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Phenylalanine prevents acute poisoning by ochratoxin-A in mice
Toxicology Letters, 6 (1980) 77-80 Q Elsevier/North-Holland Biomedical Press
PHENYLALANINE A IN MICE
PREVENTS
ACUTE POISONING
77
BY OCHRATOXIN-
EDMOND E. CREPPY, MONIQUE SCHLEGEL, ROBERT R&CHENTHALER* GUY DIRHEIMER
and
Institut de Biologie Mollculaire et Cellulaire de CNRS, I5 rue Descartes, Strasbourg (France) and Laboratoire de Toxicologic et de Biologie Molkculaires, Facults de Pharmacie, Universit& Louis Pasteur, Strasbourg (France), and *Institut fGr Mikrobiologie, Universitiit Miinster, Westfalen (FRG). (Received January 27th, 1980) (Accepted February 26th, 1980)
SUMMARY
Ochratoxin-A (OT-A) a mycotoxin isolated from Aspergillus ochruceus is toxic for animals and man causing a fatal chronic kidney disease and liver damages. OT-A is a competitive inhibitor of phenylalanine in the phenylalanyl-tRNA synthetase-catalyzed reaction thus inhibiting protein synthesis. This inhibition can be reversed by phenylalanine. When injected intraperitoneally (i.p.) to mice a dose of 0.8 mg of OT-A is lethal within 24 h. However, when this lethal dose is injected together with 1 mg of phenylalanine 100% of the animals survive. When phenylalanine was injected 30 min after OT-A the doses required for the survival of 92% of the animals had to be about 25 times higher.
INTRODUCTION
OT-A, a mycotoxin isolated from Aspergillus ochraceus is composed of a 3,4-dihydro-3-methylisocoumarin moiety linked by an a-amide bond to L-& phenylalanine. The isocoumarin is chlorinated in the 5’ position [l] . OT-A is toxic for animals, causing kidney and liver damage [2] . There is evidence to support the hypothesis that it is a disease determinant of Balkan endemic nephropathy, a fatal chronic kidney disease affecting rural populations in Bulgaria, Romania and Yugoslavia [3] . OT-A inhibits protein synthesis by competition with phenylalanine in the phenylalanyl-tRNA synthetase catalysed reaction [4, 51. We have also been able to demonstrate that phenylalanine is capable of reversing the toxic effect of OT-A in HTC cells [6] . Abbreviations:
HTC, hepatoma tissue culture; OT-A, Ochratoxin-A.
78
In view of the practical importance of OT-A to the health of livestock [ 71 and possibly even of man [3] , we consider it of great interest to find out if phenylalanine is capable of preventing acute OT-A poisoning. Our results demonstrate a high survival rate of OT-A-poisoned mice when these animals were treated with phenylalanine by i.p. injection. MATERIAL
AND METHODS
The conditions for OT-A isolation have been described elsewhere [ 81. Conventional Swiss mice of 19.5 to 20.5 g weight (C.E.S.A.L., France) were randomly distributed in lots of 10 animals. They were injected i.p. with a dose of 0.8 mg OT-A per mouse (i.e., 40 mg/kg), which was initially established to be 100% lethal within 24 h. For all the subsequent experiments this dose was applied. Control animals without toxin were injected with saline containing various amounts of phenylalanine as indicated. The toxin controls received OT-A solutions only, The total volume of the injections never exceeded 1 ml. RESULTS
AND DISCUSSION
Mice injected with a lethal dose of OT-A showed hypothermia, decreased motor-activity, and loss of appetite 1 h after injection. After 12-16 h severe diarrhoea occurred. All the animals died between 23 and 24 h after OTA injection. However, when this lethal dose was injected i.p. together with 0.8 mg of phenylalanine, 97% of the animals survived, and 100% survived when 1 mg of phenylalanine was injected (Table I). Thus phenylalanine doses, only slightly higher than the lethal doses of OT-A, can efficiently counteract the toxic effect of OT-A, when given simultaneously. The effect of these relatively low doses may be explained by the higher affinity of phenylalanine for the phenylalanyl-tRNA synthetase compared with OT-A, as already reported [ 51, Also in cell cultures, doses only slightly higher than those of OT-A were able to reverse the toxic action [6]. However, if phenylalanine was injected 30 min after OT-A, a dose about 10 times higher than that of OT-A was necessary for the survival of 85% of the animals, and a dose 25 times higher for the survival of 92% (Table I). Finally, if a dose of 8 mg of phenylalanine was injected 60 min after OT-A (0.8 mg), only 10% of the mice survived. Therefore the reversion of the OT-A effect observed in HTC cells 2 h after addition of the mycotoxin [6] was not found. Thus, phenylalanine can prevent the lethal poisoning by OT-A when given simultaneously with the mycotoxin, whatever the precise mechanism of the counteraction may be. It is conceivable that farm animals and even man may be protected from poisoning by OT-A, through supplementation of feed or food in contaminated commodities. Experiments to find out whether such a protection is feasible, are in progress.
79 TABLE I SURVIVAL OF POISONED MICE (19.5-20.5 DIFFERENT DOSES OF PHENYLALANINE 0.25
0.50
g) INJECTED WITH 0.8 mg OCHRATOXIN-A
Doses of phenyl~~ine simultaneously injected with OT-A per mouse (mg)
0
Percentage of survival after 24 h
0
80
90
97
100
100
100
100
100
Percentage of survival after 28 days
0
20
50
97
100
100
100
100
100
Doses of phenylalanine injected per mouse 30 min after OT-A injection (mg)
0
0.50
0.80
AND
1.0
1.0
5.0
5.0
8.0
8.0
10.0
20.0
10.0
20.0
Percentage of survival after 24 h
11
21
63
88
90
92
Percentage of survival
7
15
53
82
85
92
Phenytalanine (Merck, Darmstadt) or OT-A were dissolved in saline (0.9% NaCl in bidistilled water, adjusted to pH 7.2 with 0.1 M NaOH), and the solutions were sterilized by Mi~ipore filtration (SLGS 02505,0.22 pm pore size). Injection materials (1.0 ml hypodermic syringes and needles) were obtained from Terumo and were sterilized by ethylene oxide, 20 mg phenylalanine was injected by 3 injections: 10 mg, 30 min after OT-A and twice 5 mg at: 60 min and at 120 min; or at 120 min and at 240 min; or at 180 min and at 360 min. The percentages of survivors were approximately the same: 92%, 90% and 90%, respectively.
ACKNOWLEDGEMENTS
This research was supported by grants from NATO (research grant No. 1749), from the “Institut National de la Santi et de la Recherche Mddicale” (contract No. 79.1.154.3) and from the “Deutsche Forschungsgemeinschaft” (Ro. 291/5). REFERENCES K.J. van der Merwe, P.S. Steyn and L. Fourie, Mycotoxins, Part II. The constitution of ochratoxin A, B and C, metabolites of Aspergillus ochraceus wilh.; J. Chem. Sot., (1965) 7083-7093; K.J. van der Merwe, P.S., Steyn, L. Fourie, D.B. Scott and J.J. Theron, Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus wilh., Nature, 205 (1965) 1112-1113 J. Harwig, in I.F.H. Purchase (Ed.) Mycotoxins, Elsevier, Amsterdam, 1974, pp. 345-367. P. Krogh, B. Hald, R. Plestina and Ceovic, S. Nephropathy and foodborne Ochratoxiri A: preliminary results of a survey of foodstuffs, Acta Pathol. Microbial. Stand. Sect. B 85 (1977) 238-240
80
I. Bunge, G. Dirheimer, and R. Raschenthaler, In vivo and in vitro inhibition of protein synthesis in Bacillus stearothermophilus by Ochratoxin A, Biochem. Biophys. Res. Commun., 83( 1978) 398-405. 5 E.E. Creppy, A.A.J. Lugnier, F. Fasiolo, K. Heller, R. Rijschenthaler, and G. Dirheimer. In vitro inhibition of yeast phenylalanyl-tRNA synthetase by ochratoxin A, Chem. Biol. Interact., 24 (1979) 257-261. 6 E.E. Creppy, A.A.J. Lugnier, F. Fasiolo, K. Heller, R. Raschenthaler, and G. Dirheimer. In vitroof Ochratoxin A on cultured hepatoma cells. Reversion of inhibition by Ochratoxin A, FEBS Lett. 104 (1979) 287-290. 7 P. Krogh, In J.V. Rodricks, C.W. Hesseltine and M.A. Mehlman (Eds.), Mycotoxins in Human and Animal Health. Pathotox, Park Forest South, IL, 1977, pp. 489-498. 8 K. Heller, C. Schultz and R. Riischenthaler. The inhibition of bacterial growth of Ochratoxin A, Can. J. Microbial. 21 (1975) 972-979.
4
PHENYLALANINE A IN MICE
PREVENTS
ACUTE POISONING
77
BY OCHRATOXIN-
EDMOND E. CREPPY, MONIQUE SCHLEGEL, ROBERT R&CHENTHALER* GUY DIRHEIMER
and
Institut de Biologie Mollculaire et Cellulaire de CNRS, I5 rue Descartes, Strasbourg (France) and Laboratoire de Toxicologic et de Biologie Molkculaires, Facults de Pharmacie, Universit& Louis Pasteur, Strasbourg (France), and *Institut fGr Mikrobiologie, Universitiit Miinster, Westfalen (FRG). (Received January 27th, 1980) (Accepted February 26th, 1980)
SUMMARY
Ochratoxin-A (OT-A) a mycotoxin isolated from Aspergillus ochruceus is toxic for animals and man causing a fatal chronic kidney disease and liver damages. OT-A is a competitive inhibitor of phenylalanine in the phenylalanyl-tRNA synthetase-catalyzed reaction thus inhibiting protein synthesis. This inhibition can be reversed by phenylalanine. When injected intraperitoneally (i.p.) to mice a dose of 0.8 mg of OT-A is lethal within 24 h. However, when this lethal dose is injected together with 1 mg of phenylalanine 100% of the animals survive. When phenylalanine was injected 30 min after OT-A the doses required for the survival of 92% of the animals had to be about 25 times higher.
INTRODUCTION
OT-A, a mycotoxin isolated from Aspergillus ochraceus is composed of a 3,4-dihydro-3-methylisocoumarin moiety linked by an a-amide bond to L-& phenylalanine. The isocoumarin is chlorinated in the 5’ position [l] . OT-A is toxic for animals, causing kidney and liver damage [2] . There is evidence to support the hypothesis that it is a disease determinant of Balkan endemic nephropathy, a fatal chronic kidney disease affecting rural populations in Bulgaria, Romania and Yugoslavia [3] . OT-A inhibits protein synthesis by competition with phenylalanine in the phenylalanyl-tRNA synthetase catalysed reaction [4, 51. We have also been able to demonstrate that phenylalanine is capable of reversing the toxic effect of OT-A in HTC cells [6] . Abbreviations:
HTC, hepatoma tissue culture; OT-A, Ochratoxin-A.
78
In view of the practical importance of OT-A to the health of livestock [ 71 and possibly even of man [3] , we consider it of great interest to find out if phenylalanine is capable of preventing acute OT-A poisoning. Our results demonstrate a high survival rate of OT-A-poisoned mice when these animals were treated with phenylalanine by i.p. injection. MATERIAL
AND METHODS
The conditions for OT-A isolation have been described elsewhere [ 81. Conventional Swiss mice of 19.5 to 20.5 g weight (C.E.S.A.L., France) were randomly distributed in lots of 10 animals. They were injected i.p. with a dose of 0.8 mg OT-A per mouse (i.e., 40 mg/kg), which was initially established to be 100% lethal within 24 h. For all the subsequent experiments this dose was applied. Control animals without toxin were injected with saline containing various amounts of phenylalanine as indicated. The toxin controls received OT-A solutions only, The total volume of the injections never exceeded 1 ml. RESULTS
AND DISCUSSION
Mice injected with a lethal dose of OT-A showed hypothermia, decreased motor-activity, and loss of appetite 1 h after injection. After 12-16 h severe diarrhoea occurred. All the animals died between 23 and 24 h after OTA injection. However, when this lethal dose was injected i.p. together with 0.8 mg of phenylalanine, 97% of the animals survived, and 100% survived when 1 mg of phenylalanine was injected (Table I). Thus phenylalanine doses, only slightly higher than the lethal doses of OT-A, can efficiently counteract the toxic effect of OT-A, when given simultaneously. The effect of these relatively low doses may be explained by the higher affinity of phenylalanine for the phenylalanyl-tRNA synthetase compared with OT-A, as already reported [ 51, Also in cell cultures, doses only slightly higher than those of OT-A were able to reverse the toxic action [6]. However, if phenylalanine was injected 30 min after OT-A, a dose about 10 times higher than that of OT-A was necessary for the survival of 85% of the animals, and a dose 25 times higher for the survival of 92% (Table I). Finally, if a dose of 8 mg of phenylalanine was injected 60 min after OT-A (0.8 mg), only 10% of the mice survived. Therefore the reversion of the OT-A effect observed in HTC cells 2 h after addition of the mycotoxin [6] was not found. Thus, phenylalanine can prevent the lethal poisoning by OT-A when given simultaneously with the mycotoxin, whatever the precise mechanism of the counteraction may be. It is conceivable that farm animals and even man may be protected from poisoning by OT-A, through supplementation of feed or food in contaminated commodities. Experiments to find out whether such a protection is feasible, are in progress.
79 TABLE I SURVIVAL OF POISONED MICE (19.5-20.5 DIFFERENT DOSES OF PHENYLALANINE 0.25
0.50
g) INJECTED WITH 0.8 mg OCHRATOXIN-A
Doses of phenyl~~ine simultaneously injected with OT-A per mouse (mg)
0
Percentage of survival after 24 h
0
80
90
97
100
100
100
100
100
Percentage of survival after 28 days
0
20
50
97
100
100
100
100
100
Doses of phenylalanine injected per mouse 30 min after OT-A injection (mg)
0
0.50
0.80
AND
1.0
1.0
5.0
5.0
8.0
8.0
10.0
20.0
10.0
20.0
Percentage of survival after 24 h
11
21
63
88
90
92
Percentage of survival
7
15
53
82
85
92
Phenytalanine (Merck, Darmstadt) or OT-A were dissolved in saline (0.9% NaCl in bidistilled water, adjusted to pH 7.2 with 0.1 M NaOH), and the solutions were sterilized by Mi~ipore filtration (SLGS 02505,0.22 pm pore size). Injection materials (1.0 ml hypodermic syringes and needles) were obtained from Terumo and were sterilized by ethylene oxide, 20 mg phenylalanine was injected by 3 injections: 10 mg, 30 min after OT-A and twice 5 mg at: 60 min and at 120 min; or at 120 min and at 240 min; or at 180 min and at 360 min. The percentages of survivors were approximately the same: 92%, 90% and 90%, respectively.
ACKNOWLEDGEMENTS
This research was supported by grants from NATO (research grant No. 1749), from the “Institut National de la Santi et de la Recherche Mddicale” (contract No. 79.1.154.3) and from the “Deutsche Forschungsgemeinschaft” (Ro. 291/5). REFERENCES K.J. van der Merwe, P.S. Steyn and L. Fourie, Mycotoxins, Part II. The constitution of ochratoxin A, B and C, metabolites of Aspergillus ochraceus wilh.; J. Chem. Sot., (1965) 7083-7093; K.J. van der Merwe, P.S., Steyn, L. Fourie, D.B. Scott and J.J. Theron, Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus wilh., Nature, 205 (1965) 1112-1113 J. Harwig, in I.F.H. Purchase (Ed.) Mycotoxins, Elsevier, Amsterdam, 1974, pp. 345-367. P. Krogh, B. Hald, R. Plestina and Ceovic, S. Nephropathy and foodborne Ochratoxiri A: preliminary results of a survey of foodstuffs, Acta Pathol. Microbial. Stand. Sect. B 85 (1977) 238-240
80
I. Bunge, G. Dirheimer, and R. Raschenthaler, In vivo and in vitro inhibition of protein synthesis in Bacillus stearothermophilus by Ochratoxin A, Biochem. Biophys. Res. Commun., 83( 1978) 398-405. 5 E.E. Creppy, A.A.J. Lugnier, F. Fasiolo, K. Heller, R. Rijschenthaler, and G. Dirheimer. In vitro inhibition of yeast phenylalanyl-tRNA synthetase by ochratoxin A, Chem. Biol. Interact., 24 (1979) 257-261. 6 E.E. Creppy, A.A.J. Lugnier, F. Fasiolo, K. Heller, R. Raschenthaler, and G. Dirheimer. In vitroof Ochratoxin A on cultured hepatoma cells. Reversion of inhibition by Ochratoxin A, FEBS Lett. 104 (1979) 287-290. 7 P. Krogh, In J.V. Rodricks, C.W. Hesseltine and M.A. Mehlman (Eds.), Mycotoxins in Human and Animal Health. Pathotox, Park Forest South, IL, 1977, pp. 489-498. 8 K. Heller, C. Schultz and R. Riischenthaler. The inhibition of bacterial growth of Ochratoxin A, Can. J. Microbial. 21 (1975) 972-979.
4