TOXICOLOGY
AND
APPLIED
Histopathological
MASAYOSHI
Department of Pathology, Tokyo 173, and Research
42,55-64
PHARMACOLOGY
(1977)
Studies on the Toxicity of Ochratoxin Rats. I. Acute Oral Toxicity KANISAWA,’
SHIGETOSHI SUZUKI, YOSHIMICHI AND MIKIO Y AMAZAKI
A in
KOZUKA,
Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-rho, ltabashi-ku, Institute for Chemobiodynamics, Chiba University, Izumi-rho, Narashino-shi, Chiba 275. Japan
Received
October
15, 1976; accepted
April
21. I977
Histopathological
Studies on the Toxicity of Ochratoxin A in Rats. 1. Acute Oral Toxicity. S.. KOZUKA, Y., AND YAMAZAEI, M. (1977). Toxicol. Appl. Pharmacol. 42. 55-64. The acute oral toxicity of ochratoxin A was studied in adult male Wistar rats using single or multiple doses. The oral LD50 was estimated to be 28 mg/kg. The most striking pathologic changes following treatment occurred in the duodenum and jejunum, characterized by severe catarrhal or erosive enteritis which appeared within 4 hr after dosing and subsided within 24 hr. Although near-lethal single doses did not elicit any evidence of renal damage. after several consecutive daily doses. massive acidophilic degeneration with necrosis and desquamation of epithelium occurred in the proximal tubules. Necrosis of cells in germinal centers of the spleen and lymph nodes occurred, indicating a sensitivity of lymphatic tissues to the toxin. Hepatotoxicity was minimal, limited principally to depletion of hepatic glycogen. KANISAWA.
M..
SUZUKI.
Ochratoxin A (OCT A) is a mycotoxin produced by several speciesof Aspergillus (Scott, 1965; Hesseltineet al., 1972) and Penicillium (Van Walbeek et al., 1969; Ciegler et al., 1972). The distribution of thesefungi in the natural environment is extensive. The presenceof this toxin has been demonstrated in sorghum (Scott, 196.5),corn (Shotwell et al., 1969), and wheat (Scott et al., 1970). Previous studiesby others indicated that OCT A is harmful to a variety of laboratory animals, such as ducklings (Theron et al., 1966), rats (Theron et al., 1966; Purchase and Theron, 1968; Purchase and van der Watt, 1971; Munro et al., 1974), chicks (Peckham et al., 1971). hens(Choudhury et al., 1971), beagle dogs (Szczech et al., 1973a,b), pigs (Szczech et al., 1973c), and rainbow trout (Doster er ai., 1972). In Denmark, this toxin is considered to be one of the causative factors in mold nephrosisof pigs (Krogh and Hasselager, 1968; Carlton and Tuite, 1970; Elling and Mailer, 1973) and much attention has beenfocused on this toxin asa possibleenvironmental hazard to humansand various domestic animals. This report dealswith the determination of the oral LDSO in young adult rats and the histopathology of acute lesionsinduced by single or multiple dosesof OCT A. Other investigations on the acute and chronic toxicity of OCT A are in progress. ’ Address Metropolitan
reprint Institute
requests to of Gerontology.
(‘opygllt Q 1977 by Academic Press. Inc. Ail rights of rrproductlon m any frrn rrrrrvrd. Prmted in Great 8rlt:il”
Masayoshi Kanisawa. M.D.. 35-2 Sakae-cho. Itabashi-ku. 55
Department of Pathology, Tokyo 173, Japan.
Tokyo
ISSN OOJI-00X\
56
KANISAWA
ET
AL
METHODS OCT A used in this study was obtained from the culture medium of Aspergillus ochraceus Wilhelm (IMF 4443). The methods of purification and quantification were described in a previous report (Suzuki et al., 1975b). Male Wistar rats, 210 & 10 g, were given standard commercial diet and water ad libitum. Desired dosesof OCT A, freshly suspendedin 0.4 ml/l00 g of 0.1% aqueous carboxymethyl cellulose-Na (CMC-Na), were administered to nonfasted rats by gastric intubation. Control rats were given vehicle only. The animals were killed by decapitation. For histological examination, specimens of the liver. kidney, spleen. adrenal, stomach, intestine, and other organs were fixed in 10% buffered formalin. For demonstration of liver glycogen, pure ethanol was used for fixation. Tissues were embedded in paraffin and stained with hematoxylin and eosin, PAS, and alcian blue PAS. Toxicity and LD50 of single doses. OCT A was given in single oral dosesof 15. 20. 25, 30, 35, 40, 45, and 50 mg/kg to 10 rats of each group, respectively. Mortality was recorded during a 72-hr post-treatment period, and the oral LD50 was estimated by the method of Litchfield and Wilcoxon (1949). Gross examination was made of all dead rats, and tissues of about half of the rats of each group were examined histologically. Surviving rats were killed 1 week later for examination. In addition, 10 rats each were given 15, 20, or 40 mg/kg of OCT A in order to study early changesin the intestine and kidney. For this purpose, rats were killed 4 hr after the treatment except for 2 that were given 40 mg/kg of the toxin and killed after 24 hr. Toxicity of repeated doses. Ten rats each were given three dosesof 5 mg/kg or four doses of 10 mg/kg of OCT A or aqueous CMC-Na only (control) by gastric intubation. All the animals were killed 24 hr after the last dose.
RESULTS
Oral LD50. Doses of 15, 20, 25, 30, 35, 40, 45, and 50 mg/kg resulted in a 72.hr mortality rate of O/10, l/10, 3/10, 6/10, 8/10, 9/10, 9/10, and lo/IO, respectively. Rats began dying 12 hr after dosing. The acute oral LD50 in male Wistar rats was calculated to be 28.0 mg/kg with 95.5 confidence limits of 24.3-32.2 mg/kg. The slope function was 1.31. Toxicity of single doses. Catarrhal enteritis was the most conspicuous and constant finding among the initial changes in rats that received single oral dosesof more than 5 mg/kg. The small intestine showed marked dilatation due to accumulation of seromucousexudate in the lumen. Edema and hyperemia of the mucosa was noted 4 hr after dosing (Fig. 1). The intestinal contents appeared to be largely composed of exudate; the stomach and cecum were also greatly dilated and contained copious amounts of serous exudate, although no inflammatory changes were apparent. The large intestines were essentially normal. A small quantity of clear ascitic fluid was present in most of the rats that received large dosesof OCT A. Grossly, the liver appeared slightly paler than normal. The kidneys were slightly swollen and pale. On cut surface, the medulla was congestedand the corticomedullary junction was accentuated. The adrenals of rats that received more than 40 mg/kg of
HISTOPATHOLOGY
OF
OCHRATOXIN
A
TOXICITY
59
Acute toxicity of repeated doses. No deaths occurred in rats given three doses of 5 mg/kg of OCT A. Three of five rats given daily doses of 10 mg/kg were found dead 24 hr after the fourth dose. Grossly, the small intestine had slight swelling and moderate hyperemia of the mucosa. Feces appeared normal and no apparent exudation was observed in either
FIG. 3. Swollen villi of the jejunum of a rat administered 40 mg/kg of OCT A. Epithelial cells are markedly flat and elongated. Cellular debris and products of necrosis can be seen in the edematous lamina propria. H&E. x 625.
group. The liver was pale and the kidneys were slightly hyperemic. Congestion was marked in the renal medulla of rats given 10 mg/kg and the adrenals of the latter group had marked congestion of the medulla and inner cortex. Other viscera were grossly normal. At daily doses of 5 and 10 mg/kg for 3 to 4 days, severe renal tubular damage occurred. The proximal tubular epithelium had scattered or semimassive necrosis and fine or coarse hyaline droplets. The earliest and most severe damage occurred in the pars recta of proximal tubules. The lumina of proximal tubules contained desquamated cells and casts (Fig. 7). No obvious change was observed in Henle’s loops and further distal parts of the nephron, except for occasional nuclear pyknosis in the epithelium.
60
KANISAWA
ET
AL.
FIG. 4. Proximal tubules of the kidney 4 hr after a single dose of 40 mg/kg of OCT A. Tubular epithelial cells contain hyaline droplets. Some nuclei are pyknotic. although no frank necrosis is \een H&E. :< 1000.
FIG. 5. Liver of a rat 24 hr after a single dose of 15 mg/kg of OCT A. A f ecus of single-cell with leukocytic infiltration is shown in the upper right part of the photograph. Two mitotic figures are also shown. H&E. x625.
necrosis (arrows)
HISTOPATHOLOGY
FIG;. 6. Germinal
center of a spleen follicle
OF
OCHRATOXIN
A TOXICITY
4 hr after a dose of I5 mg/kg
ofOCT
6
A. H&E.
Y 625
‘I(;. 7. Kidney of a rat 24 hr after the final dose of four consecutive daily doses of IO mg/kg 01 T .A No glomerular change is observed. Necrosis of the epithelium of the proximal tubules associated 1 ~jesquamation ofepithelial cells is noted. H&E. (675.
62
KANISAWA
1, I /iI
In the liver. there were scattered foci of single-cell necrosis surrounded b> a tew leukocytes and occasional mitotic figures. Such necrotic foci were present mostly, in the periportal region. The hepatic cellular cytoplasm appeared rather opaque and full of tine granular particles. No glycogen was demonstrable to PAS reaction. Fatty change ~a~ minimal in either group (Fig. 8). In the small intestine. mild edema and cellular infiltration were observed in the lamina propria of the duodenum and jejunum. The central lacteal of villi appeared moderatel>
EKG. 8. Lwer of a rat 24 hr after the tinal dose of four consecutive daily doses of 10 mgikg Focal necrosts of hepatic cells and scattered acidophilic change arc seen. H&h. S625.
OI’O~‘!
\
distended. The epithelial lining of intestinal villi was continuous. and degeneration 01 epithelial cells was not apparent 24 hr after the last dose. Transepithelial migratron o!‘ lymphocytes and eosinophiles appeared moderately active. In all groups of animals, products of necrosis were conspicuous in germinal centers ot the lymphoid follicles of spleen, mesenteric lymph nodes. and the intestinal wall. DlSCUSSlON The oral LD50 value for OCT A in our study, 28 mg/kg, is slightly higher than that previously reported by Purchase and Theron (1968). i.e.. 22 mg/kg. This difference might reflect the differences in experimental conditions. such as the age or body weight of animals and vehicle for OCT A, and is probably insignificant. OCT A has been demonstrated to be a nephrotoxin in several species of animals. and our findings in the rat are consistent with previous data. in that the primary site of renal inJury is the proximal tubular epithelium (Purchase and Theron. 1968). In our study.
HISTOPATHOLOGY
OF
OCHRATOXIN
A
TOXICITY
63
Lhe earliest and most severe damage was seen in the pars recta of the proximal tubule. Such iocalization of renal damage was clearly demonstrated in the multiple-dose experiment. What is characteristic of this sort of renal lesion is that a single administration of the toxin, even at sublethal doses by gastric intubation, caused only negligible change in the tubular epithelium during a 7-day observation period. and three or more repetitive doses were required to induce recognizable changes. This finding is substantiated by the data from renal function tests presented in our previous report i 1975b). These results differ from those of Purchase and Theron (1968), in which a smgle dose produced tubular necrosis. The mechanism of this difference is not clear, but may be related to the different ages of animals in the two studies. Possible causes of the delayed onset of renal damage after repeated administration might Include :he participation of some metabolite(s). yet unidentified, or the biotransformation products or’0C-f A. OCT A is a minor hepatotoxin in the rat. The major change found was depletion of bepatic glycogen, a finding which is in agreement with that reported by others (Suzuki &!Iai.. I975a; Munro et al., 1973). Acute enteritis due to OCT A has been noted in the rat (Purchase and Theron. 1968). &.li. metabolism in rat liver. Tosicol. App/. Pharmaco/. 32. I lb-- 122. S~‘X~Ihl. S.. Koz~:ss. \i.. C,\roti. T.. ,\NI) \r’.+rir,\%.Zhl. M. ( lQ75b). Studieb \Ifi 111~.iiephrc~r~~il Iheaglc 3.lL.t ii Pathology. I ‘A. Parhoi, 10. 7! 9 --33 I. DOSTER,
AND
APPLIED
Histopathological
MASAYOSHI
Department of Pathology, Tokyo 173, and Research
42,55-64
PHARMACOLOGY
(1977)
Studies on the Toxicity of Ochratoxin Rats. I. Acute Oral Toxicity KANISAWA,’
SHIGETOSHI SUZUKI, YOSHIMICHI AND MIKIO Y AMAZAKI
A in
KOZUKA,
Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-rho, ltabashi-ku, Institute for Chemobiodynamics, Chiba University, Izumi-rho, Narashino-shi, Chiba 275. Japan
Received
October
15, 1976; accepted
April
21. I977
Histopathological
Studies on the Toxicity of Ochratoxin A in Rats. 1. Acute Oral Toxicity. S.. KOZUKA, Y., AND YAMAZAEI, M. (1977). Toxicol. Appl. Pharmacol. 42. 55-64. The acute oral toxicity of ochratoxin A was studied in adult male Wistar rats using single or multiple doses. The oral LD50 was estimated to be 28 mg/kg. The most striking pathologic changes following treatment occurred in the duodenum and jejunum, characterized by severe catarrhal or erosive enteritis which appeared within 4 hr after dosing and subsided within 24 hr. Although near-lethal single doses did not elicit any evidence of renal damage. after several consecutive daily doses. massive acidophilic degeneration with necrosis and desquamation of epithelium occurred in the proximal tubules. Necrosis of cells in germinal centers of the spleen and lymph nodes occurred, indicating a sensitivity of lymphatic tissues to the toxin. Hepatotoxicity was minimal, limited principally to depletion of hepatic glycogen. KANISAWA.
M..
SUZUKI.
Ochratoxin A (OCT A) is a mycotoxin produced by several speciesof Aspergillus (Scott, 1965; Hesseltineet al., 1972) and Penicillium (Van Walbeek et al., 1969; Ciegler et al., 1972). The distribution of thesefungi in the natural environment is extensive. The presenceof this toxin has been demonstrated in sorghum (Scott, 196.5),corn (Shotwell et al., 1969), and wheat (Scott et al., 1970). Previous studiesby others indicated that OCT A is harmful to a variety of laboratory animals, such as ducklings (Theron et al., 1966), rats (Theron et al., 1966; Purchase and Theron, 1968; Purchase and van der Watt, 1971; Munro et al., 1974), chicks (Peckham et al., 1971). hens(Choudhury et al., 1971), beagle dogs (Szczech et al., 1973a,b), pigs (Szczech et al., 1973c), and rainbow trout (Doster er ai., 1972). In Denmark, this toxin is considered to be one of the causative factors in mold nephrosisof pigs (Krogh and Hasselager, 1968; Carlton and Tuite, 1970; Elling and Mailer, 1973) and much attention has beenfocused on this toxin asa possibleenvironmental hazard to humansand various domestic animals. This report dealswith the determination of the oral LDSO in young adult rats and the histopathology of acute lesionsinduced by single or multiple dosesof OCT A. Other investigations on the acute and chronic toxicity of OCT A are in progress. ’ Address Metropolitan
reprint Institute
requests to of Gerontology.
(‘opygllt Q 1977 by Academic Press. Inc. Ail rights of rrproductlon m any frrn rrrrrvrd. Prmted in Great 8rlt:il”
Masayoshi Kanisawa. M.D.. 35-2 Sakae-cho. Itabashi-ku. 55
Department of Pathology, Tokyo 173, Japan.
Tokyo
ISSN OOJI-00X\
56
KANISAWA
ET
AL
METHODS OCT A used in this study was obtained from the culture medium of Aspergillus ochraceus Wilhelm (IMF 4443). The methods of purification and quantification were described in a previous report (Suzuki et al., 1975b). Male Wistar rats, 210 & 10 g, were given standard commercial diet and water ad libitum. Desired dosesof OCT A, freshly suspendedin 0.4 ml/l00 g of 0.1% aqueous carboxymethyl cellulose-Na (CMC-Na), were administered to nonfasted rats by gastric intubation. Control rats were given vehicle only. The animals were killed by decapitation. For histological examination, specimens of the liver. kidney, spleen. adrenal, stomach, intestine, and other organs were fixed in 10% buffered formalin. For demonstration of liver glycogen, pure ethanol was used for fixation. Tissues were embedded in paraffin and stained with hematoxylin and eosin, PAS, and alcian blue PAS. Toxicity and LD50 of single doses. OCT A was given in single oral dosesof 15. 20. 25, 30, 35, 40, 45, and 50 mg/kg to 10 rats of each group, respectively. Mortality was recorded during a 72-hr post-treatment period, and the oral LD50 was estimated by the method of Litchfield and Wilcoxon (1949). Gross examination was made of all dead rats, and tissues of about half of the rats of each group were examined histologically. Surviving rats were killed 1 week later for examination. In addition, 10 rats each were given 15, 20, or 40 mg/kg of OCT A in order to study early changesin the intestine and kidney. For this purpose, rats were killed 4 hr after the treatment except for 2 that were given 40 mg/kg of the toxin and killed after 24 hr. Toxicity of repeated doses. Ten rats each were given three dosesof 5 mg/kg or four doses of 10 mg/kg of OCT A or aqueous CMC-Na only (control) by gastric intubation. All the animals were killed 24 hr after the last dose.
RESULTS
Oral LD50. Doses of 15, 20, 25, 30, 35, 40, 45, and 50 mg/kg resulted in a 72.hr mortality rate of O/10, l/10, 3/10, 6/10, 8/10, 9/10, 9/10, and lo/IO, respectively. Rats began dying 12 hr after dosing. The acute oral LD50 in male Wistar rats was calculated to be 28.0 mg/kg with 95.5 confidence limits of 24.3-32.2 mg/kg. The slope function was 1.31. Toxicity of single doses. Catarrhal enteritis was the most conspicuous and constant finding among the initial changes in rats that received single oral dosesof more than 5 mg/kg. The small intestine showed marked dilatation due to accumulation of seromucousexudate in the lumen. Edema and hyperemia of the mucosa was noted 4 hr after dosing (Fig. 1). The intestinal contents appeared to be largely composed of exudate; the stomach and cecum were also greatly dilated and contained copious amounts of serous exudate, although no inflammatory changes were apparent. The large intestines were essentially normal. A small quantity of clear ascitic fluid was present in most of the rats that received large dosesof OCT A. Grossly, the liver appeared slightly paler than normal. The kidneys were slightly swollen and pale. On cut surface, the medulla was congestedand the corticomedullary junction was accentuated. The adrenals of rats that received more than 40 mg/kg of
HISTOPATHOLOGY
OF
OCHRATOXIN
A
TOXICITY
59
Acute toxicity of repeated doses. No deaths occurred in rats given three doses of 5 mg/kg of OCT A. Three of five rats given daily doses of 10 mg/kg were found dead 24 hr after the fourth dose. Grossly, the small intestine had slight swelling and moderate hyperemia of the mucosa. Feces appeared normal and no apparent exudation was observed in either
FIG. 3. Swollen villi of the jejunum of a rat administered 40 mg/kg of OCT A. Epithelial cells are markedly flat and elongated. Cellular debris and products of necrosis can be seen in the edematous lamina propria. H&E. x 625.
group. The liver was pale and the kidneys were slightly hyperemic. Congestion was marked in the renal medulla of rats given 10 mg/kg and the adrenals of the latter group had marked congestion of the medulla and inner cortex. Other viscera were grossly normal. At daily doses of 5 and 10 mg/kg for 3 to 4 days, severe renal tubular damage occurred. The proximal tubular epithelium had scattered or semimassive necrosis and fine or coarse hyaline droplets. The earliest and most severe damage occurred in the pars recta of proximal tubules. The lumina of proximal tubules contained desquamated cells and casts (Fig. 7). No obvious change was observed in Henle’s loops and further distal parts of the nephron, except for occasional nuclear pyknosis in the epithelium.
60
KANISAWA
ET
AL.
FIG. 4. Proximal tubules of the kidney 4 hr after a single dose of 40 mg/kg of OCT A. Tubular epithelial cells contain hyaline droplets. Some nuclei are pyknotic. although no frank necrosis is \een H&E. :< 1000.
FIG. 5. Liver of a rat 24 hr after a single dose of 15 mg/kg of OCT A. A f ecus of single-cell with leukocytic infiltration is shown in the upper right part of the photograph. Two mitotic figures are also shown. H&E. x625.
necrosis (arrows)
HISTOPATHOLOGY
FIG;. 6. Germinal
center of a spleen follicle
OF
OCHRATOXIN
A TOXICITY
4 hr after a dose of I5 mg/kg
ofOCT
6
A. H&E.
Y 625
‘I(;. 7. Kidney of a rat 24 hr after the final dose of four consecutive daily doses of IO mg/kg 01 T .A No glomerular change is observed. Necrosis of the epithelium of the proximal tubules associated 1 ~jesquamation ofepithelial cells is noted. H&E. (675.
62
KANISAWA
1, I /iI
In the liver. there were scattered foci of single-cell necrosis surrounded b> a tew leukocytes and occasional mitotic figures. Such necrotic foci were present mostly, in the periportal region. The hepatic cellular cytoplasm appeared rather opaque and full of tine granular particles. No glycogen was demonstrable to PAS reaction. Fatty change ~a~ minimal in either group (Fig. 8). In the small intestine. mild edema and cellular infiltration were observed in the lamina propria of the duodenum and jejunum. The central lacteal of villi appeared moderatel>
EKG. 8. Lwer of a rat 24 hr after the tinal dose of four consecutive daily doses of 10 mgikg Focal necrosts of hepatic cells and scattered acidophilic change arc seen. H&h. S625.
OI’O~‘!
\
distended. The epithelial lining of intestinal villi was continuous. and degeneration 01 epithelial cells was not apparent 24 hr after the last dose. Transepithelial migratron o!‘ lymphocytes and eosinophiles appeared moderately active. In all groups of animals, products of necrosis were conspicuous in germinal centers ot the lymphoid follicles of spleen, mesenteric lymph nodes. and the intestinal wall. DlSCUSSlON The oral LD50 value for OCT A in our study, 28 mg/kg, is slightly higher than that previously reported by Purchase and Theron (1968). i.e.. 22 mg/kg. This difference might reflect the differences in experimental conditions. such as the age or body weight of animals and vehicle for OCT A, and is probably insignificant. OCT A has been demonstrated to be a nephrotoxin in several species of animals. and our findings in the rat are consistent with previous data. in that the primary site of renal inJury is the proximal tubular epithelium (Purchase and Theron. 1968). In our study.
HISTOPATHOLOGY
OF
OCHRATOXIN
A
TOXICITY
63
Lhe earliest and most severe damage was seen in the pars recta of the proximal tubule. Such iocalization of renal damage was clearly demonstrated in the multiple-dose experiment. What is characteristic of this sort of renal lesion is that a single administration of the toxin, even at sublethal doses by gastric intubation, caused only negligible change in the tubular epithelium during a 7-day observation period. and three or more repetitive doses were required to induce recognizable changes. This finding is substantiated by the data from renal function tests presented in our previous report i 1975b). These results differ from those of Purchase and Theron (1968), in which a smgle dose produced tubular necrosis. The mechanism of this difference is not clear, but may be related to the different ages of animals in the two studies. Possible causes of the delayed onset of renal damage after repeated administration might Include :he participation of some metabolite(s). yet unidentified, or the biotransformation products or’0C-f A. OCT A is a minor hepatotoxin in the rat. The major change found was depletion of bepatic glycogen, a finding which is in agreement with that reported by others (Suzuki &!Iai.. I975a; Munro et al., 1973). Acute enteritis due to OCT A has been noted in the rat (Purchase and Theron. 1968). &.li. metabolism in rat liver. Tosicol. App/. Pharmaco/. 32. I lb-- 122. S~‘X~Ihl. S.. Koz~:ss. \i.. C,\roti. T.. ,\NI) \r’.+rir,\%.Zhl. M. ( lQ75b). Studieb \Ifi 111~.iiephrc~r~~il Iheaglc 3.lL.t ii Pathology. I ‘A. Parhoi, 10. 7! 9 --33 I. DOSTER,
