oxidative stress