Fd Cosmer. Toxicof. Vol. IS. pp. 187-193. Pergamon Press 1977. Printed in Great Britain

EFFECT OF BENOMYL AND BENOMYL HYDROLYSIS PRODUCTS ON TETRAHYMENA PYRIFORMIS*’ P. W. RANKIN, Pesticide

Research

Laboratory,

J. G. Su’a,&tct and N. P. THOMPSON Food

Science Department, Florida 3261 I, USA

University

of Florida,

Gainesville,

(Received 6 November 1976) Abstract-The toxicity of the fungicide benomyl (methyl-1-(butylcarbamoyl)-2-benzimidazol-2-yl carbamate) was evaluated at the molecular level using the ciliated protozoan Tetrahymena pyriformis as a model eukaryotic cell system. Growth of T. pyriformis was inhibited in a dose-related manner by benomyl and two major hydrolysis products, methylbenzimidazol-2-yl carbamate (MBC) and butyl isocyanate (BIC). A minor degradation product, 2-aminobenzimidazole (2-AB) had no effect on growth. Light-microscopic observations of the organisms revealed a slowing of cellular motility and the development of rounded cells after exposure to 20ppm benomyl or BIC in the incubation medium. After the cells became immobile, part of the cell membrane appeared to separate, swell and form a bubble along the side of the cell. Similar concentrations of MBC and 2-AB had no apparent morphological effect upon the cells. Increasing the concentration of benomyl decreased the incorporation of [14C]acetate and [i4C]amino acids into protein and [‘Hluridine into RNA. The incorporation of [“Clacetate into lipids and of [“Hlthymidine into DNA increased with benomyl concentrations of 5 ppm, but a decrease in incorporation occurred with higher concentrations.

to rats at levels of 0.25 or 0.50% of the diet. However, when Chang cells were exposed to benomyl at a level of 10T5 M in the medium, metaphase arrest and cytochrome damage were observed (Styles & Garner, 1974). These mutation studies were initiated because of the structural similarity of benzimidazoles with purine bases. Sieler (1972) reported that MBC and 2-AB caused forward mutations in Salmonella typhimurium. Tetrahymena pyrijix-mis is a single-cell organism, which has nutritional requirements, organelles and biochemical pathways similar to those of mammalian cells (Hill, 1972). In addition, several drugs (e.g. cycloheximide, actinomycin D and triparanol) have molecular biological effects on T. py@rmis similar to their effects in higher species (Elliot, 1973). Since the data on benomyl toxicity are somewhat conllicting, the use of T. pyrijkmis offers an opportunity for the effects of benomyl to be studied in a simplified system.

INTRODUCTION

The systemic fungicide benomyl (methyl-l-(butylcarbamoyl)-2-benzimidazol-2-yl carbamate), the active ingredient in Benlate@, was first reported to be fungitoxic by Delp & Klopping (1968). Benomyl has been reported to be the most effective of the benzimidazole fungicides and has the widest spectrum of fungitoxic activity among the newer systemic fungicides (Erwin, 1973). Benomyl is hydrolysed to methylbenzimidazol-2-yl carbamate (MBC) and butyl isocyanate (BIC) in aqueous solution (Fig. 1). In a model system of methanol-water (50:50, v/v), the half-life of benomyl was 4.28 hr (Cahnon & Sayag, 1976). The MBC is hydrolysed in turn to 2aminobenzimidazole (2-AB) and BIC is converted to n-butylamine (Fig. 1). Baude, Gardiner & Han (1973) reported that the principal residues on treated crop samples were benomyl, MBC and 2-AB. The oral LD,c of benomyl in fasted rats was reported to be > log/kg body weight (Sherman, Culik &Jackson, 1975). When [14C]benomyl was administered to rats in a single oral dose, 99% was excreted in the urine. and faeces within 72hr (Gardiner, Kirkland, Klopping & Sherman, 1974). The principal metabolites were the glucuronide and sulphate conjugates of 5-hydroxy-2-benzimidazolecarbamate (Douch, 1973; Gardiner, Brantley & Sherman, 1968; Gardiner et al. 1974). Sherman et al. (1975) reported no pathological tissue changes, teratogenic effects or mutagenic effects when benomyl was fed

EXPERIMENTAL

Materials. Analytical-grade benomyl and MBC were obtained from E. I. du Pont de Nemours & Co., Wilmington, Del., reagent-grade BIC and 2-AB from Eastman Chemical Co., New York, proteose peptone and yeast extract from Difco Laboratories, Detroit, Mich., Soluene 100 from Packard Instrument Co., Chicago, Ill., and sodium [2-14C]acetate, hydrolysate, 14C-labelled reconstituted protein [Me-3H]thymidine and [5-3H]uridine from New England Nuclear, Boston, Mass. All other reagents used were reagent grade. Measurement of cell growth. T. pyriformis strain E was incubated at 23 + 1°C in the dark in stationary 1-litre Roux flasks containing 200ml autoclaved medium consisting of 2% (w/v) proteose peptone and

*Florida Agricultural Experiment Station Journal Series No. 180. tTo whom correspondence should be addressed. @Registered trade name of E. I. du Pont de Nemours & Co., Inc., Wilmington, Del. 187

P. W. RANKIN.

188

J. G. SURAK

C, H,-N-C=0

0 ‘: \ *rN-c4H9 Co

and N. P. THOMPSON

-C

Hz0

H NH 49 2

EIC

+ co,

n-butylomlne

0

, N-C:

OCHx

‘N,!,

Benomyl

co2 t

7

0 e

OH-LOCH,

t

CH,OH

2-A% Fig.

1. Hydrolysis

extract. For experimental purposes of early log growth cells was used. Benomyl, MBC, 2-AB and BIC were prepared as solutions of various concentrations in dirnethylsulphoxide (DMSO) and were added to 20-hr-old 200-m] cultures, so that the final concentration of DMSO in the medium was 1%. This level of DMSO did not affect cell growth (Surak, Bradley, Branen & Shrago, 1976). Fresh solutions of the pesticide or derivative in DMSO were prepared immediately before their 0.1”” (w/v) yeast a l”b inoculum

addition

to the

test

culture.

Growth

of

products

of benomyl.

determine the incorporation of C3H]thymidine into DNA, C3H]uridine into RNA and [“C]amino acids into protein. The protein or nucleoprotein precipitate was dissolved in Soluene 100 prior to the determination of radioactivity. The samples were counted in scintillation fluid (toluene-PPO-POPOP, 3.5 litres: 10.5 g:350 mg) using a Packard Tri-Carb Liquid Scintillation Spectrometer Model 3375. The data presented are the means of three experiments with three replications at each concentration of benomyl.

T. pyriformis

was measured by drying a washed aliquot of cells to constant weight or by counting cell numbers with a Neubauer haemocytometer. The data presented are the means of five growth trials with four determinations at each concentration of the test compound in each trial. Microscopic observations. DMSO solutions of benomyl. MBC, BIC and 2-AB were prepared and added to aliquots of early log growth cells to yield a final concentration of 20 ppm of test compound in the medium. After an initial incubation period of 1 min. aliquots of 0.1 ml were removed at various intervals for phase-interference microscopic observation through a Nikon SUR-KE research microscope equipped with a phase-interference unit. Radioactivity meusrrrements. Benomyl was added to 20-hr cultures, and incubated for 5 hr, after which radioisotopes were added for an additional 3 hr of incubation. The incorporation of sodium [2-14C]acetate into lipid, protein and 14C-labelled amino acids into protein, [5-3H]uridine into RNA and [Me-3H]thymidine into DNA were measured. Cells were then harvested by centrifugation and washed three times with cold de-ionized water. The procedure of Shug, Elson & Shrago (1969) was used to fractionate cellular components and to determine the quantity of label from CL4C]acetate incorporated into protein and lipid. The procedure of Milner (1967) was used to

RESULTS

Growth

and

morphology

The growth of T. pyrformis was inhibited by increasing concentrations of benomyl (Fig. 2). When benomyl was added to cultures in concentrations of 20 or 25 ppm, there was a decrease in cell count in the first 8-9 hr. At 12 hr, during the log growth phase. the benomyl dose effecting 50% growth inhibition (ED,,) was 9.10ppm in the medium (Table 1). In addition, compared with benomyl-free control cultures there was a decrease in cell numbers in the stationary growth phase, with an EDso at 36 hr of 8.04 ppm. The initial reduction in cell number was detected 45 min after addition of 20 ppm benomyl to an early log growth culture. Microscopic examination showed swelling of cells and nuclei with an increase in cytoplasmic vacuoles and loss of pyriform shape (Fig. 3a). T. pyriformis then became non-motile with part of the cell membrane separating and forming a bubble along the cell (Fig. 3a). Cells so affected were never observed to recover. T. pyriformis growth was inhibited also in the presence of BIC, the EDs0 of which was 10.96ppm at 12 hr (Table 1; Fig. 4). Addition of BIC to an early log growth culture resulted in an initial decrease in cell concentration, the decrease being proportional to

189

Fig. 3. Morphological changes in T. pyrijormis demonstrated by phase-interference microscopy following exposure to a test compound, added to the culture medium in DMSO solution, and subsequent immobilization with methylcellulose; (a) after 45-min exposure to 20 ppm benomyl ( x 200) and (b) after 40-min exposure to 20 ppm BIC ( x 400).

Benomyl and Tetrahymena

Time

after

benomyl

oddltion.

hr

Fig. 2. Growth curves of T. pyrgormis in media containing 0 (0). 5 (A), 10 (O), 15 (O), 20 (A) or 25 (B) ppm benomyl added to an early log growth culture. Each point is the mean of 20 values (four replicates from each of Eve trials). the BIC concentration. Recovery to the initial cell concentration occurred within 10 hr. When compared to the control group, BIC decreased the cell density in the stationary growth phase, with an EDso of 12.05 ppm at 36 hr (Table 1). Cellular changes similar to those induced by benomyl were observed microscopically with BIC (Fig. 3b). Within 40min of the addition of 20ppm BIC to an early log growth culture, the cells and nuclei swelled and there was membrane separation with bubble formation. As in the case of benomyl treatment, individual cells did not recover. MBC inhibited the growth of T. pyriformis during both the log and stationary growth phases (Fig. 5), the EDso values being 10.86 and 6.38 ppm, respectively. Cell populations in media containing 1Oppm MBC or more showed an initial decrease in cell density, which returned to the initial level in 7 hr. Within 25 min of the initial exposure of early log growth cultures to 20ppm MBC, microscopic observations Table

1. Effective products

pyrqormis

191

lo4L--0

IO

Time

20

ofter

30

BIG

addition,

40

50

hr

Fig. 4. Growth curves of T. pyrijormis in media containing 0 (O), 5 (A), 10 (Cl), 15 (O), 20 (A) or 25 (W) ppm BIC, added to an early log growth culture. Each point is the mean of four replicates from each-of five trials. revealed an apparent increase in the motility of most cells in the culture. The few cells that became nonmotile contained cytoplasmic granules. Growth of T. pyriformis was not inhibited in media containing 15-25 ppm 2-AB (Fig. 6), and no morphological changes were observed when 20 ppm 2-AB was added to early log growth cultures.

doses of benomyl and its hydrolysis on the growth of T. pyriformis

EDs0 (ppm) values at Chemical

12 hr

36 hr

Benomyl BIC MBC 2-AB

9.10 + 1.02 10.96 f 144 10.86 f 2.17 >25

8.04 + 0.89 1205 f 2.31 6.38 f 1.10 >25

BIC = Butyl isocyanate MBC = Methyl 2-benzimidazolecarbamate 2-AB = 2-Aminobenzimidazole *Added as a concentrated solution dissolved in DMSO to an early log growth culture of T. pyriformis. Means + SEM were calculated as the concentration in ppm in the media needed to cause 50% inhibition of the growth of T. pyriformis (from Figs. 2, 4, 6 and 7).

lO’0-I

50 Time

ofter

MBC addition,

hr

Fig. 5. Growth curves of T. pyriformis in media containing 0 (01, 5 (A), 10 (O), 15 (OX 20 (A) or 25 (m) porn MBC added to an early log growth culture. Each point is the mean of four replicates from each of Eve trials.

P. W. RANKIN,

J. G. SURAK

and N. P.

THOMPSON

1200

r

0 Benomyl Time

ofter

2-AB

addition,

hr

Fig. 6. Growth curves of T. pyriformis in media containing 0 (o), 5 (A), 10 (II), 15 (0) 20 (A) or 25 (M) ppm 2-AB added to an early log growth culture. Each point is the

mean of four replicatesfrom each of five trials.

Radioisotope

measurements

Benomyl added to an early log growth culture decreasedthe incorporation of [i4C]acetate into protein with 32% inhibition at 15 ppm (Fig. 7). The incorporation of [i4C]acetate into lipids increased.with 5 ppm benomyl but decreasedwith benomyl concentrations of 10 ppm or more (Fig. 7). A decrease in the incorporation of [‘Hluridine at all levels of benomyl studied (Fig. 7) was paralleled by the incorporation of [‘4C]amino acids into protein. However, 5 ppm benomyl stimulated DNA synthesis as well as lipid synthesis compared with the control level (Fig. 7). Addition of higher benomyl concentrations to the cultures decreased the incorporation of C3H]thymidine into DNA.

DISCUSSION

Sherman et al. (1975) did not observe any pathological changes, teratogenic effects or mutagenic effects when three generations of rats were fed benomyl at dietary levels of 0.25%. However, mammalian cells in culture have been reported to be affected by benomyl. Styles & Garner (1974) reported EDSo values of 6.8 and 5.0 ppm for benomyl and MBC, respectively, in cultures of Chang cells. The data reported here are consistent with this finding, in that . . similar ED,, values were found for each of these compounds in cultures of non-replicating T. pyriformis (Table 1). Benomyl and BIC have similar effects on the morphology of T. pyrifbrmis (Figs. 3a,b), but the swelling and membrane changeswere not observed with MBC. In addition, the primary toxic effect of benomyl appears to be an inhibition of protein synthesis and

concn.

ppm

Fig. 7. Effect of benomyl (added to ‘an early log growth culture of T. pw(formis and incubated for 5 hr prior to the addition of radioactive isotope) on the synthesis of macromolecules. Protein synthesis (0) and lipid synthesis (0) were determined from the amount of 14C incorporated when r”Clacetate (05 uCi) was added to the cultures and incubated for 3 hr. DNA synthesis (0) and RNA synthesis (W) were determined from the amount of ‘H incorporated when [‘Hlthymidine or C3H]uridine (TO/.KJi) was added to the cultures and incubated for 3 hr.

RNA synthesis (Fig. 7). These inhibitory effects were dose dependent; 15 ppm benomyl inhibited protein synthesis by SO%,and 1Oppm inhibited RNA synthesis by 50%. Synthesisof both lipids and DNA was increased by 5 ppm benomyl and inhibited by 15 ppm (Fig. 7). Hammerschlag & Sisler (1972) reported that the mode of action of benomyl differed from that of MBC. While 15 ppm benomyl inhibited DNA, RNA and protein synthesis, 10ppm MBC inhibited DNA synthesisafter exposure for 1 hr but only moderately inhibited RNA synthesis and protein synthesis after exposure for 4 hr. Later it was postulated that the difference could be attributed to the release of BIC (Hammerschlag & Sisler, 1973). However, these authors did not report the effect of 5 ppm benomyl on DNA synthesis in Saccharomyces pastorians (Hammerschlag & Sisler, 1972). Styles & Garner (1974) reported that benomyl and MBC can interfere with mitosis in Chang cells; DNA replication was unaffected by either compound, but microscopic observations showed that mother-daughter cells often failed to separate as a result of lagging anaphase bridges, producing an abnormal number of doublet cells. This phenomenon was not observed in T. pyriformis exposed to benomyl. Results of experiments reported here indicate that 2-AB is not toxic to T. pyriformis under the conditions studied (Fig. 6). Toxicity of 2-AB to mammalian cells in culture has not been reported, and 2-AB is not toxic to fungi (Edgington, Khew & Barron, 1971). These results form .a basis for establishing the action of benomyl on a model cellular system.

Benomyl and Tetrahymena REFERENCES

Baude, F. J., Gardiner, J. A. & Han, J. C.-Y. (1973). Characterization of residues on plants following spray applications of benomyl. J. agric. Fd Chem. 21, 1084. Calmon, J. P. & Sayag, D. R. (1976). Instability of methyl1-(butylcarbamoyl)-2-benzimidazol-carbamate (benomyl) in various solvents. J. agric. Fd Chem. 24, 426. Davidse, L. C. (1973). Antimitotic activity of methyl benzimidazol-2-yl carbamate CMBCI in Aspergillus nidu[ans. Pestic. Biochem. Physiol. 3, 317. Delp. C. J. & Klopping, H. L. (1968). Performance attributes of new fungicide and mite ovicide candidate. PI. Dis. Reptr 52, 95. Douch, P. G. C. (1973). The metabolism of benomyl fungicide in mammals. Xenobiotica 3, 367. Edgington, L. V., Khew, K. L. & Barron, G. L. (1971). Fungitoxic spectrum of benzimidazole compounds. Phytopathology 61, 42. Elliot, A. M. (1973). Biology of Tetrahymena. Dowden, Hutchingson & Ross, In&~ Stioudsburg, Pa. Erwin, P. C. (1973). Svstemic fungicides: disease control. translocatioh, anb mode of action. A. Rev. Phytopathol: 11, 389. Gardiner, J. A., Brantley, R. K. & Sherman, H. (1968). Isolation and identification of a metabolite of methyl-l(butylcarbamoyl)-2-benzimidazolecarbamate in rat urine. J. agric. Fd Chem. 16, 1050. Gardiner, J. A., Kirkland, J. J., Klopping, H. L. & Sherman, H. (1974). Fate of benomyl in animals. J. agric. Fd Chem. 22, 419.

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pyriformis

Hammerschlag, R. S. & Sisler, H. D. (1972). Differential action of benomyl and methyl-2-benzimidazolecarbamate (MBC) in Saccharomyces pastorianus. Pestic. Biothem. Physiol. 2, 123. Hammerschlaa. R. S. & Sisler. H. D. (1973). Benomvl and methyl-2-be&imidazole-‘carbamate (MBd): Biochemical, cytological and chemical aspects of tokicity to Ustilago maydis and Saccharomyces cerevisiae. Pestic. Biochem. Physiol.

3, 42.

’ Hill, D. J. (1972) The Biochemistry and Physiology of Terrahymena. Academic Press, New York. Milner, S. M. (1967) Effects of the food additive butylated hydroxytoluene on monolayer cultures of primate cells. Nature Lond. 216, 557. Sherman, H., Culik, R. & Jackson, R. A. (1975). Reproduction, teratogenic, and mutagenic studies with benomyl. Toxic. appl. Pharmac. 32, 305. Shua A. L., Elson, C. & Shrago, E. (1969). Effect of iron on growth, cytochromes, giycogen, and fatty acids of Tetrahvmena

ovriformis.

J. Nutr.

99, 379.

Sieler, J. P. (197$. Mutagenicity of beniimidazole and benzimidazole deratives. Part I. Forward and reverse mutations in Salmonella typhimurium caused by benzimadazole and some of its derivatives. Mutation Res. 15, 273. Styles, J. A. & Garner, R. (1974). Benzimidazolecarbamate methyl ester-Evaluation of its effect in uiuo and in vitro. Mutation Res. 26, 177. Surak, J. G., Bradley, R. L., Jr., granen, A. L. & Shrago, E. (1976). Effects of butylated hydroxyanisole on Tetrahymena pyriformis. Fd Cosmet. Toxicol. 14, 277.