Lipid peroxidation and morphological changes in mammalian cells treated with the glutathione oxidant, diamide

Experimental

LIPID

PEROXIDATION MAMMALIAN GLUTATHIONE

Cell Research

AND

105 (1977) 455460

MORPHOLOGICAL

CELLS

TREATED OXIDANT,

J. A. POWER.’ J. W. HARRIS’

CHANGES WITH

IN

THE

DIAMIDE

and D. F. BAINTON2

‘Laboratory of Radiobiology, and 2Department of Pathology, University of California, San Francisco. CA 94143. USA

SUMMARY Chinese hamster ovary cells treated with the glutathione oxidant diamide formed large amounts of lipid peroxide. This effect was greater at 18°C than at 0°C and was apparently not a direct consequence of glutathione oxidation because it occurred at concentrations well above those needed to oxidize cellular glutathione. The reagent was toxic at 18°C but not at 0°C and caused extensive blebbing in 50% of the treated cells at this temperature. Electron microscopic examination of rabbit polymorphonuclear neutrophils disclosed that diamide caused formation of a large, organelle-free bleb and a band of fibrogranular material. It also inhibited phagocytosis of yeast particles by these cells. These effects were reversed when the cells were incubated at 37°C in the absence of diamide. The results indicate that, although diamide is relatively specific for glutathione under some circumstances, effects observed with intact cells under most experimental conditions may reflect processes other than oxidation of endogenous glutathione.

Diamide (diazenedicarboxylic acid bis (N,N’-dimethylamide)) is a glutathione oxidant used by many investigators to study the function of reduced glutathione (GSH) in mammalian cells [I, 21, including its role in various membrane functions [3-g]. In these studies, it is usually assumed that diamide is absolutely specific for GSH and that any functional perturbations it induces indicate that GSH is directly or indirectly involved in that particular function. In the course of a study designed to examine the role of GSH in protecting cells against various environmental insults, we treated Chinese hamster cells with diamide to remove endogenous GSH and observed that this caused extensive peroxidation of membrane lipids and distortion of cell mem-

branes. Certain features of these phenomena suggested that they were direct effects of the reagent on cell membranes and were not due primarily to the absence of GSH. We therefore examined the conditions under which diamide caused lipid peroxidation in these cells, as well as the finestructural changes.

MATERIALS

AND METHODS

Chinese hamster ovary (CHO) cells were grown in spinner culture in Ham’s FlO medium supplemented with 10% calf serum and 5 % fetal calf serum. Cells in late exponential growth were washed twice, resuspended in calcium-free phosphate-buffered saline (PBS), and incubated with diamide. In some experiments, cells were made hypoxic by gassing with nitrogen as described elsewhere [9]. X-irradiation was performed with a GE Maxitron 300 unit at a dose rate of 900 radslmin. Exp

Cell

Res

105 (1977)

456

Power,

Harris

and Bainton .

04 -

03 .

,/

for 16 h) or in glutaraldehyde-osmium [IS] (4°C for 30 mitt). The fixed cells were nacked into blocks bv centrifugation, fixed again in- 1% 0~0, in acetate: Veronal buffer. incubated for I h at 22°C with buffered 0.5% uranyl acetate containing 4% sucrose, dehydrated in graded ethanols, and embedded in Araldite.

RESULTS

02 -

Lipid peroxidation &

.

01 0 0 00

0

IO

I 20

I 30

I 40

I 50

I 60

Fig. 1. Abscissa: incubation time (min); ordinate: increase in A 53o. Time course of formation of lipid peroxides in CHO cells treated with 100 nmoles of diamide/lOs cells at 18°C (0) or at 0°C (0) and in untreated cells at 18°C (A).

Lipid peroxides were measured by a minor modification of the thiobarbituric acid (TBA) method [lO121. Treated cells were washed free of diamide, resuspended to 5~ 10B/ml in PBS, sonicated, and then incubated at 37°C for 3 h with ascorbic acid (0.9 mM) and FeCI, (0.002 mM). The TBA assay was carried out before and after this incubation by adding 2 ml of cell sonicate to I ml of 35% trichloroacetic acid, mixing, and adding 2 ml of 0.75 % TBA; this mixture was then heated to-100°C for 1S mitt, cooled, and centrifuged. The absorbance of the supemate was measured at 530 nm (A& and the results were expressed as increase in A,, durine the 3 h incubation neriod. Cytotoxiczy was measured by platmg control or diamide-treated CHO cells in McCov’s Sa medium SUDplemented with 15% fetal calf serum and scoring colony formation after incubation at 37°C for 10 days. In some experiments, viability was assessed by trypan blue exclusion. Morphological changes were examined by electron microscopy of rabbit polymorphonuclear neutrophils (PMNs) obtained from peritoneal exudates 4 h after injection of 200 ml of 0.1% glycogen [ 131. (These cells were chosen for study because they contain numerous sulfhydryl-rich intracellular granules [14] (fig. 4) that might be affected bv diamide, and because a functional parameter, phagocytosis, could also be measured.) PMNs were washed, resuspended in Hanks’ medium containing 20% rabbit serum at 4”C, and treated with a quantity of diamide sufficient to oxidize cellular GSH completely (100 nmoles/106 cells). The cells were held at 4°C for 10 min and then split into two portions, one of which was held at 4°C for 15 min more and the other of which was held at 37°C. The cells were then fixed in 1.5 % distilled glutaraldehyde in 0.1 M sodium cacodylate-HCI buffer (pH 7.4) with 1% sucrose (4°C E.rp Cdl

Res 105 (1977)

When CHO cells were incubated with diamide, they formed large amounts of lipid peroxide (fig. 1). Peroxidation was apparent after incubation for as little as 10 min and increased with time of exposure; it was more prominent at 18°C than at 0°C. The amount of lipid peroxidation at 18°C increased as the diamide concentration was raised (fig. 2). Twice as much peroxide formed in air as in nitrogen, but X-irradiation (35 or 75 krad) did not increase the amount under either condition (data not shown). Cytotoxicity

Treatment with diamide at 18°C reduced the colony-forming ability of CHO cells, even after short exposures (fig. 3). In contrast,

I

O”O

1W

I

200

I

I

300

4co

I

500

Fig. 2. Abscissa: diamide cont. (nmoles/106 cells); ordinate: increase in A..,. Concentration dependence of lipid peroxide formation in CHO cells incubated with diamide for 10 min at 18°C. “I”

Diamide-induced

Fin. 3. Abscissa: Gviving fraction. Survival of CHO diamide/lOs cells at cells held in PBS at in survival.

incubation

time (mink or&are:

cells incubated with 100 nmoles of 18°C (0) and at 0°C (0). Untreated 18°C for 90 min showed no decline

diamide was not toxic to cells treated at 0°C. About 50 % of the cells treated at 18°C (but not at O’C) became blebbed and vacuolated within the first 3-5 min of incubating; neither the size of the blebs nor the proportion of cells bearing them changed once they had formed, even if the cells were subsequently chilled to 0°C. Trypan blue staining indicated that blebbing was not accompanied by cell death during the 30 min treatment. Because diamide penetrates cells and reacts with cellular GSH at the same rate at 18°C and O”C, the dependence of killing on temperature must reflect metabolic responses to the reagent, including but not limited to lipid peroxidation. Cells can accumulate some peroxide without dying (e.g., at O’C) (compare fig. I with fig. 3), but it is not clear whether the coincident increase of peroxidation and cell killing at 18°C indicates that peroxides cause cell death or simply that dying cells are unable to prevent such damage. Fine structure of neutrophils

Normal jections

PMNs have small membrane proand contain segmented nuclei and

changes in mammalian

cells

457

cytoplasmic organelles disnumerous tributed throughout the cell (fig. 4). Incubation of PMNs with diamide at 4°C caused no change in the normal morphology. However, cells treated at 37°C exhibited loss of microvilli and redistribution of cytoplasmic organelles (fig. 5). These cells extruded a large bleb from one end, usually that opposite the nucleus; organelles were excluded from this area, leaving only matrix, a few clear vacuoles, and an indiscrete band of fibrogranular material (fig. 6). Although the dose of diamide used was somewhat greater than that required for complete oxidation of GSH [5], neither the specific granules nor the azurophil granules were altered by the treatment. The morphological changes were accompanied by a functional one: treated PMNs were unable to phagocytize opsonized yeast particles. Both normal morphology and phagocytic ability returned when diamide was removed and the cells were incubated at 37°C for 20 min, indicating that the effects were reversible. DISCUSSION Lipid peroxides accumulate in the tissues of animals exposed to oxidizing agents such as hyperbaric oxygen [ 161 and ozone [ 171, and they have been linked to the seleniumdeficiency syndrome in fowl [18], to aging in Drosophila [ 193, and to toxicity from vitamin E deficiency in man [20]. Peroxidation of lipids also occurs after exposure to high doses of ionizing radiation [21-231. We had hoped to assess the importance of endogenous GSH in protecting mammalian cells against peroxidative insults by temporarily removing GSH with diamide, but this approach proved infeasible because diamide caused lipid peroxidation at concentrations only slightly greater than those required for Evp

Cdl

Rr.\

105 (1977)

458

Power,

Harris

and Bainton

Diamide-induced

changes in mammalian

cells

459

Fig. 6. High-power electron micrograph of a diamidetreated PMN, showing a band of dense fibrogranular material 0‘) between the cytoplasmic granules (s) and

the extruded matrix. Tissue was fixed in glutaraldehyde-osmium. x20000.

complete oxidation of GSH (10-20 nmolesl lo6 CHO cells) [5]. That this effect was a direct one and was not due to the absence of GSH is indicated by the finding that peroxidation increased with diamide con-

centration in the range well above that required for GSH oxidation (fig. 2). Although it is not clear how diamide causes membrane peroxidation, the protection afforded by hypoxia indicates that oxygen is involved in the process. It is possible that diamide reacts directly with membrane components such as protein SH groups [24], perhaps facilitating peroxidation by changing the architecture of the membrane. Whatever the mechanism by which diamide causes lipid peroxidation. this effect imposes an important constraint on the experimental use of the reagent, particularly for studies of membrane function. Although diamide can be used as a reasonably specific GSH oxidant at low concentrations

4. Appearance of untreated PMNs showing multiple small plasma membrane projections (arrows), segmented nuclei (n), and numerous azurophil granules (ug) and specific granules (sg). Islands of glycogen appear as empty spaces (gl). Because these cells were processed at 4”C, no microtubules were seen. The tissue was fixed in I .5 % glutaraldehyde. x 12 500. Fig. 5. PMNs incubated with diamide (100 nmoles/106 cells) at 37°C. showing loss of the multiple small projections and general smoothing out of the plasma membranes (pm). Cytoplasmic granules and nuclei are excluded from one pole of the cells and numerous vacuoles (v) are present. Indiscrete bands of fibrogranular material (f, appear near the cytoplasmic granules. No microtubules could be seen in these cells. Tissue preparation as in fig. 4. X I2 500. Fig.

E.rpCell RCA/f.( (1977)

460

Power,

Harris

and Bainton

under carefully controlled conditions [2, 51, it is important to realize that experimental results obtained with the reagent may reflect events other than oxidation of endogenous GSH. The morphological consequences of diamide treatment in CHO cells and in PMNs are of particular interest. We are unaware of any agent besides diamide that induces unipolar, organelle-free, reversible blebs, leaving a dense band of fibrogranular material between the granules and the extruded matrix (figs 5, 6). The identity of this fibrogranular material is uncertain. It could be either actin-containing filaments (see ref. [25]) or tubulin. The possibility that it is actin-containing filaments is supported by the observation that 10% of the total protein in PMNs is actin [26] and by recent reports that diamide causes the filaments in cornea1 epithelial cells to form dense bands as the cells abruptly change shape [6]. Evidence that this material might be tubulin is that diamide dissolves the mitotic apparatus of sea urchin eggs, thereby preventing mitosis [3], and that it prevents the polymerization of isolated tubulin [27]. In view of the current interest in the roles of microfilaments and microtubules in cellular function, these possibilities should be tested directly with antibody labeling techniques

WI. After this manuscript was submitted, we became aware of a study by Oliver et al. [29] on the effects of diamide on ConA capping in PMNs. These authors show that diamide promotes capping in PMNs treated with ConA and that microtubules cannot be assembled or maintained in GSH-depleted cells. Their electron micrograph of PMNs treated with diamide and ConA shows an accumulation of electron-dense material beneath the capped membrane similar to that illustrated in fig. 6 of our study. This work was performed in part under the auspices of the US Energy Research and Development Administration. D. F. B. was supported by NIH grant CA-14264.

Exp Cell

RPS IO5 (1977)

REFERENCES I. Kosower, N S, Kosower, E M, Wertheim, B & Correa, W S, Biochem biophys res commun 37 (l%9) 593. 2. Kosower, E M, Correa, W, Kinon, B J & Kosower, N S, Biochim biophys acta 264 (1972) 39. 3. Nath, J & Rebhun, L I, J cell biol 68 (1976) 440. 4. Epstein, D L & Kinoshita, J H, Invest ophthalmol 9 (1970) 629. 5. Harris, J W, Allen, N P & Teng, S S, Exp cell res 68 (1971) I. 6. Edelhauser, H F, Van Horn, D L, Miller, P & Pedersen, H J, J cell bio168 (1976) 567. 7. Werman, R, Carlen, P L, Kushnir, M & Kosower, E M, Nature new bio1233 (1971) 120. 8. Hewitt, J, Pillion, D & Leibach, F H, Biochim biophys acta 363 (1974) 267. 9. Harris, J W, Power, J A & Koch, C J, Radiat res 64 (1975) 270. IO. Wills, E D &Wilkinson, A E, Radiat res 31 (1967) 732. II. Wilbur, K M, Bernheim, F & Shapiro, 0 W, Arch biochem biophys 24 (1949) 305. 12. Ottolenghi, A, Arch biochem biophys 79 (1959) 355. 13. Bainton. D F. J cell biol58 (1973) 249. 14. Harris, J W, Aust j exp biol‘medsci 46 (1958) 209. 15. Hirsch. J G & Fedorko. M E. J cell biol 38 (1968) 615. ’ 16. Haueaard. N. Phvsiol rev 48 (1%8) 31 I. 17. Chow, C K i Tappel, AL, Lipids7 (1972) 518. 18. Omave. S T & TaDDel. A L. J nutr 104 (1974) 747. 19. Sheldahl, J A & ‘Tappel, A L, Exp gerontol 9 (1974) 33. 20. Mukai, F H & Goldstein, B D, Science 191 (1976) 868. 21. Sutherland, R M & Pihl, A, Radiat res 34 (1968) 300. 22. Myers, D K &Bide, R W, Radiat res 27 (1%6) 250. 23. Harris. J W. Adv biol med nhvs 13 (1970) 273. 24. Harris, J W’& Biaglow, J E, Biochem biophys res commun 46 (1972) 1743. 25. Tilney, L G, J cell bio169 (1976) 51. 26. Stossel, T P & Pollard, T D, J biol them 248 (1973) 8288. 27. Mellon. M G & Rebhun. L I. J cell biol 70 (1976) 226. 28. Lazarides, E, J cell bio168 (1976) 202. 29. Oliver, J M, Albertini, D F & Berlin, R D, J cell bio171 (1976) 921. Received August 19, 1976 Accepted November 18, 1976