Simultaneous Occurrence of Fumonisin B1 and Other Mycotoxins in

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1994, p Vol. 6, No /94/$4. + Copyright 1994, American Society for Microbiology Simultaneous Occurrence of Fumonisin B1 and Other Mycotoxins in Moldy Corn Collected from the People's Republic of China in Regions with High Incidences of Esophageal Cancer FUN S. CHU* AND GUO Y. LIt Food Research Institute and Department of Food Microbiology and Toxicology, University of Wisconsin-Madison, Madison, Wisconsin 5376 Received 3 August 1993/Accepted 1 December 1993 A total of 31 corn samples collected from households in the counties of Cixian and Linxian of the People's Republic of China, where high incidences of esophageal cancer have been reported, were analyzed for fumonisin B1 (FB1), aflatoxin, and total trichothecene mycotoxins. High levels of FB, (18 to 155 ppm; mean, 74 ppm) were found in 16 of the samples that showed heavy mold contamination. FB,, at lower levels (2 to 6 ppm; mean, 35.3 ppm), was also found in 15 samples, collected from the same households, that did not show any visible mold contamination. The levels of aflatoxin in the samples were low (1 to 38.4 ppb; mean, 8.61 ppb). High levels of total type-a trichothecenes were also found in the moldy corn samples (139 to 2,3 ppb; mean, 627 ppb). Immunochromatography of selected samples revealed that these samples contained T-2 toxin, HT-2 toxin, iso-neosolaniol, monoacetoxyscirpenol, and several other type-a trichothecenes. The concentration of total type-b trichothecenes in 15 moldy corn samples was in the range of 47 to 5,826 ppb (mean, 2,359 ppb). High levels (3.7 to 5. mg/g) of FBI were produced in corn in the laboratory by five Fusarium moniliforme strains isolated from the moldy corn. These fungi were also capable of forming various nitrosamines (5 to 16,ug per flask) in the presence of nitrate and precursor amines. The present data indicate that the presence of high levels of FB1 (a cancer promoter) and other mycotoxins in the moldy corn samples and the capability of producing nitrosamines (carcinogens) by F. moniliforme may play an important role in carcinogenesis in humans in Cixian and Linxian counties. Fusarium moniliforme is one of the most common fungi colonizing corn throughout the world. For years, South African researchers have associated the cause of equine leukoencephalomalacia with horses consuming moldy corn that was infested with this fungus. This fungus has also been isolated frequently from foods and feed both in Linxian County of People's Republic of China and in Transkei of South Africa, two regions where the highest incidences of esophageal cancer have been reported. The level of contamination of this fungus in corn in these two regions has been found to be closely correlated to the esophageal cancer incidence in those regions (2, 21, 28). Attempts to isolate the carcinogenic metabolites produced by F. moniliforme led to the isolation of a potent mutagen and genotoxic mycotoxin, fusarin C (2, 8, 9, 3), in the early 198s. Several other fusarin C-related mycotoxins with different degrees of mutagenicity have since been identified (1, 5, 18, 26, 33). However, carcinogenic effects of fusarin C have not been demonstrated. Recent studies have led to the isolation of fumonisin B1 (FBI), fumonisin B, (FB2), and four other structurally related fumonisins. They are propane-1,2,3-tricarboxylic acid diesters of an amino-dimethyl-tetra- or aminodimethyl-pentapentahydroxyeicosane (6). FBI is the major mycotoxin produced by F. moniliforme and related fungi and is found most frequently in corn. It has been found to be a potent cancer promoter and also an etiological toxic agent responsible for the disease equine leukoencephalomalacia (6, 22, 25, 28). * Corresponding author. Mailing address: Food Research Institute, University of Wisconsin, 1925 Willow Dr., Madison, WI Phone: (68) Fax: (68) Electron mail address: fschu@ macc.wisc.edu. t Permanent address: The Cancer Institute of the Chinese Academy of Medical Science, Beijing, People's Republic of China. 847 Although the question regarding the carcinogenicity of FB, remains to be answered even in some reports indicating that it is carcinogenic (7, 13, 21, 28), recent reports on the worldwide occurrence of large amounts of this mycotoxin in foods and feeds, generally at the parts-per-million level, have prompted us to carry out the present study. Over the years, scientists at the Cancer Institute of the Chinese Academy of Medical Sciences have been involved extensively in studying the role of nitrosamines in the induction of esophageal cancer in humans in People's Republic of China. They have found several nitrosamines in the diet of people living in Linxian County, People's Republic of China. A number of fungi from foods in these regions, including F. moniliforme, were capable of reducing nitrate to form nitrite and nitrosamines (14, 16, 32). The objective of this study was to examine the possible role of fumonisins on human carcinogensis by (i) determining whether FB, and other mycotoxins including aflatoxin are present simultaneously in the corn collected from the regions where a high incidence of esophageal cancer has been reported and (ii) testing whether FB, and nitrosamines could be produced simultaneously by F. moniliforme if nitrate and precursor amines are present in the culture media. The results are presented in this article. MATERLALS AND METHODS Materials. Standard nitrosamines were supplied by G. M. Singer, Center of Cancer Research, National Cancer Institute (Frederick, Md.). FB, and FB2 standards were obtained from C. J. Mirocha, University of Minnesota (St. Paul, Minn.). o-phthaldialdehyde (OPA) was purchased from Sigma Chemical Co. (St Louis, Mo.). Naphthalene-2,3-dicarboxaldehyde (NDA) was purchased from Oread Lab, Inc. (Lawrence,

2 848 CHU AND LI Kans.). The Sep-Pak C-18 reverse-phase cartridge was obtained from Waters Associates (Milford, Mass.). Specific antibodies against mycotoxins and related immunochemical reagents were prepared in our laboratory (4, 15, 31). All other chemicals and organic solvents were either analytical grade or high-performance liquid chromatography (HPLC) grade. Sample collection. Corn samples were collected from households in Cixian County of Hebei Province and Linxian County of Henan Province of People's Republic of China between 18 and 2 November The samples were subdivided into two groups, one with heavy mold contamination and the other with no visual mold contamination (i.e., fine corn). A total of 18 samples (1 with heavy mold contamination and 8 fine corn) were obtained from Cixian, and 16 (9 with heavy mold contamination and 7 fine corn) were obtained from Linxian. All of the samples were milled to 12/25 mesh with a laboratory mill and kept at - 2 C before analyses. HPLC analysis of fumonisins. (i) Sample treatment and cleanup. For analysis of fumonisins, 1 g of the ground samples was shaken with 5 ml of 5% acetonitrile (MeCN) for 1 h, filtered through a Whatman no. 4 filter paper, and then subjected to a cleanup treatment as described in the procedure of Ware et al. (29). For each sample, 2 ml of the filtrate plus 4 ml of distilled water with the ph adjusted to 4.5 was loaded onto a C-18 Sep-Pak cartridge that had been washed previously with 2 ml of MeCN and 2 ml of acidified distilled water (ph 4.5) in sequence. After the sample was loaded, the cartridge was washed with 4 ml of acidified distilled water (ph 4.5), 4 ml of 2% MeCN, and then with 2 ml of distilled water. Fumonisins were eluted from the cartridge with 2 ml of 7% MeCN. Two approaches were used for derivatization of the samples before HPLC analysis. One approach involved the use of OPA in which.1 ml of the cleanup samples was reacted with.1 ml of OPA solution (.13 mg of OPA per ml of MeCN with.5% mercaptomethanol) at room temperature for 1 min and then injected into the HPLC within 3 min. The other approach involved the use of NDA in which 5,ul of the cleanup sample plus.5 ml of sodium borate (.5 M) was reacted with.5 ml of NaCN and 5,ul of NDA (.5 mg/ml of MeCN) in a heating block (6 C) for 15 min (12, 29). The reaction mixture was then used in the HPLC. An analytical recovery experiment was carried out by spiking standard FB, into a control sample at 5-, 1-, and 2-ppm levels. The control sample, which was kindly analyzed by G. A. Bennett of the National Center for Agricultural Utilization Research, USDA Agricultural Research Service (Peoria, Ill.), contained less than 6 ppb of FB1. (ii) HPLC analysis of samples derivatized by the OPA method. For all of the HPLC analyses, the method of Shephard et al. (27) was followed by using a,u-bondapak C-18 reversephase column (3.9 mm by 25 cm; Waters Associates) in a Waters Associates model A6 HPLC system equipped with a Shimadzu model RF-53 fluorescence monitor. The column was equilibrated with solvent A (MeCN-H2-acetic acid [39: 6:1]) at a flow rate of 1 ml/min. After injection of the samples and running the column in solvent A for 1 min, the column was run for 3 min with a mixture of 5% solvent A-5% solvent B (MeCN-H2-acetic acid [6:39:1]) and then for 2 min with solvent B, and finally for another 2 min with solvent A before another sample was injected. The emission and excitation wavelengths of the fluorometer were set at 44 and 335 nm, respectively. (iii) HPLC analysis of samples derivatized by the NDA method. For analysis of FB, by the NDA derivatization method (12, 29), a Waters Associates model 6A HPLC system equipped with a model 68 controller and a model U6K injector was used. Instead of a,i-bondapak C-18 column, a APPL. ENVIRON. MICROBIOL. Beckman Ultrasphere octyldecyl silica reverse-phase column (4.6 mm by 25 cm; Beckman Instruments, Fullerton, Calif.) was used. A Shimadzu model RF 535 fluorometer with excitation and emission wavelength settings at 246 and 418 nm, respectively, was used. The mobile phase was MeCN-H2-acetic acid (55:45:1), and it was run at a flow rate of 1 ml/min. For quantitation, the peak areas of a series of standard FB1 (5 to 23 ng per injection) was used to establish the standard curve. FB2 was also included in the analysis. However, because of uncertainty of the quantity of FB2 standard, the concentration of FB2 in the samples was not determined. Analyses of total TCTC and aflatoxin Bl. For the analysis of total trichothecenes (TCTC) and aflatoxins, various immunochemical methods that have been established in our laboratory for various investigations were used. The toxins were extracted from the samples with 5% MeCN in a manner similar to that for FB, analysis. For total type-a TCTC, an enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies that have a wide specificity to most type-a TCTC was used (15, 24). The samples were subjected to hydrolysis and acetylation before ELISA. For the analysis of deoxynivalenol (DON)- related type-b TCTC, a polyclonal antibody-based radioimmunoassay was used. The antibodies have high specificity to DON triacetate; thus, all of the sample extracts were also subjected to acetylation before radioimmunoassay (31). For aflatoxin, a sensitive, direct, competitive ELISA using polyclonal antibodies specific for aflatoxin B1 (1% cross-reactive) and G1 (33% cross-reactive relative to B1) was used (4). Production of fumonisins and nitrosamines by F. moniliforme. To test the production of fumonisins and formation of nitrosamines by F. moniliforme, five strains (CL1 to CL5) previously isolated from the moldy corn from Linxian County were selected. The fungi were grown in potato dextrose agar slants at 28 C for 1 week before the preparation of the conidial suspension. Production of fumonisin. For the production of fumonisins, 3 ml of the conidial suspension was inoculated into the sterilized yellow corn grits (5 g of yellow corn grits plus 25 ml of H2 in a 5-ml Erlenmeyer flask autoclaved at 121 C for 3 min). After incubation at 28 C for 4 weeks, 15 ml of methanol-h2 (3:1) was added, and the flask was shaken for 1 h before filtration. The filtrates, equivalent to 2 g of sample, were subjected to Sep-Pak C-18 reverse-phase cleanup treatment as described above. The final HPLC analysis for FB1 was carried out with the NDA derivatization method. Formation of nitrosamines. Production of nitrosamines by F. moniliforme was carried out in a 5-ml Erlenmeyer flask containing 1 ml of glucose ammonium nitrate medium enriched with 1.7 mm of isobutylamine and 1.7 mm of benzylamine (14). After inoculation with 3 ml of the conidial suspensions, the cultures were incubated at 28 C for 8 days, and then 5.8 mm of NaNO2 was added. The cultures continued to incubate at 28 C for an additional 15 h. The reaction of isobutylamine and methylbenzylnitrosamine with NaNO2 was stopped by adding 1 ml of 2% ammonium sulfamate to which an internal standard dipropylnitrosamine (4 ng) was added. Nitrosamines in culture medium were extracted by distillation (1). The first 4-ml fraction was collected and then extracted twice with 2 ml of CH2Cl2. The dichloromethane extract was concentrated to.4 ml for subsequent gas chromatography (GC)-thermoelectron analyzer (TEA) analysis (14, 23). For GC-TEA analysis, a Perkin-Elmer Sigma (model I) GC and a TEA (model 52A) instrument together with a stainlesssteel column (2 m by 2 mm) packed with 5% FFAP-Chrom Q (8/1 mesh) were used. The GC was run under the following

3 VOL. 6, 1994 FUMONISIN B1 AND OTHER MYCOTOXINS IN MOLDY CORN 849 TABLE 1. Samplea HPLC analysis of FB, in selected moldy corn samples by two derivatization methods (OPA and NDA) FBI recovered (ppm) by: NDA method OPA method 2C C C C L L L L L Avg 66.7 ± ± 5.44 a C, Cixian; L, Linxian. conditions: carrier gas, Ar at 25 ml/min; oven temperature, 12 C for 2 min and then increased to 19 C at a rate of 1 C/min. The temperature of the injection inlet was set at 2 C. Conditions for the TEA were set as follows: cracking chamber temperature, 48 C; cold well temperature, - 13 C; 2 flow rate, 2 ml/min; vacuum chamber,.35 to.7 torr (1 torr = Pa). The final nitrosamine concentrations were corrected for the loss during the sample preparation with an internal standard dipropylnitrosamine. RESULTS Analysis of fumonisins in moldy corn. Two HPLC methods for the analysis of fumonisins were tested. An examination of various chromatograms revealed that a number of other peaks in addition to that of FBI were observed. For example, FB2 was also frequently found in the samples. Because standards for other fumonisins were not available, quantitation and identification of these peaks were not made. Fumonisins were not found by either method when a clean corn sample from Wisconsin was used as a blank control. The analytical recoveries of FBI added to the blank control corn sample at 5-, 1-, and 2-ppm levels (NDA method) were found to be 85, 92.8, and 1.8%, respectively. A total of nine samples were analyzed by both methods, and the results are presented in Table 1. Whereas the average data from these two methods showed no apparent differences, linear regression analyses revealed that there is a general trend that the OPA method tends to give higher values at higher toxin levels. In contrast, the NDA method tends to give higher values at lower toxin levels. Since the OPA method gave a better resolution for FB1, it was selected for the analysis of all the samples collected. Results for the FB, levels in the samples collected in two different regions are shown in Fig. 1. The mean levels of FB, in the fine corn from Linxian County (LF), moldy corn from Linxian County (LM), fine corn from Cixian County (CF), and moldy corn from Cixian County (CM) were found to be 3.4 (range, 19.8 to 6 ppm), 49.3 (range, 17.9 to ppm), 39.5 (range, 3.3 to 47.9 ppm), and 93.8 (range, 28.4 to ppm) ppm, respectively. The overall means for FB, in the fine corn and moldy corn were found to be 35.3 and 74 ppm, respectively. Analysis of variance revealed that there was a significant difference between FB, levels in LF and CM (P <.1). Significant differences were also observed for LM versus CM (P <.5) and CF versus CM (P <.1). No significant differences were found for the LF versus LM, LF versus CF, and LM versus CF pairs. Immunochemical analyses of other mycotoxins. Several cl IL W1. 15 m 1 z En z LZ. Lz. o 5 z C.) LF LM CF CM SAMPLES COLLECTED FIG. 1. FBI levels in corn samples collected from the counties of Cixian and Linxian. immunochemical approaches were used to examine the possible presence of other mycotoxins in the corn samples. A direct competitive ELISA was used for the determination of aflatoxins, and the results are shown in Fig. 2. Low levels of aflatoxins, with a range of.7 to 38.4 ppb, were found in these samples. The analytical recovery of aflatoxins in corn at levels between 5 and 1 ppb by this method in various previous studies conducted in our laboratory was generally around 91% (3, 4). The mean levels of aflatoxins in the LF, LM, CF, and CM were found to be 7.71 (range, 3.3 to 15 ppb), 4.57 (range,.95 to 9.95 m %-O z x C.) 31 2 I F LF LM CF SALS ~~~ U 8 ~~~~~ : B U OL FIG. 2. Aflatoxin levels in corn samples collected from the counties of Cixian and Linxian. to U -e- EJ UN 8-6 CM

4 85 CHU AND LI APPL. ENVIRON. MICROBIOL. 1. m 1 En a., o9 cd ct uz C.,O 1o O [ 5 es ~~ ~~~ LF LM CF SAMLE COLLTED FIG. 3. Total type-a TCTC levels in corn samples collected from the counties of Cixian and Linxian. o ~~~~ U *@@ ~ * flr-lri m CM Lii zm IC, En m 4c :> _ RETENTION TIME (Min.) FIG. 4. Immunochromatogram of a typical moldy corn sample obtained from Linxian. HPLC and ELISA conditions were the same as described in Materials and Methods. Samples collected from HPLC were hydrolyzed, acetylated, and diluted before the ELISA was done. Identification of individual peaks was achieved by comparison of the retention time (flow rate,.5 ml per fraction per 3 s) with the standards. Abbreviations: MAS, monoacetoxyscirpenol; ISO-NES, isoneosolaniol; Ac-T4ol, 4-acetyl-T-2 tetraol; T4ol, T-2 tetraol. ppb), 8.58 (range,.7 to 35.8 ppb), and 11.4 (range, 4.15 to 38.4 ppb) ppb, respectively. Since the antibodies used in this assay are highly specific to aflatoxin Bl, the data represent the presence of this aflatoxin. For the determination of total type-a TCTC, an indirect ELISA was used. This assay quantitatively determines all of the type-a TCTC after converting them to T-2 tetraacetate (15, 24). The analytical recoveries of T-2 toxin added to a blank corn sample at.1-,.5-, 2-, and 5-ppm levels were 123, 94, 98, and 83.5%, respectively. As shown in Fig. 3, type-a TCTC were found in all of the samples analyzed (range, 3 to 2,3 ppb). Several samples had levels above 1 ppm. The mean levels of total type-a TCTC in the LF, LM, CF, and CM were found to be 31 (range, 3 to 673 ppb), 572 (range, 14 to 1,25 ppb), 367 (range, 31 to 1,33 ppb), and 666 (range, 95 to 2,3 ppb) ppb, respectively. The results of the determination of individual type-a TCTC in a representative sample by immunochromatography, i.e., a combination of HPLC and ELISA, are shown in Fig. 4. The individual TCTC were identified as T-2 toxin (16.8% of total), HT-2 (9.7% of total), monoacetoxyscirpenol (4.5% of total), iso-neosolaniol (15.3% of total), 4-acetyl-T-2 tetraol (25.5% of total), and T-2 tetraol (19% of total). Analysis of variance revealed no difference among the pairs in various groups of samples for both aflatoxin and TCTC levels. For total type-b TCTC, a radioimmunoassay was used. This assay quantitatively determines all of the type-b TCTC after converting them to DON triacetate, and a tritiated DON triacetate was used as the marker. The analytical recoveries of DON added to a blank corn sample at.1-,.5-, 2-, and 5-ppm levels were 89.2, 94, 81.3, and 97%, respectively. The results, shown in Fig. 5, indicate that DON-related type-b TCTC were found in all of the samples analyzed. High levels of type-b TCTC were also found in some samples. The mean levels of total type-b TCTC in the LF, LM, CF, and CM were found to be 1,7 (range, 535 to 2,5 ppb), 2,83 (range, 473 to 5,826 ppb), 1,681 (range, 263 to 4,14 ppb), and 2,63 (range, 64 to 4,635 ppb) ppb, respectively. Three samples were excluded from the average because of their extremely high DON-related toxin levels (LF, 1.5 ppm; LM, 67.2 ppm; CM, 12.4 ppm). Analysis of variance revealed no difference among the pairs in various groups of samples for the type-b TCTC levels. Production of fumonisins and nitrosamines by F. moniliforme. Results of the production of FB1 and formation of nitrosamines by five F. moniliforme strains are presented in Table 2. Large amounts of FB, were produced by these strains. As much as 3.7 to 5. mg of FB, was produced in 1 g of corn grits after incubation for 4 weeks at 28 C. The ability to form _ m w %.,O E En I-.,. c) J 5 F 4 F 3 F 2 F 1 F LF LM CF CM SAMPLES COLLECTED FIG. 5. Total DON-related TCTC in corn samples collected from the counties of Cixian and Linxian. U U U

5 VOL. 6, 1994 FUMONISIN B1 AND OTHER MYCOTOXINS IN MOLDY CORN 851 TABLE 2. Production of FBI and formation of nitrosamines by five F. monilifome strains isolated from moldy coma Strain FB, Amt of nitrosamine formed (ng/flask)b no. produced (ppm) NPIP NMBzA Total NA 1 4,658 7,665 1,15 8, ,712 15, ,3 3 4,587 4, ,77 4 4,965 11, , ,657 5, ,866 a Production of FB, was carried out in corn grits, and formation of nitrosamines was carried out in the glucose-ammonium nitrate medium. b Abbreviations: NPIP, nitrosopiperidine; NMBzA, N-methylbenzylnitrosamine. secondary amines and nitrosamines by these strains was also evident when the culture media were enriched with nitrite and primary amines. Neither FB, nor nitrosamines were found in the control medium, in which F. moniliforme was not inoculated. DISCUSSION The present study shows that a number of mycotoxins produced by fusaria are present in the corn collected from households in two regions of People's Republic of China where high incidences of esophageal cancer in humans are reported (16). Among many types of mycotoxins, a high concentration of FB1, at a mean level of 94 ppm, was found in CM. Fumonisin Bl, at relatively high levels, was also found in the corn samples with no apparent mold contamination collected from the same households. These data indicate that the corn collected from the households in these two regions was heavily contaminated with fumonisins. These results are consistent with early mycological examinations showing that F. moniliforme is one of the most prevalent fungi found in moldy corn in these regions (16). Earlier epidemiological studies from one of our laboratories also found that the degree of contamination of this fungus in corn in these regions was closely correlated with the esophageal cancer incidence (16). Similar observations of the presence of high levels of fumonisins and frequent contamination of F. moniliforme in the corn samples collected from the Transkei region of South Africa have also been reported (2, 21, 28). In a search for etiological factors that may contribute to the esophageal cancers in humans, some Chinese scientists have found that the presence of fusaria mold and nitrosamines in the diet are among the major factors (14, 16, 32). High levels of nitrosamines are found in the diet. A number of fungi, including F. moniliforme, are also capable of forming secondary amines and nitrosamines (14, 16). We confirmed that, in addition to producing large amounts of fumonisins, F. moniliforme strains isolated from these regions indeed are capable of forming nitrosamines including N-methylbenzylnitrosamine, one of most potent nitrosamines inducing esophageal cancer in test animals (14). Limited work conducted at the Cancer Institute of the Chinese Academy of Medical Sciences (16a) has found that the some moldy corn samples containing FB1 also contained various nitrosamines. Because nitrosamine formation is more complex, mold contamination alone may not be the sole factor that leads to the high level of dietary nitrosamines. Results showing detection of both type-a and type-b TCTC, including T-2 toxin, HT-2 toxin, isoneosolaniol, hydrolyzed products of T-2 toxin, and DON-related mycotoxins (3), in these samples indicate that molds other than F. moniliforme were also present. T-2 toxin and other TCTC have been found previously in the corn collected from Linxian County; however, the levels were lower than those in the present study (11, 19). While the role of T-2 toxin in carcinogenesis both in humans and animals is still not clear, several laboratory studies have shown that T-2 toxin enhanced cancer formation in a rat model (3, 17). Li et al. (17) suggested that TCTC causing significant tissue damage may promote tumor formation in animals and humans. Since FB1 and FB2 have been shown to be potent cancer promoters and possibly carcinogens (7, 22, 28), the simultaneous presence of FB1, nitrosamines, and other carcinogenic mycotoxins may play an important role in carcinogenesis. Our finding of the presence of type-a TCTC suggests that this group of mycotoxins may also be a contributing factor. The presence of DON-related mycotoxins in the samples also suggests that zearalenone might also be present. The role of aflatoxin is unclear because the levels were generally low. Except for two samples, one at 35 ppb and the other at 38 ppb, most of the samples had less than 1 ppb of aflatoxin B1. However, the interaction of fumonisins with other different factors, including some mycotoxins (3) that were not analyzed in this present study, such as fusarin C (also produced by F. moniliforme), Alternaria mycotoxins, and other carcinogenic mycotoxins (such as ochratoxin A and sterigmatocystin), cannot be overlooked. Since the interaction of fumonisins with these factors is very complicated, the results obtained from the present study suggest that there is a need for an extensive systematic epidemiological study, including the analysis of a larger sample population for various mycotoxins as well as other environmental factors. Only through such a vigorous study could we then gain more knowledge to further our understanding of such an interaction. ACKNOWLEDGMENTS This work was supported by grant NC-129 from the College of Agricultural and Life Sciences and by funds from the various food company affiliates of the Food Research Institute, the University of Wisconsin at Madison. We thank C. J. Mirocha and I-C. Xu of the University of Minnesota for their help in the HPLC analysis for FB, by the OPA method and Carole Ayres for her help in preparing the manuscript. REFERENCES 1. Barrero, A. F., J. F. Sanchez, J. E. Oltra, N. Tamayo, E. Cerda- Olmedo, R. Candau, and J. Avalos Fusarin C and 8Zfusarin C from Gibberella fujikuroi. Phytochemistry 3: Cheng, S. J., Y. Z. Jiang, M. H. Li, and H. Z. Lo A mutagenic metabolite produced by Fusarium moniliforme isolated from Linxian county, China. Carcinogenesis 6: Chu, F. S Mycotoxins: food contamination, mechanism, carcinogenic potential and preventive measures. Mutat. Res. 259: Chu, F. S., T. S. L. Fan, G. S. Zhang, Y. C. Xu, S. Faust, and P. L. 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