JOURNAL oF BACTERIOLOGY, Mar., 1965Copyright 0g 1965 American Society for Microbiology

Vol. 89, No. 3Printed in U.S.A.

Antibiotic Action of Griseofulvin on DermatophytesMOUSTAFA A. EL-NAKEEB, W. L. McLELLAN, JR., AND J. 0. LAMPEN

Institute of Microbiology, Rutgers, The State University, New Brunswick, New Jersey

Received for publication 20 June 1964

ABSTRACTEL-NAKEEB, MOUSTAFA A. (Rutgers, The State University, New Brunswick, N.J.),

W. L. McLELLAN, JR., AND J. 0. LAMPEN. Antibiotic action of griseofulvin on derma-tophytes. J. Bacteriol. 89:557-563. 1965.-The concentrations of griseofulvin requiredto inhibit growth and to produce the characteristic morphological distortions were de-termined for dermatophytes (highly sensitive), fungal plant pathogens (moderatelysensitive), filamentous nonpathogenic fungi (poorly sensitive), and for yeasts and Esch-erichia coli (insensitive). Addition of griseofulvin to small inocula of the dermato-phytes Microsporum gypseum and Trichophyton mentagrophytes produced complete andapparently permanent growth inhibition. If the antibiotic was added to actively grow-ing cultures, the inhibition was only temporary, even with the most sensitive derma-tophytes. During growth inhibition, griseofulvin temporarily halted the net synthesisof protein and nucleic acids, and of the amino acid and nucleotide pools. It decreasedsubstantially the incorporation of C14-uridine or C'4-thymidine into nucleic acids of M.gypseum, but not that of C'4-leucine or C14-valine into protein. With a less sensitive cul-ture, T. mentagrophytes x8, the uptake of uridine was inhibited only to a slight extent;the incorporation of leucine was unaffected. A partial protective effect of purine nucleo-tides against growth inhibition by griseofulvin was observed with one strain of T.mentagrophytes, but not with another strain or with M. gypseum.

Brian, Curtis, and Hemming (1946) isolatedfrom culture filtrates of Penicillium janczewskiiZal. a material they called "curling factor," sinceit produced severe stunting and distortion of thegerminating spores of Botrytis allii. Grove andcollaborators (1952) proved the identity of thecurling factor with griseofulvin-an antifungalantibiotic discovered earlier by Oxford, Raistrick,and Simonart (1939).

Studies on the griseofulvin-sensitivity of differ-ent organisms showed that dermatophytes werevery sensitive, whereas bacteria and yeasts wereinsensitive (Brian, 1949, 1960; Roth, Sallman,and Blank, 1959). There was disagreement con-cerning the sensitivity of filamentous fungi otherthan the dermatophytes. The effectiveness ofgriseofulvin in curing experimental ringworminfections in guinea pigs (Gentles, 1958) em-phasized the unusual sensitivity of the derma-tophytes, and has led to the use of griseofulvinin the treatment of superficial skin infectionscaused by these organisms.The chitinous cell walls of fungi were suggested

by Brian (1949, 1960) to be the site of griseofulvinaction. He observed that growth reduction andmorphological changes were produced by theantibiotic only in those fungi which possessedchitinous walls. Exceptions to this correlationwere noted, however, by Abbot and Grove

(1959) and by McNall (1960). As part of a gen-eral investigation of the action of griseofulvin onfungi, groups of organisms with diverse levels ofsensitivity were selected for subsequent bio-chemical study. Certain general features of theantibiotic action of griseofulvin are presented,especially the effect on the total synthesis of cellconstituents and on the incorporation of C14-nucleosides and amino acids into nucleic acidsand proteins.

MATERIALS AND METHODSOrganisms. The strains and their sources are

listed in Table 1. Stock cultures on slants werestored at 4 C. Before cultivation in liquid media,subcultures were made on solid media (1.5%,w/v, agar) and incubated at the appropriatetemperatures.Media. All filamentous fungi, with the exception

of Neurospora crassa, were usually grown on aNeopeptone medium which contained 10 g ofNeopeptone (Difco) and 20 g of glucose per literof tap water. In some experiments, where indi-cated, the following synthetic medium (AGS) wasused (g per liter of distilled water): arginine mono-hydrochloride, 2.0; glucose, 20.0; K2HP04, 1.0;NaCl, 1.0; MgSO4-7H20, 0.5; citric acid, 0.4;Fe2(S04)3. 6H20, 0.010; CuS04-5H20, 0.001;ZnSO4-7H20; 0.001; MnSO4-H20, 0.001. For thecultivation of yeasts and N. crassa, vitamin-en-riched Vogel's N medium (Marini, Arnow, and

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Lampen, 1961) was supplemented with 2% glucose(w/v). Escherichia coli was cultured in M-9 me-dium (Roberts et al., 1955) to which 1.5% (w/v)glucose was added.

Culture conditions. For bacteria and yeast, thecells from a 24-hr slant culture were suspended in2 ml of medium and added to 100 ml of the samemedium in a 250-ml flask. The culture was incu-bated on a reciprocal shaker (70 rev/min, 8.9-cmstroke) at 30 C for 48 hr. With filamentous fungi,10-ml volumes of the appropriate medium in 50-mlflasks were inoculated with 0.1 ml of a suspensionof spores or a mycelial homogenate and shaken(70 rev/min, 8.9-cm stroke) at 28 C for 72 hr.Inocula of filamentous fungi. A spore suspension

was prepared by adding a few milliliters of sterilemedium to a 1- to 2-week-old culture of the fungus.The spores were dislodged gently, and the mycelialfragments were removed by straining throughsterile glass wool. The suspension was then dilutedto give 106 spores per milliliter.For preparation of the cell homogeniates, 3-day-

old shaken cultures of the organism were used.The mycelia were harvested, washed twice, andsuspended in saline. The suspension was shakenwith glass beads (3 mm in diameter) on a rotaryshaker for 30 to 45 min at 28 C. The homogenatewas passed through cotton gauze and the my-celiumn was collected by cenitrifugation. The sedi-merit was washed twice and suspended in saline togive a 2.5% (v/v) suspension. Aseptic conditionswere maintained throughout the procedure.

Griseofulvin stock solution. Griseofulvin wasdissolved in dimethylsulfoxide (Stepan Chemi-cal Co., Northfield, Ill.) at 10 mng/ml, and thesolution was stored at 4 C. The required amountsof the antibiotic in microvolumes of the stocksolution were added directly to sterile media.Similar volumes of dimethylsulfoxide were incor-porated into the control cultures.

Estimation of dry weight. The fungi used in thiswork did not form a homogeneous diffuse growth.Adequate repetitive sampling from a single cultureflask was, therefore, almost impossible. The fol-lowing procedure produced reasonably repro-ducible results. Individual 10-mil cultures (flasks)were centrifuged. The mycelium was washed withdistilled water and blotted with filter papers,dried in an oven at 100 C for 15 to 30 min, and,finally, desiccated under vacuum to constantweight. For bacteria and yeast, 20-ml sampleswere taken at the stated times from a singleflask culture for each griseofulvin concentration.The cells were collected on Millipore discs, washedtwice with distilled water (4 C), and dried. Allmycelial weights are given on a dry-weight basis.

Extraction of nucleic acid and protein. A mod-ification of Schneider's extraction procedure(Schneider, 1957) was developed for use with thedermatophytes. The washed mycelia were ex-tracted three times with 2 ml of boiling water for5 min, followed by two extractions under each ofthe following conditions: 10% (w/v) trichloro-acetic acid at 4 C for 5 min, 5%/O (w/v) trichloro-

acetic acid at 90 C for 15 min, and, finally, 1 NNaOH at 90 C for 30 min. This extraction pro-cedure was found to be the best among severaltested for fractionating the cell constituents ofdermatophytes.

Analytical methods. The protein content of theNaOH extracts was determined by the method ofLowry et al. (1951) with bovine serum albumin asa standard. Ribonucleic acid (RNA) and deoxy-ribonucleic acid (I)NA) in the hot trichloroaceticacid extracts were estimated by the procedures ofMejbaum (Schneider, 1957) and Burton (1956),respectively. Values are expressed as equivalentsof yeast RNA or calf thymus DNA (WorthingtonBiochemical Corp., Freehold, N.J.). The aminoacids and nucleotides in the soluble pool (hotwater extracts) were measured as leucine andadenylic acid equivalents, respectively, accordingto the modified ninhydrin (Rosen, 1957) and or-cinol (Schneider, 1957) methods.

Radioactive tracer experiments. The followingC'4-amino acids arid nucleosides were used: L-leu-cine-1-C'4, L-valine-1-C'4, thymidine-2-C'4, anduridine-2-C'4 (specific activity, 21.0, 7.9, 25.7,and 30.0 mc/mmole, respectively). The labelednucleosides were obtained from New EnglandNuclear Corp., Boston, Mass., and the amino acidsfrom Nichem Inc., Bethesda, Md. Microamountsof these C'4-compounds were diluted with 100 mgof the corresponding unlabeled substances in 1.0ml of distilled water and sterilized by filtration.The solutions were then added, with or without 100,ug of griseofulvin, to 10-ml cultures (flasks) ofMicrosporum gypseum (44 hr) or Trichophytonmentagrophytes x8 (72 hr) grown from spore inoculain the AGS medium. At the times specified inTables 4 and 5, the amino acid and nucleotidepools, nucleic acids, and proteins of individualcultures were extracted, as described above.The radioactivity was measured by adding the

different extracts (0.1 ml) to the dioxane scintil-lation mixture (15 ml) of Bray (1960) and countingin Tri-Carb liquid scintillation spectrometer series314 E (Packard Instrument Co., La Grange, Ill.).All counts were corrected for background activity.

RESULTS

Griseofulvin action on growth and cell structure.The animal pathogens, Il1. gypseum and theTrichophyton strains, were more sensitive togriseofulvin than any of the other microorganismstested (Table 1). The growth (as determined bydry weight) of A Iternaria tenuis, Cercosporamelonis, and Glomerella cingulata (plant invaders)was partially inhibited by moderate levels ofgriseofulvin. The saprophytic fungi Aspergillusniger, Fusarium nivale, and N. crassa showedonly slight inhibition. Griseofulvin producedsevere morphological alterations (stunting andcurling) in the mycelia of the dermatophytes atall concentrations tested. The other filamentousfungi showed much less distortion, especially at

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V9GRISEOFULVIN ACTION ON DERMATOPHYTES

the lower concentrations of the antibiotic (Table1). Neither the cell mass nor the morphology ofSaccharomyces cerevisiae, Candida albicans, or E.coli was affected with levels of griseofulvin ashigh as 100 .tg/ml.

Kinetics of the inhibition of dermatophytes bygriseofulvin. When griseofulvin was added toMl. gypseum cultures at the time of inoculation, itinhibited the growth of the fungus to a greaterextent and for a longer period than when it wasadded at 44 hr, by which time considerable cellmass had developed (Fig. 1). There was almostcomplete inhibition at either time by 5 ,ug ofgriseofulvin per milliliter. The effect was onlytemporary, however, with the 44-hr cultures.The growth rate increased after a lag period,which was dependent on the concentration ofgriseofulvin. In a similar experiment, permanentinhibition of III. gypseum was observed onlywhen griseofulvin, in concentration higher than2 ,ug/ml, was incorporated into the medium at0 hr together with a small inoculum. Comparableresults were obtained with T. mentagrophytesstrains K and x8.The cells from cultures which had resumed

growth in the presence of the antibiotic did notappear to have developed resistance to its action.Sufficient mycelial suspension (4 mg/10 ml) toproduce growth after a 2-day lag was added tomedium containing 10 ,ug of griseofulvin p)ermilliliter. After 5 days, the cells were washed and

diluted in fresh medium to the original cell con-centration. Growth occurred without lag in un-supplemented medium, but was again inhibitedfor at least 48 hr by 10 ,ug/ml of the antibiotic.The explanation for this temporary insensitivitymay lie in the fact that, under these circum-stances, uptake of griseofulvin from the mediumhad ceased in the original cultures (El-Nakeeband Lampen, 1965).

Ribonucleotides and griseofulvin action. McNall(1960) reported that the purine ribonucleotidesprevented the inhibition of Al. canis by griseoful-vin. A comparable reduction in griseofulvinaction could be shown with T. mentagrophytesstrain x8 (Table 2). Adenylic and guanylic acidsdecreased the inhibition of growth from 56 to19%, when both were present at 10ltg/ml. Lowerconcentrations had little effect. The individualnucleotides were much less effective, even at 20Ag/ml. In contrast to the l)ositive results withstrain x8, the reversing effect of purine nucleo-tides for T. mentagrophytes strain 171 was slight,and the pyrimidine derivatives were withouteffect.

In another experiment, 12 nucleotides andnucleosides (including deoxy derivatives) wereincorporated at 10 gAg/mnl into cultures of M.gypseum, in liquid and solid media, withoutaltering the inhibitory action of griseofulvin.It should also be noted that essentially identicalconcentrations of griseofulvin were required to

TABLE 1. In vitro growth inhibition of microorganisms by griseofulvin

Microorganism

Microsporum gypseum 1334-9 ..............1'richophyton mentagrophytes (k) ..........T. mentagrophytes x8.....................T. interdigitalis Cg9o......................T. persicolor C654.........................Alternaria tenuis C807.....................Cercospora melonis C601 ................Glomerella cingulata C586..................Aspergillus niger C116.....................Fusarium nivale C589.....................Neurospora crassa A-17.................Saccharomyces ceo-evisiae LK2G12.Candida albicans 204.....................Escherichia coli B........................

Source*

SKSCCCCCCC

HLV

Griseofulvin concn (jg/ml)

0.5

38 Mt61 M29 M65 M60 M606000000

1.5

948587837820 M20 M25 M0000

5.0

979594908926265060100

10.0

9595973933(sa9M10 M20 M000

50.0

99

37000

100.0

000

* C = A. A. Campbell, Glaxo Laboratories Ltd., Stoke Poges, England; H = Harlyn Halvorson,University of Wisconsin, Madison; K = E. A. Konopka, Ciba Pharmaceutical Products, Inc., Summit,N.J., L = H. A. Lechevalier, Institute of Microbiology; V = H. J. Vogel, Institute of Microbiology;S = M. Solotorovsky, Rutgers, The State University, New Brunswick, N.J.

t Results, expressed as per cent inhibition of growth, are the average of three estimations. Sporeinocula were used for all the fungi. The yeasts and E. coli were incubated 48 hr; all other organismswere incubated 72 hr. M = morphological distortion observed.

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o 120

0

80

J

I,( 4001-j

01

E 20

A

/

HOURSFIG. 1. Inhibition by griseofulvin of the growth

of Microsporum gypseum. Neopeptone medium(30 ml in 125-ml flasks) was inoculated with 0.1ml of the mycelial homogenate. Symbols: 0, no

griseofulvin added; A, 1.5 ,usg/ml; O, 5.0 iig/ml.Griseofulvin was added (j ) at 0 hr (solid lines) or

at 44 hr (broken lines).

inhibit the growth of M. gypseum in the syntheticmedium (AGS), in Neopeptone medium, and inVogel's medium supplemented with 0.5% caseinhydrolysate. Nutritional factors do not appear,

therefore, to be significant in determining theresponse of M. gypseum to the antibiotic.

Effect of griseofulvin on protein and nucleic acidsynthesis. Addition of griseofulvin (10 ,Ag/ml) togrowing cultures of M. gypseum inhibited netprotein, RNA, and DNA synthesis for about 6hr (Table 3). The level of amino acids and nucleo-

TABLE 2. Effect of nucleotides on the inhibition ofTrichophyton mentagrophytes x8 by griseofulvin*

Mycelial wtt Per cent ofcontrol growth:Nucleotide

With Without With Withou tgriseo- griseo- griseo- griseo-fulvin fulvin fulvin fulvin

,g/ml0 17 37 46 10010 A 16 4310 G 17 465 A, 5 G 21 56

20 A 22 39 59 10520 G 19 35 51 9510 A) 10 G 30 39 81 105

* Neopeptone medium was inoculated with 1%of a 20% (v/v) mycelial homogenate. A and Grepresent the 5'-ribonucleotides of adenine andguanine which were added to the inoculatedmedium as sterile solutions. Cultures receivedeither 1 ,ug of griseofulvin/ml (with griseofulvin)or no antibiotic (without griseofulvin).

t Average of two estimations. Amounts areexpressed as milligrams per 10 ml.

t Growth in culture without added nucleotidesor griseofulvin.

tides in the water-soluble pool and of proteinand nucleic acids then began to rise. This wasfollowed, after 24 hr, by a resumption of growth.The incorporation of C14-uridine and C14-

thymidine into nucleic acid (hot trichloroaceticacid extracts) of M. gypseum was inhibited bygriseofulvin (Table 4), although the uptake wasnot eliminated. Incorporation of C'4-leucine andC14-valine into protein (hot NaOH extract) was,however, unaffected.The nature of the C14-labeled material in the

hot NaOH extract was examined, since griseoful-vin inhibited net protein synthesis but not the

TABLE 3. Inhibition by griseofulvin of nucleic acid, nucleotide, amino acid,and protein synthesis in Microsporum gypseum*

Mycelial wt Protein SolublNepooD(Nmoles/10ml) RNADNA(mg/bo Ml) (mg/b l) oui ol Amlsml) (mg/b0 Ml) (jsg/b0 Ml)

Time Amino acids Nucleotides-Gr + Gr -Gr + Gr -Gr + Gr -Gr -Gr

-Gr + Gr - Gr ± Gr

hr

0 3 0.96 4.7 1.34 0.27 123 - 3 1.32 0.84 10.4 4.9 2.00 0.40 0.34 0.20 18 86 4 3 1.62 0.80 16.5 12.8 3.34 1.00 0.54 0.30 27 1612 6 3 2.1 1.4 19.0 13.9 5.68 2.17 1.0 0.50 40 1824 12 4 4.4 2.1 28.0 16.4 6.35 3.00 1.5 0.74 60 2772 34 12 8.0 6.4 42.0 45.0 12.9 14.0 3.2 5.0 116 158

* M. gypseum was grown from spores in AGS medium for 72 hr before the addition of griseofulvin atzero time. -Gr = no griseofulvin; +Gr = 10lg of griseofulvin per ml.

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incorporation of C@4-amino acids. No C'4 (< 1%)was found as lipid. Acid hydrolysis and paperchromatography showed that, at 3 hr, 75%ly, ofthe count in the fraction was C'4-leucine; 25%was found in other amino acids. At 15 hr, theradioactive label was randomized, and less than40% was in amino acids. At 3 hr, 35% of theradioactivity added to the culture was lost asvolatile compounds; at 15 hr, 75% of the addedradioactivity had been lost. With C'4-valine at3 and at 6 hr, 80% of the radioactivity appearedas valine, after acid hydrolysis and chromatog-raphy.The incorporation of C'4-uridine and C14-

leucine was also determined with strain x8 ofT. mentagrophytes, which has the unusual prop-erty (among the dermatophytes) of accumulatinggriseofulvin preferentially into its protein, ratherthan into its nucleic acid fraction (El-Nakeeb,1963). Even with this organism, griseofulvin didnot prevent the incorporation of C14-leucine intoprotein (Table 5). Although actively-growingcultures of strain x8 were less sensitive to griseo-fulvin than comparable cultures of M. gypseum,a partial inhibition of uridine uptake was stillobserved.

DISCUSSIONThe data presented on the antimicrobial

spectrum of griseofulvin confirm and extend those

TABLE 4. Effect of griseofulvin on the incorporationof C'4-labeled uridine, thymidine, leucine, andvaline into nucleic acid and protein of Micro-

sporum gypseum*

Uridine Thymidine Leucine ValineTime

-Gr + Gr-Gr + Gr -Gr +Gr -Gr + Gr

hr

0 05t 0.01 0.4 00.5 1.6 0.6 - 10.4 7.21.0 2.4 1.6 - 16.6 14.416.4 19.43 28.1 7.0 0.14 0.08 76 74 26.9 38.76 57.9 17.1 0.17 0.12 134 164 96 9812 154 58.9 0.29 0.18 131 129 -

24 314 57.9 1.42 0.92 118 118 97 97

* For details, see Materials and Methods.Count/min X 10-3 added to 10-mi cultures were850, 789, 890, and 1,314 for uridine, thymidine,leucine, and valine, respectively. Data for uridineand thymidine represent uptake into the total hottrichioroacetic acid extract. Inhibition of growthby griseofulvin was comparable to that in theexperiment of Table 3. -Gr: no griseofulvin;+Gr: 10,ug of griseofulvin per ml.

t Expressed as counts per minute X 10-3 per10-ml culture.

TABLE 5. Effect of griseofulvin on the incorporationof C'4-uridine and C14-leucine into nucleic acidand protein of Trichophyton mentagrophytes x8*

Uridine LeucineTime

- Gr + Gr - Gr + Gr

hr

0 0.2t - 2.80.5 6.3 4.3 40 411 11.8 9.2 100 852 38 29 125 1294 88 66 104 1308 102 84 96 114

* Conditions same as for Table 4. Counts perminute X 10-3 added to 10-mi cultures were 722and 490 for uridine and leucine, respectively.

t Expressed as counts per minute X 10-3 per10-mi culture.

reported by Brian (1949, 1960) and by Roth et al.(1959). The degree of inhibition of the variousfungi, noted at a particular griseofulvin concen-tration, depends to a large extent on the methodused for evaluating the inhibition. Measurementsof the diameter of fungal pellets (Roth et al.,1959) or of radial growth (Brian, 1949) primarilyindicate the morphological distortions producedby the antibiotic, and give sensitivities differingfrom those obtainable by the dry-weight methodadopted in this work. The morphological distor-tions occur at much lower antibiotic concentra-tions than those required to prevent an increasein cell mass. Growth inhibition is primarily acharacteristic of the highly sensitive dermato-phytes, and, even among this group, someTrichophyton strains (El-Nakeeb, 1963) show celldistortion without restriction of cell mass.Secondly, the inhibition is greatly affected bythe size of the inoculum and the amount of cellmaterial present before the addition of griseoful-yin. With dermatophytes, the per cent inhibitionproduced by griseofulvin was inversely propor-tional to the log of the number of the spores usedto initiate the cultures (El-Nakeeb, 1963).The proposal of Brian (1949, 1960) that griseo-

fulvin interferes with chitin biosynthesis is notdirectly supported by our experimental data,although it is not excluded. It should be notedthat A. niger contains large amounts of chitin(Blumenthal and Roseman, 1957), and yet it isrelatively insensitive to griseofulvin. Also, cell-wall formation by N. crassa protoplasts (pre-pared according to Bachmann and Bonner, 1959)was not prevented by the antibiotic even at 100,ug/ml, although the resulting cells were distorted(El-Nakeeb, 1963).

Neither the glycolysis nor the respiration of

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the sensitive dermatophytes (M11. gypseum andT. mentagrophytes) or of the poorly sensitiveN. crassa was affected by griseofulvin (El-Nakeeb, 1963). The respiration of Botrytis allii(Brian, 1949) and Trichophyton rubrum (Rothet al., 1959) is also insensitive to the antibiotic.The concept that nucleic acid biosynthesis,

especially the nucleotide polymerization step,might be the target of griseofulvin action wasintroduced by McNall (1960), who observed thatthe inhibition of lllicrosporum canis by griseoful-vin could be prevented in part by ribonucleotides.In support of his postulate, McNall cited theantimitotic colchicine-like effect reported forgriseofulvin in rat and bean root cells (Paget andWalpole, 1958, 1960). Mitotic interference wasnot observed, however, in hair roots of humanscalp (Burgoon et al., 1960).

Although it was possible with T. mentagrophytesx8 (but not with related dermatophytes) todemonstrate a partial protective effect of ribonu-cleotides, the specific suggestion that the poly-merization step is a major site of interference isprobably not correct. Incorporation of uridineand thymidine into nucleic acid was reduced,but there was no accumulation of soluble nucleo-tides in inhibited cells (Table 3) nor in culturefiltrates (El-Nakeeb, 1963), as might have beenanticipated if the main block was at the poly-merization stage. It can also be concluded thatthere is no major breakdown of preformed poly-nucleotides, such as that produced by mitomycinC (Reich, Shatkin, and Tatum, 1961). Inhibitionof the net synthesis of both nucleic acid and pro-tein (Table 3) can be considered to occur rapidly;the doubling time of the organism is about 12 hr.No definite explanation can be provided for theobservation that net l)rotein synthesis was haltedduring a 3 to 6 hr period following addition ofgriseoful6in, althou,-h incorporation of C'4-aminoacids into protein was unaffected. Griseofulvinmay have initiated an increased turnover ofproteins; however, a detailed exaamination of thekinetics of protein formation and bieakdown willbe needed to resolve this apparent discrepancy.No specific site of action of griseofulvin is

demonstrated by the findings presented here.Special interest is drawn, however, to the altera-tions in nucleic acid and protein metabolism,because of the observation (El-Nakeeb andLampen, 1964, 1965) that large amounts ofgriseofulvin are taken up by sensitive organismsand are present as complexes with the cellularnucleic acid and protein.

ACKNOWLEDGMENTSThis investigation was supported by Public

Health Service grant AI-04572-02 from the Na-

tional Institutes of Health, and by a grant fromthe American Friends of the Middle East, Inc.

LITERATURE CITED

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BLUMENTHAL, H. J., AND S. ROSEMAN. 1957.Quantitative estimation of chitin in fungi. J.Bacteriol. 74:222-224.

BRAY, G. A. 1960. A simple efficient liquid scintil-lator for counting aqueous solutions in a liquidscintillation counter. Anal. Biochem. 1:279-285.

BRIAN, P. W. 1949. Studies on the biological ac-tivity of griseofulvin. Ann. Bot. London 13:59-77.

BRIAN, P. W. 1960. Griseofulvin. Trans. Brit.Mycol. Soc. 43:1-13.

BRIAN, P. W., P. J. CURTIS, AND H. G. HEMMING.1946. A substance causing abnormal develop-ment of fungal hyphae produced by Penicilliumjanczewskii Zal. I. Biological assay, productionand isolation of "curling factor." Trans. Brit.Mycol. Soc. 29:173-187.

BURGOON, C. F., H. J. GRAHAM, R. J. KEIPER, F.URBACH, J. S. BU RGOON, AND E. B. HELWIG.1960. Histopathologic evaluation of griseofulvinin Microsporum audouini infections. A. M. A.Arch. Dermatol. 81:724-732.

BURTON, K. 1956. A study of the conditions andmechanism of the diphenylamine reaction forthe colorimetric estimation of deoxyribonucleicacid. Biochem. J. 62:315-323.

ElL-NAKEEB, M. A. 1963. Antibiotic action andcellular binding of griseofulvin. Ph.D. Thesis,Rutgers, The State University, New Brunswick,N.J.

EiL,-NAKEEB, M\1. A., AND J. 0. LAMPEN. 1964.Complexing of griseofulvin by nucleic acids offungi and its relation to griseofulvin sensitivity.Biochem. J. 92:59-60 P.

EL-NAKEEB, M. A., AND J. 0. LAMPEN. 1965.Uptake of griseofulvin by the sensitive derma-tophyte, Microsporum gypseum. J. Bacteriol.89:564-569.

GENTLES, J. C. 1958. Experimental ringworm inguinea pigs: oral treatment with griseofulvin.Nature 182:476-477.

GROVE, J. F., J. MACMILLAN, T. P. C. MULHOL-LAND, AND M. A. T. IROGERS. 1952. Griseofulvin.IX'. Structure. J. Chem. Soc., p. 3977-3987.

LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measurementwith the Folin phenol reagent. J. Biol. Chem.193:265-275.

MIARINI, F., P. ARNOW, AND J. 0. LAMPEN. 1961.The effect of monovalent cations on the inhibi-tion of yeast metabolism by nystatin. J. Gen.Microbiol. 24:51-62.

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