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Toxicologic and histopathologic studies of Pleurotus ostreatus mushroom in mice
297
Joumul of ~t~~op~~~o~g~, 21&w) 297 - 305 Elsevier Scientific Publishers Ireland Ltd.
TOXICOLOGIC AND HISTOPATHOLOGIC STUDIES OF PI,EUROTUS OSTREATUS MUSHROOM IN MICE
IHSAN H.S. AL-DEEN’, HUSNI A.A. TWAIJ”, AMMAR A. AL-BADR” and TAHSIN A.W. ISTARABADIb “3liol&xl Reseamh Centre, Scientific Research Cow& bCentral Pathogen Institzcte, ~~n~t~ of Health, Bagh~
P.O. Box %71, Jadiriyah, Baghdad and iI~q~
(Accepted August 28,1987)
Summary The edible mushroom Pleurotus ostreatus (with locally reported toxic properties) was identified and collected 1 - 4 days after raining in the city of Baghdad. It was freshly extracted with distilled water at room temperature. Both oral and intraperitoneal routes were used for acute and subacute toxicity studies on mice. In spite of important toxicologic signs and findings on treated animals, the estimated 24-h LD, values exceeded 3 g/kg for both routes of administration. However, the 39day LD, values were 319 mg/kg for oral and 1143 mgkg for i.p. administration. Gross examination of the dissected organs revealed marked haemorrhages in the intestine, liver, lung and the kidney. Histopathologic examination revealed significant changes mainly in the liver, which took the form of inflammation and microabscesses. The present results were consistent with clinical findings available after human and animal ingestion of this mushroom in Iraq.
Introduction Toxic mushrooms represent a health hazard to both humans and animals in many areas of the world. The medical records of the city of Baghdad contain occasional cases of mushroom poisoning, especially when the mushrooms have been eaten in their fresh form. The signs and symptoms of poisoning attributed to toxic mushrooms may take the form of weakness, fatigue, vomiting, haemorrhage, damage to the liver, kidney and the nervous system, coma and eventual death. The lack of information regarding the toxicologic properties of local fungi (Lange and Hora, 1978; Rumack and Salzman, 19’78; Seeger, 1980; Smith, 19751 was the motive behind the present study, which was aimed at performing a systematic toxicologic screening of Iraqi mushrooms in order to identify the potentially poisonous species. The screening phase is to be followed by further research in order to 0378~?~1/87/$03.~ 0 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
298
characterize the toxic principle(s) in the mushrooms in the hope of preparing suitable antidotes. The present work reports the toxicologic and histopathologic studies on Pleurotus ostreatus (Jacq. Fr.1 Kummer. Materials
and methods
Mushroom
samples
Samples of P. ostreatzls were freshly collected in December, l-4 days after raining, from a farm located just outside the city of Baghdad. Fresh specimens were immersed in 70% ethanol, 2- 4 h after collection, and dispatched to the Royal Botanic Gardens at Kew, Surrey, U.K. for identification. All specimens of P. ostreatus were cut from the lower part (ground level) of the trunk of a single tree (Populus alba Linn., Salicaceael which was infected with termites. Preparation
of the extract
Bearing in mind the reports of detoxification of mushrooms upon cooking or heating and in order to avoid denaturation of any proteinaceous toxin, the whole fresh mushrooms were chopped and extracted overnight with distilled water at room temperature (Twaij et al., 1983) immediately after collection. The filtrate was then evaporated under vacuum at low temperature. One gram of the dry extract was equivalent to 8.1 g of the crude fresh fungi. Animals
Swiss albino male mice, 25-30 g in weight and in-bred in our animal house facilities, were used for all the experiments. Groups of 6-8 mice were kept in opaque plastic cages (12 x 13 x 44 cm1 at room temperature. Food (standard rat chow, Ministry of Agriculture, Baghdad) and water were freely available to animals. Behavioural
effects
and toxicity studies
Gross behavioural effects of the extract on animals were recorded along with the toxicity studies (Turner, 1965). Animals were continuously observed during the first hour and then intermittently for the next 4 h. Further observations were made after 24 and 48 h. The LD, was determined by giving graded doses of the total extract of the fungus, dissolved in normal saline at a dose volume of 10 ml/kg body wt. to groups of mice using both the oral and intraperitoneal routes. Acute mortalities were recorded during the first 24 h and the LD, estimated accordng to the method of Litchfield and Wilcoxon (19491. In order to conserve extract, a preliminary estimation of the LD, was obtained using a small number of animals by the up-and-down technique of Dixon (19651.Since
299
the onset of toxic signs of mushroom poisoning can take several days (see Discussion), mortalities among animals which survived the first 24 h were also recorded every 24 h for 30 days. The toxicity of the extract was studied using five groups of treated mice and one group of control animals. Each group consisted of 8 animals. Groups of treated mice received different doses ranging between 100 and 3000 mgl kg of the fungus extract for both oral and intraperitoneal administration. The control mice received normal saline. Animals were continuously observed for 2- 4 h after the administration and then intermittently every 24 h thereafter for 30 days. Mice weight was recorded before and after treatment. Post-mortem examination of the animals was done after the completion of the behavioural study or in cases where death appeared imminent, prior to the completion of the study. Following cervical dislocation, the abdomen was opened and signs of haemorrhages and necrosis were recorded. Each sample of the various organs was kept individually in a small bottle filled with 10% formalin. Histopathologic examination of the organ specimens preserved in formalin was done by cutting sections of about 5 pm in thickness using a rotary microtome. Slides were stained with haematoxylin and eosin. For amyloidosis, Congo red stain was used. Only animals that survived the behavioural study were used for this examination. Results
Behavioural
effects and toxicity
studies
Behavioural effects of the extract using either route of administration took a generalized form of poisoning. The toxic signs were: rapid respiration, increased salivation, loss of locomotor activities, sharp clonic convulsions, tremors, paralysis of hind-paws, loss of co-ordination and eventual death. Surviving animals showed a scrawny appearance. The investigated organs (kidney, lung, liver, spleen, heart and small intestine1 of the sacrificed mice were congested and displayed general cyanosis. Treatment with the mushroom did not affect significantly the weight of animals in comparison with controls. The estimated LD, values for the aqueous extract of the mushroom after various periods of time following either route of administration are presented in Table 1, together with 95% confidence limits. It was not possible to estimate the exact LD, values 24 h following the administration of the mushroom, as the lethal doses all exceeded 3000 mglkg. Histopathologic
examination
No significant changes were seen in the organs of control mice. However, there were occasional incidences of very mild hepatic lesions in the form of
300 TABLE I ESTIMATED LD, VALUES FOR THE AQUEOUS EXTRACT OF P. OSTREATUS FOR VARYING PERIODS OF TIME FOLLOWING EITHER ORAL OR INTRAPERITONEAL ADMINISTRATION TO MICE Days after administration
mg/hg LD, (95% C.L.) Oral
+1 +7 +30
> 5000 5888 (3098- 11,629) 319 ( 32-3192)
N” 0 24 24
I.P. > 3000 2460 (1902-3173) 1143 (607 - 2145)
N 0 25 20
“N = Total number of animals with expected effects between 16% and 84O/o(Litchfield and Wilcoxon, 19491.
focal leucocytic cell infiltration. All treated mice, on the other hand, showed definite pathologic changes, mainly in the liver. These changes consisted of various forms of degenerative changes in the parenchyma, which ranged from cloudy swelling to fatty changes of the hepatic cells. The liver of one mouse treated orally with the mushroom showed amyloid degeneration (Fig. 1). In addition, there were acute suppurative inflammatory lesions of the liver which took the form of a diffuse hepatitis accompanied by microabscesses (Fig. 21 and sinusoidal and portal leucocytic cell infiltration (Fig. 11. Focal
Fig. 1. Amyloidosis with sinusoidal and portal leucocytic cell infiltration in the liver of a mouse treated orally with an aqueous extract of P. ostreatw (Congo red, x 5601.
301
necrosis was seen, together with occasional cases of extensive necrotizing suppurative inflammatory lesions (Fig. 31. The lungs of treated mice suffered occasional incidences of a slight inflammatory reaction. The lung of one mouse treated orally with the mushroom showed areas of haemorrhage and necrosis. In the heart of another mouse treated intraperitoneally, there was focal leucocytic cell infiltration. There was extramedullary haemopoiesis in the spleens of all treated and control mice, but amyloidosis was evident occasionally among treated animals (Fig. 41. In most of the treated and control mice, the intestinal mucosa showed slight leucocytic cell infiltration. The major histopathologic findings of the various organs are summarized in Table 2. It was difficult to correlate the established pathologic changes with either the dose or the route of administration. Discussion
There is clear evidence from the results furnished by the present study that the edible mushroom P. ostreatus is potentially toxic when taken in large quantities in its fresh form. Previous work has indicated that the toxins of this mushroom are mostly proteinaceous in nature. For example, Bernheimer and Avigad (19791 have isolated pleurotolysin, a cytolytic watersoluble protein, from this fungus. Also, Seeger (19801 has reported
Fig. 2. Typical microabscess seen in the liver of mice treated either orally or intraperitoneally with an aqueous extract of P. ostreatus (H & E, x 500).
Fig. 3. An extensive necrotizing suppurative inflammatory lesion in the liver of a mouse treated intraperitone~ly with an aqueous extract of F. ostreatus /II & E, x 125).
Fig. 4. Typical amyloidosis seen in the spleens of mice treated with an aqueous extract of P. ostreatus (Congo red, x 125).
either orally or intraperitone~ly
Key: - , absent (018); +. occasional O-4/8f;
Intestine
Spleen
Heart
Lung
+ f , frequent
(5-818).
Focal necrosis and microabscesses Extensive necrotizing supperative inflammatory lesions Various degrees of degenerative changes Amyloidosis Focal round and leucocytic cell infiltration Slight inflammatory reactions Focal haemorrhage and necrosis Focal acute and chronic inflammatory cell infiltration Amyloidosis Extramedullary haemopoiesis Slight leucocytie cell infiltration of the mucosa
Liver
changes
Histopathologie
Organ
+ +
+ +
Control
Oral
SUMMARY OF HISTOPATHOLOGIC CHANGES FOLLOWING ORAL (100-3000 ADMINISTRATION OF p. OSTREATUS AQUEOUS EXTRACT TO MICE
TABLE 2 mgkg)
+ + ++
i++
+
+ + ++
+
++ +
++
++ + ++ + +
Treated ++ +
Control
I.P.
(100--1MM mg/kgl
++
Treated
OR INTRAPERITONEAL
304
proteinaceous toxins with direct haemolytic and cytolytic actions. The main clinical evidence for the proteinaceous nature of the toxin(s) in the samples of P. ostreatus collected for this study comes from locally reported decreases in the extent of intoxication after cooking or heating this mushroom before eating. In the present extraction procedure, care was taken not to heat the extract beyond 3i’OC or to use any chemical or process that might denature any protein. Because the onset of the signs and symptoms of mushroom poisoning in man may take up to a few days (Mitchel and Rumack, 19781, the signs of poisoning among treated animals and their mortalities were recorded in the present work for a period of 30 days. The period for a chronic or subchronic toxicity study usually exceeds 3 months. In the present study, however, it was not possible to extend it beyond 30 days. This was because of the generalized debilitation of the intoxicated (treated) animals, even when the smallest dose (100 mg/kgl was used. Since a room temperature aqueous extraction procedure was adopted in this work, it may be stated that a water-soluble protein, such as pleurotolysin, could be responsible for the haemorrhagic and necrotic conditions noted on post-mortem examination following parenteral administration of the extract. Such toxicity following oral ingestion is somewhat more difficult to explain since gastric acid and the gastrointestinal digestive enzymes would be expected to destroy most orally ingested proteins, at least when taken in reasonable quantities. The presently estimated oral 24-h LD, value for the aqueous extract was greater than 5 gl kg, each gram of which represents 8.1 g of the crude fresh mushroom. Extrapolation to, for example, a 70-kg man presumes the eating of the equivalent of at least 2.8 kg of mushrooms. Ingestion of such a large quantity could disrupt normal metabolism. Fortunately, however, humans do not normally eat such large quantities of fresh and uncooked mushroom at one time. This could explain the absence of serious manifestations after eating smaller amounts of this edible species. On the other hand, animals (herbivorous) and birds can eat large quantities of such a fresh natural product and caution must be drawn on allowing them, when possible, to feed on this mushroom. Acknowledgements The authors are grateful to Dr. D.N. Pegler, Royal Botanic Gardens at Kew, Surrey, U.K for identification of mushroom samples and to Dr. Wafa I. Al-Azawi for assistance in the preparation of histopathologic slides and in microphotography. References Bernheimer, Pburotus
A.W. and Avigad, L.S. (1979) A cytolytic protein from ostreatus. Biochimica et Biophysics Acta 585, 451-461.
the
edible
mushroom,
Dixon, W.J. (19651 The up-anddown method for small samples. Journd of American Statistical Association 60,967 - 978. Lange, M. and Hora, F.B. (1978) Mushrooms and Toadstook. Collins, London, p. 106. Litchfield, J.T. aud Wllcoxon, F. (1949) A simplified method of evaluating dose-effect experiments. Journul of Phannacobgy and Experimental Therapeutics 96,99- 113. Mitchel, D.H. and Rumack, B.H. (19’781Symptomatic diagnosis and treatment of mushroom poisoning. In: B.H. Rumack and E. Salzman (Eds.1, Mushroom Poisoning: Diugnosti and Treatment CRC Press, West Palm Beach, Florida, pp. 171- 179. Rumack, B.H. and Salzman, E. (1978) Mushroom Poisoning: Diagnosis and Treatment. CRC Press, West Palm Beach, Florida. Seeger, R. (1980) Cytolytic toxins of Basidiomycetes. In: D. Eaker and T. Wadstrom (Eds.1, Natural Totins. Pergamon Press, Oxford, pp. 165- 171. Smith, A.H. (19751 A Field Guide to Western Mushrooms. The University of Michigan Press, Ann Arbor, p. 124. Turner, R.A. (19661 Screening Methods in Pharmacology. Academic Press, New York, pp. 2241. Twaij, H.A.A., Kery, A. and Al-Khazarji, N.K. (19831 Some pharmacological, toxicological and phytochemical investigations on Centaurea phyllocephala Journal of Ethnopharmacology 9, 299-314.
Joumul of ~t~~op~~~o~g~, 21&w) 297 - 305 Elsevier Scientific Publishers Ireland Ltd.
TOXICOLOGIC AND HISTOPATHOLOGIC STUDIES OF PI,EUROTUS OSTREATUS MUSHROOM IN MICE
IHSAN H.S. AL-DEEN’, HUSNI A.A. TWAIJ”, AMMAR A. AL-BADR” and TAHSIN A.W. ISTARABADIb “3liol&xl Reseamh Centre, Scientific Research Cow& bCentral Pathogen Institzcte, ~~n~t~ of Health, Bagh~
P.O. Box %71, Jadiriyah, Baghdad and iI~q~
(Accepted August 28,1987)
Summary The edible mushroom Pleurotus ostreatus (with locally reported toxic properties) was identified and collected 1 - 4 days after raining in the city of Baghdad. It was freshly extracted with distilled water at room temperature. Both oral and intraperitoneal routes were used for acute and subacute toxicity studies on mice. In spite of important toxicologic signs and findings on treated animals, the estimated 24-h LD, values exceeded 3 g/kg for both routes of administration. However, the 39day LD, values were 319 mg/kg for oral and 1143 mgkg for i.p. administration. Gross examination of the dissected organs revealed marked haemorrhages in the intestine, liver, lung and the kidney. Histopathologic examination revealed significant changes mainly in the liver, which took the form of inflammation and microabscesses. The present results were consistent with clinical findings available after human and animal ingestion of this mushroom in Iraq.
Introduction Toxic mushrooms represent a health hazard to both humans and animals in many areas of the world. The medical records of the city of Baghdad contain occasional cases of mushroom poisoning, especially when the mushrooms have been eaten in their fresh form. The signs and symptoms of poisoning attributed to toxic mushrooms may take the form of weakness, fatigue, vomiting, haemorrhage, damage to the liver, kidney and the nervous system, coma and eventual death. The lack of information regarding the toxicologic properties of local fungi (Lange and Hora, 1978; Rumack and Salzman, 19’78; Seeger, 1980; Smith, 19751 was the motive behind the present study, which was aimed at performing a systematic toxicologic screening of Iraqi mushrooms in order to identify the potentially poisonous species. The screening phase is to be followed by further research in order to 0378~?~1/87/$03.~ 0 1987 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
298
characterize the toxic principle(s) in the mushrooms in the hope of preparing suitable antidotes. The present work reports the toxicologic and histopathologic studies on Pleurotus ostreatus (Jacq. Fr.1 Kummer. Materials
and methods
Mushroom
samples
Samples of P. ostreatzls were freshly collected in December, l-4 days after raining, from a farm located just outside the city of Baghdad. Fresh specimens were immersed in 70% ethanol, 2- 4 h after collection, and dispatched to the Royal Botanic Gardens at Kew, Surrey, U.K. for identification. All specimens of P. ostreatus were cut from the lower part (ground level) of the trunk of a single tree (Populus alba Linn., Salicaceael which was infected with termites. Preparation
of the extract
Bearing in mind the reports of detoxification of mushrooms upon cooking or heating and in order to avoid denaturation of any proteinaceous toxin, the whole fresh mushrooms were chopped and extracted overnight with distilled water at room temperature (Twaij et al., 1983) immediately after collection. The filtrate was then evaporated under vacuum at low temperature. One gram of the dry extract was equivalent to 8.1 g of the crude fresh fungi. Animals
Swiss albino male mice, 25-30 g in weight and in-bred in our animal house facilities, were used for all the experiments. Groups of 6-8 mice were kept in opaque plastic cages (12 x 13 x 44 cm1 at room temperature. Food (standard rat chow, Ministry of Agriculture, Baghdad) and water were freely available to animals. Behavioural
effects
and toxicity studies
Gross behavioural effects of the extract on animals were recorded along with the toxicity studies (Turner, 1965). Animals were continuously observed during the first hour and then intermittently for the next 4 h. Further observations were made after 24 and 48 h. The LD, was determined by giving graded doses of the total extract of the fungus, dissolved in normal saline at a dose volume of 10 ml/kg body wt. to groups of mice using both the oral and intraperitoneal routes. Acute mortalities were recorded during the first 24 h and the LD, estimated accordng to the method of Litchfield and Wilcoxon (19491. In order to conserve extract, a preliminary estimation of the LD, was obtained using a small number of animals by the up-and-down technique of Dixon (19651.Since
299
the onset of toxic signs of mushroom poisoning can take several days (see Discussion), mortalities among animals which survived the first 24 h were also recorded every 24 h for 30 days. The toxicity of the extract was studied using five groups of treated mice and one group of control animals. Each group consisted of 8 animals. Groups of treated mice received different doses ranging between 100 and 3000 mgl kg of the fungus extract for both oral and intraperitoneal administration. The control mice received normal saline. Animals were continuously observed for 2- 4 h after the administration and then intermittently every 24 h thereafter for 30 days. Mice weight was recorded before and after treatment. Post-mortem examination of the animals was done after the completion of the behavioural study or in cases where death appeared imminent, prior to the completion of the study. Following cervical dislocation, the abdomen was opened and signs of haemorrhages and necrosis were recorded. Each sample of the various organs was kept individually in a small bottle filled with 10% formalin. Histopathologic examination of the organ specimens preserved in formalin was done by cutting sections of about 5 pm in thickness using a rotary microtome. Slides were stained with haematoxylin and eosin. For amyloidosis, Congo red stain was used. Only animals that survived the behavioural study were used for this examination. Results
Behavioural
effects and toxicity
studies
Behavioural effects of the extract using either route of administration took a generalized form of poisoning. The toxic signs were: rapid respiration, increased salivation, loss of locomotor activities, sharp clonic convulsions, tremors, paralysis of hind-paws, loss of co-ordination and eventual death. Surviving animals showed a scrawny appearance. The investigated organs (kidney, lung, liver, spleen, heart and small intestine1 of the sacrificed mice were congested and displayed general cyanosis. Treatment with the mushroom did not affect significantly the weight of animals in comparison with controls. The estimated LD, values for the aqueous extract of the mushroom after various periods of time following either route of administration are presented in Table 1, together with 95% confidence limits. It was not possible to estimate the exact LD, values 24 h following the administration of the mushroom, as the lethal doses all exceeded 3000 mglkg. Histopathologic
examination
No significant changes were seen in the organs of control mice. However, there were occasional incidences of very mild hepatic lesions in the form of
300 TABLE I ESTIMATED LD, VALUES FOR THE AQUEOUS EXTRACT OF P. OSTREATUS FOR VARYING PERIODS OF TIME FOLLOWING EITHER ORAL OR INTRAPERITONEAL ADMINISTRATION TO MICE Days after administration
mg/hg LD, (95% C.L.) Oral
+1 +7 +30
> 5000 5888 (3098- 11,629) 319 ( 32-3192)
N” 0 24 24
I.P. > 3000 2460 (1902-3173) 1143 (607 - 2145)
N 0 25 20
“N = Total number of animals with expected effects between 16% and 84O/o(Litchfield and Wilcoxon, 19491.
focal leucocytic cell infiltration. All treated mice, on the other hand, showed definite pathologic changes, mainly in the liver. These changes consisted of various forms of degenerative changes in the parenchyma, which ranged from cloudy swelling to fatty changes of the hepatic cells. The liver of one mouse treated orally with the mushroom showed amyloid degeneration (Fig. 1). In addition, there were acute suppurative inflammatory lesions of the liver which took the form of a diffuse hepatitis accompanied by microabscesses (Fig. 21 and sinusoidal and portal leucocytic cell infiltration (Fig. 11. Focal
Fig. 1. Amyloidosis with sinusoidal and portal leucocytic cell infiltration in the liver of a mouse treated orally with an aqueous extract of P. ostreatw (Congo red, x 5601.
301
necrosis was seen, together with occasional cases of extensive necrotizing suppurative inflammatory lesions (Fig. 31. The lungs of treated mice suffered occasional incidences of a slight inflammatory reaction. The lung of one mouse treated orally with the mushroom showed areas of haemorrhage and necrosis. In the heart of another mouse treated intraperitoneally, there was focal leucocytic cell infiltration. There was extramedullary haemopoiesis in the spleens of all treated and control mice, but amyloidosis was evident occasionally among treated animals (Fig. 41. In most of the treated and control mice, the intestinal mucosa showed slight leucocytic cell infiltration. The major histopathologic findings of the various organs are summarized in Table 2. It was difficult to correlate the established pathologic changes with either the dose or the route of administration. Discussion
There is clear evidence from the results furnished by the present study that the edible mushroom P. ostreatus is potentially toxic when taken in large quantities in its fresh form. Previous work has indicated that the toxins of this mushroom are mostly proteinaceous in nature. For example, Bernheimer and Avigad (19791 have isolated pleurotolysin, a cytolytic watersoluble protein, from this fungus. Also, Seeger (19801 has reported
Fig. 2. Typical microabscess seen in the liver of mice treated either orally or intraperitoneally with an aqueous extract of P. ostreatus (H & E, x 500).
Fig. 3. An extensive necrotizing suppurative inflammatory lesion in the liver of a mouse treated intraperitone~ly with an aqueous extract of F. ostreatus /II & E, x 125).
Fig. 4. Typical amyloidosis seen in the spleens of mice treated with an aqueous extract of P. ostreatus (Congo red, x 125).
either orally or intraperitone~ly
Key: - , absent (018); +. occasional O-4/8f;
Intestine
Spleen
Heart
Lung
+ f , frequent
(5-818).
Focal necrosis and microabscesses Extensive necrotizing supperative inflammatory lesions Various degrees of degenerative changes Amyloidosis Focal round and leucocytic cell infiltration Slight inflammatory reactions Focal haemorrhage and necrosis Focal acute and chronic inflammatory cell infiltration Amyloidosis Extramedullary haemopoiesis Slight leucocytie cell infiltration of the mucosa
Liver
changes
Histopathologie
Organ
+ +
+ +
Control
Oral
SUMMARY OF HISTOPATHOLOGIC CHANGES FOLLOWING ORAL (100-3000 ADMINISTRATION OF p. OSTREATUS AQUEOUS EXTRACT TO MICE
TABLE 2 mgkg)
+ + ++
i++
+
+ + ++
+
++ +
++
++ + ++ + +
Treated ++ +
Control
I.P.
(100--1MM mg/kgl
++
Treated
OR INTRAPERITONEAL
304
proteinaceous toxins with direct haemolytic and cytolytic actions. The main clinical evidence for the proteinaceous nature of the toxin(s) in the samples of P. ostreatus collected for this study comes from locally reported decreases in the extent of intoxication after cooking or heating this mushroom before eating. In the present extraction procedure, care was taken not to heat the extract beyond 3i’OC or to use any chemical or process that might denature any protein. Because the onset of the signs and symptoms of mushroom poisoning in man may take up to a few days (Mitchel and Rumack, 19781, the signs of poisoning among treated animals and their mortalities were recorded in the present work for a period of 30 days. The period for a chronic or subchronic toxicity study usually exceeds 3 months. In the present study, however, it was not possible to extend it beyond 30 days. This was because of the generalized debilitation of the intoxicated (treated) animals, even when the smallest dose (100 mg/kgl was used. Since a room temperature aqueous extraction procedure was adopted in this work, it may be stated that a water-soluble protein, such as pleurotolysin, could be responsible for the haemorrhagic and necrotic conditions noted on post-mortem examination following parenteral administration of the extract. Such toxicity following oral ingestion is somewhat more difficult to explain since gastric acid and the gastrointestinal digestive enzymes would be expected to destroy most orally ingested proteins, at least when taken in reasonable quantities. The presently estimated oral 24-h LD, value for the aqueous extract was greater than 5 gl kg, each gram of which represents 8.1 g of the crude fresh mushroom. Extrapolation to, for example, a 70-kg man presumes the eating of the equivalent of at least 2.8 kg of mushrooms. Ingestion of such a large quantity could disrupt normal metabolism. Fortunately, however, humans do not normally eat such large quantities of fresh and uncooked mushroom at one time. This could explain the absence of serious manifestations after eating smaller amounts of this edible species. On the other hand, animals (herbivorous) and birds can eat large quantities of such a fresh natural product and caution must be drawn on allowing them, when possible, to feed on this mushroom. Acknowledgements The authors are grateful to Dr. D.N. Pegler, Royal Botanic Gardens at Kew, Surrey, U.K for identification of mushroom samples and to Dr. Wafa I. Al-Azawi for assistance in the preparation of histopathologic slides and in microphotography. References Bernheimer, Pburotus
A.W. and Avigad, L.S. (1979) A cytolytic protein from ostreatus. Biochimica et Biophysics Acta 585, 451-461.
the
edible
mushroom,
Dixon, W.J. (19651 The up-anddown method for small samples. Journd of American Statistical Association 60,967 - 978. Lange, M. and Hora, F.B. (1978) Mushrooms and Toadstook. Collins, London, p. 106. Litchfield, J.T. aud Wllcoxon, F. (1949) A simplified method of evaluating dose-effect experiments. Journul of Phannacobgy and Experimental Therapeutics 96,99- 113. Mitchel, D.H. and Rumack, B.H. (19’781Symptomatic diagnosis and treatment of mushroom poisoning. In: B.H. Rumack and E. Salzman (Eds.1, Mushroom Poisoning: Diugnosti and Treatment CRC Press, West Palm Beach, Florida, pp. 171- 179. Rumack, B.H. and Salzman, E. (1978) Mushroom Poisoning: Diagnosis and Treatment. CRC Press, West Palm Beach, Florida. Seeger, R. (1980) Cytolytic toxins of Basidiomycetes. In: D. Eaker and T. Wadstrom (Eds.1, Natural Totins. Pergamon Press, Oxford, pp. 165- 171. Smith, A.H. (19751 A Field Guide to Western Mushrooms. The University of Michigan Press, Ann Arbor, p. 124. Turner, R.A. (19661 Screening Methods in Pharmacology. Academic Press, New York, pp. 2241. Twaij, H.A.A., Kery, A. and Al-Khazarji, N.K. (19831 Some pharmacological, toxicological and phytochemical investigations on Centaurea phyllocephala Journal of Ethnopharmacology 9, 299-314.