Degradation of Pure Aflatoxins by Tetrahymena pyriformis

Transcription

1 APPLIED MICROBIOLOGY, Sept. 1967, p Copyright 1967 American Society foi Microbiology Vol. 15, No. 5 Printed In US.A Degradation of Pure Aflatoxins by Tetrahymena pyriformis DOROTHEA J. TEUNISSON AND JAMES A. ROBERTSON Southern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, New Orleans, Louisiana Received for publication 3 April 1967 Tetrahymena pyriformis W with nutrients, ca. 22 X 106 cells, decreased the concentration of aflatoxin B, 58 % in 24 hr and 67% in 48 hr. An unknown, bright-blue fluorescent substance was produced, with intensity about one-half that of the unchanged B1, with an Rf of 0.52 compared with 0.59 for B1 and 0.55 for B2 on a thinlayer chromatography plate, and with an ultraviolet spectrum showing maxima of 253, 261, and 328 m,u. In a separate assay, the cells with nutrients did not degrade pure Gi. Starved, washed cells, ca. 11 X 106, decreased the concentration of B, 50% in 10 hr, 70% in 22 hr, and 75% in 30 hr, producing the same unknown component. Ethyl alcohol, 1.96% (v/v), decreased cell populations and size, but the cells remained actively motile in broth plus the alcohol for 96 hr. In 72 hr, neither toxin (ca. 2 ppm) in combination with ethyl alcohol had more inhibitory effect on cell numbers, with or without nutrients, than was produced by alcohol alone. Aflatoxin B1 had no observed effect on the viability of the starved cells for 30 hr or on the nourished cells for 72 hr. There was no noticeable effect of G1 on the starved cells in 30 hr or on the nourished cells in 48 hr. After 72 hr with GI plus nutrients, many of the cells were round with blisters, nonmotile, and apparently dead or dying. Exploratory experiments in this laboratory showed that extracts of two substances containing aflatoxin (a Brazilian toxic peanut meal and a diet toxic to fish), and a crude aflatoxin extract, all known to cause hepatomas or other deleterious conditions in animals, enhanced the growth of Tetrahymena pyriformis W. Therefore, it seemed reasonable to expect that this organism might degrade aflatoxins. This is a report of this effect with some information on the growth response of the organism to pure aflatoxins B1 and G1. Aflatoxin has been shown to be partially degraded by fungi common in peanut pods and kernels, the amount degraded being governed by the initial concentration of the toxin (2). Aflatoxin was also degraded in a culture of Aspergillus flavus containing B1 and B13 when the inoculum was well dispersed, the culture was highly aerated, and when the mycelium was lysed (5). Aflatoxins were removed from a liquid medium (E. B. Lillehoj et al., Bacteriol. Proc., p. 5, 1966), and from aqueous solutions, milk, and naturally contaminated seeds (E. B. Lillehoj, A. Ciegler, and H. H. Hall, Proc. Ann. Meeting Am. Soc. Plant Physiol., p. lxxvi, 1966); they were detoxified in milk, oil, peanut butter, peanuts, and corn, and were partially detoxified in contaminated soybean (4) by Flavobacterium aurantiacum. Aflatoxin B, was partially transformed to new fluorescing compounds by some molds and mold spores (4) and was reduced by Nocardia asteroides (1) Ȧflatoxins have been reported to have a deleterious effect on growth or on morphology of animal cells such as rat fibroblasts (6), monkey kidney cells (11), calf kidney cells (8), human liver and HeLa cells (7), and cultured embryonic lung cells (9). They also have an adverse effect on microbial cells including A. flavus and Mucor (13), Escherichia coli (13; J. Wragg, and M. Legator, personal communication), F. aurantiacum (11), and strains of Streptomyces and Nocardia (1). Growth of an unidentified protozoan (3) was not inhibited by 30,ug of crude, mixed aflatoxins (36% pure) per ml of broth. MATERIALS AND METHODS Aflatoxin preparationi. Crystalline aflatoxin B, or G, was dissolved in chloroform in a 10-mi volumetric flask and made up to volume with chloroform. Samples were transferred to a 25-ml glass-stoppered Erlenmeyer flask, and the chloroform was evaporated under a stream of nitrogen. The toxin was dissolved in ethyl alcohol by gently warming on a steam bath. After cooling, 1 ml of the alcoholic toxin solution was added to 50 ml of medium. Previously, it was determined that the test organism survived in this concentration of alcohol. Cell preparation and assay. The test organism was 1099

2 1100 TEUNISSON AND ROBERTSON APPL. MICROBIOL. maintained in proteose-peptone broth (ph 7.1 prior to autoclaving), and the inoculum was prepared and used by the methods previously described by Teunisson (12), with slight modifications. The cells were exposed to the toxins with and without nutrients. For the assay of cells with nutrients, the organism was grown in 50 ml of broth in 300-ml De Long flasks without shaking for 3 days before addition of the toxins. The flasks were covered aseptically with sterile polypropylene film (4 mils thick) secured with rubber bands, and were incubated at 25 i 1 C before and after addition of the toxins. After adding toxins, the cultures were incubated on a reciprocating shaker (approximately 62 to 63 strokes/min). For the assay of cells without nutrients, the organism was grown in four stationary cultures, each consisting of 300 ml of broth in a 1-liter wide-mouth Erlenmeyer flask, covered as noted above and incubated at 25 1 C for 4 days. The cells were then harvested aseptically (without microscopically visible injury) by cooling and sedimentation. The flask cultures were placed in an ice bath until the cells settled, and then each suspension was transferred aseptically to a chilled, 500-mi separatory funnel. The sediments from these funnels were each washed with ca. 60 ml of sterile, cold phosphate buffer, ph 7.15 (175,ug each of KH2PO4 and K2HPO4/Ml) in cold 250-ml separatory funnels. The sedimented cells were resuspended in buffer to about 550 ml in a sterile, graduated bottle. Subsamples were taken for a microscopic examination of the living cells and for a count of preserved cells. The washed-cell suspension was dispensed aseptically in 50-ml lots into 300-ml De Long flasks, and toxins in ethyl alcohol or ethyl alcohol alone was added. These suspensions were covered and incubated on a shaker as noted above. The numbers of organisms were determined by visual counting of preserved cells (12) on at least 36 mm2 areas in a hemocytometer for each subsample tested. Each subsample was from one flask for each incubation period. Aflatoxin analysis. For aflatoxin determination, 50 ml of chloroform was added to the flasks containing the cells and aflatoxin in 50 ml of phosphate buffer or proteose-peptone broth. The contents of one flask were analyzed for each incubation period. The flasks were swirled gently to effect extraction of the toxin. The contents of each flask were transferred to a 250-ml separatory funnel, rinsing the flask with chloroform from a wash bottle. The separatory funnel was inverted gently six to eight times, and the phases were allowed to separate. Vigorous shaking was avoided because it results in the formation of an emulsion. The lower chloroform layer was drawn off through ca. 40 g of anhydrous sodium sulfate in a Butt extractor tube. The chloroform extract was collected in a 300-ml round-bottom flask. The extraction was repeated twice more with 50-ml portions of chloroform. After the last extraction the sodium sulfate was rinsed with approximately 10 ml of chloroform. The combined chloroform extracts were evaporated almost to dryness with a rotary evaporator, and then were quantitatively transferred to a 25- or TABLE 1. Effect of actively growing Tetrahymena pyriformis W on aflatoxins B1 and G1 plus nutrients, and its growth responsea Time of Growth response Substrate incuba- Toxin tion Cells/ml P DCb hr jig Controlc Od X X 106f X Aflatoxin Od 120 Ble X >X Gle X X a One flask of cells and broth was analyzed for each incubation period. b Percentage of joined pairs of dividing cells. c Alcohol was not added to control cultures. See Table 3. d Cells 3 days old in 50 ml of glucose, proteosepeptone broth. e Toxins in 1 ml of 95% ethyl alcohol were added at 0 hr of incubation. growth response deter- f Subsample taken for mination from same culture sampled at 0 hr. g Very actively motile cells. 50-ml volumetric flask and made up to volume with chloroform. The aflatoxin concentration of the chloroform solution was estimated by the thin-layer chromatographic technique described by Robertson et al. (10), except that the plates were developed with 15% acetone in chloroform in an unlined developing chamber. RESULTS AND DISCUSSION Effect ofthe cells on the aflatoxins. The effect of initially actively growing T. pyriformis W on aflatoxins B1 and G1 with nutrients present is shown in Table 1. The aflatoxin B1 concentration was decreased 58% in 24 hr and 67% after 48 hr of incubation. After both of these periods of incubation with T. pyriformis W, a bright-blue fluorescent compound was produced from aflatoxin B1. On a silica gel thin-layer chromatography plate developed with chloroform-acetone-2-propanol (825:150:25), the compound had an RF of 0.52 as compared with an RF of 0.59 for aflatoxin B1 and an RF of 0.55 for aflatoxin B2. This unidentified spot had a fluorescent intensity approximately one-half that of the unchanged aflatoxin B1 spot.

3 VOL. 15,y 1967 AFLATOXIN DEGRADATION BY T. PYRIFORMIS TABLE 2. Effect of Tetrahymena pyriformis W without nutrients on aflatoxins B1 and G1, and its growth responsea Time of Substrate incuba- Toxin Growth response tioii Cells/ml PDC6 hr jag Controlc Od X X 106e X 106f 0 Bic X X 106e X Gic X X 106e X One flask of cell suspension was analyzed for each incubation period. b Percentage of joined pairs in dividing cells. c Plus 1 ml of 95% ethyl alcohol. d Cells 4 days old washed and suspended in 50 ml of buffer. e Very actively motile cells. f At 96 hr after addition of alcohol, the cells were still viable but only sluggishly motile. T. pyriformis W appeared to have no effect on aflatoxin G1. The effect of starved cells on aflatoxin B1 and G, is shown in Table 2. There was a 50% decrease in the aflatoxin B1 concentration after 10 hr of incubation with starved T. pyriformis cells and a further 25% decrease after 30 hr of incubation. The unknown bright-blue fluorescent compound, noted in the previous experiment, again was found at each of the three incubation times. Extracts containing this unknown fluorescent compound from each of the incubation times from both experiments were combined and passed through a silica gel column to separate the unknown fluorescent component from the unchanged aflatoxin B1, with chloroform containing 0.5% ethyl alcohol as the solvent. The fractions containing the unknown compound which eluted from the column ahead of aflatoxin B1 were combined, the solvent was evaporated, and the dried extract was dissolved in methanol. The ultraviolet absorption spectrum of the compound as shown in Fig. 1 exhibited maxima at 253, 261, and 328 m,4. The spectrum z 0 a- 0 C,) co X(mIa) FIG. 1. Ultraviolet spectrum of the unknown compoundformed by Tetrahymena pyriformis Wfrom pure aflatoxin B1. was obtained with an automatic recording spectrophotometer. There appeared to be a 20% decrease in the aflatoxin G1 concentration after 10 hr of incubation with T. pyriformis. However, there was no further decrease after 22 and 30 hr of incubation. This decrease may be due to experimental variation, since the 0-hr control was diluted directly rather than being added to 50 ml of buffer and then extracted and diluted. There were also no detectable degradation products of aflatoxin G, noted on the thin-layer chromatography plates. Effect of the aflatoxins on the cells. The growth response of the cells to the toxins with nutrients is shown in Table 1 and that of the starved cells is shown in Table 2. It was desired to assay the aflatoxins in ethyl alcohol solution at as high a concentration as was feasible. Results of exploratory work showed that the toxins should be in solution. The concentration of toxins used was the approximate maximal amount of each that was soluble in 1 ml of 95% ethyl alcohol. Results of preliminary assays showed that washed cells remained viable and actively motile in buffer plus this concentration of the solvent for at least one day. To determine the effects of the toxins in alcohol on the test organisms, one must compare the results obtained with these substrates with those obtained with alcohol alone.

4 1102 TEUNISSON AND ROBERTSON APPL. MICROBIOL TABLE 3. Response ofactively growing Tetrahymena pyriformis W to 1.96% ethyl alcohol and to pure aflatoxins B1 and G1 plus nutrientsa Ethyl Aflatoxins Time of alcohol incuba- Cells/ml PDCb 95% Type Amt tion ml JAg hr None None - Oc 0.38 X X X 106d,e 0 1 None _ X X X 10 6d X B X X X 106d 0 1 GI X X X 106f 0 a One flask of cells was analyzed for each incubation period. b Percentage of joined pairs of dividing cells. Cells 3 days old in 50 ml of glucose, proteosepeptone broth. d Very actively motile cells. e Subsample for count taken from same culture sampled at 48 hr. f Round, nonmotile cells with blisters present. Alcohol was omitted from the first control cultures of nourished cells (Table 1). The results of another set of control cultures of cells with nutrients (Table 3) show that ethyl alcohol inhibited increase in cell populations. The cells observed after 48 hr of incubation were also smauer than in the control cultures without alcohol. The cells with alcohol were still actively motile after 96 hr. Starved cells in the buffer plus alcohol were still viable, but only sluggishly motile, after 96 hr. A comparison of the results obtained with the addition of alcohol alone and alcohol plus the toxins (Table 3) shows that the aflatoxins had no effect on cell numbers for the periods reported. Aflatoxin B1 had no observed effect on the viability of the nourished cells (6-day-old) for 72 hr or on the starved cells for 30 hr. There was no noticeable effect of G1 on nourished cells after 48 hr or on the starved cells in 30 hr. However, after 72-hr exposure to G1 with nutrients, many of the cells (6-day-old) were round, with blisters, nonmotile, and apparently dead or dying. It is not known whether this abnormality is due to the G, or perhaps to an interaction of G1 with some other component of the medium. The effect of either toxin on starved cells in 72 hr is not known. The results of this study of the effects of approximately 2,ug of aflatoxin B1 per ml on ca. 44 X 104 nourished T. pyriformis W cells per ml and ca. 22 X 104 starved cells per ml are different from those reported for cell tissue cultures exposed to known concentrations of toxins. Smith (11) reported distinct vacuolation for monolayer growth of monkey kidney cells exposed to 0.03,ug of B1 per ml of growth medium and cell destruction and almost complete inhibition of growth with 3.0,ug of this toxin per ml for unspecified periods. Daniel (6) showed that 1,ug of mixed B1 (36 li 2%), G1 (52 i 2%), B2 (3%), and G2 (2%) per ml of culture medium killed all the rat fibroblasts in a culture within 48 hr in a preliminary experiment. In her assay for aflatoxin, she used a final cell density of 5 X 104 to 10 X 104 cells per ml, and found that as little as 0.02 MAg of the mixed toxin had a clearly demonstrable cytotoxicity. Gabliks et al. (7) found that 1 to 5 Mg of B1 destroyed human liver and HeLa cells, as well as primary cell cultures of whole duck and chick embryos. In summarizing the results of studies by his group, Legator (9) reported vacuolation, abnormal production of giant cells, and an inhibition of mitosis of human embryonic lung cells, when ca. 104/ml were exposed for more than 48 hr to 0.05, 0.1, 0.5, and 1.0 ppm of mixed aflatoxins (15% B1, 9% G1, and less than 1% B2 and G2). The final concentration used was based on the known amounts of B1 and G, in the mixture. At 5.0 ppm, only a negligible amount of growth was found up to 90 hr. The control cells divided at a rapid rate with a mitotic index of 5.4%. A 43% reduction in mitosis was observed with crystalline B1 at 0.5 ppm. Approximately 2 ppm of pure aflatoxins B1 and G, had no significant effect in 48 hr on the mitosis of T. pyriformis W, based on results of analyses of total populations and percentage of dividing cells, and on observations of unstained cells at 100 times magnification. In 72 hr, aflatoxin G, had a toxic effect on nourished cells of this organism, many of the cells being round, nonmotile, and with blisters, whereas the nourished cells treated with B1 for the same period were still actively motile. Lillehoj et al. (Bacteriol Proc., p. 5, 1966) found that in a much higher concentration, 20 MAg of aflatoxin per ml (type not stated), cells of F. aurantiacum developed aberrant

5 VOL. 15, 1967 AFLATOXIN DEGRADATION BY T. PYRIFORMIS 1103 morphological forms. Lillehoj, Ciegler, and Hall (Proc. Ann. Meeting Am. Soc. Plant Physiol., p. lxxvi, 1966) reported that ceus of this organism grown in the presence of 5 ppm or more of B1 developed these aberrant forms, and they suggested that the toxin was interfering with cell wall synthesis. In summary, T. pyriformis W can degrade pure aflatoxin B1, but this compound had no marked observable effect of increasing or decreasing population growth or cell division. This organism did not degrade pure aflatoxin G1 under the conditions tested, but the substrate containing this compound did have a toxic effect on the cells. It is not known whether the abnormal morphology is due to the G1, or to an interaction of GI with some other component of the medium. ACKNOWLEDGMENTS We thank Leo A. Goldblatt, Chief of Oilseed Crops Laboratory, and Frank G. Dollear, Head, Peanut Products Investigations of the Oilseed Crops Laboratory, Southern Utilization Research and Development Division, U.S. Department of Agriculture, for their advice and encouragement and for very helpful suggestions in the preparation of the report. LITERATURE CITED 1. ARAI, T., I. TATSUYA, AND Y. KOYAMA Antimicrobial activity of aflatoxins. J. Bacteriol. 93: ASHWORTH, L. J., JR., H. W. SCHROEDER, AND B. C. LANGLEY Aflatoxins: Environmental factors governing occurrence in Spanish peanuts. Science 148: BURMEISTER, H. R., AND C. W. HESSELTINE Survey of the sensitivity of microorganisms to aflatoxin. Appl. Microbiol. 14: CIEGLER, A., E. B. LILLEHOJ, R. E. PETERSON, AND H. H. HALL Microbial detoxification of aflatoxin. Appl. Microbiol. 14: CIEGLER, A., R. E. PETERSON, A. A. LAGODA, AND H. H. HALL Aflatoxin production and degradation by Aspergillus flavus in 20-liter fermentors. Appl. Microbiol. 14: DANIEL, M. R In vitro assay systems for aflatoxin. Brit. J. Exptl. Pathol. 46: GABLIKS, J., W. SCHAEFFER, L. FRIEDMAN, AND G. WOGAN Effect of aflatoxin B, on cell cultures. J. Bacteriol. 90: JUHASZ, S., AND E. GRECZI Extracts of mould-infected groundnut samples in tissue culture. Nature 203: LEGATOR, M Biological effects of aflatoxin in cell culture. Bacteriol. Rev. 30: ROBERTSON, J. A., JR., L. S. LEE, A. F. CUCULLU, AND L. A. GOLDBLATr Assay of aflatoxin in peanuts and peanut products using acetonehexane-water for extraction. J. Am. Oil Chemists Soc. 42: SMITH, R. H The influence of toxins of Aspergillus flavus on the incorporation of (C14) leucine into proteins. Biochem. J. 88: 50P-51P. 12. TEUNISSON, D. J Microbiological assay of intact proteins using Tetrahymena pyriformis W. I. Survey of protein concentrates. Anal. Biochem. 2: WILDMAN, J. D Note on occurrence of giant cells in Aspergillus flavus Link. J. Assoc. Offic. Agr. Chemists 49: