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Acute toxicity studies on roquefortine and pr toxin, metabolites of Penicillium roqueforti, in the mouse
370
D. L. ARNOLD.P. M. Sco-rr. P. F. MCGUIRE.J. HARWIG and E. A. NERA Table 1. Acute
toxic&v
o/ P. roqueforti roxins and onalogues . . LD,o (mg/kg) values In mace Swiss-Webster*
c57 Toxin
Route
Males
Roquefortine PR toxin
ip oral
166 72 2
PR toxin + t,-leucine + L-a-alanine PR imine
ip ip ip
ip
-
Females 184 low C.l#
loo-200(( 200-300/j loo-200/l
Males 189 >140$ 4.6 -
Females 184 >140$ 3.5 -
*The Swiss-Webster mice given roquefortine ip weighed 18 f 2 8 those given PR toxin ip weighed 21 + 2 g and those given PR toxin orally weighed 35 f 3 g (males) and 27 + 2 g (females). tEstimated value: S/S mice given 121mg PR toxin/kg orally and l/5 given 81 mgFg died, but all given the lower doses survived. $Of the three animals/group dosed with 0, 80, 110 or 14Omg/kg in propylene glycol, none died within 2 wk. #Estimated value: 5/5 mice given 6.0, 3.0 or 1.5mg/kg and 2/5 given 0.75mgFg died. [FlIrree animals/group were dosed with 0. 100,200 or 3OOmgAcg. Acute toxiciry rests. The acute toxicological effects of roquefortine and PR toxin as affected by strain and sex, were examinedusingmale and femaleC57 miceweighing16 f 2 g and male and femaleSwissWebstermiceweighing18-35g (Table 1).The ip toxicity of PR toxin wascomparedwith that of its analoguesin C57 females.The animalswere obtained from a closedcolony with line breedingmaintained at the Health Protection Branch, Ottawa. The toxins wereall dissolvedin a 4: 1 (v/v) mixture of propylene glycol (laboratory grade) and dimethylsulphoxide (DMSO; spectral grade). Control animals received only the solvent mixture. The volume of solvent or solventplustest compoundadministeredwas5 ml/kg body weight. All animalswere maintainedin a controlled environment (21 f 1°C and 50-55% relative humidity). Animals dosed ip had accessto feed (commercial chow)and tap-waterad lib. at all times,whereasanimals given the toxin by gavage had their feed removed 16-17hr before dosing and, were then allowed accessto feed immediately after dosing. A 2-wk observation period followed dosing, during which time behavioural and clinical changeswere studied.The LD,, valueswere calculatedaccording to the method of Weil (1952). Severalanimalsgiven PR toxin ip were subjected to a detailed grossautopsy by a pathologist. Some of the animalsexamined had died on test, while othershad survived the 2-wk observationperiod. All organsand tissueswere stored in 10%neutral buffered formalin prior to preparation for histological examination.
Rt?SllltS
The LD,, valuesobtainedfor roquefortineand PR toxin and its reaction products are shown in Table 1. PR toxin wasfound to be more toxic on an aeute basisthan roquefortine following ip administration. ReactingPR toxin with L-leucineor L-a-alanineor converting it to the imine greatly reducedits acute ip toxicity. Regardless of the test compoundadministered,the behavioural changesand,clinical signsobserved in
treatment-affectedanimals were similar for both strainsand sexes.Within 15min of dosing,affected animals becamehypokinetic, a reaction eventually accompaniedby adipsia and anorexia. Often these animalsassumedcatatonic-like postureswhile standing on their hind legsfor prolongedperiodsof time. External auditory stimulusproduced by passinga rigid object acrossthe wire meshcages,causedthe animalsto return to their normal walking posture and walk severalsteps,but after that most animals returned to the standing position. This sequenceof events could be repeatedthree or four times. The auditory stimuluswas not repeatedmore than four times without an interval of at least 30min. Frequently, a clear nasaldischargewas observed,possibly associatedwith respiratory difficulties. Animals that did not succumbto the lethal effect of these toxins recoveredin 4-6 hr after dosing; those dying on test becamequiescent,adipsicand anorexic, and usually died within 24-48hr. Gross autopsy of animalsdosedip with PR toxin revealedno lesions.Histological examinationof sections from brain, lungs, heart, liver and kidneys revealedonly interstitial oedemain the lung with increasednumbersof mononuclearcellsand atelectasis in the animalsthat died. The other tissueswere not remarkable. There were no significant histological findings in tissuesof animals surviving the 2-wk observationperiod. Discussion
Previously observed neurotoxic properties attributed to roquefortineby C. Frayssinetand Christiane Frayssinet (cited by Scott et al. 1976) were not observed during this series of experiments.Their reported ip LD,e of 15-20mg/kg in male mice was about one tenth of the value determinedin this study as well as in a previous experiment when DMSG@9%NaC1(1:2, v/v) wasusedas the solvent mixture (D.L. Arnold, unpublishedobservation1977).The use of DMSG-O*9%NaCl(l:2. v/v) as the solvent for the roquefortineor of ,propyleneglycol for the PR toxin preparedin our laboratory resulted in suspensions of the toxins rather’ than solutions. Consequently,
Acute
toxicities
of roquefortine
propylene glycol-DMSO (4: 1. v/v) was used in the experiments reported here. The ip LD,, values for PR toxin determined in this study were similar to the value of 5.8 mg/kg reported for mice by Wei et al. (1976). Previously, Wei et al. (1973) had reported that the ip and oral LDso values for PR toxin administered to weanling rats were 11 and 115 mg/kg, respectively. Many of the clinical and histological findings reported by Wei et al. (1973) for the rat were not observed in this study, but whether such differences may be attributable to species or procedural differences is unknown. The marked reduction in the acute toxicity of PR toxin observed on its conversion to PR imine is comparable to the absence of deaths among male Swiss mice given 15 mg PR imine/kg ip (Mot&. Moreau & Bousquet, 1977). In addition, Moule et al. (1977) and Wei et al. (1976) have reported that PR toxin inhibits in uitro protein and nucleic acid synthesis, while the imine analogue has a similar but diminished inhibitory effect. However, some of the acute toxicity of the PR-toxin analogues (imine, L-a-alanine or L-leucine) seen in this study may be attributable to the small (2% or less) residue of PR toxin. It may be speculated that most or all of the PR toxin produced by P. roqueforti during the maturation of blue cheese would react with amino compounds present to produce derivatives similar to those studied here. As these derivatives are much less toxic than PR toxin, it appears that the potential toxicological hazard from PR toxin would thus be reduced. Acknowledgements-We thank S. Kanhere and B. J. Blanchfield for technical assistance and W. F. Miles for
the mass spectrum
of PR imine.
amino
acids
during
cheese ripening
Danish cheese. Milchwissenschaf
cultures and from biotransformation J. org. Chem. 42, 2632. Moreau. S.. Gaudemer. A., Lablache-Combier. A. Biguet. J. (1976). Metabolites de Penicillium royueforri: PR toxine et mitabolites associes. Tetrahedron Lerr. hum
roqueforri
27,
of some 566.
of free types
of
of
PR toxin.
et p.
833.
Moule. Y.. Moreau. S. & Bousquet. J. F. (1977). Relationships between the chemical structure and the biological properties of some eremophilane compounds related to PR ~toxin. Chemico-Biol. inreracrions i7. 185. Ohomo. S. Utaaawa. T. & Abe. M. (1977). Identification of roquefortine C produced by Pmicillium rdyuejorfi. Agric.
biol.
Chem.
41,
2097.
Ottogalli. G., Resmini. P., Bianchi. B.. Galli. A., Rondinini. G., Salvadori. P.. Saracchi. S. i Volonterio, G. (1971). Ricerche sulla maturazione del Gorgonzola sotto il profilo chimico e microbiologfco. Latre 45, 776.
Scott, P. M. & Kennedy. B. P. C. (1976). Analysis of blue cheese for roquefortine hum
roquejorri.
J. agric.
and other
alkaloids
Fd Chem.
24.
from
Penicil-
865.
Scott. P. M.. Kennedy. B. P. C.. Harwig, J. & Blanchfield. B. J. (1977). Study of conditions fortine and other metabolites Appt.
envir.
Microbial.
for production of Penicillium
of roqueroqueforri.
33. 249.
Scott. P. M., Merrien, M-A. & Polonsky. J. (1976). Roquefortine and isofumigaclavine A. metabolites from Penicillium roqueforri.
Experientia
32,
140.
Wei, R.-D., Schnoes, H. K., Hart. P. A. I% Strong. F. M. (1975).
The
Penicillium
structure roquejorti.
of PR toxin, Tetrahedron
a mycotoxin 31,
from
109.
Wei, R.-D., Schnoes, H. K., Smalley. E. B.. Lee. S.-S.. Chang. Y.-N. & Strong F. M. (1976). Production, isolation, chemistry, and biological properties of Penicillium roquejorti toxin.. In Animal. Planr. and Microbial Toxins. Voj. i. Chemistry,
Pharmacology,
and
Immunology.
Edited
bv A. Ohsaka. K. Havashi and Y. Sawai. D. 137. Plenum Press. New York. Wei, R.-D., Still, P. E.. Smalley, E. B., Schnoes. H. K. & Strong, F. M. (1973). Isolation and partial characterizaof a mycotoxin
from
Penicillium
roqueforti.
Appl.
1I I. Weil, C. S. (1952). Tables for convenient calculation of median effective dose (LDso or ED,,) and instructions Microbial.
Ismail, A. A. & Hansen, K. (1972). Accumulation
371
Moreau. S., Cacan. M. & Lablache-Combier. A. (1977). Eremofortin C. A new metabolite obtained from Prnicil-
tion
REFERENCES
and PR toxin
in their
25,
use. Biometrics
8. 249.
D. L. ARNOLD.P. M. Sco-rr. P. F. MCGUIRE.J. HARWIG and E. A. NERA Table 1. Acute
toxic&v
o/ P. roqueforti roxins and onalogues . . LD,o (mg/kg) values In mace Swiss-Webster*
c57 Toxin
Route
Males
Roquefortine PR toxin
ip oral
166 72 2
PR toxin + t,-leucine + L-a-alanine PR imine
ip ip ip
ip
-
Females 184 low C.l#
loo-200(( 200-300/j loo-200/l
Males 189 >140$ 4.6 -
Females 184 >140$ 3.5 -
*The Swiss-Webster mice given roquefortine ip weighed 18 f 2 8 those given PR toxin ip weighed 21 + 2 g and those given PR toxin orally weighed 35 f 3 g (males) and 27 + 2 g (females). tEstimated value: S/S mice given 121mg PR toxin/kg orally and l/5 given 81 mgFg died, but all given the lower doses survived. $Of the three animals/group dosed with 0, 80, 110 or 14Omg/kg in propylene glycol, none died within 2 wk. #Estimated value: 5/5 mice given 6.0, 3.0 or 1.5mg/kg and 2/5 given 0.75mgFg died. [FlIrree animals/group were dosed with 0. 100,200 or 3OOmgAcg. Acute toxiciry rests. The acute toxicological effects of roquefortine and PR toxin as affected by strain and sex, were examinedusingmale and femaleC57 miceweighing16 f 2 g and male and femaleSwissWebstermiceweighing18-35g (Table 1).The ip toxicity of PR toxin wascomparedwith that of its analoguesin C57 females.The animalswere obtained from a closedcolony with line breedingmaintained at the Health Protection Branch, Ottawa. The toxins wereall dissolvedin a 4: 1 (v/v) mixture of propylene glycol (laboratory grade) and dimethylsulphoxide (DMSO; spectral grade). Control animals received only the solvent mixture. The volume of solvent or solventplustest compoundadministeredwas5 ml/kg body weight. All animalswere maintainedin a controlled environment (21 f 1°C and 50-55% relative humidity). Animals dosed ip had accessto feed (commercial chow)and tap-waterad lib. at all times,whereasanimals given the toxin by gavage had their feed removed 16-17hr before dosing and, were then allowed accessto feed immediately after dosing. A 2-wk observation period followed dosing, during which time behavioural and clinical changeswere studied.The LD,, valueswere calculatedaccording to the method of Weil (1952). Severalanimalsgiven PR toxin ip were subjected to a detailed grossautopsy by a pathologist. Some of the animalsexamined had died on test, while othershad survived the 2-wk observationperiod. All organsand tissueswere stored in 10%neutral buffered formalin prior to preparation for histological examination.
Rt?SllltS
The LD,, valuesobtainedfor roquefortineand PR toxin and its reaction products are shown in Table 1. PR toxin wasfound to be more toxic on an aeute basisthan roquefortine following ip administration. ReactingPR toxin with L-leucineor L-a-alanineor converting it to the imine greatly reducedits acute ip toxicity. Regardless of the test compoundadministered,the behavioural changesand,clinical signsobserved in
treatment-affectedanimals were similar for both strainsand sexes.Within 15min of dosing,affected animals becamehypokinetic, a reaction eventually accompaniedby adipsia and anorexia. Often these animalsassumedcatatonic-like postureswhile standing on their hind legsfor prolongedperiodsof time. External auditory stimulusproduced by passinga rigid object acrossthe wire meshcages,causedthe animalsto return to their normal walking posture and walk severalsteps,but after that most animals returned to the standing position. This sequenceof events could be repeatedthree or four times. The auditory stimuluswas not repeatedmore than four times without an interval of at least 30min. Frequently, a clear nasaldischargewas observed,possibly associatedwith respiratory difficulties. Animals that did not succumbto the lethal effect of these toxins recoveredin 4-6 hr after dosing; those dying on test becamequiescent,adipsicand anorexic, and usually died within 24-48hr. Gross autopsy of animalsdosedip with PR toxin revealedno lesions.Histological examinationof sections from brain, lungs, heart, liver and kidneys revealedonly interstitial oedemain the lung with increasednumbersof mononuclearcellsand atelectasis in the animalsthat died. The other tissueswere not remarkable. There were no significant histological findings in tissuesof animals surviving the 2-wk observationperiod. Discussion
Previously observed neurotoxic properties attributed to roquefortineby C. Frayssinetand Christiane Frayssinet (cited by Scott et al. 1976) were not observed during this series of experiments.Their reported ip LD,e of 15-20mg/kg in male mice was about one tenth of the value determinedin this study as well as in a previous experiment when DMSG@9%NaC1(1:2, v/v) wasusedas the solvent mixture (D.L. Arnold, unpublishedobservation1977).The use of DMSG-O*9%NaCl(l:2. v/v) as the solvent for the roquefortineor of ,propyleneglycol for the PR toxin preparedin our laboratory resulted in suspensions of the toxins rather’ than solutions. Consequently,
Acute
toxicities
of roquefortine
propylene glycol-DMSO (4: 1. v/v) was used in the experiments reported here. The ip LD,, values for PR toxin determined in this study were similar to the value of 5.8 mg/kg reported for mice by Wei et al. (1976). Previously, Wei et al. (1973) had reported that the ip and oral LDso values for PR toxin administered to weanling rats were 11 and 115 mg/kg, respectively. Many of the clinical and histological findings reported by Wei et al. (1973) for the rat were not observed in this study, but whether such differences may be attributable to species or procedural differences is unknown. The marked reduction in the acute toxicity of PR toxin observed on its conversion to PR imine is comparable to the absence of deaths among male Swiss mice given 15 mg PR imine/kg ip (Mot&. Moreau & Bousquet, 1977). In addition, Moule et al. (1977) and Wei et al. (1976) have reported that PR toxin inhibits in uitro protein and nucleic acid synthesis, while the imine analogue has a similar but diminished inhibitory effect. However, some of the acute toxicity of the PR-toxin analogues (imine, L-a-alanine or L-leucine) seen in this study may be attributable to the small (2% or less) residue of PR toxin. It may be speculated that most or all of the PR toxin produced by P. roqueforti during the maturation of blue cheese would react with amino compounds present to produce derivatives similar to those studied here. As these derivatives are much less toxic than PR toxin, it appears that the potential toxicological hazard from PR toxin would thus be reduced. Acknowledgements-We thank S. Kanhere and B. J. Blanchfield for technical assistance and W. F. Miles for
the mass spectrum
of PR imine.
amino
acids
during
cheese ripening
Danish cheese. Milchwissenschaf
cultures and from biotransformation J. org. Chem. 42, 2632. Moreau. S.. Gaudemer. A., Lablache-Combier. A. Biguet. J. (1976). Metabolites de Penicillium royueforri: PR toxine et mitabolites associes. Tetrahedron Lerr. hum
roqueforri
27,
of some 566.
of free types
of
of
PR toxin.
et p.
833.
Moule. Y.. Moreau. S. & Bousquet. J. F. (1977). Relationships between the chemical structure and the biological properties of some eremophilane compounds related to PR ~toxin. Chemico-Biol. inreracrions i7. 185. Ohomo. S. Utaaawa. T. & Abe. M. (1977). Identification of roquefortine C produced by Pmicillium rdyuejorfi. Agric.
biol.
Chem.
41,
2097.
Ottogalli. G., Resmini. P., Bianchi. B.. Galli. A., Rondinini. G., Salvadori. P.. Saracchi. S. i Volonterio, G. (1971). Ricerche sulla maturazione del Gorgonzola sotto il profilo chimico e microbiologfco. Latre 45, 776.
Scott, P. M. & Kennedy. B. P. C. (1976). Analysis of blue cheese for roquefortine hum
roquejorri.
J. agric.
and other
alkaloids
Fd Chem.
24.
from
Penicil-
865.
Scott. P. M.. Kennedy. B. P. C.. Harwig, J. & Blanchfield. B. J. (1977). Study of conditions fortine and other metabolites Appt.
envir.
Microbial.
for production of Penicillium
of roqueroqueforri.
33. 249.
Scott. P. M., Merrien, M-A. & Polonsky. J. (1976). Roquefortine and isofumigaclavine A. metabolites from Penicillium roqueforri.
Experientia
32,
140.
Wei, R.-D., Schnoes, H. K., Hart. P. A. I% Strong. F. M. (1975).
The
Penicillium
structure roquejorti.
of PR toxin, Tetrahedron
a mycotoxin 31,
from
109.
Wei, R.-D., Schnoes, H. K., Smalley. E. B.. Lee. S.-S.. Chang. Y.-N. & Strong F. M. (1976). Production, isolation, chemistry, and biological properties of Penicillium roquejorti toxin.. In Animal. Planr. and Microbial Toxins. Voj. i. Chemistry,
Pharmacology,
and
Immunology.
Edited
bv A. Ohsaka. K. Havashi and Y. Sawai. D. 137. Plenum Press. New York. Wei, R.-D., Still, P. E.. Smalley, E. B., Schnoes. H. K. & Strong, F. M. (1973). Isolation and partial characterizaof a mycotoxin
from
Penicillium
roqueforti.
Appl.
1I I. Weil, C. S. (1952). Tables for convenient calculation of median effective dose (LDso or ED,,) and instructions Microbial.
Ismail, A. A. & Hansen, K. (1972). Accumulation
371
Moreau. S., Cacan. M. & Lablache-Combier. A. (1977). Eremofortin C. A new metabolite obtained from Prnicil-
tion
REFERENCES
and PR toxin
in their
25,
use. Biometrics
8. 249.