Metabolic Studies of the Effect of Griseofulvin and Candicidin on Fungi

METABOLIC STUDIES OF THE EFFECT OF GRISEOFULVIN AND CANDICIDIN ON FUNGI* WALTER G. LARSEN, M.D.** AND D. JOSEPH DEMIS, Pn.D., M.D. Griseofulvin is known to inhibit the growth of certain filamentous fungi, including the dermato-

phytes, while not inhibiting the growth of bacteria, yeasts or many other filamentous fungi. The polyene antifungal antibiotics (Fig. 1), however, inhibit the growth of a great number of yeasts and protozoa as well as the filamentous fungi (including the dermatophytes), but are ineffective ngainst bacteria.

determination of oxygen consumption the gas

phase was air; carbon dioxide was absorbed by 0.2

cc of 20% KOH placed in the center well. For measurement of anaerobic glycolysis the flasks were flushed with water-dried pure nitrogen for 10 minutes and CO2 production was measured. Each cup contained 1 ml or 2 ml of cell suspension, tris buffer, substrate, and antibiotic as appropriate, to give a total volume of 3 ml. The homogeneous fungus suspension was pre-

pared essentially after Gale et at (4). The fungal The initial studies of the effects of griseofulvin spores were collected from 1 or 2 agar plate culon respiration of fungi were reported by Brian in tures of Trichophyton mentagrophytest, inoculated 1949 (1). Using mats of fungi and mycelial sus- into 250 ml Sabouraud's broth (pH 5.7) in an Erlenmeyer flask, and placed on a gyrotary shaker pensions, griseofulvin was found to have no effect at room temperature for 3—5 days. The medium on the oxygen consumption of Botrytis allii, an was then removed by centrifugation, the pellets imperfect fungus sensitive to the antibiotic. Later washed twice with H20 or buffer and finally the Roth et al (2), working with homogenized sus- mycelium from one flask was suspended in a total volume of 100 ml. This was transferred to a Waring pensions and whole pellets of a sensitive strain of blender. A blending time of 30—50 seconds was Trichophyton rubrum, also showed griseofulvin to adequate to produce a homogenous suspension have very little effect on the oxygen consumption without clumps which was easily pipetted. Saceharomyces cerevisiee (Bakers yeast, Standof these preparations. The present study extends Brands Inc.) was starved by aerating the susthese investigations and compares the effects of ard pension for 2 hours at room temperature. The griseofulvin with those of an antifungal antibiotic, yeast was then washed twice and suspended in the

candicidin. Measurements were made of the

appropriate buffer. Glucose was added as subrespiration and anaerobic glycolysis of the yeast strate to give a final concentration of 0.1 M. Candicidin (521-RTF, 156% of reference standSaccharomyces cerevisiac, which served as a standard, S. B. Penick & Co., New York, N. Y.) was ard for comparison of manometric effects, and prepared by initially dissolving in dimethyl sulfthe respiration of a sensitive dermatophyte, oxide and subsequently adding distilled water to Trichophyton mentaqrophytes, prepared as a obtain the proper dilutions. Griseofulvin (Grifulvin microcrystalline, Batch homogenous suspension. Griscofulvin was found McNeil Laboratories, Inc., Fort Washington, to have no effect on the respiration of either the 0423, Pa.) was prepared in the same way. In addition, yeast or dcrmatophytc; conversely, candicidin in- griseofulvin was made up in distilled water hibited the respiration of both organisms and also without dimethyl sulfoxide for concentrations of inhibited anaerobic yeast glycolysis. Homoge- 5 mcg/ml and below. The maximum concentrations nized suspensions were found to provide repro- of dimethyl sulfoxide in any reaction mixture was to have no effect on the metabolism ducibility and to facilitate study of the demonstrated of either dermatophyte or yeast. metabolism of the filamentous fungi. Tris-HCIt and tris-succinate tartrate were used to provide buffering at pH 7.0 and pH 5.8 respectively. The 0.03 M tris-succinate tartrate buffer METHOD ANI) MATERIAL5 was prepared by addition of solid tris-(hydroxyManometric measurements were carried out at methyl) aminomethane to the acids until pH 5.8 30° C in standard 15 ml Warburg vessels (3). For was reached (5). * From the Department of Dermatology, Walter

Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, D. C. ** Present Address: Division of Dermatology, University of Oregon Medical School, Portland,

Oregon.

Presented at the Twenty-fourth Annual Meet-

ing of The Society for Investigative Dermatology, Inc., Atlantic City, N. J., June 19, 1963. 335

EE5TJLT5

T. mentagrophytes suspensions prepared with this technic consumed oxygen at fairly uniform f Fresh isolates courtesy of Dr. Lucille Georg, Communicable Disease Center, Atlanta, Georgia Trizma, Sigma Chemical Co., St. Louis, Mo.

336

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

ANTIFUNGAL ANTIBIOTICS I. GRISEOFULVIN CH3O

Co H2

II. POLYENE ANTIBIOTICS

A. TETRAENES I. NYSTATIN 2. PIMARICIN CH2OH

H

CH3

CHOH o

CH3

H2NH B. HEPTAENES I. AMPHOTERICIN B 2. CANDICIOIN 3. CANDIDIN 4. TRICHOMYCIN

Fxo. 1 A brief classification of some antifungal antibiotics. Over 40 polyenes have been identified. Only the structure of pimaricin has been elucidated (21). rates for more than five hours. Oxygen consump-

inhibited endogenous respiration of sensitive

tion could not be increased or decreased by the addition of glucose as substrate nor by allowing the suspensions to stand overnight at 4 C. Car-

strains of T. mentagrophytes in approximately the same concentrations (1—10 mcg/ml) as required

bon dioxide was not produced anaerobically by T. mlntagrophyte.s in marked contrast to yeast. Griseofulvin: Griseofulvin, in concentrations of 1 mcg/ml, 5 mcg/ml, 10 mcg/ml and 353 meg/mi (1 x 10—3M) was found to have no effect over a five hour period on the endogenous oxygen consumption of various griseofulvin-sensitive strains of T. mentagrophytes either in unbuffered solution

sumption was greater at pH 5.8 than at pH 7 (Fig. 2). Concentrations of candicidin (1—10

to inhibit growth; the inhibition of oxygen con-

meg/mi) which inhibited oxygen consumption of T. mentagrophytes produced similar effects on yeast (Fig. 3).

In addition, candicidin (1—10 mcg/ml) inhibited the exogenous anaerobic glycolysis of yeast (Fig. 4). Candicidin produced a more rapid

or buffered at pH 5.8 and pH 7. Under similar and profound effect on anaerobic glycolysis as experimental conditions no effect of griseofulvin compared to its effect on respiration. Concenon either the respiration or anaerobic glycolysis trations of the drug (1 mcg/ml), which produced of the yeast was detected. Candicidin: Candicidin but slight inhibition of oxygen consumption

ANTIFUNGAL ANTIBIOTICS

337

THE INFLUENCE OF pH AND CONCENTRATION ON CANOICIDIN INHIBITION OF OERMATOPHYTE RESPIRATION

240

230

T mentagrophytes + Candicidin PH 7.0

220 mentagrophytes + Candicidln

210

pH 5.8

200

100

90 80

90

Control

ISO

Candleldin 0.Ig/mI Candicidln I jog/mi

I 70

Candicldin 5 jog/mI

ISO

Candicldin 10 jog/mi

150

70

0

140 130

60

120 110

50

100

90 40

SO

70 30

60 50 40 30

.2

4

5

Hours

I

2

5

Hours

Fm. 2 Effect of candicidin on the endogenous oxygen consumption of T. mentagrophytes (isolate 45855-62). Each Warburg vessel contained T. mentagrophytes in 2 ml of buffer; 0.5 ml of eandicidin solution; 0.5 ml of distilled HuO; and 0.2 ml of 20% KOH in the center well. Gas phase: air. Temperature: 30° C.

Drug tipped at 0 minutes.

after several hours, lead to rapid and complete inhibition of glycolysis within a few minutes.

Parallelism of the effects of candicidin on the oxygen consumption of S. cerevisiae and T. mentag-

lithium chloride did not produce this reversal. Addition of the potassium or ammonium ions prior to the addition of candicidin prevented the inhibiting effect of eandicidin on yeast glycolysis.

roph pies was noted at pH 5.8 and pH 7; at a Conversely, addition of these cations did not concentration of 5 mcg/ml the percentage in- protect against nor reverse the inhibition of hibition produced by candicidin virtually over- oxygen consumption produced by candicidin in either the yeast or the dermatophyte. lapped at both pH 5.8 and pH 7 (Fig. 5). Monovalent cations have been shown to preYeast does not normally anaerobically devent or reverse the inhibition of glycolysis by carboxylate pyruvate; however Lampea et al (8) certain polyenes, including nystatin and candici- have shown that this decarboxylation occurs in din (6, 7). When potassium chloride (0.009 and the presence of added nystatin and that the effect

0.05 M final concentration) or ammonium was greater at an acid pH (pH 5.8). Consechloride (0.009 and 0.05 M final concentration) quently, nystatin* (5 and 50 mcg/ml) was added was added to the medium the inhibition of yeast to S. cerevisiae in the presence of pyruvate and glycolysis produced by candieidin was reversed decarboxylation, as measured by CO2 production, (Fig. 6). Larger concentrations of eations caused * Myeostatin Batch CID-008. Courtesy Miss greater reversal; however, calcium chloride and Barbara Sterns, Squibb, New Brunswick, N. J.

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338

THE INFLUENCE OF pH AND CONCENTRATION ON CANDICIDIN INHIBITION OF YEAST RESPIRATION

Yeast + Candicidin

Yeast + Candicidin

pH 7.0

PH 5.8

850 800

Control

Candicidin 0.1 g/ml

750

Candicidin I og/ml

700

Candicidin 5 jg/ml Candicidin 0 jog/mI

650

600 550 500

450

- 400 350 300 250

200 I 50 100

50 10

30

50

70

9

110

10

30

50

70

90

110

Minutes FIG. 3 Effect of candicidin on oxygen consumption of yeast respiring exogenous substrate. Each vess contained 20 mg (wet weight) of S. cerevisiae in 1 ml of buffer; 0.5 ml of candicidin solution; 0.5 ml of glucose; 0.5 ml of buffer; 0.5 ml of distilled water; and 0.2 ml of 20% KOH in center well. Gas phase: Air. Temperature: 300 C. Drug tipped at 0 minutes.

was found. In the presence of pyruvate (0.5 M)

inability to prepare uniform aliquots of mycelia.

and 5 and 50 mcg/ml of nystatin, yeast (6.67 Gale et al (4) point out that the objections to the meg/mi) produced 161 u 1 and 331 u 1 of CO2 use of mats cut into strips or pellets grown on a in the first hour, respectively, at pH 7. Candicidin, shaker are: (1) non-uniformity of aliquots (2) in concentrations as great as 50 meg/mi, did lack of homogeniety of age and thickness of the not lead to detectable CO2 formation by yeast mat or pellet (3) difficulty of diffusion of gases at either pH 5.8 or pH 7. and substrates to the interior of the mat or pellet. Spores of T. mentagrophyte.s were found un- The technic developed by Gale et al (4) for suitable for study under the conditions employed. Despite collection from 20 culture plates and suspension in a minimum volume of water (2 cc), the

homogenization of filamentous fungi proved satis-

factory for preparing mycelial suspensions for

respiratory study in the Warburg apparatus.

turbid suspension failed to produce significant After homogenization, long hyphal elements were found which showed no apparent mechanioxygen consumption in the Warburg apparatus. cal rupture or disintegration. These investigators DISCUSSION presented further evidence that the mycelium One of the major obstacles in the study of the was physiologically intact and also reported that metabolism of the filamentous fungi has been the dry weight determinations of the fungal suspen-

ANTIFUNGAL ANTIBIOTICS

339

THE INFLUENCE OF pH AND CONCENTRATION ON CANDICIDIN INHIBITION OF YEAST GLYCOLYSIS Yeast + Candlcidin

525

pH 5.8

pH 7.0

500 475

— Control Candicidin 0.1 ig/ml Candicidis I j'g/mI

450 425 400

—— Candicidin 5 ;'g/mI Candicldin 0 sg/mI

375

350 325 04

0 C)

25 100

75 50 25

5

IS

25

35

50

60

5

IS

25

35

45

55

Minutes FJG.

4 Effect of candicidin on the anaerobic glycolysis of yeast. Each vessel contained 10 mg (wet

weight) of S. cerevisiae in 1 ml of buffer; 0.5 ml of candicidin solution; 0.5 ml of glucose solution; 0.5 ml of buffer; and 0.5 ml of distilled water. Gas phase: N2. Temperature: 300 C. Drug tipped at 0 minutes.

sions gave small deviations from the mean. The hyphae in our suspensions appeared intact micro-

scopically and our dry weight determinations also showed but small deviations from the mean. Grieeofulcin: The present studies demonstrate that griseofulvin has no effect on the respiration of sensitive fungi under prolonged incubation with control of pH, thus corroborating prior findings (1, 2). Conceivably, griseofulvin when added to spores as they are germinating may have an effect

who demonstrated the partial reversal of griseofulvin activity by purines, pyrimidines and their nucleotides. This work deserves further corroboration. Griseofulvin has been shown to be fun gici-

dat against a mixed inoculum of spores and hyphae of various dermatophytes (11) and exerts its fungicidal effect under conditions conducive to active growth of the fungi. Blanket at (12), on the basis of electron microscopic observations, hypothesized that the youngest actively metabolizing fungal cells are killed by griseofulvin, while

on respiration. Meyer-Rohn (9) has suggested that in very high concentrations, griseofulvin the older more dormant elements are not so does inhibit the oxygen uptake of germinating drastically affected. spores; the data however is difficult to interpret Griseofulvin is well known to have a colehicineas the oxygen consumption was miniscule. The mode of griseofulvin action remains speculative. Interference with nucleic acid synthesis by

like effect, causing arrest of mitosis in metaphase

griseofulvin has been proposed by MeNall (10)

has been shown for griseofulvin; this activity ap-

in rat testes, plant cells (13) and Hela cells in tissue culture (14). Anti-inflammatory activity

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

340

PERCENTAGE INHIBITION OF OXYGEN CONSUMPTION PRODUCED BY 5pg/mI OF CANDICIDIN 00 90

80 C

0

a

70

r

E U,

C

00

60

0

CU

4-

0 50 C 0

1-o

40

-C

C

— pH 7.0

30

—--—..pH5.8 • I mentagrophytes

o Yeast

20

I0

I

2

3

4

5

Hours FIG. 5 Percentage inhibition of oxygen consumption of 7'. mentagrophytes and S. cerevisiae produced by 5 meg/ce of candicidin at pH 5.8 and pH 7. pears to be an action of an unknown nature at the site of inflammation (15).

of the different polyenes are fundamentally the same and that the primary vulnerable, physiologi-

Candicidin: Candicidin was found to have cal locus is the cellular permeation system (17, effects similar to those of nystatin on yeast 18, 19). Rapid loss of potassium ions occurs with respiration and anaerobic glycolysis including low levels of nystatin, ammonium ions leak out at greater activity at acid pH and the reversal of somewhat higher concentrations (8). It is the loss of these ions which appears to be the critical and ammonium ions. However, yeast did not de- factor in the inhibition of glycolysis. This appears carboxylate pyruvate in the presence of candi- to be true also for candieidin despite the differeidin whereas it has been shown that in the pres- ence in relation to decarboxylation of pyruvate. ence of nystatin (8) or amphotericin B (16) yeast The findings with the polyenes parallel those obwill decarboxylate pyruvate. This finding illus- tained with surfactants such as benzalkonium trates a difference in the metabolic effects of chloride (5, 18). eandicidin and suggests a mechanism of action Lampen and co-workers (20) have recently presented several lines of evidence that the binddistinct from the other polyenes. It has been concluded that the modes of action ing site for nystatin on the cell membrane eondrug induced inhibition of glycolysis by potassium

341

ANTIFUNGAL ANTIBIOTICS

Reversal by KCI of the Inhibition

Of Glycolysis by Candicidin

pH 7.0 1100

— Control Candlcidin 41.6 pg/mI

1000

+ 50 mMKCI Candicidin 41.6 pg/mI + 9 mMKCI

900

800 700

N

0 0

600 500

400

KCI

300 200 I 00

50 10

30

50

70

90

110

130

Minutes

FIG. 6 Reversal by KC1 of the inhibition of glycolysis by eandicidin at pH 7.0. Same conditions as in Fig. 4 except for the addition of 0.5 ml of KC1 at 40 minutes (arrow). KC1 was added to achieve a final concentration of 0.009 M and 0.05 M.

tains sterols, mostly unesterified ergosterol. The polyene apparently reaches the cell membrane and is bound by the ergosterol. This binding has been considered the critical event in cell damage for it has been demonstrated that only sensitive

organisms adsorb nystatin. The sterol content can be correlated with sensitivity since bacteria contain only traces, if any, of sterol and are insensitive to the polyenes whereas fungi, algae and certain protozoa contain considerable quantities of sterol, and are sensitive in some degree (20).

In view of the similar pattern of inhibition pro-

duced by candicidin with S. cerevisiae and T. mentagrophytes it is probable that candicidin also binds to the cell membrane of T. mentagrophytes with subsequent alteration of permeability. SUMMARY

Griseofulvin was found to have no effect on the respiration of the sensitive dermatophyte, Trichophyton mentagrophytes, nor on the respiration and

anaerobic glycolysis of an insensitive yeast,

342

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

antifungal antibiotics: microbiological stud-

Saceharomyces cerevisiae. Candicidin inhibited the

ies. Antibiot. Chemother. (NY), 11: 640,

respiration of T. mentagrophytes as well as the

respiration and anaerobic glycolysis of S.

1961. S. SUTTON, D. D., ANNOW, P. M. AND LAMPEN,

0.: Effect of high concentrations of nystatin upon glycolysis and cellular permeability in yeast. Proc. Soc. Exp. Biol. Med.

cerevisiae in concentrations which approximate those necessary for in vitro inhibition (1—10 mcg/ ml). The inbibition of anaerobic glycolysis produced

J.

108: 170, 1961. 9. MEvEE-RONN, J.: Manometrischc messungen

au dermatophyten und Candida albicans

by candicidin was reversed by the addition of potassium or ammonium ions, as has been shown for certain other polyenes. Yeast in the presence

of nystatin and amphotericin B decarboxylates pyruvate anaerobically; however, candicidin failed to produce this effect. This finding suggests

a difference in the metabolic effects of these polyenes. The mechanism of griseofulvin action remains

unter der

einwirkung von griseofulvin, nys-

trichomycin. Chemotherapia (Basel), 4:563, 1962. McNALL, E. G.: Biochemical studies on the metabolism of griseofulvin. Arch. Derin. tatin, amphotericin B und

10.

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mode of action of griseofulvin. Antibiot. Ann., 670, 1959—1960.

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Electron microscopic observations of the effects of griseofulvin on dermatophytes.

Arch. Derm. (Chicago), 81: 667, 1960. speculative whereas the fungicidal activity of G. E. AND WALPOLE, A. L.: Some cycandicidin appears to be due to its effect on cell 13. PAOET, tological effects of griseofulvin. Nature

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