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The acute toxicity of ochratoxin A to rats
Fd Cosmet. Toxicol. Vol. 6, pp. 479-483. Pergamon Press 1968. Printed in Great Britain
The Acute Toxicity of Ochratoxin A to Rats I. F. H. PURCHASE and J. J. THERON Division of Toxicology, National Nutrition Research Institute, South African Council for Scientific and Industrial Research, Pretoria, South Africa (Received 1 March 1968)
Abstract--The acute oral toxicity of ochratoxin A, one of three chemically-relatedmetabolites from Aspergillas ochraceus, has been determined in rats. The LDs0 values obtained, 22 mg/kg for males and 20 mg/kg for females, indicate that the acute toxicity of ochratoxin A is about half that ofaflatoxin Bt in this species. The main pathological changes associated with fatal doses of ochratoxin A were severe necrosis of the renal tubules and necrosis of the periportal cells of the liver, usually affecting single cells although occasionally there were larger foci of necrotic cells. Accumulations of glycogen were found in the cytoplasm of the liver cells, accompanied in some cells by mitochondrial degeneration. It is .suggested that ochratoxin A may interfere with the phosphorylase enzyme system to produce this increase in liver glycogen. INTRODUCTION Ochratoxin A is one o f three chemically related metabolites isolated from Aspergillus ochraceus (Van der Merwe, Steyn & Fourie, 1965). This fungus was originally isolated f r o m s o r g h u m and was shown to be toxic to rats, mice a n d d a y - o l d ducklings (Scott, 1965). Theron, Van der Merwe, Liebenberg, J o u b e r t & Nel (1966) described the lesions p r o d u c e d by a single oral dose of 100 t~g o f ochratoxin A to ducklings a n d rats. This p a p e r describes the estimation of the oral L D s o of ochratoxin A in rats. A preliminary r e p o r t o f this w o r k was presented at the " S y m p o s i u m on Microbial T o x i n s " held by the A m e r i c a n Chemical Society in September 1966. EXPERIMENTAL Male or female W i s t a r rats from our own strain weighing a p p r o x i m a t e l y 80 g were separated into groups o f four and dosed by stomach tube with 10, 15, 25 or 40 mg o c h r a t o x i n A / k g b o d y weight. Crystalline ochratoxin A was dissolved in 0 . 1 N - N a H C O a and the concentration o f ochratoxin A was adjusted so that between 0" 3 and 0-6 ml o f solution was given to each rat. C o n t r o l rats were given 0 . 5 ml N a H C O 3 solution by s t o m a c h tube. All rats were given food and water ad lib. Surviving rats were killed on day 10. A p o s t - m o r t e m examination was p e r f o r m e d on all rats, a n d p o r t i o n s o f liver, kidney, heart and lung were fixed in buffered formalin. Paraffin sections o f these tissues were stained with h a e m a t o x y l i n and erythrosine and by Pasini's m e t h o d for connective tissue. F r o z e n sections were stained with Oil R e d 0 for fat. The LD5o values were derived from the m o r t a l i t y in each group by using the tables d r a w n up by Weft (1952). A further six female rats were dosed orally with ochratoxin A (5 or 10 mg/kg) and killed by cervical dislocation 4 - 6 days later in order to have fresh material for electron microscopy. 479
480
I. F. H. P U R C H A S E
and
J. J. T H E R O N
Small portions of liver were fixed in glutaraldehyde, post-fixed in l ~ osmium tetroxide in phosphate buffer and embedded in epon-araldite (Mollenhauer, 1964). Silver-to-grey sections were cut on a Porter-Blum or LKB ultramicrotome equipped with glass knives, placed on uncoated 400-mesh copper grids and stained with lead hydroxide as described by Karnovsky (1961). Thicker sections (0.5-1.0t~) of the same blocks were stained with toluidine blue or by the periodic acid-Schiff technique for light microscopy. RESULTS
Estimation of LDso The LDso values (and 95 ~o fiducial limits) were 22 mg/kg (15-30) for males and 20 mg/kg (9-41) for females. Rats dying between 24 and 73 hr General observations. These rats received the highest doses of ochratoxin A (25 or 40 mg/ kg) and, because they died so soon after dosing, showed an average weight loss of only 4 g. Post-mortem examination revealed a marked inflammation of the duodenum and jejunum with signs of diarrhoea. The livers were slightly paler than normal. The kidneys were pale and friable and the lungs showed signs of congestion. Histology. The liver had a normal architecture, but certain periportal cells were necrotic, showing karyorrhexis and disintegration of the cytoplasm. This necrosis usually affected single cells, but occasionally a larger focus of necrotic cells was seen (Fig. 1). The nuclei of the remaining cells were normal, but some of the cells had granular cytoplasm. The distribution of fat and connective tissue was normal. The kidneys showed a tubular necrosis. The cells of the proximal convoluted tubules had pycnotic nuclei (or occasionally karyorrhexis) and cytoplasm which had disintegrated and did not stain (Fig. 2). The glomeruli were normal. Other convoluted-tubular cells showed signs of cloudy swelling, and in some cases cast formation was evident. The necrotic tubules were seen mainly in the cortex, but in some cases necrotic tubules were present in the outer zone of the medulla (Fig. 3). These were presumed to be the tubules forming Henle's loop, but because of the necrotic changes it was difficult to ascertain the cell types forming the tubule. In the most severe cases, necrosis occurred not only in these tubules but in other (collecting) tubules in the medulla. The lungs of some animals showed signs of hyperaemia and a necrotic focus was seen in the myocardium of one rat. Rats dying between days 4 and 6 General observations. Rats from each dose range died at this stage, but it was remarkable that only females died. The rats showed an average weight loss of 14 g and were in a poor condition when they died. There was no diarrhoea or enteritis in this group. The kidneys and livers appeared similar to those in the previous group. Congestion of the lungs was observed in some rats. Histology. Under the low power objective ( x 10), the liver parenchymal cells in the periportal areas had slightly darker-staining nuclei, but their cytoplasm did not stain (Fig. 4). This gave the impression of nuclei floating free in otherwise empty cells. Under higher magnification it was seen that the areas which were not eosinophilic in the cytoplasm were more or less rounded, with thin trabeculae separating them (Fig. 5). The nuclei were darker
FIG. 1. Focal necrosis in liver of rat dying 16 hr after a single (40 mg/kg) dose of ochratoxin A. Haematoxylin and erythrosine x 880.
FIG. 2. Section of renal cortex of rat dying after single (25 mg/kg) dose of ochratoxin A, showing normal glomerulus but necrosis of the proximal convoluted tubule. Haematoxylin and erythrosine x 880.
Fie. 3. Marked tubular necrosis in medulla of kidney of rat dying after single (25 mg/kg) dose of ochratoxin A. Haematoxylin and erythrosine x 220.
FIG. 4. Marked periportal vacuolation due to gl2(cogen accumulation in liver of rat killed 6 days after single (10 mg/kg) dose of ochratoxin A. Haematoxylin and erythrosine x 220.
Fie. 5. Area of glycogen accumulation in liver of rat killecL6 days after single (10 mg/kg) dose of ochratoxin A, showing thin trabeculae separating cytoplasmic vacuoles. Haematoxylin and erythrosine x 1100.
F~o. 6. Markedly dilated tubules in kidney of rat killed 10 days after single (15 mg/kg) dose of ochratoxin A. Haematoxylin and erythrosine x 220.
FIo. 7. Lakes o/' glycogen seen as dark-staining masses in cytoplasm of hepatocytas in thick section of el:onembedded liver from rat killed 6 days after single (10 mg/kg) dose of ochratoxin A. Periodic acid-Schiff and toluidine blue x 1100.
FIG. 8. Electron-micrograph of part of two liver cells from rat killed 6 days after single (10 mg/kg) dose of ochratoxin A. Accumulation of glycogen in the monoparticulate form and in rosettes is evident. The mitochondria show degenerative changes consisting of swelling (arrow) and then formation of a structureless area in the cytoplasm (double arrow), x 50,000.
ACUTE TOXICITY OF OCHRATOXIN A TO RATS
481
staining, but chromatin granules were still present. Two nuclei were frequently seen in each cell (Fig. 5) more especially on the periphery of the areas containing vacuoles. The nonstaining areas of the cytoplasm did not stain with Oil Red 0 or Pasini's stain, but stained dark red with periodic acid-Schiff. A variable but small number of fat-laden cells were observed in the frozen sections. The kidneys had normal glomeruli. A variable number of convoluted tubules in the cortex showed marked dilation. In some sections, usually in those kidneys in which dilation was marked, dilated tubules were seen in the medulla (Fig. 6). The dilated tubules were usually arranged in a wedge shape with the apex of the wedge in the medulla. Individual tubular cells in the dilated tubules were flattened and in some cases the nuclei were also fiat, giving the impression that the wall of the tubule had been stretched. Rats killed on day 10 General observations. Most of these rats received the lower doses of ochratoxin A, although three receiving 25 mg/kg survived until killed on day 10. The change in body weight was variable, ranging from a loss of 12 g to a gain of 40 g. On post-mortem examination the liver appeared normal in most rats, but in some rats it was paler than normal. The kidneys were paler than normal, with light grey streaks arranged radially in the cortex. The intestine and lungs were normal. Histology. In some liver sections no abnormalities were observed, but some showed lesions similar to those described in the previous group. Similarly some kidneys showed mild dilation in the collecting tubules, while in others marked dilation occurred. In general, however, the lesions were less extensive than in the previous group. No abnormalities were observed in the lungs and hearts. Control rats No macro- or microscopic changes were seen. Epon-embedded material Light microscopy of the thick sections revealed the same structureless areas in the liver cells as were observed in the paraffin sections. These areas were lightly pink when stained with toluidine blue and were positive to periodic acid-Schiff (Fig. 7). Thin sections viewed under the electron microscope showed large accumulations of glycogen in the cytoplasm with displacement of the organelles (Fig. 8). The glycogen was a mixture of the monoparticulate form and rosettes consisting of many particles. No glycogen was observed within the nucleus and the smooth and rough endoplasmic reticulum and mitochondria appeared normal. Cells which contained less glycogen, presumably from the centrilobular area (Fig. 4), contained mitochondria which showed degenerative changes. Some mitochondria were dilated, but contained normal cristae; others had lost their limiting membrane and most of their cristae, becoming structureless areas in the cytoplasm. DISCUSSION The acute toxicity of ochratoxin A to rats is very approximately half that of the most extensively studied mycotoxin, aflatoxin BI, the oral LDso of which has been estimated (Butler, 1964) as 7 mg/kg in male and 16 mg/kg in female rats. In the case of ochratoxin there was no significant difference between the LDso value for males and females.
482
I. F. H. PURCHASE
and
J. J. THERON
Renal necrosis was the most striking lesion observed after a high dose of ochratoxin A. Lower doses produced dilation of renal tubules between 4 and 10 days after dosing. In its mild form, where a wedge-shaped group of tubules was affected, the tubular dilation appeared to be due to blockage of a tubule in the medulla, with resulting dilation of a single nephron. The more severe lesions affecting a larger number of tubules were presumably due to blockage in a number of nephrons. Tubular dilation produced by ochratoxin A is similar to chronic nephritic changes, e.g. those produced by phenacetin (Abrahams, Rubenstein, Levin & Wunderlich, 1964) or by bacteria (Breslau, Gonick, Sommers & Guze, 1964), except that the chronic interstitial changes were absent. Single cell necrosis of the liver was observed in animals dying soon after dosing with ochratoxin A. A similar lesion was described in rats dosed with 100/zg ochratoxin A (Theron et al., 1966) and was accompanied by hyalin degeneration. The hyalin was found to consist of proliferating smooth endoplasmic reticulum forming "fingerprints". Lesions of this type were not observed in the present experiment, probably because of the difference in the dose used. A decrease in the glycogen content of the liver has been observed after administration of aflatoxin (Shank & Wogan, 1966), 3'-methyl-4-dimethylaminoazobenzene (Porter & Bruni, 1959) or ethionine (Steiner, Miyai & Phillips, 1964) and it is the expected response to toxin administration. The increase in the glycogen content of the cell after ochratoxin A dosing is not so common, although it has been described after administration of N-2-fluorenyldiacetamide (Mikata & Luse, 1964). The transient increase in glycogen caused by this carcinogen is accompanied by dramatic changes in the smooth endoplasmic reticulum. The observations on ochratoxin A are not extensive enough to provide information on the duration of the increase in glycogen, but simultaneously with the glycogen response, degenerative changes do occur in the mitochondria. The accumulation of glycogen is the most prominent feature of a group of diseases known as glycogen storage disease. Of the nine types of glycogen storage disease, each characterized by a specific enzyme deficiency, eight may occur in the liver (Hug, Garancis, Schubert & Kaplan, 1966). In Type I disease, the ultrastructure of the cytoplasm looks remarkably like that observed in ochratoxin poisoning (Baudhuin, Hers & Loeb, 1964). However, glycogen is also present in the nucleus (Sheldon, Silverberg & Kerner, 1962) in Type I disease. In Type II disease, the glycogen is present in membrane-surrounded vacuoles which differ from the glycogen in ochratoxin poisoning (Hug et al., 1966). Glycogen occurs in the heart in Type III disease and in Type IV disease there is a generalized distribution. Of the remaining diseases, Type VIII and Type IX (Hug et al., 1966) have lesions similar to those observed with ochratoxin A. Both Types VIII and IX are due to a decrease in phosphorylase activity. Recent observations in this laboratory indicate that ochratoxin A interferes with the phosphorylase enzyme system to produce the increase in liver glycogen (Pitout, 1968).
Acknowledgement--We wish
to thank Mr. N. Liebenberg for assistance with the photography.
REFERENCES Abrahams, C., Rubenstein, A. H., Levin, N. W. & Wunderlich, U. (1964). Experimentallyinduced analgesic nephritis in rats. Archs Path. 78, 222. Baudhuin, P., Hers, H. G. & Loeb, H. (1964). An electron microscope and biochemical study of type II glycogenosis. Lab. Invest. 13, 1139.
ACUTE TOXICITY OF OCHRATOXIN A TO RATS
483
Breslau, A. M., Gonick, M. C. Sommers, S. C. & Guze, L. B. (1964). Pathogenesis of chronic pyelonephritis. An1. J. Path. 44, 679. Butler, W. H. (1964). Acute toxicity of aflatoxin B~ in rats. Br. J. Cancer 18, 756. Hug, G., Garancis, J. C., Schubert, W. K. & Kaplan, S. (1966). Glycogen storage disease, types II, 1II, VIII, IX. An1. J. Dis. Child 111,457. Karnovsky, M. J. (1961). Simple methods for "Staining with Lead" at high pH in electron microscopy. J. biophys, biochem. Cytol. 11,729. Mikata, A. & Luse, S. A. (1964). Ultrastructural changes in the rat liver produced by N-2-fluorenyldiacetamide. Am. J. Path. 44, 455. Mollenhauer, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111. Pitout, M. J. (1968). The effect of ochratoxin A on glycogen storage in the rat liver. Toxic. appl. Pharmac. 13. In press. Porter, K. R. & Bruni, C. (1959). An electron microscope study of the early effects of 3'-Me-DAB on rat liver cells. Cancer Res. 19, 997. Scott, De B. (1965). Toxigenic fungi isolated from cereal and legume products. Mycopath. Mycol. appl. 25, 213. Shank, R. C. & Wogan, G. N. (1966). The acute effects of aflatoxin Bt on liver composition and metabolism in the rat and duckling. Toxic. appl. Pharmac. 9, 468. Sheldon, H., Silverberg, M. & Kerner, I. (1962). On the differing appearance of intranuclear and cytoplasmic glycogen in liver cells in glycogen storage disease. J. Cell. Biol. 13, 468. Steiner, J. W., M iyai, K. & Phillips, M. J. (1964). Electron microscopy of membrane--Particle arrays in liver cells of ethionine intoxicated rats. Am. J. Path. 44, 169. Theron, J. J., Van der Merwe, K. J., Liebenberg, N., Joubert, H. J. B. & Nel, W. (1966). Acute liver injury in ducklings and rats as a restllt of ochratoxin poisoning. J. Path, Bact. 91,521. Van der Merwe, K. J., Steyn, P. S. & Fourie, L. (1965). Mycotoxins. Part II. The constitution of ochratoxins A, B & C, metabolites of Aspergilh~s ochraceus Wilh. J. chem. Soc. 7083. Weft, C. S. (1952). Tables for convenient calculation of median effective dose (LDso or EDso) and instructions in their use. Biometrics 8, 249.
Toxicit~ aigui~ de rochratoxine A chez ie rat R~sum~--La toxicit~ orale aigu~ de l'ochratoxine A, un des trois m6tabolites chimiquement apparent~s d'Aspergillus ochraceus, a 6t6 d6termin~e chez des rats. Les valeurs de DLso obtenues, 22 mg/kg pour les raffles et 20 mg/kg pour les femelles, indiquent que la toxicit~ aigu~ de l'ochratoxine A est inf~rieure d'environ la moiti~ de celle de l'aflatoxine BI/t l'~gard de ces m~mes animaux. Les modifications pathologiques majeures associ6es aux doses fatales d'ochratoxine A sont la n6crose grave des tubuli r6naux et la n~crose des cellules p6riportales du foie; ces cellules sont g6n~ralement afl'ect~es individuellement, mais on observe parfois des foyers plus consid6rables de cellules n6cros6es. Des accumulations de glycog~ne ont 6t6 trouv~es dans le cytoplasme des cellules h6patiques; dans certaines cellules elles ~taient associ6es ',3. une d~g6n~rescence mitochondriale. L'auteur ~met l'hypoth~se que I'ochratoxine A agit sur le syst~me enzymatique de la phosphorylase pour provoquer cette augmentation du glycog~ne du foie.
Die akute Toxizit~it yon Ochratoxin A in Ratten Zusammenfassung--Die akute orale Toxizit~it von Ochratoxin A, eines von drei chemisch verwandten Stoffwechselprodukten von Aspergillus ochraceus, wurde an Ratten bestimmt. Die erhaltenen LDso-Werte, 22 mg/kg ftir m~innliche und 20 mg/kg ftir weibliche Tiere, lassen darauf schliessen, dass bei dieser Spezies die akute Toxizit/it von Ochratoxin A ungef~ihr die H~ilfte der von Aflatoxin Bt betriigt, Die nach t~Sdlichen Dosen yon Ochratoxin A gefundenen haupts~ichlichen pathologischen ,~nderungen waren schwere Nekrose der Nierenkan~lchen und Nekrose der periportalen Leberzellen, gew6hnlich nur bei einzelnen Zellen, wenn es auch gelegentlich gr6ssere Herde nekrotischer Zellen gab. Ansammlungen yon Glykogen wurden im Cytoplasma der Leberzellen gefunden und waren bei einigen Zellen von einer Degeneration der Mitochondrien begleitet. Vermutlich beeinflusst Ochratoxin A das Phosphorylaseenzymsystem u-ld verursacht so diese Zunahme des Leberglykogens.
The Acute Toxicity of Ochratoxin A to Rats I. F. H. PURCHASE and J. J. THERON Division of Toxicology, National Nutrition Research Institute, South African Council for Scientific and Industrial Research, Pretoria, South Africa (Received 1 March 1968)
Abstract--The acute oral toxicity of ochratoxin A, one of three chemically-relatedmetabolites from Aspergillas ochraceus, has been determined in rats. The LDs0 values obtained, 22 mg/kg for males and 20 mg/kg for females, indicate that the acute toxicity of ochratoxin A is about half that ofaflatoxin Bt in this species. The main pathological changes associated with fatal doses of ochratoxin A were severe necrosis of the renal tubules and necrosis of the periportal cells of the liver, usually affecting single cells although occasionally there were larger foci of necrotic cells. Accumulations of glycogen were found in the cytoplasm of the liver cells, accompanied in some cells by mitochondrial degeneration. It is .suggested that ochratoxin A may interfere with the phosphorylase enzyme system to produce this increase in liver glycogen. INTRODUCTION Ochratoxin A is one o f three chemically related metabolites isolated from Aspergillus ochraceus (Van der Merwe, Steyn & Fourie, 1965). This fungus was originally isolated f r o m s o r g h u m and was shown to be toxic to rats, mice a n d d a y - o l d ducklings (Scott, 1965). Theron, Van der Merwe, Liebenberg, J o u b e r t & Nel (1966) described the lesions p r o d u c e d by a single oral dose of 100 t~g o f ochratoxin A to ducklings a n d rats. This p a p e r describes the estimation of the oral L D s o of ochratoxin A in rats. A preliminary r e p o r t o f this w o r k was presented at the " S y m p o s i u m on Microbial T o x i n s " held by the A m e r i c a n Chemical Society in September 1966. EXPERIMENTAL Male or female W i s t a r rats from our own strain weighing a p p r o x i m a t e l y 80 g were separated into groups o f four and dosed by stomach tube with 10, 15, 25 or 40 mg o c h r a t o x i n A / k g b o d y weight. Crystalline ochratoxin A was dissolved in 0 . 1 N - N a H C O a and the concentration o f ochratoxin A was adjusted so that between 0" 3 and 0-6 ml o f solution was given to each rat. C o n t r o l rats were given 0 . 5 ml N a H C O 3 solution by s t o m a c h tube. All rats were given food and water ad lib. Surviving rats were killed on day 10. A p o s t - m o r t e m examination was p e r f o r m e d on all rats, a n d p o r t i o n s o f liver, kidney, heart and lung were fixed in buffered formalin. Paraffin sections o f these tissues were stained with h a e m a t o x y l i n and erythrosine and by Pasini's m e t h o d for connective tissue. F r o z e n sections were stained with Oil R e d 0 for fat. The LD5o values were derived from the m o r t a l i t y in each group by using the tables d r a w n up by Weft (1952). A further six female rats were dosed orally with ochratoxin A (5 or 10 mg/kg) and killed by cervical dislocation 4 - 6 days later in order to have fresh material for electron microscopy. 479
480
I. F. H. P U R C H A S E
and
J. J. T H E R O N
Small portions of liver were fixed in glutaraldehyde, post-fixed in l ~ osmium tetroxide in phosphate buffer and embedded in epon-araldite (Mollenhauer, 1964). Silver-to-grey sections were cut on a Porter-Blum or LKB ultramicrotome equipped with glass knives, placed on uncoated 400-mesh copper grids and stained with lead hydroxide as described by Karnovsky (1961). Thicker sections (0.5-1.0t~) of the same blocks were stained with toluidine blue or by the periodic acid-Schiff technique for light microscopy. RESULTS
Estimation of LDso The LDso values (and 95 ~o fiducial limits) were 22 mg/kg (15-30) for males and 20 mg/kg (9-41) for females. Rats dying between 24 and 73 hr General observations. These rats received the highest doses of ochratoxin A (25 or 40 mg/ kg) and, because they died so soon after dosing, showed an average weight loss of only 4 g. Post-mortem examination revealed a marked inflammation of the duodenum and jejunum with signs of diarrhoea. The livers were slightly paler than normal. The kidneys were pale and friable and the lungs showed signs of congestion. Histology. The liver had a normal architecture, but certain periportal cells were necrotic, showing karyorrhexis and disintegration of the cytoplasm. This necrosis usually affected single cells, but occasionally a larger focus of necrotic cells was seen (Fig. 1). The nuclei of the remaining cells were normal, but some of the cells had granular cytoplasm. The distribution of fat and connective tissue was normal. The kidneys showed a tubular necrosis. The cells of the proximal convoluted tubules had pycnotic nuclei (or occasionally karyorrhexis) and cytoplasm which had disintegrated and did not stain (Fig. 2). The glomeruli were normal. Other convoluted-tubular cells showed signs of cloudy swelling, and in some cases cast formation was evident. The necrotic tubules were seen mainly in the cortex, but in some cases necrotic tubules were present in the outer zone of the medulla (Fig. 3). These were presumed to be the tubules forming Henle's loop, but because of the necrotic changes it was difficult to ascertain the cell types forming the tubule. In the most severe cases, necrosis occurred not only in these tubules but in other (collecting) tubules in the medulla. The lungs of some animals showed signs of hyperaemia and a necrotic focus was seen in the myocardium of one rat. Rats dying between days 4 and 6 General observations. Rats from each dose range died at this stage, but it was remarkable that only females died. The rats showed an average weight loss of 14 g and were in a poor condition when they died. There was no diarrhoea or enteritis in this group. The kidneys and livers appeared similar to those in the previous group. Congestion of the lungs was observed in some rats. Histology. Under the low power objective ( x 10), the liver parenchymal cells in the periportal areas had slightly darker-staining nuclei, but their cytoplasm did not stain (Fig. 4). This gave the impression of nuclei floating free in otherwise empty cells. Under higher magnification it was seen that the areas which were not eosinophilic in the cytoplasm were more or less rounded, with thin trabeculae separating them (Fig. 5). The nuclei were darker
FIG. 1. Focal necrosis in liver of rat dying 16 hr after a single (40 mg/kg) dose of ochratoxin A. Haematoxylin and erythrosine x 880.
FIG. 2. Section of renal cortex of rat dying after single (25 mg/kg) dose of ochratoxin A, showing normal glomerulus but necrosis of the proximal convoluted tubule. Haematoxylin and erythrosine x 880.
Fie. 3. Marked tubular necrosis in medulla of kidney of rat dying after single (25 mg/kg) dose of ochratoxin A. Haematoxylin and erythrosine x 220.
FIG. 4. Marked periportal vacuolation due to gl2(cogen accumulation in liver of rat killed 6 days after single (10 mg/kg) dose of ochratoxin A. Haematoxylin and erythrosine x 220.
Fie. 5. Area of glycogen accumulation in liver of rat killecL6 days after single (10 mg/kg) dose of ochratoxin A, showing thin trabeculae separating cytoplasmic vacuoles. Haematoxylin and erythrosine x 1100.
F~o. 6. Markedly dilated tubules in kidney of rat killed 10 days after single (15 mg/kg) dose of ochratoxin A. Haematoxylin and erythrosine x 220.
FIo. 7. Lakes o/' glycogen seen as dark-staining masses in cytoplasm of hepatocytas in thick section of el:onembedded liver from rat killed 6 days after single (10 mg/kg) dose of ochratoxin A. Periodic acid-Schiff and toluidine blue x 1100.
FIG. 8. Electron-micrograph of part of two liver cells from rat killed 6 days after single (10 mg/kg) dose of ochratoxin A. Accumulation of glycogen in the monoparticulate form and in rosettes is evident. The mitochondria show degenerative changes consisting of swelling (arrow) and then formation of a structureless area in the cytoplasm (double arrow), x 50,000.
ACUTE TOXICITY OF OCHRATOXIN A TO RATS
481
staining, but chromatin granules were still present. Two nuclei were frequently seen in each cell (Fig. 5) more especially on the periphery of the areas containing vacuoles. The nonstaining areas of the cytoplasm did not stain with Oil Red 0 or Pasini's stain, but stained dark red with periodic acid-Schiff. A variable but small number of fat-laden cells were observed in the frozen sections. The kidneys had normal glomeruli. A variable number of convoluted tubules in the cortex showed marked dilation. In some sections, usually in those kidneys in which dilation was marked, dilated tubules were seen in the medulla (Fig. 6). The dilated tubules were usually arranged in a wedge shape with the apex of the wedge in the medulla. Individual tubular cells in the dilated tubules were flattened and in some cases the nuclei were also fiat, giving the impression that the wall of the tubule had been stretched. Rats killed on day 10 General observations. Most of these rats received the lower doses of ochratoxin A, although three receiving 25 mg/kg survived until killed on day 10. The change in body weight was variable, ranging from a loss of 12 g to a gain of 40 g. On post-mortem examination the liver appeared normal in most rats, but in some rats it was paler than normal. The kidneys were paler than normal, with light grey streaks arranged radially in the cortex. The intestine and lungs were normal. Histology. In some liver sections no abnormalities were observed, but some showed lesions similar to those described in the previous group. Similarly some kidneys showed mild dilation in the collecting tubules, while in others marked dilation occurred. In general, however, the lesions were less extensive than in the previous group. No abnormalities were observed in the lungs and hearts. Control rats No macro- or microscopic changes were seen. Epon-embedded material Light microscopy of the thick sections revealed the same structureless areas in the liver cells as were observed in the paraffin sections. These areas were lightly pink when stained with toluidine blue and were positive to periodic acid-Schiff (Fig. 7). Thin sections viewed under the electron microscope showed large accumulations of glycogen in the cytoplasm with displacement of the organelles (Fig. 8). The glycogen was a mixture of the monoparticulate form and rosettes consisting of many particles. No glycogen was observed within the nucleus and the smooth and rough endoplasmic reticulum and mitochondria appeared normal. Cells which contained less glycogen, presumably from the centrilobular area (Fig. 4), contained mitochondria which showed degenerative changes. Some mitochondria were dilated, but contained normal cristae; others had lost their limiting membrane and most of their cristae, becoming structureless areas in the cytoplasm. DISCUSSION The acute toxicity of ochratoxin A to rats is very approximately half that of the most extensively studied mycotoxin, aflatoxin BI, the oral LDso of which has been estimated (Butler, 1964) as 7 mg/kg in male and 16 mg/kg in female rats. In the case of ochratoxin there was no significant difference between the LDso value for males and females.
482
I. F. H. PURCHASE
and
J. J. THERON
Renal necrosis was the most striking lesion observed after a high dose of ochratoxin A. Lower doses produced dilation of renal tubules between 4 and 10 days after dosing. In its mild form, where a wedge-shaped group of tubules was affected, the tubular dilation appeared to be due to blockage of a tubule in the medulla, with resulting dilation of a single nephron. The more severe lesions affecting a larger number of tubules were presumably due to blockage in a number of nephrons. Tubular dilation produced by ochratoxin A is similar to chronic nephritic changes, e.g. those produced by phenacetin (Abrahams, Rubenstein, Levin & Wunderlich, 1964) or by bacteria (Breslau, Gonick, Sommers & Guze, 1964), except that the chronic interstitial changes were absent. Single cell necrosis of the liver was observed in animals dying soon after dosing with ochratoxin A. A similar lesion was described in rats dosed with 100/zg ochratoxin A (Theron et al., 1966) and was accompanied by hyalin degeneration. The hyalin was found to consist of proliferating smooth endoplasmic reticulum forming "fingerprints". Lesions of this type were not observed in the present experiment, probably because of the difference in the dose used. A decrease in the glycogen content of the liver has been observed after administration of aflatoxin (Shank & Wogan, 1966), 3'-methyl-4-dimethylaminoazobenzene (Porter & Bruni, 1959) or ethionine (Steiner, Miyai & Phillips, 1964) and it is the expected response to toxin administration. The increase in the glycogen content of the cell after ochratoxin A dosing is not so common, although it has been described after administration of N-2-fluorenyldiacetamide (Mikata & Luse, 1964). The transient increase in glycogen caused by this carcinogen is accompanied by dramatic changes in the smooth endoplasmic reticulum. The observations on ochratoxin A are not extensive enough to provide information on the duration of the increase in glycogen, but simultaneously with the glycogen response, degenerative changes do occur in the mitochondria. The accumulation of glycogen is the most prominent feature of a group of diseases known as glycogen storage disease. Of the nine types of glycogen storage disease, each characterized by a specific enzyme deficiency, eight may occur in the liver (Hug, Garancis, Schubert & Kaplan, 1966). In Type I disease, the ultrastructure of the cytoplasm looks remarkably like that observed in ochratoxin poisoning (Baudhuin, Hers & Loeb, 1964). However, glycogen is also present in the nucleus (Sheldon, Silverberg & Kerner, 1962) in Type I disease. In Type II disease, the glycogen is present in membrane-surrounded vacuoles which differ from the glycogen in ochratoxin poisoning (Hug et al., 1966). Glycogen occurs in the heart in Type III disease and in Type IV disease there is a generalized distribution. Of the remaining diseases, Type VIII and Type IX (Hug et al., 1966) have lesions similar to those observed with ochratoxin A. Both Types VIII and IX are due to a decrease in phosphorylase activity. Recent observations in this laboratory indicate that ochratoxin A interferes with the phosphorylase enzyme system to produce the increase in liver glycogen (Pitout, 1968).
Acknowledgement--We wish
to thank Mr. N. Liebenberg for assistance with the photography.
REFERENCES Abrahams, C., Rubenstein, A. H., Levin, N. W. & Wunderlich, U. (1964). Experimentallyinduced analgesic nephritis in rats. Archs Path. 78, 222. Baudhuin, P., Hers, H. G. & Loeb, H. (1964). An electron microscope and biochemical study of type II glycogenosis. Lab. Invest. 13, 1139.
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Breslau, A. M., Gonick, M. C. Sommers, S. C. & Guze, L. B. (1964). Pathogenesis of chronic pyelonephritis. An1. J. Path. 44, 679. Butler, W. H. (1964). Acute toxicity of aflatoxin B~ in rats. Br. J. Cancer 18, 756. Hug, G., Garancis, J. C., Schubert, W. K. & Kaplan, S. (1966). Glycogen storage disease, types II, 1II, VIII, IX. An1. J. Dis. Child 111,457. Karnovsky, M. J. (1961). Simple methods for "Staining with Lead" at high pH in electron microscopy. J. biophys, biochem. Cytol. 11,729. Mikata, A. & Luse, S. A. (1964). Ultrastructural changes in the rat liver produced by N-2-fluorenyldiacetamide. Am. J. Path. 44, 455. Mollenhauer, H. H. (1964). Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39, 111. Pitout, M. J. (1968). The effect of ochratoxin A on glycogen storage in the rat liver. Toxic. appl. Pharmac. 13. In press. Porter, K. R. & Bruni, C. (1959). An electron microscope study of the early effects of 3'-Me-DAB on rat liver cells. Cancer Res. 19, 997. Scott, De B. (1965). Toxigenic fungi isolated from cereal and legume products. Mycopath. Mycol. appl. 25, 213. Shank, R. C. & Wogan, G. N. (1966). The acute effects of aflatoxin Bt on liver composition and metabolism in the rat and duckling. Toxic. appl. Pharmac. 9, 468. Sheldon, H., Silverberg, M. & Kerner, I. (1962). On the differing appearance of intranuclear and cytoplasmic glycogen in liver cells in glycogen storage disease. J. Cell. Biol. 13, 468. Steiner, J. W., M iyai, K. & Phillips, M. J. (1964). Electron microscopy of membrane--Particle arrays in liver cells of ethionine intoxicated rats. Am. J. Path. 44, 169. Theron, J. J., Van der Merwe, K. J., Liebenberg, N., Joubert, H. J. B. & Nel, W. (1966). Acute liver injury in ducklings and rats as a restllt of ochratoxin poisoning. J. Path, Bact. 91,521. Van der Merwe, K. J., Steyn, P. S. & Fourie, L. (1965). Mycotoxins. Part II. The constitution of ochratoxins A, B & C, metabolites of Aspergilh~s ochraceus Wilh. J. chem. Soc. 7083. Weft, C. S. (1952). Tables for convenient calculation of median effective dose (LDso or EDso) and instructions in their use. Biometrics 8, 249.
Toxicit~ aigui~ de rochratoxine A chez ie rat R~sum~--La toxicit~ orale aigu~ de l'ochratoxine A, un des trois m6tabolites chimiquement apparent~s d'Aspergillus ochraceus, a 6t6 d6termin~e chez des rats. Les valeurs de DLso obtenues, 22 mg/kg pour les raffles et 20 mg/kg pour les femelles, indiquent que la toxicit~ aigu~ de l'ochratoxine A est inf~rieure d'environ la moiti~ de celle de l'aflatoxine BI/t l'~gard de ces m~mes animaux. Les modifications pathologiques majeures associ6es aux doses fatales d'ochratoxine A sont la n6crose grave des tubuli r6naux et la n~crose des cellules p6riportales du foie; ces cellules sont g6n~ralement afl'ect~es individuellement, mais on observe parfois des foyers plus consid6rables de cellules n6cros6es. Des accumulations de glycog~ne ont 6t6 trouv~es dans le cytoplasme des cellules h6patiques; dans certaines cellules elles ~taient associ6es ',3. une d~g6n~rescence mitochondriale. L'auteur ~met l'hypoth~se que I'ochratoxine A agit sur le syst~me enzymatique de la phosphorylase pour provoquer cette augmentation du glycog~ne du foie.
Die akute Toxizit~it yon Ochratoxin A in Ratten Zusammenfassung--Die akute orale Toxizit~it von Ochratoxin A, eines von drei chemisch verwandten Stoffwechselprodukten von Aspergillus ochraceus, wurde an Ratten bestimmt. Die erhaltenen LDso-Werte, 22 mg/kg ftir m~innliche und 20 mg/kg ftir weibliche Tiere, lassen darauf schliessen, dass bei dieser Spezies die akute Toxizit/it von Ochratoxin A ungef~ihr die H~ilfte der von Aflatoxin Bt betriigt, Die nach t~Sdlichen Dosen yon Ochratoxin A gefundenen haupts~ichlichen pathologischen ,~nderungen waren schwere Nekrose der Nierenkan~lchen und Nekrose der periportalen Leberzellen, gew6hnlich nur bei einzelnen Zellen, wenn es auch gelegentlich gr6ssere Herde nekrotischer Zellen gab. Ansammlungen yon Glykogen wurden im Cytoplasma der Leberzellen gefunden und waren bei einigen Zellen von einer Degeneration der Mitochondrien begleitet. Vermutlich beeinflusst Ochratoxin A das Phosphorylaseenzymsystem u-ld verursacht so diese Zunahme des Leberglykogens.