Chem. Res. Toxicol. 2001, 14, 11-15
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Deamination of Fumonisin B1 and Biological Assessment of Reaction Product Toxicity Shawna L. Lemke,† Sean E. Ottinger, Charles L. Ake, Kittane Mayura, and Timothy D. Phillips* Department of Veterinary Anatomy and Public Health, College of Veterinary Medicine, MS 4458, Texas A&M University, College Station, Texas 77843-4458 Received August 4, 2000
Fumonisin B1, a potent mycotoxin found in grain, has been resistant to degradation and detoxification by a variety of methods, including milling, fermentation, ammoniation, and ozonation. The primary amine of this compound contributes significantly to its toxicity; therefore, the major aim of this research was to remove this moiety via diazotization. In this study, fumonisin B1 was deaminated in aqueous solution under conditions of acidic pH and low temperature (pH 1.0 and 5 °C) with the addition of NaNO2. The concentration of fumonisin B1 in the solution was analyzed by HPLC using o-phthaldialdehyde to derivatize the primary amine. Progress of the reaction was monitored as a loss of the derivatized peak as observed by HPLC with fluorescence detection. TLC analysis showed the disappearance of fumonisin B1 following diazotization. Further, TLC displayed at least four reaction products that were not primary amines. Matrix-assisted laser desorption/ionization mass spectrometry coupled with time-of-flight analysis of the diazotization products also showed a diminished amount of authentic fumonisin B1 and allowed identification of a product formed by the replacement of the primary amine with a hydroxyl group. The adult Hydra attenuata bioassay indicated a marked decrease in the toxicity of the products in comparison to parent fumonisin B1. Optimization of this reaction could result in a rapid and practical method for the reclamation of fumonisin B1-contaminated feeds.
Introduction Fumonisins are mycotoxins produced by Fusarium moniliforme that are found worldwide as natural contaminants in a variety of agricultural products (1-4). The most abundant member of the group, fumonisin B1 (FB1),1 has been linked to human esophageal cancer (5-7), equine leukoencephalomalacia (8), swine pulmonary edema (9), and hepatocarcinogenesis in rats (10). A variety of techniques have been applied to the remediation of FB1-contaminated foods; however, these studies have not yet elucidated an adequate means of eliminating the toxin. FB1 can be hydrolyzed but not detoxified by heat and acid (11). While highly water soluble, the toxin cannot be adequately washed off of corn or grain (12). In addition, milling, fermentation, and enzymatic detoxification were also unsuccessful (12). Detoxification methods that have been useful for the structurally unrelated mycotoxin, aflatoxin B1, have also failed to remediate FB1. In particular, ammoniation (13) and nixtamalization (12) have no effect on the compound. Ozonation does produce a chemical change but does not detoxify FB1 (14). * To whom correspondence should be addressed: Veterinary Anatomy and Public Health, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4458. Phone: (979) 845-6414. Fax: (979) 862-4929. E-mail:
[email protected]. † Present address: Department of Nutrition, One Shields Ave., University of California, Davis, CA 95616. E-mail:
[email protected]. 1 Abbreviations: AHA, adult Hydra attenuata; FA , fumonisin A ; 1 1 FA2, fumonisin A2; FB1, fumonisin B1; FB2, fumonisin B2; FB3, fumonisin B3; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MW, molecular weight; OPA, o-phthaldialdehyde; Rf, retardation factor; SPE, solid-phase extraction.
Figure 1. Chemical structures of fumonisins B1, B2, B3, A1, and A2.
Several natural and co-occurring fumonisins exist. These include FB1, FB2, FB3, and the N-acetyl derivatives, FA1 and FA2 (Figure 1). Structure-activity studies comparing members of the fumonisin class showed that compounds containing an N-acetyl group were not cancer initiators and were less cytotoxic in rat liver cells than fumonisins containing a free amino group (15). FB1 has
10.1021/tx000166d CCC: $20.00 © 2001 American Chemical Society Published on Web 12/19/2000
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Chem. Res. Toxicol., Vol. 14, No. 1, 2001
been shown to disrupt ceramide synthase, an enzyme necessary for sphingolipid metabolism (16). Studies have shown that analogues with an N-acetyl-blocked amine did not interfere with ceramide synthase (17). Hydrolysis of the two propane-1,2,3-tricarboxylic acid side chains from FB1 diminished this effect, but did not eliminate toxicity. These results suggest that the side chains play a role in the effects of FB1 but that the presence of an intact primary amine is necessary for toxicity. Detoxification of FB1 by blocking the primary amine has been previously studied. For example, reaction of reducing sugars such as fructose or glucose with the primary amine of FB1 to form a Schiff base has been achieved in the laboratory (12, 18). Toxicity studies have shown that FB1 modified by reaction with fructose did not produce altered hepatic foci in rats, suggesting that the hepatocarcinogenicity of FB1 had been eliminated (19). These studies demonstrate the potential for FB1 detoxification by blocking or eliminating the primary amine moiety. Diazotization is the reaction of a primary or secondary amine with a nitrosating agent, such as HONO, that results in a diazonium salt. The reaction can be accomplished in aqueous solution using NaNO2 and acid to produce HONO. The reaction is most notably used for the production of diazo dyes, but may also be incorporated into preparative synthesis. When the reaction is performed on primary aliphatic amines, the resulting diazonium compound is unstable, often resulting in an SN1 deamination and the formation of a variety of products (20). As the reaction is conducted in aqueous solution and may allow excess reagent to be washed away, the procedure has the potential to be utilized as a reclamation technique for contaminated feeds and foods. The objectives of this study, therefore, were (1) to determine if diazotization could be used to deaminate FB1 and (2) to investigate the comparative toxicity of the diazotization products versus parent FB1.
Experimental Section Materials. FB1, mercaptoethanol, o-phthaldialdehyde (OPA), phosphomolybdic acid, and ninhydrin spray reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The hydrochloric acid was obtained from J. T. Baker (Phillipsburg, NJ). The sodium tetraborate was a product of Matheson, Coleman & Bell (Norwood, OH). The OPA solution was prepared according to previously described methods (11). The FB1 antibody columns and phosphate-buffered saline (PBS), 10 mM at pH 7, were products of Vicam (Watertown, MA). Experimental Conditions. (1) NaNO2 Titration Experiment. An aqueous solution of FB1 (15 µg/mL) was adjusted to pH 1 with concentrated HCl. Aliquots (1 mL, 20.8 nmol) were placed in 4 mL amber glass vials (Supelco, Bellefonte, PA) with 2 mm × 7 mm microstir bars (VWR, Houston, TX). The vials were set in a rack and placed in an ice bath over a stir plate until the temperature reached 5 °C. Solid NaNO2 was added (1-100 mg), and the solution was allowed to react for 2 h. Three controls were also included: pH 7 with no NaNO2, pH 7 with 100 mg of NaNO2, and pH 1 with no NaNO2. The supernatants were analyzed for FB1 using a modification of the Vicam FumoniTest HPLC procedure for corn (1 g of sample equivalent, 0-10 ppm, Vicam). Since the samples did not originate from corn, extraction and dilution was not necessary. An aliquot of each sample (0.5 mL) was diluted with 4 mL of PBS and applied to a Vicam column. This was followed by a rinse with 10 mL of PBS and elution with 1.5 mL of methanol. The methanol eluate was dried, and the sample was resuspended in 500 µL of water. An aliquot of 100 µL was removed, and 400 µL of the OPA
Lemke et al. solution was added. This mixture was then vortexed for 30 s, and 20 µL was injected and analyzed by a Waters HPLC system (Milford, MA), using a model 510 pump and a model 420 fluorescence detector. A 3.9 mm × 300 mm, 10 µm particle size, steel µBondapak C-18 column (Waters) was used with a mobile phase of water, acetonitrile, and acetic acid (50:50:1) (21). The experiment was performed in triplicate. (2) Deamination of FB1 for Structural Analysis and Toxicity Testing. FB1 (24 mg) was divided into two 12 mg (16.6 µmol) lots. One quantity was reserved as starting material. The second quantity was dissolved in 150 mL of distilled, deionized water and placed in a 500 mL flat-bottom flask. The solution was adjusted to pH 1 using concentrated HCl. A stir bar was added to the flask, and the solution was placed in an ice bath over a stir plate until the temperature reached 5 °C. NaNO2 was added in increments of 500 mg every 20 min, and HCl was added to maintain a solution pH of 1. Prior to each addition, a 200 µL aliquot of solution was removed for analysis to monitor the FB1 concentration. The supernatants were analyzed for FB1 using the Vicam FumoniTest HPLC procedure for corn described above. From each sample, 200 µL was mixed with 600 µL of PBS and applied to the column. This was followed by a rinse with 10 mL of PBS and elution with 1.5 mL of methanol. The sample was dried, resuspended in water (200 µL), derivatized with OPA, and analyzed by HPLC as described above. Diazotization was considered complete when FB1 was no longer detected by HPLC. After diazotization, the sample was isolated by C-18 solidphase extraction (SPE). Briefly, the Supelclean ENVI-18 SPE tube (6 mL, Supelco) was washed with 10 mL of methanol followed by 10 mL of water. The sample was applied and washed with 50 mL of water to remove excess NaNO2. Acetonitrile containing 0.1% trifluoroacetic acid (50 mL) was used to elute the sample, which was subsequently dried under nitrogen and stored at 5 °C. Analysis of Diazotization Reaction Products. The starting material and diazotization products were applied (20 µg) to duplicate silica TLC plates (Uniplate, Newark, DE) and eluted with an 85:15 acetonitrile/water mixture (22). One plate was developed with the phosphomolybdic acid spray reagent, and the second plate was developed with the ninhydrin spray reagent. Retardation factors (Rf) were measured for all visible bands. The two samples were also analyzed by a Voyager Elite XL matrix-assisted laser desorption/ionization (MALDI) mass spectrometer with a time-of-flight (TOF) detector (PerSeptive Biosystems, Framingham, MA). A 337 nm nitrogen laser was utilized. Samples were prepared by the overlay method using a matrix of R-cyano-4-hydroxycinnamic acid with 0.1% formic acid. Data were collected in the positive ion mode. Hydra attenuata Bioassay. Adult H. attenuata (AHA) were obtained from E. Marshall Johnson, Jefferson Medical College (Philadelphia, PA). Maintenance of cultures and feeding of AHA were carried out according to previously described methods (23). AHA were not fed for 24 h before initiating the experiments and were maintained clean and free from bacteria and fungi contamination by treating with a dilute iodine solution (2.7 ppm) periodically. The assay was performed by exposing the AHA to FB1 (150 µg/mL) at a dose 1.5 times higher than the previously determined minimum concentration needed to produce the toxic end point to ensure a toxic response (14). NaNO2 was also tested for toxicity at this concentration to control for the possibility that all the salt was not removed during the SPE cleanup. Each test dish (5 cm diameter Petri dish) contained 2.0 mL of test solution and three normal healthy AHA. The experiment was run in triplicate (n ) 9). Hydra were examined for signs of toxicity at 0, 4, 24, 48, 72, and 96 h. The toxic end point was determined by death, i.e., disintegration of the AHA. The total number of dead AHA in each treatment group at each time point was counted. Data from each observation time were subjected to an R × C χ2 test followed by a Fisher’s exact test (24) to determine differences in the live:dead ratio between treatment groups.
Deamination and Detoxification of Fumonisin B1
Chem. Res. Toxicol., Vol. 14, No. 1, 2001 13
Figure 2. Titration of the decrease in FB1 concentration in an acidic solution with increasing levels of NaNO2. Aqueous solutions of FB1 at pH 1 or 7 (15 µg/mL) were chilled to 5 °C, and NaNO2 was added as a solid. Samples were allowed to react for 2 h. FB1 was extracted with Vicam antibody columns, derivatized with OPA, and analyzed by HPLC. Controls at pH 1 (0 mg of NaNO2) and at pH 7 (100 mg of NaNO2) were included to demonstrate that pH and excess salt did not interfere with FB1 analysis. ND denotes nondetectable levels of FB1 (
