Fd Chem. Toxic. Vol. 22, No. 4, pp. 275 281, 1984 0278-6915/84 $3.00+0.00 Printed in Great Britain Pergamon Press Ltd

TOXICITY OF RHIZONIN A, ISOLATED FROM RHIZOPUS MICROSPORUS, IN LABORATORY

ANIMALS

T. WILSON Animal Disease Diagnostic Laboratory, Veterinary Science Department, The Pennsylvania

State University, University Park, PA 16802, USA

C. J. RABIE and J. E. FINCHAM

National Research Institute for Nutritional Diseases, South African Medical Research Council, P.O. Box 70, Tygerberg 7505, South Africa

P. S. STEYN

National Chemical Research Laboratory, P.O. Box 395, Pretoria 0001, South Africa

and

M. A. A. SCHIPPER Centraalbureau voor Schimmelcultures, Baarn, The Netherlands

(Received 14 September 1983)

Abstrac~Maize culture material of 25 isolates of the genera Rhizopus and Mucor caused deaths in day-old unsexed Pekin ducklings when fed as a 50% (w/w) mixture with duckling feed. Nine of these isolates were tested for toxicity in young inbred male BD IX rats, which were fed a mixture of 50~,,~ (w/w) culture material and rat feed. Only one isolate of Rhizopus microsporus was clearly toxic, causing 100% mortality in rats within 10 days. Growth in rats was reduced by adding culture material of this isolate to the feed in concentrations of 2.5, 5, 10 or 20'~/o (w/w). The same isolate of R. microsporus was used to produce the mycotoxin rhizonin A. Pure rhizonin A was dissolved in dimethylsulphoxide and given to young male partially inbred albino rats by gavage in single doses of 70, 96, 131 or 180 mg/kg. The lowest dose exceeded the LD~00. Evaluated by light microscopy, lesions in livers and kidneys were similar in rats fed culture material and in those intubated with rhizonin A. Hepatocytes showed changes ranging from degeneration to necrosis, the liver-tissue architecture was changed by disassociation of liver cell cords and there was periportal bile-duct proliferation. Renal tubular epithelium showed changes ranging from degeneration to necrosis.

INTRODUCTION

The Rhizopus species of fungi frequently cause spoil- age of vegetables and fruits (Splittstoesser, 1979; Zycha & Siepmann, 1969) and may occur in high numbers as contaminants of products such as malts (Gyllang, S~tmark & Martinson, 1977). Extensive use is made of some Rhizopus species in Far Eastern countries for the production of a wide range of fermented food products, including t6mp6 and tap6 in Indonesia, lao-chao in China and Taiwan and meju in Korea (Beuchat, 1979).

Hepatotoxicity was reported to have followed the feeding of mice with culture material of an unspecified Rhizopus isolate (van Rensburg, Pur- chase & van der Watt, 1970). Rhizonin A produced by Rhizopus microsporus van Tieghem, is the only mycotoxin known to be produced by any of the lower fungi (Steyn, Tuinman, van Heerden et al. 1983). All other known mycotoxins are produced by the higher fungi, mostly by species in the classes of the As- comycetes and Deuteromycetes.

Lesions produced in rats by the feeding of maize culture material of R. mierosporus and by adminis- tration of rhizonin A by gavage at different dose levels are described in this report.

EXPERIMENTAL

Fungal isolates. The isolates (Rhizopus and Mucor species) used were all started from single-spored cultures and were deposited in the culture collection of the South African Medical Research Council as freeze-dried cultures. The designated M R C culture collection numbers of the isolates tested are shown in Table 1.

Bulk culturing. Yellow maize kernels in 2-1itre glass jars (400 g maize kernels and 400 ml tap-water/jar) were autoclaved at 121°C for 1 hr on each of two consecutive days. Each jar of autoclaved maize was inoculated with a spore suspension of one of the fungal isolates (Table 1) and was incubated at 2Y'C in the dark for 21 days. After incubation, the contents of the jars were dried at 4Y~C for 24 hr and finely ground in a laboratory mill. The resulting meal was stored at 5 C until the start of the experiment and during the course of the feeding trials. Maize-culture material of all the isolates was analysed for rhizonin A by prolonged chloroform methanol extraction, solvent partition and chromatography on silica gel, as previously described (Steyn et al. 1983).

Toxicity of culture material to ducklings. Nine different fungal isolates were evaluated for toxicity.

275

276 T. WILSON et al.

Table I. Toxicity of pure cultures of different Rhizopus and Mucor species to ducklings

Mean MRC time to

Fungal component no. of No, of death of diet* isolate Food source Country of origin deaths (days)t

R. o~vzae 1945 Sorghum malt South Africa 14/20+ + 5.4 R. rhizopod(/ormi.~ 1954 Sorghum malt South Africa 8/12~. 4.6 R. mi~ro.sporus§ 303 Ground nuts Mozambique 44/44 + 5.7 R. rhizoporhJ~rmis 1639 Sorghum malt South Africa 4/4 5.0 Rhizopus sp. 234 Ground nuts Mozambique 4/4 5.0 M. indicus 1629 Sorgham malt South Africa 5/8:~ 4.5 M. circinelloides 1638 Sorghum malt South Africa 4/8 + 9.0

1857 Sorghum malt South Africa 4/4 4.0 Mucor sp. 1789 Sorghum malt South Africa 5/8{ 4.0

*Diet contained 50";i commercial duckling species.

,'-From the start of the experiment. +Repetitive trials were conducted with four §The only species producing rhizonin A.

feed and 50!~o (w/w) culture material of the appropriate fungal

ducklings in each additional trial.

Diets were prepared by mixing the crude ground culture material with an equal weight of commercial duckling feed (Epol Duckling Mash; Cape Town). The control diet contained 50~/o ground autoclaved uninoculated maize in a commercial duckling feed. Groups of four unsexed day-old Pekin ducklings from a commercial breeder (Farm Fares (Poultry) Ltd, Cape Town) were balanced for initial weight. Each group was fed one of the diets ad lib. for 14 days. Duration of feeding, in days, was recorded for the ducklings that died. Surviving birds were weighed on days 7 and 14 of the experiment. Repetitive trials were conducted on several of the fungal isolates. The ducklings were maintained in an ambient tem- perature of 27_+ 0.5°C, with a relative humidity of 50%, air circulation of about 22 changes/hr and a 12-hr light/dark cycle using fluorescent lighting.

Toxicio, trials in rats

Animals and maintenance. Tests were carried out in male albino rats and male rats of the BD IX strain, inbred by mating siblings for 33 generations in the latter case and for seven generations for the albino strain. The rats were bred in the facility where experiments were conducted. Environmental controls set the temperature at 24_+ 0.5"C, and the relative humidity, air circulation and lighting were the same as for the ducklings. At the start of each treatment, each group of rats had the same average weight. The rats were observed daily and clinical signs were noted. All rats in the culture-material toxicity trials were weighed at weekly intervals and at termination. The rats were caged singly or in pairs and allowed to adapt for 6 days before experimentation commenced.

Toxicity q f culture material o f different Rhizopus and Mucor species. The fungal isolates assayed in ducklings were also tested in rats. For each treatment, six inbred male BD IX weanling rats were fed ad lib. a feed containing 50°o (w/w) ground culture material and 50~ o commercially produced rat feed (Epol Rat Mash). Rats in each group were housed two per cage. The control rats were fed a mixture of 50% (w/w) ground autoclaved uninoculated maize and 50~ of the commercial rat feed. Duration of feeding was 19 wk, unless death intervened.

Toxicity oJ Rhizopus microsporus culture material to rats. Tiaree groups each of five inbred male BD IX

rats, individually caged, were fed ad lib. a ration of either ~"/ (w/w) mouldy meal and 95"~ commercial ~/o rat feed or 2.5}o mouldy meal and 97.5'~, commercial rat feed or a control diet of 5°,o ground autoclaved uninoculated maize meal and 95),0 commercial rat feed for 40 days, after which the two test rations were adjusted to 10 and 5}{:, mouldy meal, respectively, with a corresponding reduction in the commercial rat feed, for the remainder of a 9-wk test period. The control feed was unchanged. In a second trial, three groups each of 15 inbred male BD IX rats, individu- ally caged, were fed ad lib. for 14wk rations compounded (by weight) from 10'~, mouldy meal and 90~o commercial rat feed, 20'){, mouldy meal and 80')~, commercial rat feed or 203~i commercial maize and 8070 commercial rat feed (control group).

Toxici O, ()fl rhizonin A to rats. Weanling, partially inbred male albino rats were randomly divided into five groups each of five rats, caged individually. Purified rhizonin A was dissolved in dimethyl- sulphoxide (DMSO) and administered in 0.25ml volumes by gavage to each rat in a single dose of 180, 131, 96 or 70 mg/kg. The fifth (control) group re- ceived DMSO. These rats were weighed 2, 6 and 10 days after dosing.

Histopathology. In each study, rats surviving to termination were anaesthetized with sodium pento- barbital and killed by exsanguination for autopsy. For all rats, sections of the trachea, thyroid, para- thyroid, lung, myocardium, liver, adrenal, kidney, stomach (glandular and squamous), intestine, testis and spleen were fixed in 10!~, ~, buffered formalin. Histological sections were prepared by routine meth- ods, stained with haematoxylin and eosin (H & E) and examined by light microscopy.

Liver lesions involving disruption of hepatic cell cords and lobular architecture were quantified in the pure rhizonin A study. In three randomly selected fields (x 125) disruption was graded as absent (0), mild, indicating occasional focal architectural dis- ruption (1), moderate, involving about 50°~, of the lobule (3) or severe, in which no recognizable ordered structure remained (5). Karyomegaly was defined as the presence of a large irregular nucleus containing a big nucleolus or multiple nucleoli and prominent chromatin clumping in abnormal hepatocytes. Distri- bution of this nuclear pleomorphism within lobules

Toxicity of rhizonin A in laboratory animals 277

320

280

240

._~

, t o

~ / . ¢ °

120

8 0 I I I I I I I l I 0 1 2 3 4 5 6 7 8 9

Dure t ion of feed ing (wk)

Fig. 1. Mean body weights of control rats (0) and of two groups of rats fed Rhizopus microsporus culture material at dietary levels initially of 2~2°~, ([~) and 5 ( i ) and increased after 40 days to 5 and 10~,, respectively, for the rest of the

9-wk test period.

was graded numerically as no atypia (0), occasional foci of karyomegaly comprising less than 25~o of the lobule (1), involvement of 25-50% of the lobule (3) or involvement of the entire lobule (5). Bile-duct prolif- eration was diagnosed microscopically as the occur- rence of duct-like structures lined with plump cu- boidal hyperplastic epithelial cells surrounding a lumen with minimal supporting connective tissue, and was graded as absent (0), three bile ducts per triad (1), four or five bile ducts per triad (3) or more than five bile ducts per triad (5).

R E S U L T S

Toxicity of culture material to ducklings

All nine isolates of Mucor and Rhizopus tested caused death in ducklings but varied in their toxicity (Table 1). In eleven repetitive trials, R. microsporus killed all 44 of the treated ducklings in a mean time of 5.7 days. R. microsporus was the only species that

produced rhizonin A. In five repetitive trials, R. oryzae killed 14 of 20 ducklings in a mean time of 5.4 days. One Mucor isolate (MRC 1789) killed five of eight ducklings in a mean time of 4 days.

Toxicity of culture material o/d(fferent Rhizopus and mucor species to rats

R. microsporus was the only species which was highly toxic to rats. It killed all six test animals, 9 days being the average time to death. Six isolates caused mild growth depression, weight being de- pressed by up to 9% compared with that of control rats. In rats fed culture material of M. indicus (MRC 1629) and M. circinelloides (MRC 1638), growth was slightly greater than that of control rats.

No significant gross pathology was recorded in any of the rats at autopsy. Microscopically, the six rats fed R. microsporus material all had hepatic lesions characterized by hepatocellular degeneration and ne- crosis, hepatic cord disassociation, bile-duct prolif- eration and karyomegaly in the hepatocytes. Degen- erating hepatocytes were randomly distributed throughout the liver Iobule and were detectable by cytoplasmic acidophilia and hyalinization. Necrotic hepatocytes showed pycnosis, karyorrhexis and karyolysis, in the presence of little inflammation. Small, acidophilic hepatocytes showing karyolysis were commonly disseminated throughout the liver tissue. Degenerative changes in the hepatocytes ap- peared to be randomly distributed, although entire lobules were often involved. Marked karyomegaly was evident in hepatocytes throughout the liver Iob- ules; the big nuclei contained large nucleoli and chromatin was clumped or stippled. Nuclear di- ameters regularly measured 15pro or more, com- pared with an average of 6ffm in control rats. Bile-duct proliferation was minimal and randomly distributed. Apart from mild focal disseminated renal tubular degeneration, no lesions were detected in other organs. No significant histopathological lesions were observed in the control rats.

Toxicity of R. microsporus culture material to rats

No deaths occurred in rats fed culture material at 5~,~ (w/w) of the diet, rising to 100/O after 40 days, or

300

25O

2OO J;z

150 m ~-'" | ~ m • • i / i / i _ _ n / i - - I

o t O 0

g ,

N 5 O

o I I I I I I I I I I I I I I 0 f 2 3 4 5 6 7 8 9 10 11 12 13 14

Duration of f eed ing (wk)

Fig. 2. Mean body weight o f con t ro l rats ( 0 ) and o f rats fed Rhizopus microsporus cul ture mater ia l at dietary levels of 10°~ (D) and 20~,~ (el) for ]4wk.

278 T. WILSON et al.

Table 2. Toxicity of purified rhizonin A to male weanling rats

Mean time Mean Dose level* No. of to death terminal

(mg/kg) deaths+ (days) + weight (g)

0§ 0 172.8 70 5 5 75.6 96 5 4 85.4

131 5 3 91.4 180 4 5 83.8

*Single dose given by gavage. t in groups of five rats. +From administration of dose; times of death ranged

from 1 to 10 days after dosing. §Vehicle control: 0.25 ml dimethylsulphoxide/rat.

at 2.5~ (w/w) rising to 5~ after 40 days. Growth, in terms of body weight, was depressed by 37.5% for the higher level of feeding and by 32.5 for the lower level, compared to control rats. Final average live weights achieved were 320, 216 and 200g for control and treated rats, respectively (Fig. 1). Rats fed either level of culture material were lean at autopsy. However, no remarkable histopathological lesions were observed. Control rats were normal macroscopically and micro- scopically.

One rat out of 15 died in the group fed culture material at 10~o (w/w) of the diet for 14wk, and two out of 15 died in the group fed 20~. Growth was depressed by 31~o in rats fed the 10%o level and by 53~o in rats fed the 20~ level compared to control rats (Fig. 2). Final average live weights achieved were 318, 219 and 149g for control and treated rats respectively.

Livers from rats fed either level of culture material showed mild diffuse yellowing at autopsy and small quantities of straw-coloured ascitic fluid were present. Multiple small (< 1 mm) foci of pale discol- oration were scattered through some livers. Micro- scopically, karyomegaly in hepatocytes and foci of liver cell necrosis were apparent in all rats fed the 10~o (w/w) culture-material diet. The enlarged nuclei mea- sured 15/~tn or more in diameter. The distribution of necrosis was random within the lobules and dying cells had pycnotic nuclei and rounded irregular bor- ders. The cytoplasm of necrotic cells appeared gran- ular and was deeply acidophilic. Necrosis was pre- ceded by cell shrinkage which caused disassociation of hepatic cell cords. Similar, but more severe, lesions

were observed in the livers of rats fed 20~o (w/w) culture material. Apart from mild focal renal tubular epithelial degeneration, no lesions were detected in other organs. Grossly and histologically, the control rats were normal.

Toxicity ~[" rhizonin A to rats

Rhizonin A was acutely toxic at all four dose levels and killed 19 out of 20 rats within 10 days (Table 2). There appeared to be no difference in toxicity at any of the four dose levels; all treated groups showed a marked loss in weight and lesions were present within 24 hr of gavage. While some rats died within a day, others survived up to 10 days after the single oral dose. The controls remained healthy and gained weight normally.

Autopsy findings were similar in the rats of all four rhizonin-A treated groups and included generalized subcutaneous and body icterus. Numerous pale foci (< 1 mm) were noted in the livers. Histopathological lesions were confined to the liver and kidney in all rhizonin A-dosed rats and were qualitatively similar. Numerical scoring of the liver lesions was attempted and the results are summarized in Table 3. There was mild to severe disruption of the normal hepatic cord and lobular pattern which often resulted in complete architectural effacement (Fig. 3). Atypical hepa- tocytes were common, as was hyperchromasia. He- patic cords were often disassociated, with angularity and irregularity evident in the hepatocyte cell wall. Histological disruption of the hepatic parenchyma appeared to start first in the periportal area, while more normal tissue, when present, was evident around the central vein. Necrotic hepatocytes in the more severely affected livers often contained many small cytoplasmic vacuoles.

Karyomegaly was common in the hepatocytes of all treated rats. Affected nuclei were often over 15 ,urn in diameter, irregular in outline and with large nu- cleoli (Fig. 4). Bile-duct proliferation was prominent in all treated rats and appeared as ductular structures lined by plump cuboidal cells with little supporting connective tissue (Fig. 5). The epithelial cells ap- peared hyperplastic and hyperchromatic and their nuclei often had prominent nucleoli.

Bile-duct proliferation appeared to be most severe in the highest dose group and lowest in that given 70 mg/kg. Conversely karyomegaly was most promi- nent in the 70-mg/kg group and showed the lowest

Table 3. Quantitative evaluation of major hepatic histopathological lesions in male rats dosed with rhizonin A

Mean scores for five rats/groupt Mean time Dose level* . . . . . . . . . . . . . . . to death

(mg/kg) AD Karyomegaly BDP (days)+ +

0 0 0 0 70 2.4 3.1 1.6 5 96 2.4 2.2 3.1 4

131 2.4 1.4 2.6 3 180 2.5 1.0 3.5 5

AD = Architectural disruption BDP Bile-duct proliferation *Single dose given by gavage. 4-Scores used for the three types of lesion were negative (0), mild (1),

moderate (3) and severe (5). Detailed definitions are given in Experi- mental.

~From administration of dose. §Vehicle control: 0.25 ml dimethylsulphoxide/rat.

Fig. 3. Bile-duct proliferation, portal-vein distension and hepatic architectural effacement in a rat that died 3 days after gavage with 9 6 m g rhizonin A/kg. H & E x 125.

Fig. 4. Megalocytosis of hepatocytes with karyomegaly and a multinucleated hepatocyte in a rat that died 6 days after gavage with 70 mg rhizonin A/kg. H & E x 500.

279

Fig. 5. Bile-duct proliferation in a rat that died 6 days after gavage with 131 mg rhizonin A/kg. H & E x 500.

280

Toxicity of rhizonin A in laboratory animals 281

score in the rats given the high dose of rhizonin A. Non-birefringent crystalline-like material was ob- served in vacuoles throughout the liver of all the four treated groups. The composit ion of this crystalline- like material was not determined. All the treated rats developed a mild renal tubular nephrosis. No lesions were detected in other organs.

warranted and the ability of various other species of the Mucorales to produce this mycotoxin should be investigated. Rhizonin A does not account for all the toxicity of R. microsporus. Several other minor and unrelated toxic extracts exist and are under in- vestigation. These must have contributed to the over- all toxicity of the culture material.

DISCUSSION

Rhizonin A, a metabolite from R. microsporus and the first mycotoxin isolated from a species of the lower fungi, proved to be a potent hepatotoxin in rats. No correlation could be established between the type and severity of the hepatic lesions, the day of death and the dose level. It would appear, however, that bile-duct proliferation was more pronounced in the higher dose range.

Toxic damage to the liver is a common finding with many chemicals and drugs, including mycotoxins, and the wide range of histological lesions caused by rhizonin A is consistent with a non-specific hepa- totoxicity. Although hyperchromasia, karyomegaly and bile-duct proliferation represent significant he- patic damage, a direct relationship with preneoplastic or neoplastic change should not be inferred at this stage.

Because of the widespread occurrence of Rhizopus species in foods, further long-term studies on the chronic toxicity of pure rhizonin A appear to be

REFERENCES

Beuchat L. R. (1976). Traditional fermented food products. In Food and Beverage Mycology. Edited by R. R. Beuchat. p. 224. AVI Publishing Co. Inc., Westport, CT.

Gyllang H., S~itmark L. & Martinson E. (1977). The influence of some fungi on malt quality. Proceedings of the 16th Congress, European Brewery Convention, Am- sterdam, p. 245.

Splittstoesser D. F. (1979). Fruits and fruit products. In Food and Beverage Mycology. Edited by R. R. Beuchat. p. 83. AVI Publishing Co. Inc., Westport, CT.

Steyn P. S.. Tuinman A. A., van Heerden F. R., van Rooyen P. H., Wessels P. L. & Rabie C. J. (1983). The isolation, structure, and absolute configuration of the mycotoxin rhizonin A, a novel cyclic heptapeptide containing N- methyl-3-(3-furyl)alanine, produced by Rhizopus micro- sporus. J. chem. Sot., Chem. Commun. p. 47.

van Rensburg S. J., Purchase I. F. H. & van der Watt J. J. (1970). Hepatic and renal pathology induced in mice by feeding fungal cultures. In Symposium on Mycotoxins in Human Health. Edited by I. F. H. Purchase. p. 153. Macmillan, London.

Zycha H. & Siepmann R. (1969). Mucorales. p. 75. J. Cramer, Lehre.