ARCHIVESOF BIOCHEMISTRYAND BIOPHYSICS Vol. 196, No. 1, August, pp. 270-280, 1979

The Influence of Vitamin E and Selenium on the Growth and Plasma Membrane Permeability of Mouse Fibroblasts in Culture A. S. M. GIASUDDIN Department

AND

A. T. DIPLOCK

of Biochemistry and Chemistry, Guy’s Hospital London SE1 9RT, United Kingdom

Medical

School,

Received January 29, 1979; revised March 28, 1979 The present communication describes a tissue culture system which can be used to simulate conditions of vitamin E, selenium, and essential fatty acid deficiency, and in which the effect of adding these, and other, substances can be studied. By restricting the lipid content of fetal calf serum, the effect of the addition of specific lipids on growth and on permeability to 2-deoxyglucose of the plasma membrane was determined. It was found that optimal growth and glucose -transport depended on the presence together of vitamin E, linoleic acid, and cholesterol in the medium, and the significance of this finding is discussed in relation to current ideas about the biological action of vitamin E. By incorporating only 2.5% fetal calf serum in the growth medium, conditions of selenium deficiency could be demonstrated, and the addition of 0.1 nmol Se per dish stimulated growth whereas at higher levels of inclusion selenium was found to be toxic.

The question of whether the role of vitamin E at the cellular and molecular levels is entirely due to its antioxidant action (l), or whether a-tocopherol has a more specific function, remains controversial. The suggestion (2, 3) that a-tocopherol may enter into specific physicochemical interactions with the arachidonyl residues of membrane phospholipids has found support in recent studies. Thus, experiments in which the interaction of tocopherols of varying sidechain length with phospholipids of differing unsaturation was studied in monolayers at an air-water interface showed (4) that maximum “fit” was obtained between natural a-tocopherol (with 15 carbon atoms in the side chain) and arachidonyl phosphatidyl cho line. Removal or addition of carbon atoms in the tocopherol side chain lessened the extent of the interaction, and a similar result was obtained when lecithins of less unsaturation, or of shorter fatty acid chain length, were substituted for arachidonyl lecithin. It has also been shown (5) that tocopherols either without a side chain or with a longer side chain than a-tocopherol are devoid of biological activity. In other experiments (6), liposomes were made from leci0003-9861/79/090270-11$02.00/O Copyright 0 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.

thins of different arachidonic acid content, and the effect of a-tocopherol on the permeability of the vesicles to glucose and chromate ions was determined. It was found that the permeability to both substances was increased in liposomes prepared from phospholipids containing a relatively high proportion of arachidonyl residues; cr-tocopherol decreased the permeability of these liposomes, the largest effect being seen in association with the largest proportion of arachidonic acid. In the present communication, a method is described for culturing a mouse fibroblast cell line in media from which essential fatty acids, cholesterol, and a-tocopherol have been largely removed; the effect of adding back ai-tocopherol, linoleic acid, and cholesterol on growth and on the permeability of the cell membranes to 2-deoxy-r+[1-3H]glucose was investigated. In addition, the effect of selenium upon growth of the cultured cells was investigated in media low in their content of fetal calf serum. MATERIALS

AND METHODS

Cell line. Mouse Balb/3T3,A3, (7) cells were used.

They can be kept under continuous culture and are

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normal, nontumorigenic, strongly contact-inhibited cells that are fibroblast derived but appear epithelioid in confluent culture. Culture techniques. The cell line was cultured in a humidified 5% CO* atmosphere using conventional techniques in Dulbecco’s modified Eagle medium (DMEM)’ (8) supplemented with 2.5-10% fetal calf serum (FCS) or delipidized fetal calf serum: The amount of serum added is given in the individual experiments. Preparation of delipidized serum. The method used was modified from that of Albutt (9). FCS, 50 ml, was cooled to 4°C and added with stirring to 450 ml of acetone at -20°C. The precipitated proteins were washed twice at -20°C with 100 ml of acetone and twice with 100ml of diethyl ether at -20°C. The powder was air-dried in thin layers on paper sheets, and residual solvent was removed at the pump on a Buchner funnel. The dry powder was redissolved in 50 ml of balanced salt solution and sterilized by filtration through a 0.45pm Millipore filter. The reconstituted serum was designated acetone-extracted serum (AES). Estimation of lipids. Total phospholipids were estimated by the method of Raheja et al. (lo), cholesterol by the method of Zlatkis et al. (ll), triglyceride by the method described in a Sigma Technical bulletin (12), free fatty acids by the method of Mosinger et al. (13), and a-tocopherol by the method of Bieri and Prival(14). Preparation of growth media. The medium used was DMEM (IX) with bicarbonate buffer, to which was added L-glutamine (200 mM) and a penicillinstreptomycin mixture (50- 100 units of each antibiotic in 1 ml of medium). The medium was used with the addition of FCS or AES as indicated in the individual experiments. AES was either used without the addition of lipids or after specific amounts of the individual lipids described had been added. DLa-Tocopherol (Roche Products Ltd., 15 Manchester Square, London W.l., U. K., 99% purity), linoleic acid (Sigma Chemical Co., St. Louis, MO., 99% purity), arachidonic acid (Sigma Chemical Co;, 99% purity), and cholesterol (Sigma Chemical Co., 99% purity) were dissolved individually in appropriate amounts of ethanol. The solutions were warmed to 50°C and added, in the amounts stated in the experiments, to portions of AES; the mixture was then incubated at 37°C for 1 h to allow maximum formation of serum-lipid complexes and it was sterilized by passage through a 0.45pm Millipore 6lter. The serum (either FCS, AES, or AES with added lipids) was added to a screw-cap bottle containing the appropriate volume of the DMEM-bicarbonate medium described above. The ethanol content of the media to which lipids were added was 0.3%; in control experiments, this was shown to have no effect on the growth * Abbreviations used: DMEM, Dulbeceo’s modified Eagle medium; FCS, fetal calf serum; AES, aeetoneextracted serum; BHT, butylated hydroxytoluene.

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of the cells. The possibility of contamination of the cultures with Acholeplasmu was checked at regular intervals using a standard technique (15). In preliminary experiments cell growth was monitored by counting the cells and by estimating the total protein content of the dishes by the method of Lowry et al. (16). It was found that, during the logarithmic phase of growth, the viable cell count was quantitatively related to the protein content, and in most experiments the protein content was used as a measure of the growth rate of the cells. Measurement of 2-deoxy-Bglueose transport. The procedure was based on that of Hatanaka and Hanafusa (1’7). Cells were cultured as described above and in the individual experiments; the growth media were removed from the monolayers by gentle suction and the cells washed twice with balanced salt solution, pH 7.4, at 37°C. A solution of the amounts of 2-deoxy-D-glucose indicated in the experiments, in balanced salt solution, pH 7.4 (18), was prepared containing 0.5 +i of 2-deoxy-D-[1-3H]glucose. One-milliliter aliquots of this solution were added to each dish bearing cells and the dishes incubated in the CO, incubator for the times stated. The radioactive medium was then removed and the cells were washed twice with Z-ml portions of glucose-free balanced salt solution, pH 7.4, at 37°C. The cells were lysed by incubation for 30 min at 37°C with 1 ml water and a suspension was made of the lysate, portions of which were taken for protein estimation and for liquid scintillation counting by standard techniques. Uptake of the sugar was expressed as nanomoles of 2-deoxy-D-glucose taken up per milligram of protein per 10 min. RESULTS

A, The Effect of Added Lipids on Cell Growth. In preliminary experiments it was found that a medium containing 7.5% AES without added lipids gave a convenient growth rate, and that the growth rate of the cells was stimulated by the addition of a-tocopherol and arachidonic acid but not by the addition of cholesterol or linoleic acid. The effects of these lipids on growth was therefore investigated. Media containing the lipids shown in Table I were prepared and three dishes containing each medium were seeded with 4 x lo4 cells. Growth was followed until, after 88 h, the cells in medium 8, which contained a-tocopherol, cholesterol, and linoleic acid, were nearing the end of the logarithmic phase, when all the cells were harvested and their protein content was measured. The results are given in Table I. Tocopherol,

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TABLE I

EFFECTOFWTOCOPHEROL, CHOLESTEROL, ANDLINOLEICACIDONCELLS CULTUREDINLIPIDDEPLETEDMEDIA" Protein (pg/dish)

Supplements to growth medium 1. 2. 3. 4. 5. 6. 7. 8.

112 k 141 f 104 ” 157 + 109 2 149 2 119 2

None 2.5 @g/ml DL-a-tocopherol 10 @g/ml cholesterol 2.5 pgiml DL-a-tocopherol + 10 pg/ml cholesterol 10 pg/ml linoleic acid 2.5 pg/ml DL,-cu-tocopherol+ 10 pg/ml linoleic acid 10 pg/ml cholesterol + 10 pg/ml linoleic acid 2.5 kg/ml DL-a-tocopherol + 10 pg/ml cholesterol + 10 @g/ml linoleic acid

Percentage change relative to medium 1 +26 -7 +40 -3 +33 +6

3 4b 2 5b 7 4* 6

210 ? 5**c

+88

a Each value is the mean 2 SD of triplicate determinations on three separate dishes. Three dishes, each containing DMEM with 7.5% AES and one of the supplements shown, were seeded with 4 x lo4 cells and, after 88 h of growth, the cells were harvested and their protein content measured. * Significantly different from value for medium 1: P > 0.001. c Significantly different from value for media 4 and 6: P > 0.001.

alone or in combination with other lipids, consistently stimulated growth, in contrast to cholesterol and linoleic acid which gave no such stimulation either separately or when combined together. However, the greatest effect on the growth rate of the cells was obtained when tocopherol, cholesterol, and linoleic acid were all added to the medium. The lipid content of the sera was determined and the results are given in Table II, from which it can be seen that the lipid extraction procedure resulted in a very considerable decrease in the level of all the classes of lipids tested. The level of a-tocopherol in FCS is always low because a-tocopherol can only penetrate the placenta with difkulty (19), and it can be assumed that the very small amount of tocopherol present in the original FCS was removed by the lipid extraction technique. In normal cells, substantial amounts of linoleic acid are converted into arachidonic acid which becomes incorporated into membrane phospholipids. An experiment was therefore carried out in which the effect of added arachidonic acid, and its possible interaction with cz-tocopherol, was investigated. Media containing the lipids shown in Table III were prepared and six dishes containing eachmedium were seededwith 5 x lo4 cells. The number of cells in each dish was

counted after 48 h (three dishes) and again after 120 h (three dishes) when the cells in medium 4 were almost confluent. After 48 h of growth (Table III) the inhibitory effect of arachidonic acid at the higher level was the only significant effect of the added lipids. After 120 h, however, tocopherol, either with or without added arachidonic acid, stimulated growth and the vitamin also reversed the inhibitory effect of the higher concentration of arachidonic acid. Arachidonic acid at the lower concentration stimTABLE II

LIPIDCONTENTOFFETALCALFSERUMAND ACETONE-EXTRACTEDFETALCALFSERUM" AES Lipid estimated

FCS (mg/lOO ml) mg/lOO ml % of FCS

Total phospholipid 54 + Total cholesterol 49 2 Triglyceride 69 ” 30 f Free fatty acids a-Tocopherol 0.15 2

4 1 2 1 0.01

14 t 4 522 4rl 321 N.D.b

26 + 6 10 + 4 621 11 c 5 0

a Each value is the mean ? SD of duplicate analyses on three separate lots of serum. The lipid contents of the sera were determined by the methods described in the text. b Not detected.

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TABLE III EFFECT OF (Y-TOCOPHEROL AND ARACHID~NIC ACID ON CELLS CULTURED IN LIPID DEPLETED MEDIAN Number of cells Supplements to growth medium 1. 2. 3. 4.

None 1 pg/ml DL-o-tocopherol 10 pg/ml arachidonic acid 1 pg/ml DL-cw-tocopherol+ 10 pg/ml arachidonic acid 5. 20 pg/ml arachidonic acid 6. 1 pg/ml r&-o-tocopherol + 20 pg/ml arachidonic acid

x

10m5

At 48 h

At 120 h

Percentage change at 120 h relative to medium 1

1.8 r 0.3 1.9 2 0.2 1.7 t 0.1

6.2 * 0.4 7.9 f 0.3b 9.2 2 0.2c

+27 +48

1.7 r 0.3 0.7 r O.lb

8.4 k 0.3c 3.8 + 0.2”

+35 -38

1.7 r 0.2

7.5 f 0.20

+21

a Each value is the mean +- SD of triplicate determinations on three separate dishes. Six dishes, each containing DMEM with 7.5% AES and one of the supplements shown, were seeded with 5 x lo4 cells and, after each of the times of growth stated, cells were harvested and the number of cells in each dish counted. b Significantly different from value for medium 1: P > 0.01. c Significantly different from value for medium 1: P > 0.001.

mated growth but the maximum effect was noted when a-tocopherol was added with the lower concentration of arachidonic acid. The possibility that the growth-stimulating effect of cr-tocopherol in combination with linoleic or arachidonic acids was due to a nonspecific antioxidant effect could not be ignored. An experiment was therefore done to investigate the effect of a synthetic nonphysiological antioxidant, butylated hydroxytoluene (BHT), on growth of the cells. A stock solution of BHT was made in ethanol and added to a solution of AES in DMEM as described above for a-tocopherol. Dilutions

were then made with DMEM (Table IV) to give final concentrations of 2.5,5.0, and 10.0 pg BHT/ml medium; appropriate amounts of each medium were placed in each of 12 dishes, 6 of which were seeded with 2 x lo4 cells, and the remainder with 4 x lo4 cells. Three dishes were withdrawn after 96 h for protein estimation and the others after 120 h. No consistent statistically significant effect of BHT on growth was noted (Table IV) although the trend was for the antioxidant to be slightly stimulatory. Furthermore, the molar concentration of the BHT at the highest level of inclusion was approximately

TABLE IV THE EFFECT OF BUTYLATED HYDROXYTOLUENEON GROWTHOF CELLS IN CULTURE” Protein (fig/dish) 2 Supplements to growth medium 1. 2. 3. 4.

None 2.5 gg BHT/ml 5.0 pg BHT/ml 10.0 pg BHTlml

lo4 cells seeded

x

At36h 44 48 47 48

f + 2 k

2 5 3 2

4

x

lo4 cells seeded

At 120 h

At 96 h

At 120 h

89 f 3 100 -c 12 92 k 5 101 f 3*

73 2 6 87 ” 2b 84k6 86 2 3b

128 + 126 + 126 k 129 2

18 17 6 5

a Each value is the mean 2 SD of triplicate determinations on three separate dishes. Six dishes, each containing DMEM with 7.5% AES and one of the supplements shown, were seeded with the numbers of cells shown and, after each of the times of growth stated, cells were harvested and their protein content measured. b Significantly different from cells in corresponding medium without added BHT: P > 0.01.

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AND DIPLOCK TABLE V

eight times that of the a-tocopherol used (Table I) where a-tocopherol showed a consistent statistically significant stimulator-y effect on growth.

EFFECT OF SELENITE ON CELLS IN CULTURE Protein (@g/dish)

B. The Effect of Selenium on Cell Growth A preliminary experiment was first carried out in which the effect on cell growth of the addition of a wide range of amounts of selenium was studied. A stock solution of Na,SeO, (10 PM) was made in DMEM, diluted with DMEM to give the concentration shown in the experiments, and sterilized by filtration. Six dishes containing each of the growth media shown in Table V were set up and each was seeded with 5 x lo4 cells. The protein content of each dish was measured after 72 h (three dishes) and 90 h (three dishes) when the cells in medium 6 were nearly confluent. The results (Table V) show that at both times of growth, selenium at final concentrations of 100 and 1000 nmol/ dish was toxic; lower concentrations (10, 1, and 0.1 nmol/dish) either had no effect or produced a significant stimulation of growth. Analysis of the DMEM used for their experiments, for selenium, by a sensitive fluorometric technique revealed that the only source of selenium in the growth medium is the fetal calf serum; the selenium content of the serum can vary widely, depending on the selenium level in the soil (and thence in the plants) in the region where the cattle originated. In order to obtain an estimate of the probable biologically available selenium content of the serum we devised a bioassay, based upon the fatal rat liver necrosis associated with vitamin E and selenium deficiency. This assay was then used to estimate the content of biologically avaiIable selenium in two typical batches of fetal calf serum; the experimental details were as follows. Weanling male Wistar rats (50-60 g) were given a vitamin E- and selenium-deficient torula yeast-containing diet for 7 days. They were then divided into six groups of six rats and given, respectively, 0,0.25,0.5, 1.0, and 2.5 ml orally per day of FCS while the dietary regimen was continued; a further group of six rats was given 0.1 ppm Se as Na$eO, to drink instead of water. The day on which rats in all the groups died was noted, and, where possible, dead rats were

1. 2. 3. 4. 5. 6.

Supplements to growth medium

At 72 h

At 90 h

None 1000 nmol Se/dish 100 nmol Se/dish 10 nmol Se/dish 1 nmol Se/dish 0.1 nmol Se/dish

156 + 72 69 t 165 k 178 + 189 k

306 2 15 k 108 + 310 k 328 k 335 t

9 lb 4* 1’ 8* 3”

15 4b 5b 11 I* 4*

a Each value is the mean k SD of triplicate determinations on three separate dishes. Six dishes, each containing DMEM with 7.5% AES and one of the supplements shown, were seeded with 5 x lo4 cells and, after each of the times of growth stated, cells were harvested and their protein content measured. b Significantly different from value for medium 1: P > 0.001. c Significantly different from value for medium 1: P > 0.02.

subjected to postmortem examination. Those rats surviving after 45 days were also killed and their livers examined visually. The results are given in Table VI for one of the two batches of serum tested; the other batch gave similar results. In calculating the content of biologically available selenium in the serum, two assumptions were made; first, that 0.02 ppm of selenium in the diet is the minimum amount required to give complete protection from liver necrosis (this fact has been established many times in parallel experiments in our laboratory and is in line with experience worldwide). Second, it was assumed, from the results in Table VI, that 1.0 ml BCS/rat/day is the minimum quantity required for complete protection against liver necrosis (this may be a slight overestimate). Using these facts, the approximate content of biologically available selenium in FCS was calculated to be 200 rig/ml; the content of selenium in the medium containing 7.5% FCS was thus 18 ng Se/dish (containing 1.5 ml of complete medium). Since the lipid extraction technique involved the use of acetone and ether it seems unlikely that any selenium would be extracted with these solvents. It can therefore be assumed that the amount of selenium determined in FCS represents the maximum amount of selenium that could be present in AES.

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TABLE VI

BIOASSAYFORSELENIUMOFFETALCALFSERUM~ Days rats died

Rats surviving at end of experiment

Dosage

Remarks Survivor had postnecrotic scarring

None 0.1 ppm Se as NkSeO, to drink 2.5 ml FCS/day

l/6

28,28,33,37,37

6/6 516

24

1.0 ml FCS/day 0.5 ml FCS/day

616 2/6

26, 32, 41, 42

0.25 ml FCS/day

l/6

28,29, 30,30, 32

Survivors had normal livers Survivors and dead rat had normal livers Survivors had normal livers Survivors had moderate necrosis affecting one to three lobes of liver Survivor had extensive hemorrhagic necrosis of all lobes of liver

a Thirty-six weanling Wistar male rats were given the vitamin E- and selenium-deficient diet (A. T. Diplock, H. Baum, J. A. Lucy, 1971 Biochem. J. 123, 721-729) for 1 week and then FCS orally as indicated while the diet was continued.

The effect of selenium on growth of cells was expected to depend upon the level of inclusion of FCS or AES in the medium, since serum is the only source of selenium in the medium. The effect of varying the serum content of the media was therefore investigated using 7.5,5.0, and 2.5% FCS and AES in the DMEM medium. The results, given in Table VII, show that growth was dependent on the level of FCS or AES in the medium. The effect of removing the lipids from the medium was large at the 7.5% level of inclusion of the serum, but, at the 2.5% level,

delipidation of the serum had no effect on growth. Having thus established that cells would grow, albeit slowly, in media containing low levels of FCS and AES, the effect of added selenium on growth was investigated in these media. Media containing 5.0% FCS and AES and 2.5% FCS and AES were prepared (Table VIII) and selenite added to give final concentrations of 0, 0.1, and 0.5 nmol Se/dish. Nine dishes containing each medium were seeded with 5 x lo4 cells and three dishes with each medium were withdrawn for protein estimation 2.5, 3.5, and

TABLE VII

Protein (pg/dish) 5.0%

7.5% Growth period (days)

FCS

AES

2 3 4 5 6 6.5

16 2 1 41 ” 1 120 -c 4 271 2 13 553 k 21 643 k 11

2.5%

FCS

AES

16 2 2

15 t 1

29 ” 4 69 zt 3 185 k 1 376 f 15 470 f 38

29 5 7 90 ‘- 4

I3 2 2 28 2 1 59 -c 7

221 + 1 407 ? 0 488 2 3

FCS

AES

620

720

15 -e 3

15 + 1

145 r 4

33 c 2 95 + 4

31 re_9 96 -c 3

301 f 8 375 -c 6

164 f 1.

1685 11

204 +- 10

231 2 10

a Each value is the mean * SD of triplicate determinations on three separate dishes. Dishes containing the media shown were seeded with 4 x lo4 cells and, after the time intervals shown, three dishes were removed for each medium for protein determination.

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4.5 days after beginning the experiment. Growth curves were also plotted using the time of taking the samples and the logarithm to the base 2 of the protein values. The log, was used because it has been shown (20) that an increase in one unit on the log, scale corresponds to one division by each cell. Mass doubling times (h) and generations per 24 h were therefore calculated from the linear portions of these growth curves and this information is also given in Table VIII. These results show clearly that, at 0.1 nmol Se/dish, selenium has a consistent stimulatory effect on growth of the cells, both with FCS and AES at both levels of inclusion; this effect was much larger when the medium serum concentration was 2.5% than when it was 5.0%. The effect of delipidation of the serum (AES vs FCS) was to decrease somewhat the growth of the cells grown in media to which no selenium had been added, but

AND DIPLOCK

the magnitude of the stimulatory effect of selenium was not greatly affected by delipidation. C. Transport of Glucose into Cells Grown in Media of Differing Lipid Composition The objective of these experiments was to assess the effect of the lipids studied in section (A) on the carrier-mediated transport of glucose into the cells. Media containing 0, 1, 1.5, 2, 4, and 6 mM Z-deoxy-n[l-3H]glucose were prepared and the uptake of the isotopically labeled hexose into cells grown for 72 h (approximately half-confluency) in DMEM with 7.5% AES was studied as described under Materials and Methods. The results (Fig. 1A) show that the transport system was saturated at approximately 2 mM 2-deoxy-D-glucose and this concentration of hexose was used in subsequent experiments. The time course of uptake of the

TABLE VIII EFFECT OF SELENITE ON CELLS IN CULTURES

Protein (pgldish) 5.0% FCS

5.0% AES

Growth period (days)

0 nmol Se/dish

0.1 nmol Se/dish

0.5 nmol Se/dish

0 nmol Se/dish

0.1 nmol Se/dish

0.5 nmol Se/dish

2.5 3.5 4.5 Mass doubling time (h) Generations per 24 h

29 2 1 61 k 1 195 2 14 20.1 1.19

34 2 1 85 k 3 202 ” 9 18.6 1.29

29 k 1 72 f 3 219 2 14 -

24 k 1 50 + 1 108 k 4 22.1 1.09

31 2 1 60 k 3 141 2 4

25 t 1 53 k 8 107 k 3 -

20.1

1.09

Protein (pg/dish) 2.5% FCS

2.5% AES

Growth period (days)

0 nmol Se/dish

0.1 nmol Se/dish

0.5 nmol Se/dish

0 nmol Se/dish

2.5 3.5 4.5 Mass doubling time (h) Generations per 24 h

22 5 0 36 f 4 73 + 1 28.9 0.83

29 + 3 59 2 7

22 k 0 56 +- 3

137 + 1

108 k 9

18 * 1 27 2 1 41 -+ 0

21.5 1.13

-

31.5 0.77

0.1 nmol Se/dish

0.5 nmol Se/dish

19 2 1

15 t 0 34 k 8 49 k 4 -

44 -c 3 83 2 6 22.2 1.08

a Each value is the mean + SD of triplicate determinations on three separate dishes. Dishes containing the media shown were seeded with 5 X lo4 cells each and, after the time intervals shown, three dishes were removed for each medium for protein determination.

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since it was not possible to carry out the transport experiments simultaneously on cells grown in all the media together, the experiment was done in a series of batches which always contained cells grown in DMEM with 7.5% AES as reference; the transport of 2-deoxy-D-[l-3H]glucose in this medium under our standard conditions was 92 + 8 nmol hexose taken up per milligram of protein in the lo-min experimental period. TimeCmin) The transport rate observed for the batch 3601 under investigation was, for the purposes a of comparison with other batches, called -A1.00, and the transport rates in cells grown in the other media were related to this figure; / the results are given in Table IX. cr-Tocopherol consistently increased the rate of transport in all cases in contrast to cholesterol and linoleic acid which, when added OY , , , , , , 0 6 2 alone, did not affect the transport rate; the &I DOG addition of cholesterol and linoleic acid to FIG. 1. (A) A series of dishes containing 1.5 ml of the tocopherol did not result in any increase in DMEM medium with 7.5% AES were seeded with 4 the transport rate compared to the rate in x 1W cells. After ‘72h growth, when the cells were cells grown in media containing a-tocopherol approximately half-confluent, the medium was removed only. When a-tocopherol and cholesterol and replaced by medium containing 0, I, 1.5, 2, 4, and were present in the medium together with 6 mM 2-deoxy-ti-[l-3H]glucose. Glucose uptake was either linoleic or arachidonic acids, the studied in triplicate lots of cells as described in the greatest rate of transport was observed, text, after 15 min of contact with the media containing labeled hexose. The figure shows the content of and this rate was statistically larger than 2-deoxy-D-[l-3H]glucose in the cells at the end of this the rate observed in cells grown in media period. (B) Cells were cultured as described above for containing only a-tocopherol. 60 and 80 h; after removing the growth medium, medium containing 2 mM 2-deoxy-D-[l-3H]glucose was added to cells grown for the two time periods, and uptake of the labeled hexose measured 5, 10, 15, 20, and 30 min after it was added. The figure shows the content of 2-deoxy-o[l-3H]glucose in the cells grown for 60 h (0) and 80 h (0).

DISCUSSION

The results presented in this paper confirm and establish our earlier work (21) which showed that fibroblasts could be cultured in media which are delipidated, that such media are also vitamin E deficient, and isotopically labeled hexose was then studied that selenium is often present in suboptimal in cells grown as described above for 60 and amounts in media used for tissue culture. In 80 h. After adding the 2-deoxy-D-glucose addition, it has been shown that alterations the medium was removed from triplicate in the a-tocopherol, cholesterol, and fatty lots of cells, grown for the two periods, at acid compositions of media in which the cells intervals of 5, 10, 15, 20, and 30 min. The were cultured gave rise to changes in the results are given in Fig. lB, from which it ability of the plasma membrane of the cells can be seen that uptake of the hexose was to take up a hexose, 2-deoxyglucose. The complete 20 min after it was added to the technique of delipidation that we have used cells; an incubation time of 10 min was there- depends upon acetone extraction, followed fore chosen for the subsequent experiment. by a washing of the acetone powder with Media containing the combinations of ether to remove further lipids and the residadded lipids shown in Table IX were pre- ual acetone; the resultant powder, when repared and portions of each were seeded in constituted with balanced salt solution, is triplicate with 4 x lo4 cells. In practice, then used in place of fetal calf serum in the

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TABLE IX EFFECT OF VITAMIN E, CHOLESTEROL, LINOLEIC ACID, AND ARACHIDONIC ACID ON TRANSPORT OF Z-DEOXY-D-[1-3H]~~~~~~~ IN CULTURED CELLS?

Supplements to growth medium 1. 2. 3. 4. 5. 6.

None 10 pg/ml 10 pgiml 10 pgiml 10 pgiml 10 pgiml

cholesterol linoleic acid arachidonic acid cholesterol + 10 pg/mI linoleic acid cholesterol + 10 pg/ml arachidonic acid

Transport without vitamin E 1.00 k 1.05 2 1.04 * 1.12 k 1.12 t 1.12 t

0.07 0.05 0.11 0.09 0.05 0.13

Transport with vitamin E (2.5 pg DL-cz-tocopheroYm1) 1.13 * 0.07* 1.19 2 0.06’ 1.25 -c 0.04d 1.24 * 0.13 1.42 2 O.OF’ 1.37 + 0.05”J

n Mean transport in medium 1 (without vitamin E) was 92 * 8 nmol hexose taken uplminimg protein; this value was taken as unity, and the transport in all the media was expressed relative to this. Values given are mean ? SD of triplicate measurements on three dishes. For experimental details, see text. bmPStatistically larger than -E value: bP > 0.05, ‘P > 0.02, dP > 0.01, “P > 0.001. ’ Statistically larger than value for medium 1 with added tocopherok P > 0.01.

tissue culture media. The results in Table II show that 74% of the total phospholipids, 90% of the cholesterol, 94% of the triglyceride, 89% of the free fatty acids, and 100% of the a-tocopherol were removed by this procedure. In general, the lipid composition of cells, cultured in media containing unextracted serum or serum from which lipids have been removed, is similar to that of the serum in the media (22, 23). Further, there is excellent evidence (24, 25) that cells cultured in lipid-depleted media are particularly depleted of linoleic and arachidonic acids which indicates that, in cultured cells as in the intact animal, these unsaturated fatty acids cannot be synthesized de novo. The acetone extraction technique that we have employed here can therefore be expected to provide a medium deficient in essential fatty acids, so that addition of unsaturated fatty acids can be made at will. The removal of cholesterol from the medium may also be expected to give rise to cells of restricted cholesterol content, although cholesterol can be synthesized de novo; when the cholesterol content of cells grown in a medium containing 7.5% AES was measured the cells were found to have only 60% of the cholesterol of similar cells grown in media containing 7.5% FCS. The effects of the various combinations of lipid additions to the media are given in Tables I and III. The stimulatory effect of cY-tocopherol which was seen in all the ex-

periments reflects the lack of the vitamin in the medium. In other experiments not reported here, we have found a similar stimulation of growth by a-tocopherol in media containing FCS rather than the delipidated serum and this raises the question of whether a-tocopherol should be added to all media used for tissue culture in order to obtain optimal growth. Whereas addition of cholesterol and linoleic acid severally or together had no effect on the growth rate of the cells, when a-tocopherol was added together with these two lipids a very large stimulation of growth was obtained, which was also significantly greater than the rate obtained when cu-tocopherol was added to media containing either cholesterol or linoleic acid alone. It appears, therefore that for optimal growth all three lipids are required in the medium and omission of any one results in a large fall in the growth rate. This observation supports our suggestion (2,3) that there may be a close cooperativity between a-tocopherol, unsaturated fatty acyl moieties of phospholipids, and cholesterol in biological membrane structure. The results obtained with arachidonic acid (Table III) are, however, difficult to interpret and, certainly when the fatty acid was included in the medium at a level of 20 pg/ml, it was toxic, probably because it was peroxidized. The experiment with butylated hydroxytoluene (Table IV) was carried out in order to establish whether the effect of a-tocoph-

VITAMIN

E AND SELENIUM

IN CULTURED

erol on growth was specific or whether another antioxidant exhibited a similar effect. No consistent stimulatory effect of BHT was obtained, although in some instances the antioxidant exhibited some stimulatory activity. In view of the fact that, in molar terms, the amounts of BHT tested were, respectively, approximately two, four, and eight times the amount of a-tocopherol found to give optimal growth, it seems probable that the stimulatory effect of a-tocopherol was not due to its antioxidant properties. The experiments with selenium described in Table V show that with 7.5% FCS in the medium at a concentration of 100 nmol Se/ dish or more, selenium was toxic to the cells; levels of 0.1 and 1.0 nmol Se/dish were found to be stimulatory to growth of the cells. When the selenium content of the media was restricted further by lowering their content of FCS and AES, very large stimulatory effects of selenium upon growth were noted (Table VIII); this effect was maximal when 2.5% FCS or AES were added to the medium and at a level of inclusion of selenium of 0.1 nmol/dish. It appears, therefore, that growth in media normally employed for tissue culture is suboptimal and that addition of selenium is required for maximal growth. The experiments on the transport of 2-deoxy-[l-3H]glucose were an attempt to assessthe functional integrity of the plasma membrane of cells grown in media of varying lipid composition. Transport of glucose into cells is a carrier-mediated process (26) and 2-deoxyglucose can be used as an analog for the natural hexose, because it is phosphorylated within the cell and not further metabolized (27). cY-Tocopherol either alone or in combination with other lipids (Table IX), stimulated the uptake of 2-deoxyglucose. The largest rate of transport was, however, obtained when a-tocopherol was present in the medium together with linoleic acid or arachidonic acid, and cholesterol. It would seem, therefore, that just as the rate of growth of the cells is maximal when the three lipids are present, so the rate of carrier-mediated transport depends on a similar cooperativity of the three lipids. These findings may be interpreted as meaning that the integrity and maximal functional ability of the plasma membrane in carrying out the

FIBROBLAST

MEMBRANES

279

vital process of hexose transport depend on a structural interaction of a-tocopherol, cholesterol, and linoleic acid or its derivatives in a manner similar to that proposed (2, 3). Earlier experiments (6) in which the transport across liposome membranes of glucose was studied showed that the presence of a-tocopherol with arachidonyl phosphatidyl choline decreased transport. In this case, however, a simple diffusion of the sugar through lipid lamellae was studied in the absence of any protein-dependent carrier mechanism, and both the earlier experiments (6) and those reported here are consistent with our hypothesis (2,3) concerning the possible role of cr-tocopherol in biological membranes. The experiments described here show that the tissue culture system that we have developed is a powerful tool for the study of the interaction of vitamin E, selenium, and unsaturated fatty acids and cholesterol. Using this system we have shown, in experiments to be reported, that the dependence of the cells on selenium is correlated with the level of glutathione peroxidase in the cells, and that vitamin E appears to exert a modulating effect on the conversion of 14Clabeled linoleic acid to arachidonic acid, and on the incorporation of arachidonic acid into phosphatidyl ethanolamine and phosphatidyl serine, which are known to contain high levels of this fatty acid. ACKNOWLEDGMENTS The generous financial support of the Wellcome Trust is gratefully acknowledged. The authors are also very grateful to Dr. L. M. Corwin, Boston University Medical School, for his help in devising some of the methods during his sabbatical leave in London. REFERENCES 1. TAPPEL, A. L. (1%2)Vitam. Harm. 20,493-510. 2. LUCY, J. A. (1972) hoc. N. Y. Acad. 5%. 203, 4-11. 3. DIPLOCK, A. T., AND LUCY, J. A. (1973) FEBS

Lett. 29, 205-210. 4. MAGGIO, B., DIPLOCK, A. T., AND LUCY, J. A. (1977) Biochem. J. 161, 111-121. 5. DIPLOCK, A. T., AND GIASUDDIN, A. S. M. (1979) Nutr. Rep. ht., submitted. 6. DIPLOCK, A. T., LUCY, J. A., VERRINDER, M., AND ZIELENIEWSKI, A. (1977) FEBS Lett. 82, 341-344.

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7. PORTER, R. K., TODARO, G. J., AND FONTE, V. (19’73) J. Cell. Biol. 59, 633-642. 8. MORTON, H. J. (1970) In Vitro 6, No. 2, 89-108. 9. ALBUTT, E. C. (1966) J. Med. Lab. Tech. 23, 61-82. 10. RAHEJA, R. K., KAUR, C., SINGH, A., AND SHATIA, I. S. (1973) J. Lipid Res. 14, 695-697. 11. ZLATKIS, A., ZAK, B., AND BOYLE, A. J. (1953) J. Lab. Clin. Med. 41,486-492. 12. Sigma Chemical Company (1972) Sigma Technical Bulletin No. 405, pp. 12-15. 13. MOSINGER, F. (1965) J. Lipid Res. 6, 157-164. 14. BIERI, J. G., AND PRIVAL, E. L. (1965) Proc. Sot. Exp. Biol. Med. 120, 554-557. 15. BARILE, M. F., HOPPS, H. E., GRABOWSKI, M. W., RIGGS, D. B., AND DEL GUIDICE, R. A. (1973)Ann. N. Y. Acad. Sci. 225, 251-264. 16. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 17. HATANAKA, M., AND HANAFUSA, H. (1970) Virology 41, 647-652. 18. HANKS, H. J., AND WALLACE, R. E. (1949) Proc. Sot. Exp. Biol. Med. 71, 196-200.

AND DIPLOCK 19. CORWIN, L. M., AND HUMPHREY, L. P. (1972) Proc. Sot. Exp. Biol. Med. 141, 609-612. 20. MERCHANT, D. J., KAHN, R. H., AND MURPHY, W. H. (1967) Handbook of Cell and Tissue Culture, 2nd ed., pp. 253-256, Burgess, Minneapolis, Minn. 21. GIASUDDIN, A. S. M., AND DIPLOCK, A. T. (1976) Trace Subs. Env. Health 10, 423-428. 22. KRITCHEVSKY, D., AND HOWARD, B. V. (1970) in Ageing in Cell and Tissue Culture. (Sonkopova, M., Holeckova, E., and Hnerkorsky, P., eds.), pp. 57-69, Plenum, New York. 23. BOYLE, J. J., AND LUDWIG, E. H. (1962) Nature (London) 196, 893-898. 24. MACKENZIE, C. G., MACKENZIE, J. B., AND REISS, 0. K. (1967) Lipid Metabol. Tissue Cult. Cells Symp., 63-71. 25. MACKENZIE, C. G., MACKENZIE, J. B., REISS, 0. K., AND WISNESKI, J. A. (19’70) J. Lipid Res. 11, 571-580. 26. MITCHELL, P. (1967) in Advances in Enzymology (Nerd, F. F., ed.), Vol. 29, pp. 33-88, Wiley, New York. 27. WEBER, M. J. (1973) J. Biol. Chem. 218, 29782983.