Chem. Res. Toxicol. 1993,6, 91-96
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Isolation, Structural Identification, and Characterization of a Mutagen from Fusarium modiforme Fu-Xiong Lut and Alan M. Jeffrey'PtJ Diuision of Enuironmental Sciences, Department of Pharmacology, and comprehensive Cancer Center, Columbia University, New York, New York 10032 Receiued September 8, 1992 Strains of Fusarium moniliforme produce a variety of toxins, including several uncharacterized mutagens that act directly in the Ames assay using Salmonella typhimurium, strain TA 100. The Ames assay was used to monitor isolation of the direct-acting mutagens from the F. moniliforme culture extracts. Seven strains were tested, of which strains F07 and F84 contained the highest levels of direct-acting mutagens. Extracts of strain F84 were fractionated on a silica gel column, eluted with methanol-chloroform (1:9). This fraction was then separated on a reverse-phase, C-18 column with 50% methanol in water as eluant and further purified by TLC. One compound was isolated and given the trivial name fusarin X (FX). Its structure was determined from its UV (Ama 357 nm), 500-MHz NMR, and mass spectra, and those of its diacetate, to be the 1-hydroxy analog of the previously characterized fusarin C. FX was present in culture extracts of strains F07 and F84 a t 83 and 8 pg/g, respectively, which was proportional to their relative mutagenicities. It was not detected in the other strains tested. Since exposure of FX most likely occurs in cooked corn, its thermal stability was measured; it,like FC, decomposes at 100 "C, especially a t high pH. Again, in common with FC, it was decomposed by GSH. The possible role of these Fusarium metabolites in the etiology of human cancers has still to be resolved.
Introduction Epidemiological investigationsprovide strong evidence that environmental agents, many of which are likely to be found in foods, are a major factor in human cancer induction (1,2). Fusarium moniliformeis one of the most commonly found fungi on corn. Previous studies have shown that papillomas and squamous cell carcinomas can be induced in rata fed cornbread cultures of F. moniliforme ( 3 ) and, more recently (4),that esophageal tumors in rats and mice can be so induced. Based upon the distribution of F. moniliforme and the consumption of corn contaminated with this fungus, a possible association with human esophageal cancer has been suggested, especially in the areas of Linxian County, China, and Southern Africa (57). During the past decade several compounds produced by this fungus have been isolated, some of which are toxic and mutagenic. One of these, fusarin C (FC;l Figure 1,R = H), whose chemical structure has been elucidated (491, was isolated from corn cultures of this fungus and was also found to occur naturally on corn in China, Southern Africa, and North America ($12). Several studies have demonstrated that FC was toxic, mutagenic, and capable of inducing sister chromatid exchanges, micronuclei, chromosomal aberrations, and 6-thioguanine-resistantmutants in V79 cells (5,6,9,11,13). We showed that FC increased the number of DNA breaks in bacteria in the presence of a metabolic activation system, induced asynchronous replication of the polyoma DNA sequence in H3 cells (a phenomenon also seen when these cells were exposed to
* To whom correspondence should be addressed. Tel(212) 305-6925; Fax (212) 305-5328. + Division of Environmental Sciences and Comprehensive Cancer Center. 8 Department of Pharmacology. Abbreviations: FC, fusarin C; FX, fusarin X; FME, Fusarium moniliforme culture extracts; PB, phenobarbital.
20 21 24
'4
23
.
C0PCH3
Fusarin X R-OH
OH
Figure 1. Structures of fusarin C and fusarin X. FC: R = H. FX: R = OH.
a variety of other carcinogens), and alkylated @-nitrobenzyllpyridine without an added metabolic activation system (14).
Although there is evidence of potentially genotoxic effect(@in exposed individuals, other studies have not shown FC to be carcinogenic (7, 15). To obtain the maximum mutagenic response, metabolic activation of FC is required (6,13,14),despite the fact it already contains an epoxide ring. Such activation occurs most efficiently with phenobarbital (PB)-induced microsomal enzymes (13). In the carcinogenesis studies, FC was given before PB administration, such that the PB would function as a liver promoter rather than an enzyme inducer which could have changed FC metabolism. Hence, it is unclear whether the FC was appropriately activated to genotoxic metabolites. In addition, in the areas where esophageal cancer is high, there is, for the population involved, a dietary deficiency of zinc, copper and molybdenum, as well as low intakes of vitamins A, Bz, and C and polyunsaturated fatty acids (16, 17). Thus the animal carcinogenicity studies cannot be used as definitive evidence to exclude risk to humans since,besides the above caveats, there may be species differences in metabolism,
0893-228~/93/2706-0091$04.00/0 0 1993 American Chemical Society
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92 Chem. Res. Toxicol., Vol. 6, No. I , 1993
response to FC, or the requirement for specific tumor promoters. Another study, using albeit impure FC, showed esophageal and forestomach tumors are formed in rata and mice gavaged with FC (4). Fusarium moniliforme extracts contain, besides FC, several direct-acting mutagens which in the past have been lost as purification of FC proceeded (14,18). The directacting mutagenicity of F. moniliforme extracts decreases with respect to concentration of FC during purification. Without preincubation, HPLC- and TLC-purified FC showed no mutagenic activity without S9 (14). These results indicate that, besides FC, other direct-acting mutagens are present in the column and in TLC-purified samples of FC. The major component has now been isolated from F. moniliforme strain 84 and identified. Its structure is very similar to that of FC and is present in culture extracts of a t least one other strain of F. moniliforme.
Materials and Methods Caution: The extracts from F. moniliforme are mutagenic and carcinogenic in rodents. They should be handled accordingly. Since FC ie light sensitive, all experiments were performed under gold lights or in the dark.
Fungi and Their Culture. F. moniliforme strains F14, F84, F1023, and F1131(18) were generously provided by Dr. K. Grose, Department of Nutrition Science, University of California, Berkeley. Strains F06, F07, and F19 were isolated from diseased corn in Linxian, China (3,6).Corn cultures of six strains of F. moniliforme were prepared aa previously described (6)and incubated a t 25 "C for 4 weeks. Mutagen Assay. Mutagenicity was assayed by the Ames test using Salmonella typhimurium, strain TA 100, as previously described (14,19),without activation by S9 mix or preincubation. Each sample was run in triplicate, and means and standard deviations are reported. Initial Extraction and Purification. Culture material from strain F84 (25 g) was extracted twice with 50 mL of methanol by blending and filtering. The methanol extract was evaporated to dryness (1g) and separated on a silica gel (60-100 mesh) 23 cm X 2.5 cm column, with a sample-to-gel ratio of 1:2 by weight. The column was successively eluted with 300 mL of methanolchloroform (1:9) (fraction F84-F1,100mg), methanol-chloroform (1:l)(200 mL, fraction F84-F2, 500 mg), and finally methanol (200 mL, fraction F84-F3, 300 mg). The active fraction ( F a Fl), was further purified using a Zorbax ODS column (25 cm X 1cm, Du Pont Co., Wilmington,DE), elutingwith 75% methanol in water and maintaining the flow rate at 2.5 mL/min. Fractions were collected every 1min for 20 min. The active fraction (F84F1-6, 5-6 min), eluted from the above HPLC column, was separated using the same column with a gradient from 0% methanol to 100% methanol in water over 20 min. Fractions were collected in the same way. The fraction from 19 to 20 min (Fa-F1-6-20) was further purified by silica gel TLC [methanolchloroform (1:9)], and a yellow spot, designated fusarin X (FX) (Rf0.42), was eluted with methanol. Bulk Purification of Fusarin X. After the initial fractionation, three more culture extracts were prepared and combined. These were separated on a silica gel column as described above. After it had been established that the major direct-acting mutagen haa a chromophore similar to FC, this was used to monitor purification, and the F84-Fl fraction was applied directlytoa reverse-phasePrepSep C-18column (14-mL capacity, Fisher Scientific, Springfield, NJ) with 50% methanol in water as the eluant. This fraction was then separated by reverse-phase HPLC and TLC as described above. Acetylation and Hydrolysis of Fusarin X. The TLCpurified FX showed no impurities upon reanalysis by HPLC
[pBondapak (2-18 column, Phenomenex Inc., Torrence, CA; 4 6 75% methanol in 10 mM potassium phosphate buffer (pH 7.4), over 40 min, and measuring absorption from 220 to 370 nm]. However, the 500-MHz NMRspectrum showed other componentr (8) were present. To remove these, the sample was acetylated (200pL of pyridine, 30 pL of acetic anhydride, room temperature, 30 min) and repurified using the same HPLC system (retention times: FX 14 min; FX-diacetate 28 min). The FX-diacetate was converted back to FX catalyzed by porcine liver esterase (10 pL containing 1unit coupled to acrylic beads, Sigma, St. Louie, MO), in 30 pL of 0.1 M Tris-HC1buffer (pH 5.0), with 5 pL of methanol a t 37 OC for 60 min. Mass spectra were obtained in both chemical ionization (ammonia) and electron ionization modes using a Nermag 10-10 instrument. NMR spectra were obtained a t 500 MHz (Bruker Instruments) in 100% CD2C12. D20 was added to some samples to remove exchangeable protons. Ultraviolet spectrophotometric measurements were performed a t room temperature in a Perkin Elmer Lambda 3B UV/ vis spectrophotometer except where indicated. Stability of FX to temperature,pH, and GSH was followed spectrophotometrically by complete scans from 250 to 450 nm to determine the types of products formed and a t 360 nm to determine the half-life of FX. Temperatures and buffer conditions are given with the results. FX (1pg in 20 pL of methanol) was added to 180 pL of 0.1 M potassium phosphate buffer a t pH values ranging from 6 to 10. The samples were sealed in Eppendorf tubes and heated either in a boiling water bath before cooling rapidly on ice or at the temperatures indicated in the Results. Samples were centrifuged briefly to return any fluid lost to the cap during heating. Residual FX was separated by HPLC and its concentration measured from its peak heights nt 360 nm. In reaction with GSH, 4 pg of FX or FC in 400 pL of 0.1 M phosphate buffer (pH 8.0) was incubated at 36 OC. GSH was then added to give a final concentration of 5 mM, and spectra were recorded from 250 to 450 nm a t 0, 2, 7, 20, and 30 min. Values a t 360 nm were used to calculate half-lives. Molecular Modeling. Initial coordinates for the 82 isomer of FC were taken from ref 8. For the modeling we used Discover, version 2.7.0 (Biosyn Technologies, San Diego, CA), programs. Conjugate convergence was used for energy minimization.
Results Isolation of Direct-ActingMutagens. The levels of direct-acting mutagenicity in culture material for different F. moniliforme strains in S. typhimurium, TA 100, are shown in Table I. The methanol extracts of strain F07 showed the highest mutagenicity among the seven strains tested. Unfortunately, this strain was lost and no subcultures are available. We therefore chose the next most mutagenic strain, F84, for the isolation of the direct-acting mutagens. During the first step of the purification, the majority of direct-acting mutagens were eluted from the silica gel column with methanol-chloroform (1:Q). This fraction comprised only 10% of the mass of the crude extract, but accountad for about two-thirds of the mutagenicity applied to the column (Table 11). The first HPLC analysis of fraction 84-F1 showed that fractions 6 and 7 contained about 82% of the mutagenicity (Table 111). The direct-acting mutagenic principle(s) eluted in fraction 20 (from 19 to 20 min) on second HPLC fractionation (Table IV). Further purification by TLC [silica gel, methanol-chloroform (1:9)1 indicated that a compound, designated FX ( R f 0.42), comprised about the half of the mutagenicity applied to the plate. This compound is yellow with maximal absorption at 357 nm. From 40 g of F84 culture extract was isolated 200 fig of FX
Chem. Res. Toricol., Vol. 6, No. 1, 1993 93
A Mutagen from F. moniliforme
Table I. Mutagenicity in S.typhimurium TA 100 and Levels of FX and FC in F. moniliforme Methanol Extracts ~~
~~
revertants/plateb extracts' FS4 F14 F1131 F1023 F06 F19 F07
location central California central California Greece N. Italy N. China N. China N. China
1st culture 416 f 67 299 f 51 257 f 15 171 f 15 287 f 3 142 f 18 1870 f 178 (0.6 me)
FX (0.9 pg/plate) MezSO (20 pL) styrene oxide
5th culture 211 f 15 107 f 6 129 f 29 144 f 14 141 f 12 155 f 8 634 f 23O (0.2 mg) 479 f 210 109 f 16 363 & 32 (1.5 pmol)
126 f 27 513 f 135 (5 pmol)
FX (ppm)' 8 NDd ND ND ND ND 83
FC (ppmIC 830
ND ND 33
ND
60 667
0 10 mg of methanol extract per plate, except where indicated. Sample run in triplicate. Means and standard deviations are reported. c rg/g of methanol extract in the 5th culture. none detected: detection limit 5 ppm. e Extracts from a single culture. Since this extract was more active, the smaller amounts indicated were used.
Table 11. Mutagenicity of Silica Gel Column Fractions of F84 Culture Extract ~~
fraction FS4 F84-Fl FS4-F2 FS4-F3
g/fractions 1
mdplate 20
0.1 0.5 0.3
10 6
revertants/platea (mean f SD) 224 f 16 216 f 13 149 f 10 193 & 20
2
5% survival 39 51 82 91
total Me2SO (10 pL) styrene oxide
117 f 20 425 f 50
1.66 pmol
% recover9 100 67 14 21 102
100 37
Sample run in triplicate. Means and standard deviations are repomd. Corrected for survival.
_
Table 111. Mutagenicity of HPLC Fractions of F84-F1 Fraction from Silica Gel Column _ _ ~ ~
fractions0 5c 6 7 8
revertants/plateb net mutants 190 f 19 13 1486 f 9 1309 585 f 49 408 177 f 43 0
~~
Table IV. Mutagenicity of Second HPLC Fractions ~
recovery (% ) 0.6 62.3 19.4 0
sd 10 11 12 13
143 f 25 137 f 8 319 f 15 336 f 16
0 0 142 159
0 0 6.8 7.6
167 f 5
0 2031 2100
0 96.7 100
14d
15 total F84-Fl fraction (1mg) MezSO (10 pL)
261 & 21 177 f 2
a The fractions were collected for 1min up to the time indicated. The sample was derived from 25 mg of FM-Fl. Sample run in triplicate. Means and standard deviations are reported. e Void volume of column. Not assayed in this fractionation but negative in other runs.
corresponding to 62% yield and about 37% of the mutagenicity present in the crude extracts, based upon the direct estimates of FX concentrations (Table I). Structure of Fusarin X. The color of FX suggested a structural similarity to FC which was confirmed by its NMR spectrum (Table V). Many of the protons showed very similar chemical shifts and coupling constants. The most significant change was in the H-1 protons which moved downfield from 1.77 to 4.18 ppm. In addition, the coupling pattern to H-2 changed from a triplet to a doublet. This was consistent with hydroxylation of the C-1 methyl group and was confirmed by acetylation which showed a characteristic further downfield shift of 0.43 ppm of the proton on C-1and the presence of two acetate groups rather than the one found in FC after acetylation with acetic anhydride in pyridine (20).The chemical ionization mass spectrum (NH3)of the acetatylated product was consistent
revertantslplateb net mutants recovery (%) 149 f 7 5 0.4 132 f 9 0 0 147 f 6 3 0.2 144 f 19 0 0 163 f 9 19 1.4 1470 f 31 1326 97.6 21 239 & 14 95 7 121 f 1 22 0 0 total 1448 106 fraction F84-F1-6/7 688 f 16 1360 100 144 f 16 MezSO (10 pL) fractions'
15 16 17 18 19 20
a The fractions were collected for 1 min up to the time indicated. The sample was from the firat HPLC separation (F84-F1-6/7) and was equivalent to 25 mg of F84-Fl. Sample run in triplicate. Meam and standard deviations are reported.
with the presence of two acetate groups and the proposed structure. It showed a weak molecular ion a t M + 1(532, 6 % ) and an ion corresponding to reaction with the ammonia (549,75 % 1. Both showed loss of acetic acid (472, 23 5% ,and 489,100 % ,respectively). We were unsuccessful in obtaining an electron ionization spectrum of this compound. Mutagenicity of Fusarin X. When the concentrations of FX were determined from the absorption of FX a t 357 nm and assuming the same molar extinction coefficient as FC (8),1nmol of TLC-purified FX doubled the number of mutants in TA 100 without metabolic activation. In addition, the purer FX derived from FX-diacetate showed mutagenic activity equivalent to the TLC-pure FX (413 f 22 and 436 f 24 mutant42 nmol/plate, respectively). FX-diacetate was not mutagenic (147 f 12 mutant42 nmol/plates), under these assay conditions when compared to solvent controls (119 f 12 mutantdplate). Level of FX and FC in Fusarium moniliforme Culture Extracts (FME). After FX was identified from extracts of F84, the levels of FX as well as FC in FME of
94
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Chem. Res. Toxicol., Vol. 6, No. 1, 1993
proton 1
Table V. NMR (SO0 MHz) Spectra in CDEl; of FX and FX-diacetate FC FX FX-diacetate 6 (ppm) J (Hz) 6 (ppm) J (Hz) 6 (ppm) J (Hz) 1.77 7.2 4.18 br 4.61 6.2
(+DzO)
2 4 6
8 9 10 14 18 19 21 22 23 24
6.96 6.07 6.30 6.79 6.67 7.49 4.06 2.06 2.11 4.05 3.9 3.74 1.73 2.09 1.98
7.2 15 15/11 11 2.1 151614 151814 11/8/4 111614
6.87 6.07 6.24 6.74 6.66 7.43 4.02
6.2 6.1
6.76 6.08 6.30 6.77 6.68 7.45 4.00
(+DzO)
15 11/15 11 2 0
2.15
ABX pattern
3.95
6.2 14 11/15 11
*
2.00 2.24 4.33
br, sharpens with D20
3.72 1.69 2.06 1.96
br br
3.72 1.73 2.06 1.96
FC-acetate CH3CO 19 14 18 0
Values for FC from ref 8 and FC-acetate from ref 20. br indicates a broad signal.
new cultures of the six strains, together with the original extracts of strain F07, were measured by HPLC using a Zorbax ODS column with 60% methanol in 10 mM potassium phosphate buffer (pH 7.4) (Table I). FX was detected only in extracts of strains F07 (83 pglg of FME) and F84 (8 pg/g of FME). Much higher levels of FC were detected in extracts of strains F07 (667 pglg of FME) and F84 (830 pg/g of FME) than in other strains. Stability of FX. The effect of pH on the stability of FX was measured at 100 OC from 6.0 to 8.0. Above pH 7.4, no FX was detected after heating. Samples a t pH values of 7.2, 7.0, 6.8, 6.6, and 6.0 had 13, 22, 44, 82, and 84% of the initial FX remaining after heating. From experiments in which samples were held at pH 6.0 for times up to 90 min a half-life of 41 min was calculated. At 25 OC FX was more stable at higher pH values: half-lives of 78 and 13 min were calculated for pH 9.0 and 10.0, respectively. At pH 10 and 36 OC, FX was slightly less stable than FC, with half-lives of 6.3 and 8.3 min, respectively. The concentration of solvent in which the fusarin derivatives were dissolved influenced the halflives: FC at pH 10 and 36 "C were 7.4 and 11.6 min for 0.3 and 10% methanol, respectively. Both FX and FC decomposed in the presence of GSH with haH-lives, under the conditions used, of 2.0 and 7.5 min, respectively. Without GSH both FC and FX were quite stable with half-lives >40 min. The product formed from FX was less stable and the new absorption at about 320 nm was less sharp and stable than that formed from FC (Figure 2). HPLC analysis of these samples showed FX and FC eluted at 14.6 and 27.4 min, while their decomposition products eluted at 2.8 and 3.4 min, respectively. The decomposition products formed from FX in the presence of GSH or high pH values appear identical: both eluted at 6.8 min when using a 45-75% methanol in 10 mM potassium phosphate buffer (pH 7.4) over 40 min, and no broadening of the peak was seen when they were coinjected. Similar results were obtained for FC with the buffer- and GSH-catalyzed decomposition. Both products eluted at 14.7 min. When mercaptoethanol
2.03 & 2.07
CHsCO
2.08 4.38 4.00 2.1 & 2.23 (*)
Signals obscured by methyl protons.
i 12
B n
FC
A
00 250
300
350
4W
450
Wavelength (nM)
Figure 2. Ultraviolet spectra of fusarin C and fusarin X during reactionwith glutathione. Spectrawere recordedin the presence of 5 mM GSH in potassium phosphate buffer (pH 8.0),at 36 "C. During decomposition the absorption at 360 nm decreased. Spectra were recorded at 0, 2, 7, 20, and 30 min.
replaced GSH, half-lives were 1.1and 5.9 min for FX and FC, respectively.
Possible Conformationsof FX. Energy minimization of the X-ray structure of FC were undertaken after replacing one of the hydrogens on C-1 with a hydroxyl group and converting the structure back to the BE isomer. This produces an extended polyene structure in which it is impossible for the C-1 hydroxyl group to approach the epoxide ring. The hydroxyl group had little influence on the conformation of the overall structure when compared to FC. Discussion Although FC contains an epoxide ring (8, 211, it still requires enzyme activation to obtain maximum mutagenic activity (6, 9, 13, 14). In this way, relatively stable metabolites can be produced by fungi which, in common with, for example, aflatoxins, are metabolically activated
A Mutagen from F. moniliforme
to much more reactive and unstable intermediates. It was therefore surprising to find that some culture extracts of F. moniliforme contain significant levels of direct-acting mutagens. It is possible that the bacteria themselves, as happens with nitroarenes (22,231,activate the compound. To determine the structure and possible importance of these direct-acting mutagens, we have isolated and identified the major component, designated FX, from F. moniliforme strain F84. The Ames assay was used to follow purification. Since the number of samples to be assayed was large and the quantities of active fractions limited, we checked fractions at low doses where cytotoxicity, which could have influenced the results, should have been small. Viability of bacteria was checked when assaying crude fractions. Support for the conclusion that little cytotoxicity occurred comes from the generally high recoveries of total mutagenic activities. The FX was extracted from the culture material with methanol. An initial silica gel chromatographicseparation removed about 90% of more polar compounds. Subsequent reverse-phase HPLC and TLC gave a product which appeared pure when reanalyzed by HPLC on the basis of its absorption from 220 to 370 nm. However, from ita NMR spectrum, which showed many signals in common with FC, it clearly still contained impurities. To purify the sample further, without any method besides NMR analysis to detect impurity(ies), it was acetylated in the hope that this would achieve a selectivity either at the level of reaction or at the level of subsequent chromatography between the compounds present. FX was efficiently converted into a new product having a longer retention time by reverse-phase HPLC but the same UV spectrum. However, the acetylated product was no longer mutagenic. Thus, it could be that the hydroxyl groups of FX are essential for mutagenicity or that the direct-acting mutagen(s) was(were) separated or destroyed. To reconvert the FX-diacetate to FX was difficult since, besides the acetyl groups which had to be removed, retention of the 21 methyl group was critical (24). This selectivity was achieved using porcine liver esterase which, at low pH values, allows exchange reactions to occur. The addition of 10% methanol to the enzyme reaction buffer helped drive the reaction to release the acetate groups, possibly with the formation of methyl acetate, while suppressing the overall hydrolysis of the methyl ester. FX-diacetate was converted to FX (15% ) which showed the same level of mutagenicity (413 mutants/2 nmol) as the crude FX (426 mutanta/2 nmol) when the concentration of FX was estimated from its UV absorption at 357 nm. Although levels of FC between strains F07 and F84 are similar, the culture extract of F07 contains -10 times more FX then F84, which is also reflected in their relative levels of direct-acting mutagens. In the other five less or nonmutagenic strains no FX was detected. These results suggest that FX present in FME of F07 and F84 accounts, at least in part, for the direct-acting mutagenicity of these extracts, which was confirmed during the fractionation and purification of FX. Strain F07 was isolated from corn in Linxian, China, where there is a high incidence of human esophageal cancer and the foods, including corn, consumed by residents are often contaminated by F. moniliforme. FC is unstable at high pH values (25) or when heated (25, 26) and is decomposed by GSH to a compound containing the C-1 and C-12 moiety with a characteristic absorption at 320 nm (27). Owing to the carboxyl group
Chem. Res. Toxicol., Vol. 6, No. 1, 1993 96
in this decomposition product, ita retention time by reverse-phase HPLC is quite variable depending upon the buffer conditions used. FX behaves similarly although is generally more reactive, in keeping with ita greater mutagenicity. The product is less stable and ita spectrum less clear, probably owing to the 1-hydroxyl group. The C-13to (3-19 moieties from FC and FX would be predicted to be the same. However, owing to their lack of adequate UV absorption, they are not detected. To help understand the reason(s) for the direct mutagenicity of FX in comparison to FC, molecular modeling was undertaken. The X-ray structure of the 8 2 isomer of FC (8) was taken as the starting point. The 8-double bond was rotated through 180° to give FC before energy minimization began. Substitution of a hydroxyl group on the 1-methyland reminimization gave essentially the same structure. One possible role of the hydroxyl group could be to assist opening of the epoxide ring that could occur if they were in close proximity. This can be achieved with isomerization of one or more of the double bonds which occurs relatively easily in the presence of UV light (8). Alternatively, 4-hydroxy-(E)-2-alkeneoicesters are quite reactive with nucleophiles (281,and this may explain the increased mutagenicity and reactivity of FX. FX, in spite of ita potent mutagenicity in the Ames test, does not account for all the mutagenic activity of F84 extract. Another compound(s) which was eluted from silica gel TLC plate with a higher Rf value than FX exhibited total mutagenic activity equivalent with FX. This unknown mutagen(s) has not yet been isolated. It has no detectable UV absorption, making isolation difficult.
Acknowledgment. This work was supported by Grant ACS CN7 from the American Cancer Society. Part of the work reported in this paper was undertaken during the tenure of a Research Training Fellowship awarded F.X.L. by the International Agency for Research on Cancer. We should like to thank Drs. Carlos de loa Santos for help in obtaining the NMR spectra, Vinka Parmakovich for the mass spectra, and John M. Hubbard, of the Molecular Modeling Facility for Molecular Biology, which is supported by NSF Grant DIR-8720229, for his assistance. References (1)Doll,R.,and Peto, R. (1981)The causes of cancer: Quantitative estimates of avoidable risks of cancer in the United States. J.Natl. Cancer Zmt. 66,1193-1308. (2) Doll,R.(1992)The leesons of life: Keynote addrese to the Nutrition and Cancer Conference. Cancer Res. (Suppl.) 12, 2024s-2029e. (3) Li, M. H., Tian, G. Z.,Lu, S. H., Guo, S. P., Jin, C. L., and Wang, Y. L. (1982)Forestomach carcinomas induced in rats by cornbread incubated with Fusarium moniliforme. Chin. J. Oncol. 4,241. (4) Li, M., Jiang, Y., and Bjelandee, L. F. (1990)Carcinogenicity of Fusarin C isolated from Fusarium moniliforme. Chin. J. Cancer Res. 2,1-5. (6) Gelderblom, W. C. A., Thiel, P. G., van der Merwe, K. J., Mar-, W.F.O.,andSpies,H.S. C. (1983)Amutagenproducedbyhrearium moniliforme. Toxicon 21, 467-473. (6) Cheng,S.J., Jiang, Y. Z., Li, M. H., and Lo,H. Z.(1986)A mutagenic metabolite produced by Fwarin moniliforme isolated from L d a n county, China. Carcinogenesis 6, 903-906. (7) Gelderblom, W. C.A., Thiel, P. G., Jaskiewicz, K., and Mar-, W. F. 0. (1986)Investigations on the carcinogenicity of fusarin C. A mutagenic metabolite of b a r i u m moniliforme. Carcinogenesb 7, 1899-1901. (8)Gelderblom, W. C. A., Marasas, W. F., Steyn, P. S., Thiel, P. G., van de Merwe, K., van Rooyen, P. H., Veleggaar, R., and Weasels, P. L. (1984)Structure elucidation of fusarin C, a mutagen produced by Fusarium moniliforme. J. Chem. SOC., Chem. Commun.,122-124. (9) Wiebe, L. A.,and Bjeldanes, L. F. (1981)Fusarin C, a mutagen from b a r i u m moniliforme grown on corn. J. Food Sci. 46,1424-1426.
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