Comparison of the Toxicities of Patulin and Patulin Adducts

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Neither patulin nor the adduct mixture was found to induce the repair effect in. E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1978, p. 1003-1007 0099-2240/78/0035-1003$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 35, No. 6

Printed in U.S.A.

Comparison of the Toxicities of Patulin and Patulin Adducts Forned with Cysteine SEPPO LINDROTH* AND ATTE VON WRIGHT Technical Research Centre ofFinland, Food Research Laboratory, SF-02150 Espoo 15, Finland Received for publication 7 December 1977

The toxicities of patulin and of the patulin adducts forned with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polAU and its polAl+ revertant. The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice. The adduct mixture was shown by thinlayer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components. The results showed that patulin was over 100 times more toxic to E. coli than the adduct complex. Neither patulin nor the adduct mixture was found to induce the repair effect in E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg.

Patulin is a secondary metabolite of several species of the genera Aspergillus and Penicillium and of the species Byssochlamys nivea (7, 10, 14). It is toxic to some degree to all bacterial species tested irrespective of Gram type (15). The oral, subcutaneous, intraperitoneal, and intravenous toxicities of patulin to various test animals have been investigated by several authors (2, 6, 8, 9). Patulin has also been shown to possess mutagenic (10) and carcinogenic properties (4). Patulin reacts with sulfhydryl compounds, such as cysteine, glutathione, and thioglycholate, to form adducts (3, 5, 6, 12, 13). It has been suggested that the toxicity of patulin may result from its reaction with the sulfhydryl groups of the active sites of enzyme molecules. Singh (13) on the other hand maintained that the truly toxic agent may be some modified form of patulin rather than the parent compound itself. Geiger and Conn (5) and Rinderknecht et al. (12) have reported that an excess of sulflfydryl compounds partially inactivates the bactericidal effect of patulin. Hofmann et al. (6) showed that the adduct mixture formed by patulin with glutathione is not toxic or has only a slight toxicity to mice, chicken embryos, or rabbit skin at concentrations corresponding to the 50% lethal dose (LD5o) of patulin itself. Ciegler et al. (3) did not observe lethal effects when patulin cysteine adduct mixture was injected intraperitoneally at levels corresponding to 4 times the patulin LD5o using mice and 50 times the patulin LD5o using chicken embryos. The adduct mixture was, however, found to have a teratogenic effect on

chicken embryos. Previous research (3, 5, 6, 12) has shown that adducts of patulin and sulffhydryl compounds have toxicities lower than that of patulin itself. However, very little infornation is available concerning the relative toxicities of patulin and of the adducts, since experiments have usually been carried out using equal doses of patulin and adduct as patulin equivalents, with the exception of one experiment (3) in which patulin and adduct mixture were shown to have a teratogenic effect. The aim of the present research was to clarify the relative toxicities of patulin and patulin adduct formed with cysteine using Escherichia coli and mice as test organisms, and also to determine whether free patulin is liberated from the adduct by microbial action. MATERIALS AND METHODS Patulin. Patulin was produced and purified by the method of Nordstadt and McCalla (11). The melting point of the crystalline patulin prepared was 109 to 110.5°C. Purity of the toxin was also demonstrated by ultraviolet (UV) (Hitachi Perkin-Elmer 139 UV-VIS), mass (Jeol JMS-D 100), and nuclear magnetic resonance (Jeol JNM-PMX 60) spectrometries. Patulin determination. Patulin contents in the adduct mixtures and the bacterial liquid cultivations were determined by thin-layer chromatography. Samples of solutions were taken and extracted with three times the equivalent volume of ethyl acetate. The extract was dried with anhydrous sodium sulfate for 30 min, after which it was evaporated to dryness in a Rotavapor vacuum evaporator to remove the ethyl acetate. The residue was dissolved in a suitable volume of chloroform. This chloroform extract and 1- to 7-pl samples of a patulin standard (10 ,g of CHC13 per ml)

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were pipetted onto activated (2 h, 110°C) thin-layer plates (E. Merck silica gel 60). The plates were developed in saturated chambers using toluene-ethyl acetate-formic acid (50:40:10) as the driving solvent, after which the plates were dried and sprayed with 0.5% MBTH (3-methyl-2-benzothiazolinone hydrazone, Aldrich Chemicals) solution and heated to 130°C for 15 min. The toxin concentration of the test spot was assayed by visual comparison with the fluorescence intensity of the spots of standard solutions of patulin under long-wave UV illumination. The sensitivity of the method was 0.01 ,g of patulin per spot. Production of adduct. Equimolar amounts of crystalline patulin and cysteine (cysteine HCl- H20, E. Merck, in 0.1 M citric acid-0.2 M disodium phosphate buffer, pH 6.0) were mixed, and the pH of the reaction mixture was adjusted to 6.0 with sodium hydroxide. The reaction was allowed to continue for 30 min with continuous magnetic stirring, and was then terminated by extracting the free patulin with an amount of ethyl acetate corresponding to three times the volume of the reaction solution. The amount of patulin reacted with the cysteine was defined by assaying the titer of free patulin in the ethyl acetate extract and subtracting this amount from the initial amount of patulin added. The concentration of the adduct was expressed as an amount of reacted patulin per unit volume. Spectrophotometric and thin-layer chromatographic characterization of adduct. The UV spectrum of the adduct mixture purified from free patulin by ethyl acetate extraction was measured using a Hitachi Perkin-Elmer 139 UV-VIS spectrophotometer. For thin-layer chromatographic characterization of the adducts, spots of adduct mixture, patulin standard, and cysteine solution were pipetted onto thinlayer plates (E. Merck cellulose F, silica gel 60, and silica gel 60 F2u). The plates were developed using butanol-acetic acid-water (40:10:50, upper phase) or toluene-ethyl acetate-formic acid (50:40:10). The plates were examined under UV light (254 and 365 nm) before and after staining with 0.3% ninhydrin in a 2% solution of pyridine in acetone (110°C, 5 min) or with 0.5% MBTH (1300C, 15 min). Bacteriological toxicity tests. The toxicities of patulin and of patulin adduct formed with cysteine were compared in both plate and liquid cultivation tests. In the plate tests the strain pair E. coli W3110 thy poUA (requires thymine for growth, functional DNA polymerase 1 absent) and its poUAl+ revertant (1) was used. The cultivation medium was a mineral medium (M9) containing 50 ug of thymine per ml. A 3-ml volume of soft agar (45°C), containing 0.1 ml of an overnight cultivation of the test strain, was poured onto the test plate. After the overlay had solidified, a filter paper disk (diameter, 13 mm), to which had been added 50 1d of sample solution, was placed onto its surface. Citric acid-sodium phosphate buffer (pH 6.0), extracted with ethyl acetate, was used as a blank control. The repair effect was monitored using disks containing 0.5 g of methyl methanosulfonate. Patulin levels used in the disks were 3, 5, 7, 10, 20, 30, 40, and 50 ug, while those of the adduct were 190, 750, 1,310, 1,870, 2,480, 2,990, 3,550, and 4,110,ug of patulin equivalents. The plates with their filter paper disks were incubated for 24 h at 370C, after which the inhibition

APPL. ENVIRON. MICROBIOL.

zones resulting from the samples were measured for both test strains. Thymine-supplemented M9 medium was also used in the liquid cultivations. The test organism was E. coli W3110 thy polA. Before the test, cells were cultivated in the medium overnight, and this cultivation was inoculated into a new batch of the same medium so that the cell density was approximately 107 cells per ml. The inoculated cultivation medium was divided into 35-ml samples, and to each was added 5 ml of sample solution (patulin or adduct mixture). Ethyl acetate-extracted buffer was again used as blank control. The final concentrations of patulin and of patulin equivalents of adduct in the cultivation media were 1, 3, and 10 ug/ml and 20, 190, 1,940, and 4,840 ,ug/ml, respectively. The cultivations were incubated for 20 h at 37°C, during which time samples of 1.5 ml for assay of viable cells and 1.0 ml for assay of patulin were taken at 4-h intervals. Measurement of acute toxicity with mice. The mice used in these tests were NMRI male mice, weight 30 g, obtained from Oy Orion Ab, Finland. The test procedure was based on the method of Weil (17) for measurement of LD5o. The mice were fasted overnight prior to the test to facilitate dosing. The samples were administered orally as single doses using stomach intubation. The amount of sample solution per test animal was 0.5 ml, and five animals were used at each level of dosage. Ethyl acetate-extracted buffer was used as control. Dose levels of patulin were 8.8, 16.7, 33.3, and 66.6 mg/kg, and those of the adduct were 88, 265, 790, and 2,370 mg of patulin equivalents per kg. The subsequent observation period was 2 weeks, during which time the mice were free to eat and drink ad libitum. After the test a postmortem examination was made of all the test animals.

RESULTS Reaction of patulin with cysteine. Adduct formation took place rapidly when equimolar amounts of patulin and cysteine were mixed in 0.1 M citric acid-0.2 M disodium phosphate buffer (pH 6.0). After 20 and 30 min, 92 and 94%, respectively, of the patulin had reacted with the cysteine. The reaction product mixture was of yellow color, with a UV absorption maximum at or near 300 nm (patulin X,, 275 nm). During storage of the adduct as a dilute solution in buffer, the absorption maximum slowly disappeared, without alteration in the visual appearance of the solution. In thin-layer chromatography, the fresh adduct mixture was found, by development with butanol-acetic acid-water on cellulose plates, to contain two ninhydrinpositive and three ninhydrin-negative components. Using the same developing solvent on silica gel plates, one ninhydrin-positive, one ninhydrin- and MBTH-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative spots were observed. In toluene-ethyl acetateformic acid, the adduct mixture did not migrate from its point of application when using cellulose

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Voi. 35, 1978

plates. On silica gel plates one ninhydrin- and MBTH-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative spots were observed. Bacteriological toxicity tests. In the plate cultivation tests, 5 ,ug of patulin per filter paper disk was sufficient to cause observable inhibition zones with E. coli W3110 thy poIAl and poIlA+ (Fig. 1). The patulin cysteine adduct mixture was, as anticipated, very much less toxic than the free patulin (Table 1). The relative toxicity of adduct mixture and free toxin was 1:500 in these plate cultivations. The ethyl acetate-extracted buffer control did not produce an inhibition zone. The repair effect was not observed in this experiment. In fact, the poUlAI strain was slightly more sensitive than the polAl strain to both patulin and to the adduct. The methyl methanosulfonate used as positive control for the repair effect caused, in the case of the polAU strain, an inhibition zone 2 cm greater than that in the case of the poUlA+ strain, indicating that the repair properties of the two strains fulfilled the requirements of this test. In the liquid cultivations, a patulin concentration as low as 3 ,tg/ml caused a significant reduction in the viable cell count of the bacterial suspension (Fig. 2). The lowest concentration of adduct with bactericidal properties was correspondingly between 190 and 1,940 ug of patulin equivalents per ml (Fig. 3). The bacterial strains used were not found to liberate free patulin from the adduct present in the growth medium. On the basis of these bacteriological tests, it was found that the patulin cysteine adduct mixture was slightly bactericidal, but that its toxic-

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ity was less than 1% of that of free patulin. Mouse feeding tests. The oral LD50 of patulin for male NMRI mice was found to be 29 mg/kg (95% confidence boundaries, 22 and 38 mg/kg). Those mice to which the patulin dose was lethal all died within 2 days of dosage. The TABLE 1. Bactericidal effect ofpatulin adduct formed with cysteine on E. coli W3110 thy polAU andpoL41 strains Concn of adduct (ug of patulin

Diam of inhibition zone (mm)a

equivalents/

poIAl

disk)

poIAI+

Calculated concn (ug/disk) of free patulin causing the same effect'

poIAl

190 + + 750 0.5 1.8 1,310 2.0 2.0 1,870 3.2 6.0 2,430 4.0 6.7 2,990 5.1 7.0 3,550 5.0 7.2 4,110 -, No inhibitory effect detected;

poL41+

4.3 4.2 4.9 4.2 5.4 5.8 5.8 6.2 6.4 6.3 6.3 6.4 +, slightly inhib-

itory. b Values have been obtained from Fig. 1. 10

_8 6 4 0 _2

4 8 12 16 20 Incubation period (hours) 35

FIG. 2. Effect of patulin on the growth of E. coli W3110 thy polAl in M9 broth. SYmbols: (0) buffer; (E) 1, (-) 3, and (*) 10 pg ofpatulin per ml.

E 30 -0

0

C 26 0

10

20,.16

Pol

C

_

A1*'

Pol

Al

10.

IL.

'J 2e

0

o

8 4 16 20 12 Incubation period (hours)

U

3

5

7

10

20

30 4050

Patulin concontration

(po)

FIG. 1. Bactericidal effect of patulin on E. coli W3110 thy poLAI and polAl+ strains in the plate test.

FIG. 3. Effect of the patulin adduct formed with cysteine on the growth of E. coli W3110 thy polAU in M9 broth. Symbols: (0) buffer; (O) 20, (-) 190, (*) 1,940, and (*) 4,840 pg ofpatulin equivalents per ml.

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most obvious macroscopic pathological alteration was a swollen, fluid-filled stomach. Although the highest dose of patulin cysteine adduct contained 2,370 mg of patulin equivalents per kg of body weight, corresponding to 85 times the LD50 of free patulin, the adduct did not cause a single death in the mouse feeding tests. Furthermore, no macroscopic pathological symptoms were observed during postmortem examination of the mice receiving adduct (data not shown).

DISCUSSION The patulin cysteine adduct complex formed in citric acid-sodium phosphate buffer (pH 6.0) was found by thin-layer chromatography to be composed of two ninhydrin-positive and five ninhydrin-negative components. Ciegler et al. (3) have reported that a patulin cysteine adduct complex prepared in acetic acid-ammonium acetate buffer (pH 5.4) contained at least four ninhydrin-positive and two ninhydrin-negative products. The absorption maxima and colors of the adduct solutions were the same in both cases. The results of Ciegler et al. (3) and of the present work together show that the reaction of patulin with cysteine results in several reaction products, the nature of which depends on the test conditions. Patulin has been reported to induce the repair effect in rec mutants of Bacillus subtilis (16). The repair effect was not, however, observed in the present research using E. coli poUA mutants, so that possible DNA damage caused by patulin could not be observed. The patulin adduct formed with cysteine was shown in the bacteriological experiments carried out to be slightly bactericidal, although the cysteine strongly inhibited the toxicity of the patulin. Geiger and Conn (5) and Rinderknecht et al. (12) have shown with certain gram-positive and gram-negative bacterial species that patulin cysteine adduct mixture is slightly bacteriostatic. The oral LD50 of patulin obtained in this work was somewhat lower (29 mg/kg) than that reported in the literature (35 mg/kg) (2). This difference may well be due to the use of different mouse strains and different experimental conditions. By contrast, an adduct mixture dose containing reacted patulin corresponding to 85 times the LD50 of free patulin was not found to have any acute toxic effects on the test mice. On the basis of the results obtained as well as previous reports (3, 6), it is apparent that the acute toxicity of patulin decreases radically on reaction with compounds containing sulfhydryl groups. The patulin cysteine adduct complex used in this work was not found to release free

APPL. ENVIRON. MICROBIOL.

patulin as a result of microbial metabolism. Taking into account the extremely low toxicity of the adduct mixture to mice, it also seems unlikely that the adduct can be metabolized to produce free toxin in mammals. It therefore seems probable that adducts formed in food products between patulin and SH groups, in concentrations that might reasonably be expected to occur, have no short-term toxicity to humans. However, before final conclusions concerning the toxicity of patulin-sulfhydryl adducts, and before the acceptance of safety criteria imparted by addition of sulfhydryl compound to foodstuffs, further research should be carried out to determine the possible long-tern effects of the consumption of such adducts. ACKNOWLEDGMENTS We thank Auli Murrola and Pekka Salovaara for excellent technical assistance.

LITERATURE CITED 1. Bamford, D., M. Sorsa, U. Gripenberg, I. Laamanen, and T. Meretoja. 1976. Mutagenicity and toxicity of amitrole. III. Microbial tests. Mutat. Res. 40:197-202. 2. Broom, W. A., E. Biilbring, C. J. Chapman, J. W. F. Hampton, A. M. Thomson, J. Ungar, R. Wien, and G. Woolfe. 1944. The pharmacology of patulin. Br. J. Exp. Pathol. 25:195-207. 3. Ciegler, A., A. C. Beckwith, and L. K. Jackson. 1976. Teratogenicity of patulin and patulin adducts formed with cysteine. Appl. Environ. Microbiol. 31:664-667. 4. Dickens, F., and H. E. H. Jones. 1961. Carcinogenic activity of a series of reactive lactones and related substances. Br. J. Cancer 15:85-100. 5. Geiger, W. B., and J. E. Conn. 1945. The mechanism of the antibiotic action of clavacin and penicillic acid. J. Am. Chem. Soc. 67:112-116. 6. Hofmann, K., H. J. Mintzlaff, I. Alperden, and L. Leistner. 1971. Untersuchung uber die Inaktivierung des Mykotoxins Patulin durch Sulfhydrylgruppen. Fleischwirtschaft 51:1534-1536, 1539. 7. Karow, E. O., and J. W. Foster. 1944. An antibiotic substance from species of Gymnoascus and Penicillium. Science 99:265-266. 8. Katzman, P. A., E. E. Hays, C. K. Cain, J. J. van Wyk, F. J. Reithel, S. A. Thayer, E. A. Doisy, W. L. Gaby, C. J. Carrol, R. D. Muir, L. R. Jones, and N. J. Wade. 1944. Clavacin, an antibiotic substance from Aspergillus clavatus. J. Biol. Chem. 154:475-486. 9. Lovett, J. 1972. Patulin toxicosis in poultry. Poult. Sci. 51:2097-2098. 10. Mayer, W. M., and M. S. Legator. 1969. Production of petite mutants of Saccharomyces cerevisiae by patulin. J. Agric. Food Chem. 17:454-456. 11. Norstadt, F. A., and T. M. McCalla. 1969. Patulin production by Penicillium urticae Bainier in batch culture. Appl. Microbiol. 17:193-196. 12. Rinderknecht, H., J. L. Ward, F. Bergel, and A. L. Morrison. 1947. Studies on antibiotics. 2. Bacteriological activity and possible mode of action of certain nonnitrogenous natural and synthetic antibiotics. Biochem. J. 41:463-469. 13. Singh, J. 1967. Patulin, p. 621-630. In D. Gottlieb and P. D. Shaw (ed.), Antibiotics I: mechanism of action. Springer-Verlag, Berlin. 14. Sommer, N. F., J. R. Buchanan, and R. J. Fortlage.

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1974. Production of patulin by Penicilium expansum. Appl. Microbiol. 28:589-593. 15. Stott, W. T., and L B. Bullerman. 1975. Patulin: a mycotoxin of potential concern in foods. J. Milk Food Technol. 38:695-705. 16. Ueno, Y., and K. Kubota. 1976. DNA-attacking ability

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of carcinogenic mycotoxins in recombination-deficient mutant cells of Bacillus subtilis. Cancer Res. 36:445-451. 17. Weil, C. S. 1952. Tables for convenient calculation of median-effective dose (LDw or EDO) and instructions in their use. Biometrics 8:249-263.