Oct 20, 1980 - HANS-DIETER HAUBECK,' GERHARD LORKOWSKI,2 ECKEHART KOLSCH,1 .... Creppy, E.-E., M. Schlegel, R. Roschenthaler, and G.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1981, p. 1040-1042
Vol. 41, No. 4
0099-2240/81/041040-03$02.00/0
NOTES Immunosuppression by Ochratoxin A and Its Prevention by Phenylalanine HANS-DIETER HAUBECK,' GERHARD LORKOWSKI,2 ECKEHART KOLSCH,1 AND ROBERT ROSCHENTHALER2* Institutes of Immunologyl and Microbiology,' University of Muenster, D-4400 Muenster, West Germany Received 20 October 1980/Accepted 26 January 1981
Ochratoxin A, a toxin of Aspergillus ochraceus, suppresses the immune reto sheep erythrocytes in BALB/c mice at doses as low as 0.005 pg/kg of body weight. This effect is prevented by phenylalanine.
sponse
Ochratoxin A (OT-A) has been shown to inhibit protein synthesis and, to a certain extent, ribonucleic acid synthesis in bacteria (8, 14). In hepatoma tissue culture cells, immediate inhibition of protein synthesis was observed, followed by inhibition of RNA synthesis after a lag of 2 h (5). In vitro, OT-A is a competitive inhibitor of phenylalanyl transfer ribonucleic acid synthetase from bacteria (12), yeast cells (4), and rat and guinea pig livers (E.-E. Creppy et al., unpublished data). In a cell-free peptidesynthesizing Bacillus stearothermophilus system, no inhibition was observed when charged Phe-transfer ribonucleic acid was added in excess to the reaction mixture, whereas when uncharged transfer ribonucleic acid was added, competitive inhibition of peptide synthesis by OT-A was found (1). Inhibition of growth and protein synthesis by OT-A in hepatoma tissue culture cells could be reversed by the addition of phenylalanine (5). In mice poisoned with a 100% lethal dose of OT-A, an immediate intraperitoneal injection of phenylalanine prevented the lethal effect (6, 7). Furthermore, the inhibition of macrophage migration by OT-A could be prevented or attenuated by phenylalanine, whereas this was not the case when citrinin, a toxin of Penicillium citrinum, was used as an inhibitor (W. Klinkert, G. Lorkowski, E. E. Creppy, G. Dirheimer, and R. Roschenthaler, Toxicol. Eur. Res., in press). Here, we report the strong inhibition by OTA of induction of an immune response in mice, which can be overcome through application of phenylalanine. OT-A was prepared from wheat kernels infected with Aspergillus ochraceus NRRL 3174, generously provided by A. Ciegler, Peoria, Ill. Isolation and purification were done as previ-
ously described (2) by chromatography on Sephadex LH 20 (Pharmacia, Uppsala, Sweden) and on silica gel columns (E. Merck, AG, Darmstadt, Germany). The animals used were 8- to 12-week-old BALB/c inbred mice obtained from Blomholdgard Ltd. (Ry, Denmark). Each group consisted of two animals. Sheep red blood cells (SRBC) (BehringWerke, Marburg, Germany) were used as antigens. They were washed four times in phosphate-buffered saline (containing 0.14 M NaCl and 0.003 M KCl in 0.008 M sodium phosphate buffer [pH 7.2]) before use. The mice were immunized by intraperitoneal injection of 2 x 108 SRBC in 0.5 ml of phosphatebuffered saline. The anti-SRBC response was assayed by the Jerne plaque test (11) by a direct hemolytic plaque-counting technique on day 5 after immunization. Thus, an essentially immunoglobulin M antibody response was measured. In the first experiment, the respective mice received single doses of OT-A intraperitoneally in 0.2 ml of phosphate-buffered saline, in addition to SRBC. In the second experiment, some groups of mice received intraperitoneal doses of phenylalanine in phosphate-buffered saline in addition to SRBC and OT-A. OT-A is a potent immunosuppressive agent (Fig. 1). Induction of the anti-SRBC antibody response was still suppressed by 50% after injection of only 0.005 ,ug of OT-A per kg per mouse. The animals from this stock were more sensitive than those in the next experiment, which originated from another stock. From the dose dependence curve, it can be deduced that even a lower concentration than that used may have a certain effect on the immune system. In the next experiment, the ability of phenyl-
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alanine to prevent immunosuppression by OT-A 1.6 was tested. The mice received OT-A either alone or in combination with phenylalanine. When the concentration of phenylalanine was approxi100 1.4mately twice that of OT-A (wt/wt), no immunosuppressive effect was observed or the effect .11 n was strongly attenuated (Fig. 2). Control mice 1.2k injected with different concentrations of phena 80 5 a b ylalanine alone did not show suppression (data Lfl 0 not shown). The effect of phenylalanine was 1.0k z demonstrated in two independent experiments, -J -4 llJ and the effect of OT-A on immunosuppression 60 Ulw 0.8 I was demonstrated in three others. r, The finding that a dose of 0.005 ,ug/kg suffices mLaD -i w in young adult mice for a immunosuppression of 0.6V approximately 50% seems to be of importance. -if Hult et al. (9) have reported that about 16% of a large number of blood samples from slaughter 0.4 H pigs in Sweden contained OT-A concentrations Ii. varying between 2 and 187 ng/ml. This corre20 aI 0.2 V sponds to roughly 2 to 187 ,ug/kg of blood. Therefore, the concentration of OT-A in the blood of such animals may exceed the necessary concentration for significant immunosuppression by several orders of magnitude. This would even FIG. 2. Prevention of OT-A-induced immunohold true if one assumed that almost all of the suppression by phenylalanine. Antibody producing OT-A injected intraperitoneally would end up in cells (plaque-forming cells [PFCs]) per 1i1' spleen -
.
x
u
In
cells were counted 5 days after immunization of the mice with SRBC. (a) Mice received, in addition, I pg of OT-A (white column) or I pg of OT-A plus 2 pg of phenylalanine (black column). (b) Procedure as in (a), but mice received 0.01 pg of OT-A (white column), or 0.01 pg of OT-A plus 0.02 pg of phenylalanine (black column). (c) Controls; SRBC only.
the blood plasma bound to serum albumin (3) and would thus raise the concentration in the z blood of the mice. Hult et al. (10) have further shown that the Q. disappearance of low doses of OT-A from the blood plasma is slow. If such low doses were still -i 0 able to impair immunity, this would be of contLL sequence for human nutrition, especially since (I such concentrations apparently do not exhibit other readily observable toxic effects. Richard et al. (13), on the other hand, reported that there was no difference between the antibody levels of guinea pigs given OT-A and the controls when the animals were immunized with Brucella abortus antigen. However, in contrast to our experiments, these animals were fed per os. This discrepancy could therefore be due to 1.15 15 lggkg mouse the preventive effect of phenylalanine in the Ochratoxin A animal food. Preliminary results (Creppy et al., FIG. 1. Dose dependence of suppression of the im- unpublished data) concerning the protective efmune response to SRBC by ochratoxin A. BALB/c mice received a single intraperitoneal injection of the fect of phenylalanine in the food of mice seem dose given in the abscissa. Antibody response on day to support this notion. Alternatively, there could 5 is expressed as plaque-forming cells (PFCs) per lt' be a difference in the OT-A sensitivity of the immune response to thymus-independent (B. spleen cells. 3
w w
U,
-
5
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abortus) and thymus-dependent (SRBC) antigens. The fact that phenylalanine is able to prevent immunosuppression by OT-A is interesting not only from a practical, but also from a theoretical, point of view. This effect could be expected if the mechanism of action of OT-A operates in the manner we have proposed (4, 6, 7). If the toxic effect of OT-A in animals is due to competitive inhibition of phenylalanyl transfer ribonucleic acid synthetase, a rise in the substrate concentration, i.e., of phenylalanine, should reverse the inhibition. The mode of action of OTA in animals can thus be verified if treatment with phenylalanine overcomes its biological effects. In other papers, we have shown reversive, preventive, or attenuating effects of phenylalanine (i) on inhibition by OT-A of growth and protein synthesis in hepatoma tissue culture cells (5), (ii) on lethal effects on mice of a 100% lethal dose of OT-A (6, 7), and (iii) on inhibition of guinea pig macrophage migration (Klinkert et al., Toxicol. Eur. Res., in press). We have presented here another animal system in which phenylalanine counteracts the effect of OT-A. We consider this to be a further confirmation that the toxic effect of OT-A in animals is mainly due to inhibition of phenylalanine transfer ribonucleic acid synthetase. This study has been supported by grants Ro 291/6 and Ko 379/10 from the Deutsche Forschungsgemeinschaft.
LITERATURE CITED 1. Bunge, I., G. Dirheimer, and R. Roschenthaler. 1978. In vivo and in vitro inhibition of protein synthesis in Bacillus stearothermophilus, by ochratoxin A. Biochem. Biophys. Res. Commun. 83:398-405.
APPL. ENVIRON. MICROBIOL. 2. Bunge, L, K. Heller, and R. Roschenthaler. 1979. Isolation and purification of ochratoxin A. Z. Lebensm. Unters. Forsch. 168:457-458. 3. Chu, F. S. 1971. Interaction of ochratoxin A with bovine serum albumin. Arch. Biochem. Biophys. 147:359-366. 4. Creppy, E.-E., A. A. J. Lugnier, F. Fasiolo, K. Heller, R. Roschenthaler, and G. Dirheimer. 1979. In vitro inhibition of yeast phenylalanine tRNA synthetase by ochratoxin A. Chem. Biol. Interactions 24:257-261. 5. Creppy, E.-E., A. A. J. Lugnier, R. Rischenthaler, and G. Dirheimer. 1979. Action of ochratoxin A on cultured hepatoma cells-reversion of inhibition by phenylalanine. FEBS Lett. 104:287-290. 6. Creppy, E.-E., M. Schlegel, R. Roschenthaler, and G. Dirheimer. 1979. Action pr6ventive de la phenylalanine sur l'intoxication ague par l'ochratoxine A. C. R. Acad. Sci. Paris Ser. D 289:915-918. 7. Creppy, E.-E., M. Schlegel, R. R6schenthaler, and G. Dirheimer. 1980. Phenylalanine prevents acute poisoning by ochratoxin A in mice. Toxicol. Lett. 6:77-0. 8. Hefler, K., and R. R6schenthaler. 1978. Inhibition of protein synthesis in Streptococcus faecalis by ochratoxin A. Can. J. Microbiol. 24:466-472. 9. Hult, K., E. Hokby, S. Gatenbeck, and L. Rutqvist. 1980. Ochratoxin A in blood from slaughter pigs in Sweden: use in evaluation of toxin content of consumed feed. Appl. Environ. Microbiol. 39:828-30. 10. Hult, K., E. Hokby, U. Hagglund, S. Gatenbeck, L. Rutqvist, and G. Sellyey. 1980. Ochratoxin A in pig blood: a method of analysis and use as a tool for feed studies. Appl. Environ. Microbiol. 38:772-776. 11. Jerne, N. K., A. A. Nordin, and C. Henry. 1963. The agar plate technique for recognizing antibody producing cells, p. 109. In B. Amos and H. Koprowski (ed.), Cellbound antibody. Wistar Institute Press, Philadelphia. 12. Konrad, I., and R. Roechenthaler. 1977. Inhibition of phenylalanine tRNA synthetase from Bacillus subtilis by ochratoxin A. FEBS Lett. 83:341-347. 13. Richard, J. L., J. R. Thurston, B. L. Deyoe, and G. D. Booth. 1975. Effect of ochratoxin and aflatoxin on serum proteins, complement activity, and antibody production to Brucella abortus in guinea pigs. Appl. Microbiol. 29:27-29. 14. Singer, U., and R. Roschenthaler. 1978. Induction of autolysis in Bacillus subtilis by ochratoxin A. Can. J. Microbiol. 24:563-568.
