Usefulness of MC-540 fluorescent dye as probe versus scanning electron microscopy for assessing membrane changes

Toxicology

169

Lerlers, 54 (1990) 169-I 74

Elsevier TOXLET

02460

Usefulness of MC-540 fluorescent dye as probe versus scanning electron microscopy for assessing membrane changes

Sarita Agarwal l>*, Romesh ‘Sartjay Gandhi Postgraduate Lucknow

Khardori2

Institute of‘ Medical Sciences. Department

(India) and JSouthern Illinois University,

Sprin&eld,

IL (U.S.A.)

(Received

I July 1989)

(Accepted

25 July 1990)

KeJj words: Erythrocyte

and S.S. Agarwal’

membrane;

MC-540;

School of Medicine,

of‘ Genetics and Immunology. Department

qf Medicine,

SEM; Primaquine

SUMMARY The effect of primaquine erythrocytes

was studied

quine enantiomers drug-induced

produces

alterations

that primaquine

significant

in membrane

enantiomers

and G-hPD-deficient 540, a lipophylic

enantiomers

on cell membranes

in vitro. Staining

with merocyanine

fluorescence fluidity.

altered membrane

in G-6PD-deficient

Scanning

electron

morphology

cells. The concentration-dependent

dye, than with SEM (an expensive

of glucose-6-phosphate (MC-540) showed

erythrocytes,

microscopy

(by producing

effct, however,

(G-6PD)-deficient

that exposure indicating

(SEM) studies

stomatocytes)

to primamarked confirmed

in both normal

was more pronounced

with MC-

technique).

INTRODUCTION

Previous investigations from our laboratory and other reports have shown that glucose-6-phosphate dehydrogenase (G-6PD)-deficient erythrocytes are sensitive to primaquine and a variety of other oxidative compounds, resulting in hemolysis [l-4]. However, the exact mechanism of hemolysis remains controversial, with several authors suggesting that the cell membrane is the point where first oxidative injury occurs and others arguing that oxidation of cellular contents is more important [5-71. We have previously reported leakiness of the erythrocyte membrane documented by release of hemoglobin into plasma. This observation led us to investigate the effect *Current Medical

address and correspondence: College of Georgia,

0378-4274/90/$3.50

Augusta,

Sarita

Agarwal,

Ph.D.,

Cell and Molecular

GA 30912, U.S.A.

@ 1990 Elsevier Science Publishers

B.V. (Biomedical

Division)

Biology

Department,

170

of primaquine enantiomers on the erythrocyte membrane, the results of which are presented in this paper using merocyanine (MC-540) staining versus electron microscopy * MATERIALS AND METHODS

Fresh blood (5.0 ml) from 5 normal healthy volunteers and 5 G-6PD-deficient subjects was drawn in EDTA vials. The whole blood in duplicate was incubated with either (+)- or (- )-p~maquine (PQ) at different concentrations of the drugs ranging from O-3.0 mM. To one set an equal volume of saline was added to serve as control. Incubation was carried out at 37°C for 1 h in a shaking water bath. After incubation the blood was centrifuged immediately at 1500 rpm for 10 min. The pellet was washed thrice with buffered saline (pH 7.2). The erythrocyte pellet was reconstituted to 1 ml with low-ionic-strength incubation medium (LISIM) consisting of 0.25 mM sucrose, 15 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM Tris supplemented with 5% bovine serum albumin and 1.75 mM merocyanine. The tubes were incubated at 37°C for 1 h under complete darkness. The cells were washed with LISIM to get rid of unbound MC-540. Finally, the cells were suspended in 100 ml of LISIM, loaded on slides and observed under an epifluorescence microscope (Zeiss) with BP 45W90 excitation filter and LP-520 barrier filter IX]. Orange-red fluorescent erythrocytes were recorded as a percentage of total erythrocytes. For scanning electron microscopy (SEM), a 20-ml aliquot of the suspension was taken and washed with 100 mM phosphate-buffered saline (pH 7.4) thrice at 2000 rpm for 10 min each time. The cells were resuspended in 1 ml of buffered saline and fixed with 0.25% glutaraldehyde for 30 min at room temperature. After fixation, the cells were washed thrice with buffered saline and treated with osmium tetraoxide (1%) for 2 h. The fixed cell suspension was washed and dehydrated with ascending grade alcohol concentration. The stubs were prepared, coated with gold and examined under the SEM at 15 KVA. RESULTS

MC-540 staining Alteration in membrane fluidity of erythrocytes exposed to (+)PQ and (-)PQ was evaluated by using MC-540 as fluorescence probe. No significant fluorescence was detected in saline-treated normal and G-6PD-deficient erythrocytes; however, incubation at 0.75, 1.5 and 3.0 mM of (+)PQ produced fluorescence in 44,69 and 9.596 of erythrocytes, respectively, while (-)PQ produced Huroescence in 28,46 and 8 l%, respectively, of G-6PD-deficient erythrocytes (Table I). G-6PD-deficient cells treated with the enantiomers showed very bright fluorescence on MC-540 staining. Figure 1 shows approximately 100% fluorescent cells in cases with G-6PD deficiency as against 40% in normal individuals on (+)PQ treatment at 3.0 mM concentration.

171

TABLE

I

PERCENT

FLUORESCENCE

Concentration

(+F’Q

(--F’Q

@MI Deficient

Normal 0

6.33k3.21

10.33* (9-12)

0.75

(610) 10.33 +4.5

1.50

(Gl5) 25.33 +4.5

3.0

Normal 1.52

Deficient

6.33k3.21

10.33+

(4-10) 11.33k3.21

(9-12)

(40-50)

(9-l 5)

(25-30)

68.66kd.02

10.0*2.0

46.0 k 3.6

(21-30)

(63-75)

(8-10)

(43-50)

40.33k4.5

95.0*5.0

16.66k3.05

81.Ok3.6

(3M5)

(9&100)

(1420)

(78-85)

All values are mean + SD. Numbers

44.0*

in parentheses

5.29

1.52

21.66k2.5

show the range

Scanning electron microscopy

The effect of PQ enantiomers on the surface morphology of normal and G-6PDdeficient human erythrocytes is shown in Figures 2 and 3. Both enantiomers produced a stomatocytic effect on the erythrocyte membranes irrespective of the G-6PD status of the cells. DISCUSSION

This study shows that (+)PQ produces more membrane change in erythrocytes than (-)PQ as reflected by significantly greater membrane fluidity (PC 0.01) (Table I).

GG’PD Deficient

Cells (100%) Fig. 1. MC-540

fluorescence

Normal Cells (40%) on (+) PQ exposure.

172

PRIMAQUINE t+’ TREATMENT (1.5 mm)

Ineet: Top right x 3tXM

Magnification: Background x1500 Inset: Top right x SCNMJ Lower right x 3CUM

(3.0 mM)

Inset

X3000

Background x 1500 Inset X3000

SALINE TREATED NORMAL CELLS (NO PRIMAQUINE)

Magniflcetlon: Beckground x 1500 Ineet X3000

Fig. 2. Effect of (+)PQ

enantiomer on erythrocyte surface morphology cells at 1.5and 3.0 mM concentration.

of normal

and G-6PD-deficient

PRIMAQUINE (-1 TREATMENT

Magnfflcatkm: Background x 1500 X3ooo Met

Magnlttcatlon: Background x lso0 Inset X3OW

Fig. 3. Effect of (-)PQ

enantiomer on erythrocyte surface morphology of normal and G-6PD-deficient cells at 1.5 and 3.0 mM concentration.

A concordance was found between the changes in cell morphology revealed by SEM and the MC-540 staining pattern. However, the concentration-dependent effect was more clearly seen with MC-540 staining than in SEM studies (this suggests that MC-540 affinity for membranes increases as soon as the cell membrane pattern becomes symmetrical) [9]. The differential toxicity of PQ enantiomers is also more clearly visible in MC-540 than SEM studies. Our findings with PQ enantiomers indicate that MC-540 staining is a comparatively simple, sensitive and less expensive method for assessing changes in membranes than SEM. The MC-540 dye staining technique may be useful as a model test system for the evaluation of drug toxicity on membranes, if our results are confirmed in larger studies.

174

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Nitya Arand and Dr. R.C. Gupta, CDRI, Lucknow, India, for providing primaquine enantiomers, to Mrs. Jan Hicks for secretarial assistance, and to ICMR, New Delhi, for providing funding to ICMR, Advanced Center for Genetics, KGMC, Lucknow. REFERENCES

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