Effect of thiols in reversing the inhibition by methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate on Saccharomyces cerevisiae

PESTICIDE

13IOCHE.MISTR1-

Effect

.\iVl)

of Thiols

I’HYSIOLOGY

1,

@-a08

in Reversing

the

Inhibition

by Methyl-l-

(Butylcarbamoyl)-2-Benzimidazolecarbamate Saccharomyces R. Physiology

Program,

MAILMAN,

Department Received

IL

on

cerevisiae’

HODGSON,

D.

AND

of Entomology, Department of University, Raleigh, &‘orrh Carolina August

30,

1971;

accepted

HUISINGH

Plant Pathology,

,Vorth

Curolina

State

27607

November

5, 1971

Cultures of Saccharomyces cerevisiae in minimal medium were inhibited by 1 pg/ milliliter or more of methyl-l-~butylcarbamoyl)-2-he~~~imidazole-carbamate (benomyl). The yeast could reverse t)he inhibition at concentrations less than 50 pg/ milliliter after a time proportional to the concentration of the fungicide, thus indicating t,he approximate toxicity under the conditions used. Cysteine, and other thiols of the structure IL-CH?-SH tended to reverse the inhibition. This reversal was due to a metabolic effect rather than a direct chemical combination of the thiols with benomyl or methylbenzimidazole carbamate, its breakdown product,. Fungitoxic residues were reduced in the medium during yeast growt)h if benomyl and cyst,eine were present. However, spectrophotometric comparisons of cultures grown in benomycysteine mixtures us. controls using whole washed cells suggested that neither cyt,ochrome P-450 nor any cytochrome normally present were induced. The mechanism of act,ion of the thiols remains to be demonstrated.

in controlling fungal growth. The toxicity of both compounds towards a particular species may be similar, or may differ significantly (5). Little information is available concerning the mode of action of the fungicide, or it,s metabolism. Clemons and Sisler (7) reported that a level of BCM inhibitory to the growth of ATeurospora crassa (8 pg/milliliter) did not inhibit glucose or acetate oxidation. A lower level (1 clg/milliliter) inhibited DNA synthesis, but not RSA or protein synthesis. They concluded that BCM interferes with some aspect of nuclear or cell division. Gardiner et al. (8) found that the metabolic fate of 4970 mg/kilogram doses of benomyl in rats was twofold. First, the compound was hydroxylated to ;i-hydroxyl BC>I, and second it was excreted as the glucuronidc or sulfate conjugate. The product,, 5-hydroxyl-BCM, has been shown

INTRODUCTION

The fungicide, methgl-l-(butyIcarbacarbamate (benmoyl)-2-benzimidazole omyl), has sntifugal activity against a wide spectrum of organisms (l-3). In aqueous environment,s benomyl breaks down to methyl-Zbenzimidazole-carbamate (BCAI) (4). Both compounds have fungitoxic activity (5, 6), and the use of benomyl implies that both benomyl and BCM may be important 1 Paper No. 3546 of the Journal Series of the Nort)h Carolina St,ate University Agriculture Experiment. Station, Raleigh, North Carolina. The authors gratefully acknowledge partial sapport from grants No. ES-00083 and ES-00044 of the United States Public Health Service. Preliminary experiments were performed during the tenure of one of the aut)hors (RM) as a research assistant in the Department of Food Science, North Carolina State Universit,y. 401

402

MAILMAN,

HODGSON,

to have no antifungal activity.2 These reactions have not been reported in fungi. Hastee (9) postulated that benomyl map cause certain genetic effects. He found that when Aspergillus sp. diploids were treated with benomyl, segregational changes occurred that were similar t’o those caused when mutagenic agents were used on the same hcterozygotes, although he did not detect any mut,ations. Richmond and I’ring (10) stated that. benomyl causes a metabolic imbalance when it is applied to germinating conidia of Botrytis fubue. Electron micrographs seemed to indicate that increased cell wall synthesis occurred a.t the expense of other intracellular processes. In this paper we present results of studies designed to elucidate the metabolic response of S. cerevisiae treated with benomyl. MATERIALS

AND

METHODS

Cultures. Two strains of X. cerevisiae were used interchangeably with no differences being observed. They were #477 (Champagne), and #481 (Burgundy) obtained from D. E. Carroll, Department of Food Sciences, Xorth Carolina State University, and originally from the collection of Scott Laboratories, Richmond, California. The cultures were maintained on fresh potato dextrose agar slants stored at 4 C, after 4%hr growth at 25 C. Inoculum was prepared by transferring culture material to minimal medium (see below), and incubating on a shaker for 12-20 hr, by which time the culture was in exponential growth. Minimal media of the following compositions per liter of distilled water were used: (1) 13.4 g yeast nitrogen base (YNB) (II), and 40 g glucose. (2) 6.7 g YSB and 45 g glucose. Nutrient media were sterilized prior to inoculation while grape juice was pasteurized in order to provide a comparison with the methods used for the industrial 2 J. A. Gardiner, 1970, E. I. du Pont ton, 1)elaware.

personal de Nemours

communication, & Co., Wilming-

AKD

HUISINGH

processing of grape juice. The grape: juice was pressed from red grapes, fort,ified with sugar to %2%‘, and pasteurized at 83 C for 1 min. It was t)hen held at 4 C until usod (wit’hin 24 hr). Analytical methods. For growth measurements, the yeast cultures were grown in flasks with a 13 mm OD sidearm which could be read in a Beckman DU-Gilford 2000 spectrophotometer and incubated at 25 f 1 C. Cell counts were made by the method of the American Public Health Service (12). Bioautography was done by a modification of the method of Peterson xnd Edgington (6). Aspergillus Jlavus was substitut,ed for Penicillum sp., and a solvent system of chloroform: acetone: hexanc (2: 1: 1, v:v) was used for TLC on silica gel. Amino acid analysis was done on a Beckman 116 Amino Acid Analyzer. Ultraviolet spect,ra were determined on a Gary 15 spectrophotometer. Reduced-oxidized and (reduced + CO) - reduced difference spectra of washed cells resuspended in 0.15 M phosphate buffer (pH 7.0) were detcrmined lvith a Beckman Acta V spectrophotomet’er using dithionit,e as t,he reducing agent. Yeast cells wcrc broken with an Aminco French Pressure Cell or a Branson Sonifier. Examinations for disappearance of fungitoxic residues was done by taking 10 ml samples from flasks containing 10 Fg/ml benomyl, and 0.5 mg/ml cgsteine. One flask was inoculated with a log phase yeast culture while t’he other remained sterile. The samples were ext)racted twice with 40 ml chloroform, without centrifugation. The organic phase was evaporated to 2 ml, and 20 ~1 Fas analyzed by bioautography. All thin-layer chromatography was done by the ascending techniques on Eastman #6061 silica gel sheets. Separation of ninhydrin-positive materials was effected by a solvent system of methanol: chloroform : 17 5% ammonia (2: 2: 1, v: v), followed by spraying with a solution of 2.5 g ninhydrin

EFFECT

OF

THIOLS

ON

SACCHAROXYCES

per liter of acetone and heating at 60 C for 15 min. RESULTS

The effect of benomyl (from l-100 pg/ milliliters) on the growth of S. cerevisiae was tested in minimal medium. Growth n-as inhibited by all levels tested. A correlat,ion was made between cell number and absorbancc at varying levels of benomyl. In minimal medium 5 pg/milliliters benomyl gave an initial inhibitory (toxic) period. This was also expressed as a const’ant absorbance reading. In both the control and benomyl-treated samples, the beginning of growth and reproduction as measured by cell counts paralleled the beginning of an increase in absorbance. These results were in agreement with a similar experiment, (Fig. 1) conducted in grape juice treated with varying levels of benomyl. In addition it was noted in t’hese two experiments, that at the lower levels of benomyl (<50 pg/ the yeast would eventually milliliters), reverse the effects of the fungicide and increase in cell number. However, a level of 100 pg./milliliters in this medium, prevented any detectable yeast’ growth during the

2.0 w 0 1.6 2

CEREVISIAE

103

duration of the measurement (greater t)han 150 hr). During the course of further studies it was observed that inoculum age had an influence upon the duration of the inhibition by benomyl. Older cultures reversed the inhibition more rapidly. To investigate this, a late log phase-early stationary phase inoculum (A = 1.65) was divided into halves. One part was washed twice and resuspended in 2.5 X lo-* & phosphate buffer (pH 7.2) to a cell density of 6.1 X 10’ cells/milliliter. Various inoculum sizes were used with varying fungicide concentrations. The results are shown in Table 1. A doubling of the number of cells in the inoculum should have an effect on the speed of reaching maximum cell density approximately equal to one log phase generation time. Our results (Table 1) show that increases in inoculum size have an effect far great’er than that,. Wit(h 10 pg/‘milliliter benomyl a 5X increase in unwashed inoculum volume caused a (94-24) = iO-hr decrease in the time needed to obtain maximum growth. This does not appear to be due principally to an increase in cell number since the normal log phase generation time of the yeast, was less than 2 hr. In contrast, the use of washed inoculum significantly decreased the effect of increased inoculum size. A 5X increase in

1.2

TABLE

E 0.6 z 0.4 -a 0

Yhe

e$ect of fungicide

Quantity inoculum (ml) TIME

of

1

inoculnm size and treatment concentration on the amount of inhibition.

Benomyl concentrations b%/ml)

and

Time to Achieve A = 1.5 (f10’x) (hr) UnLvashed

\Vashed

(HRS.)

FIG. 1. Relationship between absorbance and plate count of control and benomy-treated cultures in grape juice. A. control read by plate count. B. control read by absorbance. C. 10 @g/ml benomyl read by plate count. D. 10 pg,fml benomyl read by absorbance. E. 100 pg/ml benomyl read by plate count. F. 100 pg/ml benomyl read by absorbance.

1

0

1 2 5 1 2 5

5 5 5 10 10 10

11.5 38 37 14 94 17.5 24

11.5 3‘4 32 13.5 82 83.5 49.5

404

MAILMAN,

HODGSON,

inoculum in the presence of 10 ~g/millilit,er caused only a (52-49.5) = 32.5 hr decrease compared to a 70 hr decrease when the unwashed inoculum was used. In addition the washed cells had a higher viable cell density (6.8 X lo7 IAS.4.S X lo7 cells/ milliliter) indicating that this effect was not principally due to cell number. This suggested that a compound(s) which was present, in the unwashed inoculum that had been produced by the yeast cells could be readily removed. Furthermore, this compound(s) prevented inhibition. E$ect of added nuwients in overcoming inhibition,. It was found that, t’he presence of r,-cysteine in the growth medium allowed the yeast to almost totally reverse the inhibition of the fungicide. Figure 2 shows that a slight inhibition resulted when a mixture of cys teine and methionine was added to a control. However, cyst,eine alone, or cysteine and methionine together reversed the inhibition by benomyl. This resulted in a growth rate equal to the control containing both amino

8

16 TIME

24

AND

HUISIKGH

acids. Methionine alonc is ineffective in reversing the inhibition of bmomyl. Supplementation of benomyl-treatrd medium with an amino acid mixture (for composition set Table 2) caused some stimulation of growth, as would be expected. However, cyst&e and the amino acid mixture together gave maximum reversal of inhibition. Further results (Fig. 3) show that srrine, the metnbolic precursor of cysteine, had no comparable effect. Serine added to both a control and a benomyl-treat’ed sample gave slight stimulation of growth, but it, did not’ ovcrcome the inhibition to t,he same degree as cyst&e. Conversely, cyst~cinc i&elf at the concentrations used inhibit,ed the growth TABLE 2 of amino acid mixture

Composition

Amino Acid

g/l00 g mixture

Amino acid

Lysine Histidine Arg-inine Aspartic acid Threonine Serine Glutamic acid Proline Glycine

5.3 4.6 0.3 11.2 5.5 9.7 22.1 3.8 4.9

Alanine Valine Methionine Isoleucine Tyrosine Phenylalanine Cysteic acid Leucine

g/100 g mixture 6.9

5.8 3.2 4.2 0.4 1.6 0.4 10.6

32

(HRS.1

FIG. 2. Effect of an amino acid mixture, L-cysteine-HCl-HO and L-methionine in reversing inhibition by benomyl. A. 5 pgglml benomyl, 0.8 mglml amino acids, 0.4 mg/ml L-cysteine-HCLHzO, 0.4 mg/ml L-methionine. B. Control. C. Control, 0.8 mg/ml A-cysteine-HCl-HPO, 0.8 mg/ml L-methionine. D. 5 w/ml benomyl, 0.8 mg/ml L-cysteine-HCl-HzO. E. 5 fig/ml benomyl, 0.8 mg/ml L-cysteine-HCl-HzO, 0.8 mg/ml L-methionine. F. 5 pg/ml benomyl, 0.8 mg/ml amino acids. G. 5 pgglml benomyl, 0.8 mg/ml z-methionine. H. 5 rg/ml benomyl.

5

IO

15 TIME

20

25

30

35

(HRS.)

of L-cysteine-HCl-Hz0 and on reversing inhibition by benomyl. A. control, 0.25 mg/ml L-serine. B. control. C. control, 0.5 mg/ml z-cysteine-HCl-HzO. D. 5 pg/ml benomyl, 0.5 mg/ml L-cysteine-HCL-H20. E. 5 M/ml benomyl, 0.25 mg/ml L-se&e. F. 5 rg/ml benomyl. FIG.

L-serine

3. Effect

EFFECT

OF

THIOLR

ON

SICCHAROMYCES

of the control, yet permitted a nearly equal growth rate when added to a benomyltreated sample. Additional studies showed that folic acid, pyridoxal, pyridoxine, biotin, cyanocobalamin, thiamine, pantothenate, s-adenosplmethionine, L-tryptophane, L-tyrosine, Lserine, L-homoserine, L- or nn-methionine, Dor L-cystine, L-histidine, L-arginine, L-cystathionine, L-homocysteine-thiolactone, the amino acid mixture, adenosine, and xanthine did not reverse the inhibition. Reduced glutathionr, DL- and n-cysteine were found to be of the same order of effectiveness as L-cysteine. These compounds were highly effective in overcoming the inhibition of benomyl. Experiments were also run with varying levels of two other reduced sulfhydryl compounds, 2-mercaptoet’hanol (2-Me), and dithiothreitol (DTT). The results are shown in Fig. 4. The maximum reversal of DTT was at a lcvcl of 0.6 X 10e3 17il, whereas for ~-MC it was cu. 3.0 X 1OP M. Cysteine was most effective at about this latter Icvel. The DTT gave a

123456 CONCENTRATION

(M.)

Lx lo‘3 I

FIG. 1. Comparative e$ects of thiols on reversing inhibition by benomyl. A. L-cysteine-HCLH&. B. dithiothreitol. C. 2-mercaptoethanol. Control + 20 My/ml benomyl: A = 0.05, Control: A = 1.9.5.

CEREVISIAE

405

significantly better response than the 2-11~ did, but neither was as effective as cysteine. However, they did reverse the inhibition whereas control levels would have a significant inhibitory influence on the growth of the yeast. Higher levels of 2-Me and DTT than those shown in Fig. 5 caused inhibition of yeast as would be expected from tjhcir toxicity. Chemical reactivity of cysteirze arkd benomyl. Since cysteine had a remarkable effect in abolishing the inhibition of benomyl, the possibility of direct chemical combination of the two was considered. Two methods were used to test this possibility. (1) An aqueous solution of (I) 0.02 JJ cysteine, and a solution of (II) 0.002 N benomyl in methanol were mixed to yield (III) a solution with a tenfold molar excess of cysteine. The thrrc solutions were allowed to stand in t,ho dark and sampled at intervals for 72 hr. Some precipitation of benomyl was noted. Duplicates of each of the thrw solutions above were spotted on a TLC plate and dcvcloped. The plate was divided in halves and one analyzed by bioautography, the other by ninhy-drin. All analysts were identical. The results of a typical experiment arc depicted in Fig. 5. No nen ninhydrin positive compounds were observed, nor was disappearance of the fungitoxic zone detected by bioautographic analysis reducing the possibility that chemical combinat,ion occurred. (‘2) The ultraviolet spectra of benomyl, cystrine-HCL-H?O, and a mixture of the two were examined to detect any spectral shifts. The only shifts noted in the mixture were t)hose associated with the conversion of benomyl to BCRI and t’hose could bc duplicated by the addition of trace amounts of HCI. These results further reduced the possibility of this type of chemical combination. Additional evidence that t,he thiols did not effect direct, nonenzymatic combinat,ion wit,h bcnomyl or BCAI was obtained by

406

FIG . 5 . Bioa

M

L-c ,yst einc.

MAILMAN,

utog graphic II. 0.002

and ninhydrin M benomyl.

III.

HODGSOK,

AND

analyses for chemical 0.01 M I,-cysteine

analyzing medium containing 10 pg/milliliter benomyl, and 0.5 mg/milliliter cysteine, with and without an inoculum of yeast cells. Noninoculated medium showed no disappearance of BCM and benomyl at all until 43.8 hr had elapsed, whereas the inoculated medium showed distinct decreases by the time the culture had entered midexponential phase (cu. 10 hr). These decreases were manifested as a conversion of residues t’o BCM and also a decrease in total residues. Since cytochrome P-4.50 has been demonstrated to play an important role in the metabolism of xenobiotics in plants, animals and microorganisms, including fungi (13), both control and benomyl-treated cultures were examined for this cytochrome. No apparent induction of a P-450 type cytochrome was observed, nor was there a change detectable by our methods in cytochrome levels when treated cultures were compared with control cultures at equivalent optical densities.

HUISIKGH

combination and 0.001

M

of cysteine benom!]!.

amd benomyl.

I.

DISCUSSION

Our results show that D- or L-cysteine, reduced glutathione, DTT, or 2 Me reverse the toxicity of benomyl to yeast. The fact that several compounds of the general structure R-CH&SH are efficacious, though they may normally be inhibitory to the growth of X. cerevisiae, suggests that this effect is not specific for cysteine. Similarly, compounds with other sulfur linkages, Dor L-cystine, D- or L-methionine, L-cystathioninc, and L-homocysteinethiolactone, did not show a reversal of inhibition. The ineffectiveness of cysteine metabolites and cofact,ors further showed that the effect of sulfhydryl compounds is not related to the in viva metabolism of the sulfur amino acids, including the function or metabolism of the BIG vitamins (7). The occurrence, though chemically unlikely, of direct reaction of reduced sulfhydryl groups and benomyl, and/or BCM was also considered. Chromatographic be-

EFFECT

OF

THIOLS

ON

SdCCHAROMYCES

havior and ultraviolet absorption spectra of a mixture of cysteine and benomyl versus t,he individual components eliminates this possibility. The involvement of thiols in overcoming chemical inhibition has been cited previously Richmond and Somers (14) showed that the uptake of captan was mediated by sulfhydry1 groups on the cell membrane of AVeuwspora crassa, in an active t’ransport’ system. The presence of thiols in the medium competed for the fungicide and thus countered its inhibition. This type of mechanism is possible, though somewhat unlikely as the possibility of a chemical reaction between a substituent of benomyl or BC;\I and thiols is probably significant’ly less than that’ between captan (S-[trichloromcthyllthio-[4cyclohexenel-1 ,%dicarboximide) and thiols. Kaars Sijpesteyn et al. (15) have reported the detoxification of another carbamic acid ester, sodium dimethyldithiocarbama@ by yeast. This reaction involved the enzymatic conjugation of the fungicide and homoserine. The probabilit’y of a system of this type involving bcnomyl is lessened by the present finding. Two problems aw thus unanswered: the mechanism of the action of cysteine and the mechanism of the disppearancc of the fungicide. The two may be intimately related. It has been shown2 t)hat the metabolic product of rats treated with benomyl, S-hydroxylBCAI, is not fungitoxic. Further studies involving radio-labeled benomyl/BCI\I and cyst,eine should answer these questions. h detoxification of this type may be involved despite the failure to detect any cytochrome P-450 or other species in yeast. These results may have several practical consequences. Cole, Massie, and Duich (15) have reported that benomyl will only control stripe smut of ‘ ‘AIrrion” Kentucky bluegrass when it is applied during the grass dormant season. This result might be caused by differences in the concentration of exogenous cysteinc, from the grass, available to

CEREVISIAE

407

the smut in active vs. dormant growth periods. These facts, if correlated by additional field work, may greatly increase the effcctiveness of usage of benomyl. REFERENCES

1. C.

2.

3.

4.

5.

6.

7.

J. Delp and H. L. Klopping. Performance attributes of a new fungicide and mite ovicide candidate, Plant Dis. Rep. 52, 95 (1968). A. Engelhard, Preventat,ive and residual fungicidal activity of three benzimidaxole compounds and zinc ion and maneb against Diplocarpon rosae on two rose rultures, Plant Dis. Rep. 53, 537 (1969). 0. E. Schult)z, Potato Rhizoctonia disease, Fungicide and nematode tests, 23, 83, Amer. Phytopathol. Sot., St. Paul, Minn. (1969). J. J. Sims, H. Mee, and II. C. Erwin, Methyl a fungitoxic 2-benzimidazolecarbamate, compound isolated from cot,ton plants treated with methyl 1-(butylcarbamoyl)-2benzimidazole carbamate (benomyl). Phytopathology 39, 1775 (1969). Ct. P. Clemons and H. I). Sisler, Formation of a fungit,oxic derivative from Benlate, I’h,+ pathology 59, 705 (1969). C. A. Peterson and 1,. V. Edgington, Quantitative estimation of the fungicide benomgl using a bioautographic technique, 1. A gr. Food Chem. 17, 898 (1969). Cr. P. Clemons and H. I). Sisler, Localizatjion of the site of action of a fungit,oxic benomyl derivative, Pest. Biochem. Physiol., 1, 32 (1971).

8. J. A. Gardiner, IL. K. Brantley, and H. Sherman Isolation and identification of a metabolite of methyl-I-(b~~tylcarbamoyl)-2benzimidazole carbamate in rat urine, J. Agr. Food Chem. 16, 1050 (19G8). 9. A.

10.

11. 12.

13.

C. Hastee, Benlate-induced instability of Aapergillus diploids. LVnture (London) 226, 771 (1970). II. V. Richmond and IL. J. Pring. The effect of benomyl on the fine structure of Botr&~ jubae, J. Gen. Microbial. 66, 79 (1971). L. J. Wicherham, Taxonomy of yeasts, OT. S. Depart. Agr. Tech. Bull. 1029, (1951). American Public Health Service, “Standard Methods for the Examination of l)airy Prodlicts,” 11th edition, New York (1960). S. H. Ambike and It. M. Baxter, Cytochrome P-459 and Alkaloid Synthesis in Clavireps purpwea, .I. Pharm. Sci. .59, 1149 (1970).

40s 14. II.

MAILMAN,

HODGSON,

V. Richmond and E. Somers, Studies on the fungitoxicity of Captan. II. The uptake of captan by conidia of IVeurospora crassa, Ann. Appl. Biol. 50, 45 (19G2). 15. A. Kaars Sijpesteijn, J. Kaslander, and Cr. J. M. van der Kerk, On the conversion of sodium dimelhyldithiocarbamate into its

AND

HUISINGH

or-amino butyric acid derivative by microorganisms, Biochim. Biaphys. Beta 62, 5s7 (1952). 16. H. Cole, L. B. Massie, and J. L)uich, Control of stripe smut in Merion Kentucky bluegrass wit,h benomyl, Plant Dis. Rep. .j4, 146 (1970).