A mutagen produced by Fusarium moniliforme

No. 4, pp. Printed in Great Britain . Tasicon, Vol . 21,

0041-0101/83 $3 .00+ .00 © 1983 Pergamon Press Ltd .

467-473, 1983.

A MUTAGEN PRODUCED BY FUSARIUM MONILIFORME* W. C . A.

GELDERBLOM,'

P. G.

K. J. VAN DER and H. S. C. SPIESS

THIEL,'

MERWE, 2

W. F. O.

MARASAS'

'National Research Institute for Nutritional Diseases, S .A. Medical Research Council, Tygerberg 7505, 'Department of Biochemistry, University of Stellenbosch, Stellenbosch 7600 and 'Department of Chemistry, University of Stellenbosch, Stellenbosch 7600, South Africa (Accepted for publication 16 November 1982) W . C . A . GELBERBLOM, P . G . TMEL, K . J . VAN DER MERWE, W . F. O . MARAsAs and H . S . C. SPIES . A mutagen produced by Fusarium monil(forme. 7bxicon 21, 467-473, 1983 . - A mutagenic compound produced by Fusarium moniliforme on maize was isolated by CHCI, -isoPrOH extraction, solvent partitioning and liquid chromatography on silica gel and Sephadex LH-20 . HPLC studies showed that different mutagenic and non-mutagenic forms can be derived from the mutagen (P,) and that prolonged exposure to longwave u .v . light and to high temperatures causes a total loss of its u .v. absorption and mutagenic characteristics . Spectral data presented for P, include u .v  i .r., mass spectra as well as ' H NMR and "C NMR . Mass spectral data indicated a molecular formula of C 2, H 2, NO, . INTRODUCTION

Fusarium moniliforme Sheldon (=

F. verticillioides (Sacc.) Nirenberg) occurs world-wide on a great variety of plant hosts and is one of the most prevalent fungi associated with maize (Zea mays L.) kernels in most maize-producing areas of the world (BOOTH, 1971 ; MARASAS et al., 1979a) . In a comparative study of the mycoflora of home-grown maize produced in Transkei, South Africa, a significantly higher incidence of F. moniliforme was found in maize produced in an area with a high human oesophageal cancer rate than in maize from an area with a low oesophageal cancer rate (MARASAS et al., 1979b, 1981). F. moniliforme is also one of the fungi most frequently associated with foodstuffs in regions of China having a high risk for oesophageal cancer (LI et al., 1979) . Several North American and European isolates of F. moniliforme produce mutagenic substances in culture (BJELDANES et al., 1978 ; BJELDANES and THOMSON, 1979). We also employed the Salmonella mutagenicity assay to show that strains of F. moniliforme isolated from Transkeian maize are able to produce mutagenic compounds. The Salmonella assay was subsequently used as a monitoring system for the isolation of the mutagenic compounds from cultures of F. moniliforme strain MRC 826. This isolate was highly toxic to ducklings, rats, horses, pigs and sheep (KRIEK et al ., 1981a, b) . The chemical nature of the mycotoxin(s) responsible for the lesions caused in experimental animals is unknown. The main mutagenic compound purified from this strain of F. moniliforme differed from fusariogenin C (BJELDANES and WEIB, 1980), but seems to be identical to fusarin C isolated from a culture of F. moniliforme strain M-1 (WIEBE and BJELDANES, 1981).

*Adapted from a thesis submitted by the senior author during March 1982 to the Department of Biochemistry, University of Stellenbosch, in fulfilment of the requirements for the M .Sc. (Agric .) degree . 467

468

W. C. A. GELDERBLOM

et al.

This paper describes the isolation procedures as well as the interconversion between different forms of the mutagen as monitored by HPLC procedures . Spectroscopical data of the main mutagenic compound are compared with those for fusarin C.

Fungus

MATERIALS AND METHODS

Fusadum moniliforme strain MRC 826 was isolated from maize produced in an area with a high human oesophageal cancer rate in Transkei, Southern Africa (KaiEK et al., 1981x) . Stock cultures were maintained by lyophilization and used to inoculate autoclaved maize. Cultures were incubated in the dark for two weeks at 25°C, followed by one week at 10°C and finally one week at 25°C . The culture maizewas lyophilized, ground to a fine meal and stored in air-tight containers at 4°C. Mutagenwity assay

The plate incorporation assay and spot test were carried out as described by AnEs et al., (1975). Liver homogenate fractions (S-9) prepared from phenobarbitone induced BD IX female rats (200-250 g) (M ARSHAr i and McLEAN, 1969), with a protein concentration of 41 .2 mg/ml (Loway et al., 1951), was incorporated in the S-9 mix at a ratio of 0.025 ml/ml.

Isolation procedures

The lyophilized culture (5 kg) was extracted by stirring 500 g batches vigorously at room temperature for 2 hr with 1 .5 litres of distilled water and 3 litres of CHCI, -iso-PrOH (1 :1) followed by filtering. The residue was reextracted twice with 1 .5 litres of CHC1, -iso-PrOH (1 :1). Filtrates obtained from 1 kg of material were combined, dried over anhydrous Na,SO, and the solvents evaporated in vacuo at 40°C. The dried extract was successively extracted with 3 x 100 ml of petroleum ether (60° -80°C), 3 x 100 ml of CHCI, and 3 x 100ml McOH - CHCI, (1 :19) . CHCI, soluble fractions (approximately 12 g) of each 1 kg of cultured maize were further fractioned on a silica gel column (5 x 75 cm : silica gel 60,0.063-0.200 mm) by successive elution with CHCI, and McOH-CHCI, (1 :19) . Fractions (50 ml) were collected during elution with McOH-CHCI, (1 :19) and the mutagenic activity of each fraction was determined with the spot test. Mutagenic fractions were analysed by TLC (silicagel 60 F-254) using McOH -CHCI, (1 :19) as developing solvent, and combined . Further purification was achieved on a medium pressure chromatographic system consisting of two silica gel (0 .032-0.062 nmi) columns: a small scrubber column (1 .27 x 33 cm) and a main separating column (2 .52 x 109 cm). The same elution procedures as described above were followed and fractions (10 ml) were analysed by TLCand the spot test for mutagenicity . Final purification was achieved on a Sephadex LH-20 column (2 .8 x 80 cm) using CH 2 C1 2 as solvent. Fractions (5 ml) were monitored by the spot test and TLC, while mutagenic fractions were analysed by HPLC . The pure mutagen was stored in the dark at 4°C. Stability

Solutions of P, (0 .1 mg/ml CH 2 CI 2) were exposed for different time intervals to longwave u.v . light and titrated (in DMSO) in the Salmonella test. Longwave u.v. light-induced conversions of P, (0.04 mg/ml CH 3 L-12) were monitored by HPLC on silica gel after exposure for similar time intervals . The heat lability of P, was studied by exposing solutions of P, (0 .1 mg/ml H2O) to different temperatures, whereafter their mutagenic activities were determined. Each solution (50 pl) was also analysed by reverse phase HPLC . HPLC analyses were performed on a Micromeritics High Performance Liquid Chromatograph (model 700) equipped with a Micromeritics Variable Wavelength Detector (model 785) . Mutagenicity ofdifferentforms

Similar concentrations (30 Ng/ml DMSO) of the u.v . induced (P 1, P;, P,') and spontaneously derived forms (P  P,, P,) were prepared and titrated in the Salmonella test . Solutions of each sample (0 .1 mg/ml CH .Cl2) were incubated together with the test plates and analysed (20 pl) by HPLC on silica gel after the incubation period . Spectroscopkcal andyses

Ultraviolet spectra were recorded on a double beam Beckman Spectrophotometer (Model UV 5260) while the i.r . spectra were obtained on a double beam Beckman Acc:ulad 8 Spectrometer . 'H NMR (80 MHz) and "C NMR (20 MHz) spectra were obtained with a Varian FT-80 Fourier Transform NMR Spectrometer in CDCI, . External referencing using a second tube containing a solution of about 2% TMS-CDCI, was used to calculate the 6-values. Mass spectra were recorded on a Varian Mass Spectrometer MAT 311A fitted with a Varian data system 620/L-100.

F=rlum monll(forme Mutagen

469

RESULTS AND DISCUSSION

Preliminary studies on the mutagenicity of the crude CHCI, -iso-PrOH extracts using Salmonella strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 showed a mutagenic response only with strain TA 100 in the presence of S-9 (C3elderblom, W. C. A., M.Sc. (Agric .) thesis, University of Stellenbosch, South Africa). During the solvent partitioning step of the crude CHCl, -iso-PrOH extract the bulk of the mutagenic activity was recovered in the CHC1 3 soluble fraction, while inactive oily substances were removed by extraction with petroleum ether. Purification of the main mutagenic compound P, was achieved by two successive separations on silica gel and a final purification on Sephadex LH-20. TLC analyses revealed a characteristic dark spot (Rf = 0.17) under longwave u .v . light in those fractions having mutagenic activity. The purified mutagen P, eluted as a single sharp peak during HPLC on a silica column (Fig. 1). Three different forms (Pi, P,' and P,') could be resolved by HPLC when P, was exposed to longwave u.v. light (Fig. 1) . Similar HPLC chromatograms were obtained when the individual forms (P 1', P2' and P,') were re-exposed to u.v. light. After prolonged exposure all these forms, including P,, lost their u.v . absorption properties and could no longer be detected . The u.v .-induced transformation of P, coincides with a total loss in mutagenicity (Table 1). The individual forms (P 1', P z and P,') exhibited mutagenic activities almost similar to that of P, (Table 2). It therefore seems as if P, is converted to non-mutagenic forms, either directly or through the initial formation of the mutagenic intermediates P .', P2' and P,' .

î 0 .5

5 tae

30 sae

120 sec

300 sec

ie

a 0 .25

0

se t .e

5

10

5

10

5

10

Rataatiaa tim (min) FIG . 1 . EFFECT OF LONOWAVE U .V . LIGHT IRIIADIATION OF As MONrroRED By HPLC .

P, (40 ieg/ml CH,CI,)

Analyses were performed on a Microsil (7 nm, 5 nun i.d . x 300 mm) column using McOH-CH,Cl, (1 :19) as eluting solvent (1 .5 ml per min) . 50 ;Al of the sample was injected after each exposure.

470

W. C. A. GELDERBLOM et al. TABLE 1 . THE EFFECT OF LONGWAVE U .V. LIGHT ON THE MUTAGENICITY OF THE MUTADEN P 3 (0 .1 mg/ml CH, CI=)

Revertants Exposure time per 12g P3 * (sec) 530 0 5 460 10 360 20 290 30 271 60 155 55 120 5 300 1 600 were titrated against tester strain Irradiated samples TA 100 in the presence of 0.5 mg S-9 protein per plate. *Values represent the mean of triplicate determinations corrected for background reversion frequency.

Pl 14*)

à

Pl (37°)

P4 (37)

P4 (r)

A

PI

M 0.2

5

P

10

5

1a

Rsts"@

5

1a

5

10

tim (nain)

21 /art

6

10

5

10

Rmutim ùl" (uda)

5

10

FIG . 2. TEMPERATURE-DEPIEIDENT CONVERSION OF P, AND P 4 (0 .1 mg/ml CH 2C12). A: HPLC chromatograms of P, and P4 before and after an incubation period of 48 hr at 37°C . B: HPLC chromatogram of P, (kept at room temperature) periodically analysed over a period of 21 days . Analyses were performed on a Microsil (7 nm, 5 mm i.d . x 300 mm) column using Me0H-CH2 Cl 2 (1 :19) as eluting solvent (1 .5 ml per min). 20 Nl was injected in all cases.

Fusarium monUiforme Mutagen

47 1

Compounds with similar u .v. absorption properties (P,, P=, P, and Pa) were observed during the isolation procedure. The HPLC retention times of these compounds differ from those of the u.v.-induced forms (Table 2). Both P, and P, could be converted back to P, . When kept at 37°C for 48 hr the conversion of P, to P, was complete, while a substantial part of P, was converted to P, (Fig. 2A). At room temperature P, remained stable, while P, was slowly converted not ony to P, but also to P, (Fig . 2B) . No conversion of P, or P, to P, was noticed at either room temperature or at 37°C. The spontaneously derived forms P, and P, were also mutagenic, but to a lesser extent than P, (Table 2). These mutagenic responses might have resulted from the formation of P, from P, and P, during performance of the mutagenicity test. It is therefore possible that both P, and P, are in fact non-mutagenic, similar to the other spontaneously derived forms P, and P, (Table 2) which cannot be converted to P, . The disappearance of the compound P, through exposure to higher temperatures (above 60°C) coincides perfectly with a loss in mutagenicity (Fig. 3). The mutagenic compound P, can therefore be converted to both mutagenic and nonmutagenic forms by u.v . irradiation and spontaneous conversion, respectively . Both types of conversion are likely to involve structural changes in the molecule, that in the case of spontaneous conversion alters the part on the molecule responsible for its mutagenic activity . Mass spectral data of compound P, [EIMS (probe) 70 eV, m/z (rel int) : 431 (M I ), 314 (13), 281 (32), 241 (40), 213 (70), 185 (91) and 133 (95)] showed a molecular ion with an accurate mass of 431 .1970. No combustion analysis was performed but a molecular formula of CH NO, reported for fusarin C (WIEBE and BJELDANES, 1981) (required 431 .1993, found 431 .1953) seems to fit in well. A IH NMR spectrum of P, indicated signals for at least 26 hydrogen atoms. IH NMR (80 MHz, CDCI,): 6 7.54 (1H, d, J = 9 .80 Hz), 6.97 (lH, q, J = 7 .18 Hz), 6 .74 (1H, s), 6.65 (1H, d, J = 9.80 Hz), 6.28 (IH, s), 6.05 (1H, s), 4.02 (1H, s), 3 .94-3 .83 (2H, m), 3.73 (3H, s), 2 .38-2.16 (2H, m), 2.00 (3H, s), 1 .95 (3H, s), 1 .76 (3H, d, J= 7.18 Hz) and 1 .71 (3H, s). I'C NMR analysis of P,

TABLE 2 . MurAaamcrrir AND HPLC RETENTION TAMES OF THE DIFFERENT FORMS OF THE MUTAGEN P, Compound P, P, P, P, P, P, '

Spontaneous derived forms + + ? + + ?

U .V. lightinduced forms

HPLC retention time (min) 7 .50

Revenants per mg P, 570

5 .90 7.10 8 .80 10 .05

340 25 240 20

+ 5 .43 5.50 + P,' 6.60 520 + 8 .45 P,' 555 form (5 pg/plate) was assayed against strain TA 100 in the presence of 0 .5 mg Each S-9 protein per plate . * Values represent the mean of triplicate determinations . HPLC analyses of solutions (50 Mg/ml C11,0,) of the different forms were performed on a Microsil (7 run, 5 mm i .d . x 300 mm) column using McOH-CH,CI, (1 :19) as eluting solvent (1 .5 ml per min).

472

W . C . A . GELDERBLOM et al.

20 6

6

16

4

10

3

2

1

26

46

6o

6o

Z!I

106

T @sq vamn' FIG . 3 . MUTAGENICITY AND

HPLC

PEAK HEIGHT OF P, (0 .1 INCREASING TEMPERATURE.

mg/ml H?O)

AS A FUNCTION OF

Each sample was heated for 30 min at each temperature. Revertant counts represent means of triplicate determinations and spontaneous revertant counts are subtracted . HPLC analyses were performed on a Radial-Pak A cartridge using a Radial Compression Module (model RCM-100) and Me0H-H,O (55 :45) as mobile phase (3 ml per min) .

showed signals for 23 carbon atoms. A partially decoupled spectrum indicated 26 H - C bonds in the molecule . '3C NMR (20 MHz, CDC13): 6 190.29 (s), 170.36 (s), 167.56 (s), 149.21 (d), 146.18 (d), 140.8 (d), 140 .06 (d), 137.35 (s), 134.77 (s), 133 .28 (s), 130.27 (s), 126.04 (d), 123.3 (d), 85 .33 (s), 63 .73 (d), 61.93 (s), 57.85 (t), 51.8 (q), 36 .07 (t), 18.64 (q), 15.86 (q), 13 .96 (q) and 11 .31 (q). The NMR data of the mutagenic compound P3 correlates well with those published for fusarin C (WIEBE and BJELDANES, 1981). Of the 21 signals reported, one signal at 6 58 .00 in the ' 3C NMR spectrum of fusarin C was given as a singlet while for P3 a triplet was obtained at 6 57 .85. This could explain why only 14 H - C bonds were indicated in the partially decoupled "C NMR spectrum of fusarin C. The 6-values of only 12 of the 24 observed protons in the 'H NMR spectrum of fusarin C were reported. One of these signals at 6 6.78 (2H) was indicated as a multiplet, while for P3 a singlet at 6 6.74 (1H) and a doublet at 6 6.65 (1H, J = 9.80 Hz) were observed . The u.v. absorption (x EtmOH nm: 353) and i.r. spectrum (V CHE0, cm-1 : 3400, 2930, 1750, 1630, 1590, 1390, 1330, 1220, 1175, 1140, 1070, 980, 860) of P s differ only slightly from that reported for fusarin C. Further studies on the chemical structure of compound P 3 are in progress . The production of mutagenic compounds (potential carcinogens) by F. moniliforme on maize may affect human health in areas where maize is the staple diet . Although the motivation behind this study was the high incidence of F. moniliforme in an area with a

Fusarium monlliforme Mutagen

47 3

high oesophageal cancer rate, the role of the isolated mutagen is unknown at present. It's carcinogenic potential and natural occurrence must first be investigated before any conclusions can be drawn. REFERENCES AMES, B. N., MCCANN, J. and YAMAsAKI, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test . Mutat. Res. 31, 347. BJELDANEs, L . F. and THOMSON, S. V. (1979) Mutagenic activity of Fusarium monfl(forme isolates in the Salmonella typhimurium assay. Appl. environ. Mkrobiol. 37, 1118 . BJELDANES, L . F. and WEIB, L . A. (1980) Mutagenic mycotoxins from Ficsarlum monil(forme. Environ. Mutagenesis2, 240. BJELDANEs, L . F ., CHANG, G. W. and THOMSON, S. V. (1978) Detection of mutagens produced by fungi with the Salmonella typhimurium assay. Appl. environ. Microbiol. 35, 1150. BOOTH, C. (1971) The Genus Fusarium, p. 11 . Commonwealth Mycological Institute, Kew, Surrey, U.K. KRIEIc, N. P. J., KELLERMAN, T. S. and MARAms, W. F. O. (1981a) A comparative study of the toxicity of Fusarium veHicillioldes (= F. monil(forme) to horses, primates, pigs, sheep and rats . Onderstepoort J. vet. Res. 48,129. KRIEK, N. P. J., MARAsAs, W. F. O. and THIEL, P. G. (1981b) Hepato- and eardiotoxieity of Fusarium vertkillioides (F. monlltforme) isolates from Southern African maize. Fd Cosmet. Toxic. 19, 447. LI, M., Lu, S., JI, C ., WANG, M., CHENG, S. and JIN, C. (1979) Formation of carcinogenic N-nitroso compounds in corn-bread inoculated with fungi. Scientla sin. 22, 247. LowRY, O. H., ROSEBR000H, N. J., FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem . 193, 265. MARAsAs, W. F. O., KRIEK, N. P. J., WIGGINs, V. M., SrEYN, P. S., TOWERS, D. K. and HASTIE, T. J. (1979a) Incidence, geographic distribution, and toxigenicity of Fusarium species in South African corn. Phytopathology 69, 1181 . MARASAS, W. F. O., VAN RENSBURG, S. J. and MmocHA, C. J. (19796) Incidence of Fusarium species and the mycotoxins, deoxynivalenol and zearalenone, in corn produced in esophageal cancer areas in Transkei . J agric. Fd Chem . 27, 1108 . MARAsAs, W. F. O., WEHNER, F. C., VAN RENSBURG, S. J. and VAN SCHALKWYK, D. J. (1981) Mycoflora of corn produced in human esophageal cancer areas in Transkei, Southern Africa . Phytopathology 71, 792. MARSHALL, W. J. and MCLEAN, A. E. M. (1969) The effect of oral phenobarbitone on hepatic microsomal P-450and demethylation activity in rats fed normal and low protein diets. Biochem. Pharmac. 18, 153. Wn3BE, L . A. and BJELDANEs, L. F. (1981) Fusarin C, a mutagen from Fusarium moniliforme grown on corn . J. Fd Sci. 46,1424.