Free Radical Biology & Medicine, Vol. 15, pp. 661-665, 1993 0891-5849/93 $6.00 + .00 Printed in the USA. All fights reserved. Copyright © 1993 Pergamon Press Ltd.

Brief Communication

V I T A M I N E D E F I C I E N C Y I M P A I R S T H E M O D I F I C A T I O N S O F M I T O C H O N D R I A L M E M B R A N E P O T E N T I A L A N D M A S S IN

R A T S P L E N O C Y T E S S T I M U L A T E D T O P R O L I F E R A T E

CARLO PIERI, FAUSTO MORONI, and RINA RECCHIONI

Cytology Center, Gerontol. Res. Dept. of I.N.R.C.A., Ancona, Italy

(Received 25 January 1993; Revised 30 March 1993; Accepted 12 April 1993)

Abstract--This study was designed to evaluate the time-dependent changes of mitochondrial membrane potential and mass during Con-A-induced proliferation of splenic lymphocytes from rat fed a normal or a vitamin E deficient diet. Rhodamine 123 and Nonyl Acridine Orange were used as specific probes to monitor the membrane potential and mass of mitochondria, respectively, by means of flow cytometry. The results demonstrate that the increase ofRh- 123 and NAO uptake observed in cells from normally fed rats was prevented by vitamin E deficiency, at any time considered. After 72 h from Con A stimulation, 62% of cells from controls, as against 16% of cells from vitamin E deficient rats, showed hyperpolarized mitochondria. At the same time, in this last group, 60% of cells had depolarized organelles. The same pattern was observed considering the changes of mitochondrial mass, measured using NAO as a probe. These data support that mitogenic stimulation induced an increase of the respiratory activity of mitochondria with subsequent production ofsuperoxide radicals. This resulted in depolarization and loss of mass of the organelles if the intracellular level of vitamin E is not adequate.

Keywords--Vitamin E, Lymphocyte proliferation, Mitochondria, Membrane potential, Mitochondrial mass, Free radicals

INTRODUCI'ION

It is well recognised that activation of lipid peroxida- tion, regardless of the nature of prooxidant, causes a time-dependent fall of transmembrane potential, ef- flux of Ca ++, and other cations from mitochondria and swelling.l-4 Generally, in these studies, peroxida- tion was induced, adding different substances able to produce free radicals to the suspension of isolated mi- tochondria. However, there are no studies reporting whether free radicals produced during a physiological process of the cell may influence the mitochondrial transmembrane potential and mass. In principle, one has to find a model in which free radicals are physio- logically produced and in which the antioxidant de- fense system is so low that it cannot completely scav- enge these radicals.

The proliferating lymphocytes from vitamin E defi- cient rats seem to fulfill both conditions. Free radicals are produced by mitogenic stimulation, 5-7 and a de- crease of protective antioxidant system is assured by vitamin E deficiency. Indeed, the biological activity of

Address correspondence to: Carlo Pieri, Cytology Center, Geron- tol. Res. Dept. of I.N.R.C.A., Via Birarelli, 8-60121 Ancona, Italy.

vitamin E is generally believed to be due to its antioxi- dant action to inhibit lipid peroxidation in biological membranes, by scavenging the chain-propagating peroxyl-radicals, s,9 In addition, vitamin E, incorpo- rated into the cellular membranes, exerts a stabilising effect on the structure of biological membranes, thus reducing the sites available for free radical attack.I°

Depletion of dietary vitamin E has been shown to be associated with an increased formation of lipid per- oxidation product and a number of biochemical and pathological lesions in rats and other species of ani- mals. l

The immune system, too, responds adversely to severe vitamin E deficiency, ~2 and in particular, it has been reported that lymphocyte from vitamin E defi- cient rats showed marked mitochondrial swelling. 13

Transmembrane potential and mass of mitochon- dria can be determined by means of the vital stains Rhodamine-123 (Rh-123) 14,15 and 10-N-nonyl acri- dine orange (NAO),~6 respectively. Comparative stud- ies of NAO and Rh-123 uptake by lymphocytes dem- onstrated that mitochondrial binding of acridine or- ange derivatives was independent of respiratory activity.

Osmotic shock, uncoupler agents, and ionophores

661

662 C. PIER! et al.

Table 1. Composition of the Vitamin E Deficient Diet

Maize starch and sucrose 59% Extracted casein 18% Torula yeast 10% Melted filtered fat 8% Osborne-Mendel salts 5% Vitamin supplement lacking vitamin E 1%

that promptly decreased Rh-123 incorporation ~4 had no effect on NAO binding when measured within 30 min from the agent application) 6 Taking into ac- count this finding, we used these two probes to moni- tor the changes in membrane potential (i.e., in the respiratory activity) and mass of splenocyte mito- chondria in normally fed and vitamin E deficient ani- mals.

MATERIALS AND METHODS

Animals

Ten female Wistar rats from our breed were used. By the age of I month the animals were divided into two groups of five animals each. One group was fed for 10 months with commercial chow and the other with a vitamin E deficient diet (Dottori Piccioni, Bre- scia) of the composition reported in Table 1. Ten months of treatment were long enough to allow the development of the standard deficiency symptoms of vitamin E, such as weight loss and increased red blood cell hemolysis (data not shown).

Cell proliferation and flow cytometric analysis

Splenic lymphocytes were prepared by Ficol-Hy- paque gradient centrifugation according to Boyum.17 After repeated washes in Hank's solution they were resuspended (2 × 106 cells/ml) in RPMI 1640 supple- mented with 2 mM glutamine, 10% fetal calf serum, 100 units/ml penicillin, and 100 ~g/ml streptomycin.

The splenocytes were stained for mitochondrial membrane potential determination by Rh-123 (Mo- lecular Probes, Eugene, OR) as previously described by Darzynkiewicz et al. 15 Samples of cells (2 × 106/ ml) were incubated for various durations in the pres- ence and absence of Con A (5 ~g/ml). At different time points, the cells were stained with 25 #M Rh- 123 at room temperature for 20 min in the dark. The cells were washed twice with phosphate-buffered saline (pH 7.4) and were suspended in RPMI 1640.

The same procedure was followed for the staining with 5 uM NAO (Polyscience) for 15 min according to Retinaud et al. t6 Before analysis, l0 ~g/ml ethid-

ium bromide (EB) was added to each sample to moni- tor the dead cells, which were excluded from the analy- sis ofRh-123 and NAO fluorescence by gating for red fluorescence. Measurements of the fluorescence on a cell-by-cell basis were carded out in a Coulter Epics V flow cytometer (Coulter, Hialeah, FL). The argon ion laser was tuned to 488 nm. The green fluorescence of Rh-123 and NAO-stained cells was detected between 500 and 540 nm, whereas the red fluorescence emit- ted by the EB, which penetrated only dead cells, was measured at wavelengths higher than 615 nm. Data were analysed by program packages provided by the manufacturers.

RESULTS

Rh- 123 has largely been used to monitor the mito- chondrial membrane potential of proliferating lym- phocytes, ~5.16.~s:9 and this method gives a satisfactory resolution of the cell populations with different trans- membrane potential.

Figure 1 reports examples of histograms showing the distribution of Rh-123 fluorescence over the cell population, obtained with resting and Con A stimu- lated cells at 72 h. Although the data reported in the present article were calculated from the integral of probe fluorescence, to emphasise the difference be- tween the two experimental groups in the figure the logarithm of the fluorescence was reported in the ab- scissa.

In control and in mitogen stimulated cells, three distinctly different fluorescent populations could be recognised after Rh- 123 staining. The first population (between channels 0-80) binds low amounts of dye, representing cells with partially or totally depolarized mitochondria. The second population (channels 80- 140) is likely to include lymphocytes which had nor- mally polarised organelles; and the third one is a highly fluorescent (greater than channels 140) popula- tion which arises from ceils with hyperpolarized mito- chondria. Mitogenic stimulation resulted in an in- crease of the number of the cells belonging to the first and third populations at the expense of the sec- ond one.

It is of importance that the lymphocytes from vita- min E deficient animals showed a large number of cells within the first population.

A picture very similar to the one ofRh- 123 fluores- cence was seen in the case of NAO fluorescence, re- porting on the overall mass of mitochondria present in the investigated population (not shown),

The time-dependent changes in cell populations showing low, normal, and high Rh- 123 and NAO fluo- rescence are summarised in Fig. 2.

Vitamin E and mitochondria

Rh-123

663

400 Control

8

Z

300

200

100

50 I00 150 200

400 -Vit. E

300

t ~

,~ 2oo

Z 100

50 100 150 200

Log of fluorescence intensity (channel number)

Fig. 1. Histograms of Rh-123 fluorescence distribution of 72 h unstimulated (D) and Con A stimulated ( I ) lymphocytes from control and vitamin E deficient rats.

Small changes in the percentage of cells with depo- larized mitochondria occurred in the unstimulated lymphocytes, irrespective of the diet applied to the animals. After 24 h from Con A stimulation, the dif- ferences between the two models in the percentage of cells showing hyperpolarized or depolarized mito- chondria did not exceed 6-7%. However, at 48 h, 60% of lymphocytes from control rats had hyperpolarized organelles, as against 42% of cells from vitamin E defi- cient animals. At this time, depolarization of mito- chondria occurred in 35% and 22% of the lympho- cytes from vitamin E deficient and normally fed ani- mals, respectively.

The differences already observed at 48 h were stressed when 72 h of culture was taken into account. Indeed, the number of cells showing high Rh- 123 fluo- rescence reached the level of 62% in the control ani- mals, but decreased to 16% in the rats fed a vitamin E deficient diet. At the same time, in this group the per- centage of lymphocytes with depolarized mitochon- dria increased up to 60%.

The same pattern could be observed considering the NAO fluorescence. It must be noted, however, that the number of cells showing loss of mitochon- drial mass was lower when compared to the number of lymphocytes with depolarized organelles at any

664 C. P1ERI et aL

100.

80.

,~ 60.

40.

~_. 20.

0

lO0

Low

2'4 4'8 ; 2

Low

Rh-123 N o r m a l High

NAO N o r m a l High

8O

"~ 60 g ~ 40

o 20 Cu Y f , i • i i

24 48 7'2 0 24 48 7'2 T i m e a f t e r Con A s t i m u l a t i o n

24 4'8 7'2

Fig. 2. Changes of cell population showing low, normal, and high Rh-123 and NAO fluorescence during proliferation of lymphocytes from control (open symbols) and vitamin E deficient (closed symbols) rats.

given time and in both models. One must take into account, however, that only viable cells were taken into consideration in our analysis, and it is likely that lymphocytes with swelled mitochondria died and were thus excluded on the basis of EB gating.

D I S C U S S I O N

Mitochondria consume over 90% of oxygen used by most cells and are a significant source of active oxygen under normal conditions.

However, in normal conditions the cells possess a number of antioxidants to protect them against the harmful attack of free radicals. Any condition which increases this generation or decreases the protective system will most likely have deleterious consequences to the mitochondria. Both conditions are present in our experimental model. Indeed, free radicals are pro- duced by mitogenic stimulation of lymphocytes dur- ing the activation process, 5-7 and a decrease of the protective system is assured by the long-lasting feed- ing of a vitamin E deficient diet.

Several authors have addressed their studies to the effect of vitamin E on the proliferative response of lymphocytes and showed that this vitamin was neces- sary to maintain optimal T- and B-lymphocyte re-

sponse. 12 It must be noted, in this contest, that the level of dietary vitamin E which was necessary to maintain optimal lymphocytes response was greater than the level required to prevent other standard signs of vitamin E deficiency, suggesting a specific role of this vitamin in the protection of these cells against peroxidation) 2 Since a decrease of body weight as well as an increase of red cell blood hemolysis occur in our deficient animals (data not shown), it is safe to assume that in our deficient model the vitamin E con- tent of splenocytes is decreased.

Findings of the present work clearly show that vita- min E deficiency resulted in small changes of mito- chondrial membrane potential and mass of lympho- cytes when they were in resting state or after 24 h from Con A stimulation. This suggests that the production of free radicals, due to the early phase of mitogenic stimulation, 5-7 was low enough to be buffered by the antioxidant defense system present in the cell and also of vitamin E deficient animals.

However, already after 48 h, but more evidently at 72 h from Con A stimulation, the increase of Rh-123 and NAO uptake commonly observed during lym- phocyte proliferation 15'16'18'19 occurred at different ex- tents, depending on the diet applied.

The number of cells able to increase the uptake of

Vitamin E and mitochondria 665

Rh-123 a n d N A O d imin i shed , a n d the n u m b e r o f cells showing d a m a g e d m i t o c h o n d r i a inc reased with t ime in v i t amin E def ic ient an ima l s as c o m p a r e d to the n o r m a l l y fed ones.

There are no da t a in the l i te ra ture to c o m p a r e ou r results wi th since the p resen t work seems to be the first p resent ing a c o m b i n e d analys is o f m i t o c h o n d r i a l m e m b r a n e po ten t i a l and mass changes du r ing prol i f- e ra t ion o f cell f rom v i t amin E def ic ient an imals . How- ever, the present f indings suggest tha t mi togen ic s t im- u la t ion induces an increase o f the resp i ra tory ac t iv i ty o f m i tochond r i a , as d e m o n s t r a t e d by the increase o f Rh- 123 uptake , wi th subsequen t p r o d u c t i o n o f super- oxide radicals . The ha rmfu l effect o f this is d e p e n d e n t bo th on the t ime o f cu l tu r ing and on the presence o f v i t amin E. W h e n this v i t amin is lacking, excessive pe rox ida t ion m a y occur, which p r o m o t e s efflux o f Ca ++ and o f o the r ions, wi th col lapse o f m i t o c h o n - dr ia l m e m b r a n e potent ia l .

A s imi la r m e c h a n i s m m a y accoun t for the decrease o f N A O up take occur r ing in pro l i fe ra t ing l y m p h o - cytes f rom v i t amin E def ic ient rats (Fig. 2). M i t o c h o n - dr ia l swelling fol lows ra ther than preceeds or paral le ls m e m b r a n e po ten t i a l loss. 2°-22 This is also suppo r t ed by the presen t f inding tha t the n u m b e r o f cells wi th depo la r i zed m i t o c h o n d r i a is a lways higher t han tha t repor t ing the loss o f m i t o c h o n d r i a l mass.

In conclus ion , results o f the presen t work suggest tha t pro l i fe ra t ing i y m p h o c y t e s f rom v i t amin E defi- c ient an ima l s m a y be a m o d e l to s tudy the re la t ion- ship be tween phys io logica l ly p r o d u c e d free radicals and m i t o c h o n d r i a l func t ion in l iving cells. This m o d e l m a y be useful to test in l iving cells an t i ox idan t s ac t ing at the m i t o c h o n d r i a l level.

Acknowledgement - - The authors thank Mr. G. Mazzarini for edit- ing the text and Mrs. M. Glebocki for reading the manuscript.

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