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Viridicatumtoxin, a new mycotoxin from Penicillium viridicatum westling
TOXICOLOGY
AND APPLIED
PHARMACOLOGY
SHORT
Viridicatumtoxin,
(1973)
24,507-509
COMMUNICATION
a New
Mycotoxin Penicillium viridicatum Westling
from
Viridicatumtoxin, a New Mycotoxin from Penicillium viridicatum Westling. R. D. HUTCHISON, P. S. STEYNand S. J. VAN RENSBURG (1973). Toxicol. Appl. Pharmacol. 24,507-509. Extraction and fractionation of toxic cultures ofP. viridicatum led to the isolation of viridicatumtoxin. Its chemical properties are reported. The LD50 in rats was found to be 122.4 mg/kg. Hyaline droplet degeneration of the myocardium, renal tubular necrosis and changes in the hepatocytes were observed. Atrophy of the spleen occurred. Renal diseases in swine caused by the consumption of feed contaminated by P. viridicaand Tuite, 1970) led to the isolation of citrinin as a causative factor (Krogh et al., 1970). According to Carlton et al. (1970) the hepatic lesions of the mycotoxicosis induced in mice by cultures of P. viridicatum resembled those of facial eczema in sheep; recently Butiarso et al. (1970) reported that feeding a rice culture of this fungus to mice which were exposed to sunlight resulted in a phototoxic syndrome in some animals. Our studies on different isolates of P. viridicatum established that certain cultures of this fungus when grown on wet sterile maize meal were toxic to laboratory animals. Prolonged extractions of cultures of CSIR 1029, a toxigenic strain of P. viridicatum, followed by systematic fractionation (Fig. 1) and bioassay in ducklings, led to the isolation of a substance, subsequently named viridicatumtoxin, as the only acutely toxic component.
turn (Carlton
METHODS Viridicatumtoxin is a bright yellow pigment with a molecular constitution C30H31N010 as established by elemental analysis and high-resolution mass spectroscopy [m/e 565.1962 (M+, C,,H31N0,0 requires : 565.1948)]. It has mp 211°C (CHCl,hexane) ; ah3,“”- 12” (c, 1.0 EtOH); XE$” 237, 285, 317, 331, 347 and 424 nm (E 21,900, 32,900, 2760, 2590, 1900 and 8100, respectively); ~~~~‘33550-3350, 2980, 1725, 1650, 1620 and 1595 cm-‘. It gives a red-brown coloration with ethanolic FeCl, and can be detected in fungal cultures by separation on oxalic acid-impregnated silica gel plates (Steyn, 1969), on which it appears at Rf 0.35 as a yellow-brown fluorescent spot upon exposure to ultraviolet illumination (366 nm). Young male Wistar-derived rats weighing approximately 100 g were used to study the acute toxicity of the toxin. Four groups of five rats each received a single dose of the Copyrir&t 8 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
507
508
SHORT COMMUNICATION
toxin at doses ranging from 50 mg/kg to 500 mg/kg. The toxin was dissolved in dimethylsulfoxide and was administered by gastric intubation; a fifth group received the solvent only. Survivors were killed at 10 days and hematoxylin and eosin-stained sections were prepared from the heart, lungs, liver, kidneys, adrenals, and pancreas. Dried mouldy maize (3 kg) CHCld/MeOH
extn and evap
Crude extract (240 g) (TOXIC) partition
between CHCl3/H20
CHCI, layer
H,O extract freeze-dry
(NONTOXIC)
(82 g) (TOXIC) partition
Hexane layer
Hexan
MeOH
90 “/‘,’MeOH layer Wi3P
CWtlp
46 g (TOXIC)
35 g (NONTOXIC)
column chromatography on formamide-impregnated cellulose powder, elution with mixtures of hexane and benzene
Griseofulvin’ (11.0 g)
Viridicatumtoxin (4.0 g) (TOXIC)
FIG. 1. The fractionation of the toxic extract of P. viridicatum CSIR 1029. 1 Identified by direct comparison with an authentic sample of griseofulvin.
RESULTS AND DISCUSSION Deaths were generally sudden without obvious preceding clinical signs. The animals commenced dying within 24 hr in the group receiving the highest dose; the last death occurred in the lowest dose group on the seventh day after dosing. The LD50 value, determined according to the method of Weil(1952), was 122.4 mg/kg. Histopathological examination revealed the consistent presence of hyaline droplet degeneration of the myocardium in all treated animals. Renal tubular necrosis was
SHORT
COMMUNICATION
509
present in the animals that died. Hepatocytes exhibited considerable hydropic change with single cells exhibiting necrotic changes; abnormal mitotic figures were common. The spleen was atrophied and there was a paucity of small dark-staining lymphocytes in the splenic nodules. REFERENCES BUTIARSO, I. T., CARLTON, W. W. AND TUITE, J. F. (1970). Phototoxic syndrome induced in mice by rice cultures of Penicillium viridicatum and exposure to sunlight. Pathol. Vet. 7,
531-546. CARLTON, W. W. AND TUITE, J. (1970). Nephropathy and edema syndrome induced in miniature swine by corn cultures of Penicillium viridicatum. Pathol. Vet. 7,68-80. CARLTON, W. W., TUITE, J. F. AND MISLIVEC, P. (1970). Pathology of the toxicosis produced in mice by corn cultures of Penicillium viridicatum. In Proc. 1st U.S.-Japan Confi on Toxic Microorganisms (M. Herzberg, Ed.), pp. 94-106. U.S. Govt. Printing Office, Washington,
D.C. KROGH, P., HASSELAGER, E. AND FRIIS, P. (1970). Isolation of two nephrotoxic compounds from Penicillium viridicatum Westling: Citrinin and oxalic acid. Acta Pathol. Stand. (B) 78.
401. STEYN, P. S. (1969). The separation and detection of several mycotoxins by thin-layer chromatography. J. Chromatog. 45,473475. WEIL, C. S. (1952). Tables for convenient calculation of median-effective dose (LD50 or EDSO) and instructions in their use. Biometrics 8, 249-263.
R. D. HUTCHISON P. S. STEYN S. J. VAN RENSBURG* National Chemical Research Laboratory, CSIR, and National Research Institute for Nutritional Diseases, Medical Research Council Pretoria, South Africa Received July 11, 1972
’ National ResearchInstitute for Nutritional Diseases.
AND APPLIED
PHARMACOLOGY
SHORT
Viridicatumtoxin,
(1973)
24,507-509
COMMUNICATION
a New
Mycotoxin Penicillium viridicatum Westling
from
Viridicatumtoxin, a New Mycotoxin from Penicillium viridicatum Westling. R. D. HUTCHISON, P. S. STEYNand S. J. VAN RENSBURG (1973). Toxicol. Appl. Pharmacol. 24,507-509. Extraction and fractionation of toxic cultures ofP. viridicatum led to the isolation of viridicatumtoxin. Its chemical properties are reported. The LD50 in rats was found to be 122.4 mg/kg. Hyaline droplet degeneration of the myocardium, renal tubular necrosis and changes in the hepatocytes were observed. Atrophy of the spleen occurred. Renal diseases in swine caused by the consumption of feed contaminated by P. viridicaand Tuite, 1970) led to the isolation of citrinin as a causative factor (Krogh et al., 1970). According to Carlton et al. (1970) the hepatic lesions of the mycotoxicosis induced in mice by cultures of P. viridicatum resembled those of facial eczema in sheep; recently Butiarso et al. (1970) reported that feeding a rice culture of this fungus to mice which were exposed to sunlight resulted in a phototoxic syndrome in some animals. Our studies on different isolates of P. viridicatum established that certain cultures of this fungus when grown on wet sterile maize meal were toxic to laboratory animals. Prolonged extractions of cultures of CSIR 1029, a toxigenic strain of P. viridicatum, followed by systematic fractionation (Fig. 1) and bioassay in ducklings, led to the isolation of a substance, subsequently named viridicatumtoxin, as the only acutely toxic component.
turn (Carlton
METHODS Viridicatumtoxin is a bright yellow pigment with a molecular constitution C30H31N010 as established by elemental analysis and high-resolution mass spectroscopy [m/e 565.1962 (M+, C,,H31N0,0 requires : 565.1948)]. It has mp 211°C (CHCl,hexane) ; ah3,“”- 12” (c, 1.0 EtOH); XE$” 237, 285, 317, 331, 347 and 424 nm (E 21,900, 32,900, 2760, 2590, 1900 and 8100, respectively); ~~~~‘33550-3350, 2980, 1725, 1650, 1620 and 1595 cm-‘. It gives a red-brown coloration with ethanolic FeCl, and can be detected in fungal cultures by separation on oxalic acid-impregnated silica gel plates (Steyn, 1969), on which it appears at Rf 0.35 as a yellow-brown fluorescent spot upon exposure to ultraviolet illumination (366 nm). Young male Wistar-derived rats weighing approximately 100 g were used to study the acute toxicity of the toxin. Four groups of five rats each received a single dose of the Copyrir&t 8 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.
507
508
SHORT COMMUNICATION
toxin at doses ranging from 50 mg/kg to 500 mg/kg. The toxin was dissolved in dimethylsulfoxide and was administered by gastric intubation; a fifth group received the solvent only. Survivors were killed at 10 days and hematoxylin and eosin-stained sections were prepared from the heart, lungs, liver, kidneys, adrenals, and pancreas. Dried mouldy maize (3 kg) CHCld/MeOH
extn and evap
Crude extract (240 g) (TOXIC) partition
between CHCl3/H20
CHCI, layer
H,O extract freeze-dry
(NONTOXIC)
(82 g) (TOXIC) partition
Hexane layer
Hexan
MeOH
90 “/‘,’MeOH layer Wi3P
CWtlp
46 g (TOXIC)
35 g (NONTOXIC)
column chromatography on formamide-impregnated cellulose powder, elution with mixtures of hexane and benzene
Griseofulvin’ (11.0 g)
Viridicatumtoxin (4.0 g) (TOXIC)
FIG. 1. The fractionation of the toxic extract of P. viridicatum CSIR 1029. 1 Identified by direct comparison with an authentic sample of griseofulvin.
RESULTS AND DISCUSSION Deaths were generally sudden without obvious preceding clinical signs. The animals commenced dying within 24 hr in the group receiving the highest dose; the last death occurred in the lowest dose group on the seventh day after dosing. The LD50 value, determined according to the method of Weil(1952), was 122.4 mg/kg. Histopathological examination revealed the consistent presence of hyaline droplet degeneration of the myocardium in all treated animals. Renal tubular necrosis was
SHORT
COMMUNICATION
509
present in the animals that died. Hepatocytes exhibited considerable hydropic change with single cells exhibiting necrotic changes; abnormal mitotic figures were common. The spleen was atrophied and there was a paucity of small dark-staining lymphocytes in the splenic nodules. REFERENCES BUTIARSO, I. T., CARLTON, W. W. AND TUITE, J. F. (1970). Phototoxic syndrome induced in mice by rice cultures of Penicillium viridicatum and exposure to sunlight. Pathol. Vet. 7,
531-546. CARLTON, W. W. AND TUITE, J. (1970). Nephropathy and edema syndrome induced in miniature swine by corn cultures of Penicillium viridicatum. Pathol. Vet. 7,68-80. CARLTON, W. W., TUITE, J. F. AND MISLIVEC, P. (1970). Pathology of the toxicosis produced in mice by corn cultures of Penicillium viridicatum. In Proc. 1st U.S.-Japan Confi on Toxic Microorganisms (M. Herzberg, Ed.), pp. 94-106. U.S. Govt. Printing Office, Washington,
D.C. KROGH, P., HASSELAGER, E. AND FRIIS, P. (1970). Isolation of two nephrotoxic compounds from Penicillium viridicatum Westling: Citrinin and oxalic acid. Acta Pathol. Stand. (B) 78.
401. STEYN, P. S. (1969). The separation and detection of several mycotoxins by thin-layer chromatography. J. Chromatog. 45,473475. WEIL, C. S. (1952). Tables for convenient calculation of median-effective dose (LD50 or EDSO) and instructions in their use. Biometrics 8, 249-263.
R. D. HUTCHISON P. S. STEYN S. J. VAN RENSBURG* National Chemical Research Laboratory, CSIR, and National Research Institute for Nutritional Diseases, Medical Research Council Pretoria, South Africa Received July 11, 1972
’ National ResearchInstitute for Nutritional Diseases.