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Teratogenic and toxic effects of ochratoxin A in rats
TOXICOLOGY
AND
APPLIED
Teratogenic
PHARMACOLOGY
and Toxic
37,331-338
Effects
(1976)
of Ochratoxin
A in Rats
M. H. BROWN,’ G. M. SZCZECH, AND B. P. PURMALIS Pathology and Toxicology Research Unit, The Upjohn
Company, Kalamazoo, Michigan 49001
Receiced Noaember 24, 1975; accepted April 2, 1976
Teratogenicand Toxic Effects of Ochratoxin A in Rats. (1976) BROWN, SZCZECH, G. M., AND PURMALIS, B. P. Toxicol. Appl. Pharmacol. 37, 331-338. The mycotoxin ochratoxin A, is a teratogen as well as a potent nephrotoxin. Crystallineochratoxin A wasdissolvedin 0.1 N NaHCO, and given daily by gastric intubation to rats on Days 6-15 of gestation.There were10rats per group and the doselevelstestedwere0.25,0.50,0.75,1,2,4, and 8 mg/kg. Acute ochratoxicosis,characterizedmainly by signsof renal failure, occurred in damsgiven 4 or 8 mg/kg. Several damsdied and the litters were resorbedin all damsgiven the larger doses.Signsof toxicosis were not observedin damsgiven 1 or 2 mg/kg, but their litters were also resorbed. There was no sign of toxicosis in dams given 0.25, 0.50, or 0.75 mg/kg. However, at Day 20, there wasan increasedincidenceof fetal resorption in damsgiven 0.75 mg/kg. All fetusestaken on gestationDay 20 from damsgiven 0.25, 0.50 or 0.75 mg/kg weighedsignificantly lessthan control fetuses,and fetusesfrom damsgiven0.75or 1.0mg/kg werestunted. Many had open eyes,deformedsnouts,and other major alterationsincluding wavy ribs and agenesisof vertebrae. At the lower doses,ochratoxin A wasteratogenic,but at the higher dosesit wasembryocidal. M. H.,
Since 1965, when van der Merwe and associatesfirst reported the occurrence of ochratoxin A, over 100researchpapershave been published (Chu, 1975)and an international symposium focusing on ochratoxin A has been organized. Ochratoxin A is a potent nephrotoxin in several species,is produced by storage fungi in the Aspergillus and Penicillium groups, causesendemic porcine nephropathy under natural conditions, and has been found as a contaminant in a variety of cereal grains and foods including coffee beansand ham asreviewed by Chu (1975). It is essentialto know more about this potent mycotoxin sincehuman exposure is likely. Still et al. (1971) reported that ochratoxin A caused pregnant rats to resorb litters and Hayes et al. (1974) found that ochratoxin A was teratogenic in mice. Ochratoxin A is also a teratogen in hamsters (Hood et al., 1975), but aside from the report of More and Galtier (1974), there is little information on the effects of ochratoxin A on morphology of developing rats or rabbits. More and Galtier (1974) gave bred rats dosesof 4 or 5 mg/kg of crystalline ochratoxin A during various days during the period of active organogenesis.The mycotoxin was given ip and po. Although they judged ochratoxin A to be teratogenic on the basis of 1This researchis part of a SeniorIndependentProjectsubmittedto the faculty of Kalamazoo College by the senior author in partial fulfillment of requirements for the B.A. degree. Copyright 0 1976 by Academic Press, Inc. 331 All rights of reproduction in any form reserved. Printed
in Great
Britain
332
BROWN,
SZCZECH
AND
PURMALIS
fetal hemorrhage and an undefined defect termed “coelosomy,” they were unable to detect any skeletal or visceral malformations. The present study was designed to further assessthe teratogenic potential of ochratoxin A in rats dosed by gastric intubation. It is important to give this myocotoxin by the oral route because human exposure would occur by the ingestion of contaminated food. In this study, ochratoxin A was given during Days 6 through 15 of gestation because similar exposure is required by regulatory agencies responsible for assessing the teratogenic hazard of various test materials. METHODS
Bred, young adult rats were supplied by the Animal Rearing Unit of The Upjohn Company. The rats were from Sprague-Dawley-derived specific pathogen-free stock, and historical control data were available for over 2000 fetuses, since this strain has been used for teratology tests in this laboratory for many years. The day on which sperm were found in a vaginal smear was designated as Day 0 of pregnancy. Crystalline ochratoxin A was purchased commercially’ and dissolved in 0.1 N NaHCO, immediately prior to administration. The rats were dosed by gastric intubation at 0,0.25,0.50, 0.75, 1,2,4, or 8 mg/kg on Days 6 through 15 of gestation. There were 10 bred rats in each of eight groups. The physical condition of each rat was assessedeach day, and on Day 20 of gestation all dams were killed by inhalation of CO, and the fetuses were taken by uterine incision. Each fetus was carefully examined for gross anomalies. One-third of the fetuses were selected by a random method, fixed in Bouin’s solution, and examined for visceral anomalies by a modification of Wilson’s razor slicing technique (Wilson and Warkany, 1965) to include dissection of the great vessels in the thorax and the pelvic organs. The remaining fetuses were fixed in 70% ethyl alcohol, stained with alizarin red S and processed for skeletal evaluations. A necropsy was performed on all dams, and portions of liver, kidney, spleen, and mesenteric lymph node were fixed in 10 % neutral buffered formalin for histopathologic evaluation. Data for fetal and maternal body weights were statistically evaluated with an analysis of variance and the Dunnett Multiple Comparison procedure (Ostle, 1973). Data for the various malformations were analyzed using the litter as the experimental unit, with a modified Jonckheere test (Lin and Haseman, 1976). This method tests for the presence of a monotonic dose-response relationship, which is one that never varies from an upward or downward trend. Although analyses like the generalized KruskalWallis test will detect significant departure of test data from control values, they do not test for ordered (dose-related) departure. We prefer the modified Jonckheere test because it excludes outliers that are inconsistent with the trend. The comparison of each value from each test group to a control value then becomes of secondary importance. RESULTS Ochratoxicosis in dams. Acute ochratoxicosis, characterized by loss of body weight, depression, diarrhea, ruffled fur, polydipsia, and polyuria was prominent in all dams given 8 mg/kg of ochratoxin A after the third dose was administered. Loss of body z Makor Chemicals Ltd., Jerusalem, Israel.
OCHRATOXIN
A : A TERATOGEN
IN RATS
333
weight was progressive in dams given 4 or 8 mg/kg, and after five doses were administered, 2 of 10 dams at the highest dose level were found dead. Although dosing was discontinued at that point, only one rat in the high dose group survived. One rat given 8 mg/kg developed a massive hematoma involving most of the tongue. Rats given 4 mg/kg were dehydrated, apparently depressed, and had lost considerable body weight after seven doses of toxin had been administered. Dosingwas discontinued at that point; 9 of 10 rats survived and a necropsy was performed on Day 20 of gestation. The other rat was killed on gestation Day 17 because the distal one-third of its tail was infarcted and undergoing dry gangrene. Two rats given 8 mg/kg and two rats given 4 mg/kg had a bloody vaginal discharge first observed on gestation Day 13. Although rats given daily doses of 1 or 2 mg/kg gained slightly less weight than control rats, there were no overt signs of toxicosis. Rats given 0.25,0.50, or 0.75 mg/kg gained slightly more weight than control rats and were in good physical condition when dosing was completed. Gross lesions consisting of enteritis, gastric ulceration and subserosal haemorrhage, dehydration and petechial hemorrhage in the thymus were limited to the carcasses of rats given either 4 or 8 mg/kg. Clots of blood and free blood were observed in the uterus and vagina in a few rats given 1 or 2 mg/kg, but were found in most rats given 4 or 8 mg/kg. Pathoanatomic alterations occurred in cervical lymph nodes, thymus, spleen, stomach, jejunem, ileum, and kidney in dams given 4 or 8 mg/kg of ochratoxin A. Lymphoid depletion, edema, and necrosis of lymphocytes in germinal centers and in perifolicular and paracortical areas were prominent alterations in the cervical lymph nodes of treated rats. Lymphoid depletion and necrosis of lymphocytes primarily around sheathed arterioles were prominent alterations in the thymus and spleen. All rats given these higher doses of ochratoxin A had severe necrotizing, ulcerative gastritis. Subacute hemorrhagic enteritis was characterized by segmental necrosis of the mucosa in the jejunum and ileum. Many proximal convoluted tubules in the kidneys of rats given 4- or 8-mg/kg doses of ochratoxin A contained necrotic tubular epithelial cells and granular casts. Some renal tubules in the cortex were dilated and some proximal tubules consisted only of a basement membrane that was devoid of renal tubular epithelial cells. There were signs of mild toxic nephrosis in some rats given 2 mg/kg, but there were no consistent pathoanatomic alterations in rats given smaller doses. Reproductive data. Although the conception rate was similar for control and all groups treated with ochratoxin A, no rats given 2,4, or 8 mg/kg carried litters to term (Table 1). At necropsy, litters from these dams were found in various stages of resorption (Fig. 1). Only five of the eight pregnant dams given 1 mg/kg had live fetuses when the necropsy was performed and there were numerous resorptions. Ten of 10 dams given 0.50 mg/kg and 9 of 10 dams given either 0.75 or 0.25 mg/kg had live fetuses at necropsy. Eight of 10 control dams were pregnant and carried normal litters to term, The number of resorptions in dams given 0.25 or 0.50 mg/kg was comparable to the control value, but a sharp increase in resorption rate occurred at the 1 mg/kg level (Table 1). Fetuses from dams given 0.75 or 1 mg/kg were smaller than the controls (Fig. 2) and the average number of fetuses per litter was significantly decreased in dams given 1
334
BROWN,
SZCZECH
AND
PURMALIS
TABLE
1 SURVIVALANDREPRODUCTIVEDATAFOR RATSGIVENOCHRATOXINABY GASTRIC~NTUBATIONON DAYS~ THROUGH 15 OFGESTATION
Doseb-dkg) Observations
0
0.25
0.50
0.75
1
2
4”
8b
S/l0
s/10
lO/lO
s/10
s/10
s/10
S/l0
s/10
0 150 78 3 3.5 9.4 0 4.3 0.1193
0 163 93 4 4.3 10.0 0 4.1 0.0770
0 166 130 3 2.3 12.8 0 4.0 0.0669
0 168 113 16 14 10.8 0 3.1’ 0.2067
0 91’ 106 78 74 3.5 0 2.4’ 0.3158
0 74” 110 110 100 0 0 -
1 44” 120 120 100 0 0 -
No. dams pregnant
No. of dams bred No. dams killed or dead with ochratoxicosis Average body wt. gain (g) Total no. implantation sites Total no. resorptions Percentage resorbedd Average no. live fetuses per litter Total no dead fetuses Average body wt. live fetuses (g) Variance body wt. live fetuses (g)
10 80 80 100 0 0 -
a Ochratoxicosis developed, dosing discontinued on gestation Day 12. ’ Ochratoxicosis developed, dosing discontinued on gestation Day 10. Only one rat survived. ’ Significantly different from control value (p < 0.01) by Dunnett Multiple Comparison procedure (Ostle, 1973). d Significant (p < 0.05) monotonic increasing dose-response relationship by modified Jonckheere test (Lin and Haseman, 1976).
FIG. 1. Uterus from control dam (left) compared to uterus from dam given 1.O mg/kg of ochratoxin A. All fetuses are resorbed in uterus from treated dam.
mg/kg. Although dams given 0.75 mg/kg had as many live fetuses as control dams, the fetuses weighed significantly less. Teratologic Jindings. Ochratoxin A induced various gross, visceral, and skeletal anomaliesin rat fetusesfrom dams given the smaller dosesof toxin on Days 6 through 15 of gestation (Table 2).
OCHRATOXIN
FIG.
A : A TERATOGEN
335
IN RATS
2. Stunted fetus on left is from a dam given 1.0 mg/kg of ochratoxin A. Thereisa slightdepres-
sionat the bridgeof the snout.Control fetusis on theright. TABLE 2 MALFORMATIONS
IN FETUSES
FROM
RATS GIVEN
OCHRATOXIN
A
BY GASTRIC
INTUBATION
Ochratoxin A (mg/kg) Observations Gross Edema,subcutaneous“ Snout, short, bridge depressed” Eyes, open Body, foreshortened Visceral Kidney, hychonephrosis Kidney, polycystic Heart, left atrium small”
Historical controls
0
0.25
0.50
0.75
o/75 o/75 o/75
o/90 o/90 o/90 o/90
5/128 O/128 O/128 l/128
9196 5196 O/96
511,304 l/24 O/l ,304 O/24 o/1,304 O/24
o/29 o/29 o/29
o/45 o/45 o/45
2133 l/33 o/33
82/83 l/83 17/83 3/83 25183 l/83 O/63 O/63 33183
63163 O/63 lo/63 10163 21/63 3163 O/63 O/63 48163
212,788 o/75 O/2,788 012,788 012,788
Skeletal Ribs, 13 pair 2,284/2,318 51/51 61/61 Ribs, 13 pair, 14th unilateral 6/2,318 o/51 O/61 Ribs, wavy“ 5/2,318 o/51 5/61 Sternebrae,agenesis’ l/61 137/2,318 l/51 Sternebrae,asymmetry 191/2,318 7151 17/61 o/51 O/61 Long bones,major malformation l/2,318 Metacarpal bone, 5th, agenesis” 73/2,318 s/51 O/61 Metatarsal bone, 5th, agenesiP O/2,318 o/51 O/61 All bones,grossimmaturity’ 212,318 o/51 O/61
l/96
1.0
2128 16/28 28128 O/28
019 o/g g/g 18119 l/19 3/19 14/19 2119 o/19 9119 3/19 12/19
’ Significant(p < 0.05)monotonicincreasingdose-response relationshipby modifiedJonckheere test(Lin andHaseman,1976).
336
BROWN,
SZCZECH
AND
PURMALIS
Alterations observed at necropsy included small fetal size, subcutaneous edema, multiple hematomas, open eyes, and a shortened snout with a characteristic depression on the bridge of the nose (Fig. 2). Only one visceral anomaly was consistently observed in fetuses from ochratoxin A-treated rats; fetuses from dams given 1 mg/kg had very small left atria, but this alteration was not quantitated. Table 2 summarizes the skeletal anomalites found in control fetuses and in fetuses from dams given ochratoxin A. Wavy ribs occurred in several litters from dams given 0.25, 0.5, 0.75, or 1.0 mg/kg. There was a high incidence of agenesis and asymmetry of sternebrae in many fetuses from treated dams. There were major malformations of long bones in one fetus from a dam given 0.5 mg/kg and in three fetuses from dams given 0.75 mg/kg. In addition, two fetuses from treated dams had multiple bizarre anomalies. The fetus from a dam given 0.5 mg/kg had agenesis of all lumbar and sacral vertebrae, agenesis of adrenal glands, diaphragm, ribs, spinal cord, and gonads plus bilateral talipes, stenosis of the rectum, a constriction at the base of the tail, and caudal displacement of the kidneys. A fetus from a dam given 0.75 mg/kg had anterior displacement of the uterus, adrenals, and ovaries, and its kidneys were displaced caudally. That fetus also had major malformations involving most of the long bones. There was prominent skeletal immaturity in most fetuses from dams given 0.5 mg/kg or larger doses of ochratoxin A. DISCUSSION
Although the LD50 (PO) for ochratoxin A in female rats is 22 mg/kg (Purchase and Theron, 1968), we observed signs of toxicosis at dose levels as low as 1 mg/kg given for 10 consecutive days. Two unusual lesions, a large hematoma of the tongue and an infarcted tail, occurred in dams given the largest doses of ochratoxin A and were considered to be related to treatment. The larger doses of ochratoxin A also caused enteritis, renal failure, and necrosis of lymphoid tissue, all of which have been reported in other laboratory species. It is probable that lymphoid necrosis is a characteristic of ochratoxicosis in most species since, when looked for, it has been found in several animal species. As tested in this study, 0.75 mg/kg of ochratoxin A proved to be a very critical dose. There were no signs of toxicosis or pathoanatomic alterations in dams given 0.75 mg/kg, but there were signs of fetotoxicity. Dams given 0.75 mg/kg gained as much weight as control dams, but they had more resorptions and produced fetuses that weighed significantly less than controls. Still et al. (1971) previously reported that ochratoxin A caused a marked increasein the number of dead and resorbed fetuses when given to pregnant rats by gastric intubation on Day 10 ofpregnancy at doses of 6.25,12.5, or 25 mg/kg. Our results also confirm the report by More and Galtier (1974) that ochratoxin A caused an increase in the number of resorptions and a decrease in fetal body weights when given (ip) to pregnant rats at 4 or 5 mg/kg over a 2-, 4-, or 8-day period. These workers described extensive hemorrhage and edema in some fetuses and concluded that ochratoxin A was a teratogen. Since neither edema nor hemorrhage are signs of teratogenicity, they apparently made this judgment on the basis of coelosomy, which occurred in fetuses from treated
OCHRATOXIN A : A TERATOGEN IN RATS
337
dams but not in controls. They looked for, but did not find, skeletal or visceral anomalies. Hood et al. (1975) also noted that although ochratoxin A caused a variety of gross anomalies in fetuses from treated hamsters, there were no skeletal anomalies. We consider ochratoxin A to be a teratogen in rats because multiple gross, visceral, and skeletal anomalies were related to treatment and dose. Hayes et al. (1974) reported that ochratoxin A was teratogenic when given to mice at 5 mg/kg (ip) on one of gestations Days 7 through 12. They found a variety of skeletal anomalies, mostly in ribs and vertebrae. In their study, regardless of the day of exposure, treated dams had significant numbers of dead or resorbed fetuses and the body weights of live fetuses from treated dams were decreased. We conclude that multiple exposure of rats to doses of ochratoxin A larger than 1 mg/kg results in ochratoxicosis and the loss of litters approaches 100%. Multiple doses of 1 mg/kg are embryocidal, doses of 0.75 mg/kg are embryotoxic and teratogenie, and doses of 0.25 to 0.50 mg/kg are principally teratogenic. These low doses suggest that ochratoxin A is a rather potent teratogen; however, the mechanism is unknown. The teratogenic effect involved many organ systems in the three species studied (mouse, rat, and hamster), but embryolethality and embryotoxicity were common to all studies. We did not find any fetuses that had died near term. It was apparent that ochratoxin A killed the embryos soon after initial exposure. Hood et al. (1975) suggest that ochratoxin A may have a direct effect on the fetus. More and Galtier (1974) proposed that since ochratoxin A inactivates phosphorylaseP-kinase (Pitout, 1968) and impairs glycolysis in maternal liver, many of the effects may be due to decreased concentrations of glucose available to developing fetuses. However, Suzuki et al. (1975) recently reported that ochratoxin A increased serum glucose in ochratoxin A-treated rats and caused marked depletion of hepatic glycogen stores. They attributed the glycogen depletion to inhibition of active transport of glucose into the liver, suppression of glycogenesis, and acceleration of glycogenolysis. It is also known that ochratoxin A inhibits mitochondrial respiration (Moore and Truelove, 1970) and that it causes necrosis in lymphoid tissue. There may be more than one mechanism to explain its teratogenic activity. ACKNOWLEDGMENTS
Mr. William Burr is thanked for assistancewith illustrations in this paper, Mr. Marshall Brunden for the statisticalanalysis,and Ms. Kay Franz for typing. REFERENCES CHU,F. S.(1975).Studieson ochratoxins. CRC Critical Reviews in Toxicology, 2,499-524. HAYES,W. A., Hook, R. D., ANDHUMPHREY, L. L. (1974).Teratogeniceffectsof ochratoxin A in mice. Teratology 9,93-97. HOOD,R. D., NAUGHTON,M. J., ANDHAYES,A. W. (1975).Prenataleffects of ochratoxin A in hamsters.Teratology 13, 1l-14. LIN, F. O., ANDHASEMAN,J. K. (1976).A modifiedJonckheeretestagainstorderedalternatives whenties are presentat a singleextremevalue. Biometrische Zeitschrift (in press). MOORE,J. H., ANDTRUELOVE, B. (1970). Ochratoxin A: Inhibition of mitochondrial respiration. Science 168, 1102-l103. MoRB, J., AND GALTIER,P. (1974). Toxicite de l’ochratoxine A. I. effect embryotoxique et teratogenechezle rat. Ann. Rech. Veter. 5, 167-178.
338
BROWN, SZCZECH AND PURMALIS
OSTLE, B. (1963).Statistics In Research. Iowa State University Press,Ames,Iowa. PITOUT,M. J. (1968).The effect of ochratoxin A on glycogenstoragein the rat liver. Toxicol. Appl. Pharmacol. 13,299-306. PURCHASE, I. F. H., AND THERON, J. J. (1968).The acute toxicity of ochratoxin A to rats. Fd. Cosmet. Toxicol. 6,479-483. STILL, P. E., MACKLIN, A. W., R.IBELIN, W. E., AND SMALLEY, E. B. (1971).Relationship of ochratoxin A to fetal death in laboratory and domesticanimals.Nature (London) 234,563-
564. SUZUKI, S., SATOH, T., AND YAMAZAKI, M. (1975).Effect of ochratoxin A on carbohydrate metabolismin rat liver. Toxicol. Appl. Pharmacol. 32, 116-122. VAN DER MERWE, IL J., STEYN, P. S., FOURIE, L., DE B. SCOTT,AND THERON, J. J. (1965). Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus Wilh. Nature, 205:
1112-1113. WILSON, J. G., AND WARKANY, J. (1965).Teratology:
University of ChicagoPress,Chicago, Illinois.
Principles
and Techniques, pp. 271-217.
AND
APPLIED
Teratogenic
PHARMACOLOGY
and Toxic
37,331-338
Effects
(1976)
of Ochratoxin
A in Rats
M. H. BROWN,’ G. M. SZCZECH, AND B. P. PURMALIS Pathology and Toxicology Research Unit, The Upjohn
Company, Kalamazoo, Michigan 49001
Receiced Noaember 24, 1975; accepted April 2, 1976
Teratogenicand Toxic Effects of Ochratoxin A in Rats. (1976) BROWN, SZCZECH, G. M., AND PURMALIS, B. P. Toxicol. Appl. Pharmacol. 37, 331-338. The mycotoxin ochratoxin A, is a teratogen as well as a potent nephrotoxin. Crystallineochratoxin A wasdissolvedin 0.1 N NaHCO, and given daily by gastric intubation to rats on Days 6-15 of gestation.There were10rats per group and the doselevelstestedwere0.25,0.50,0.75,1,2,4, and 8 mg/kg. Acute ochratoxicosis,characterizedmainly by signsof renal failure, occurred in damsgiven 4 or 8 mg/kg. Several damsdied and the litters were resorbedin all damsgiven the larger doses.Signsof toxicosis were not observedin damsgiven 1 or 2 mg/kg, but their litters were also resorbed. There was no sign of toxicosis in dams given 0.25, 0.50, or 0.75 mg/kg. However, at Day 20, there wasan increasedincidenceof fetal resorption in damsgiven 0.75 mg/kg. All fetusestaken on gestationDay 20 from damsgiven 0.25, 0.50 or 0.75 mg/kg weighedsignificantly lessthan control fetuses,and fetusesfrom damsgiven0.75or 1.0mg/kg werestunted. Many had open eyes,deformedsnouts,and other major alterationsincluding wavy ribs and agenesisof vertebrae. At the lower doses,ochratoxin A wasteratogenic,but at the higher dosesit wasembryocidal. M. H.,
Since 1965, when van der Merwe and associatesfirst reported the occurrence of ochratoxin A, over 100researchpapershave been published (Chu, 1975)and an international symposium focusing on ochratoxin A has been organized. Ochratoxin A is a potent nephrotoxin in several species,is produced by storage fungi in the Aspergillus and Penicillium groups, causesendemic porcine nephropathy under natural conditions, and has been found as a contaminant in a variety of cereal grains and foods including coffee beansand ham asreviewed by Chu (1975). It is essentialto know more about this potent mycotoxin sincehuman exposure is likely. Still et al. (1971) reported that ochratoxin A caused pregnant rats to resorb litters and Hayes et al. (1974) found that ochratoxin A was teratogenic in mice. Ochratoxin A is also a teratogen in hamsters (Hood et al., 1975), but aside from the report of More and Galtier (1974), there is little information on the effects of ochratoxin A on morphology of developing rats or rabbits. More and Galtier (1974) gave bred rats dosesof 4 or 5 mg/kg of crystalline ochratoxin A during various days during the period of active organogenesis.The mycotoxin was given ip and po. Although they judged ochratoxin A to be teratogenic on the basis of 1This researchis part of a SeniorIndependentProjectsubmittedto the faculty of Kalamazoo College by the senior author in partial fulfillment of requirements for the B.A. degree. Copyright 0 1976 by Academic Press, Inc. 331 All rights of reproduction in any form reserved. Printed
in Great
Britain
332
BROWN,
SZCZECH
AND
PURMALIS
fetal hemorrhage and an undefined defect termed “coelosomy,” they were unable to detect any skeletal or visceral malformations. The present study was designed to further assessthe teratogenic potential of ochratoxin A in rats dosed by gastric intubation. It is important to give this myocotoxin by the oral route because human exposure would occur by the ingestion of contaminated food. In this study, ochratoxin A was given during Days 6 through 15 of gestation because similar exposure is required by regulatory agencies responsible for assessing the teratogenic hazard of various test materials. METHODS
Bred, young adult rats were supplied by the Animal Rearing Unit of The Upjohn Company. The rats were from Sprague-Dawley-derived specific pathogen-free stock, and historical control data were available for over 2000 fetuses, since this strain has been used for teratology tests in this laboratory for many years. The day on which sperm were found in a vaginal smear was designated as Day 0 of pregnancy. Crystalline ochratoxin A was purchased commercially’ and dissolved in 0.1 N NaHCO, immediately prior to administration. The rats were dosed by gastric intubation at 0,0.25,0.50, 0.75, 1,2,4, or 8 mg/kg on Days 6 through 15 of gestation. There were 10 bred rats in each of eight groups. The physical condition of each rat was assessedeach day, and on Day 20 of gestation all dams were killed by inhalation of CO, and the fetuses were taken by uterine incision. Each fetus was carefully examined for gross anomalies. One-third of the fetuses were selected by a random method, fixed in Bouin’s solution, and examined for visceral anomalies by a modification of Wilson’s razor slicing technique (Wilson and Warkany, 1965) to include dissection of the great vessels in the thorax and the pelvic organs. The remaining fetuses were fixed in 70% ethyl alcohol, stained with alizarin red S and processed for skeletal evaluations. A necropsy was performed on all dams, and portions of liver, kidney, spleen, and mesenteric lymph node were fixed in 10 % neutral buffered formalin for histopathologic evaluation. Data for fetal and maternal body weights were statistically evaluated with an analysis of variance and the Dunnett Multiple Comparison procedure (Ostle, 1973). Data for the various malformations were analyzed using the litter as the experimental unit, with a modified Jonckheere test (Lin and Haseman, 1976). This method tests for the presence of a monotonic dose-response relationship, which is one that never varies from an upward or downward trend. Although analyses like the generalized KruskalWallis test will detect significant departure of test data from control values, they do not test for ordered (dose-related) departure. We prefer the modified Jonckheere test because it excludes outliers that are inconsistent with the trend. The comparison of each value from each test group to a control value then becomes of secondary importance. RESULTS Ochratoxicosis in dams. Acute ochratoxicosis, characterized by loss of body weight, depression, diarrhea, ruffled fur, polydipsia, and polyuria was prominent in all dams given 8 mg/kg of ochratoxin A after the third dose was administered. Loss of body z Makor Chemicals Ltd., Jerusalem, Israel.
OCHRATOXIN
A : A TERATOGEN
IN RATS
333
weight was progressive in dams given 4 or 8 mg/kg, and after five doses were administered, 2 of 10 dams at the highest dose level were found dead. Although dosing was discontinued at that point, only one rat in the high dose group survived. One rat given 8 mg/kg developed a massive hematoma involving most of the tongue. Rats given 4 mg/kg were dehydrated, apparently depressed, and had lost considerable body weight after seven doses of toxin had been administered. Dosingwas discontinued at that point; 9 of 10 rats survived and a necropsy was performed on Day 20 of gestation. The other rat was killed on gestation Day 17 because the distal one-third of its tail was infarcted and undergoing dry gangrene. Two rats given 8 mg/kg and two rats given 4 mg/kg had a bloody vaginal discharge first observed on gestation Day 13. Although rats given daily doses of 1 or 2 mg/kg gained slightly less weight than control rats, there were no overt signs of toxicosis. Rats given 0.25,0.50, or 0.75 mg/kg gained slightly more weight than control rats and were in good physical condition when dosing was completed. Gross lesions consisting of enteritis, gastric ulceration and subserosal haemorrhage, dehydration and petechial hemorrhage in the thymus were limited to the carcasses of rats given either 4 or 8 mg/kg. Clots of blood and free blood were observed in the uterus and vagina in a few rats given 1 or 2 mg/kg, but were found in most rats given 4 or 8 mg/kg. Pathoanatomic alterations occurred in cervical lymph nodes, thymus, spleen, stomach, jejunem, ileum, and kidney in dams given 4 or 8 mg/kg of ochratoxin A. Lymphoid depletion, edema, and necrosis of lymphocytes in germinal centers and in perifolicular and paracortical areas were prominent alterations in the cervical lymph nodes of treated rats. Lymphoid depletion and necrosis of lymphocytes primarily around sheathed arterioles were prominent alterations in the thymus and spleen. All rats given these higher doses of ochratoxin A had severe necrotizing, ulcerative gastritis. Subacute hemorrhagic enteritis was characterized by segmental necrosis of the mucosa in the jejunum and ileum. Many proximal convoluted tubules in the kidneys of rats given 4- or 8-mg/kg doses of ochratoxin A contained necrotic tubular epithelial cells and granular casts. Some renal tubules in the cortex were dilated and some proximal tubules consisted only of a basement membrane that was devoid of renal tubular epithelial cells. There were signs of mild toxic nephrosis in some rats given 2 mg/kg, but there were no consistent pathoanatomic alterations in rats given smaller doses. Reproductive data. Although the conception rate was similar for control and all groups treated with ochratoxin A, no rats given 2,4, or 8 mg/kg carried litters to term (Table 1). At necropsy, litters from these dams were found in various stages of resorption (Fig. 1). Only five of the eight pregnant dams given 1 mg/kg had live fetuses when the necropsy was performed and there were numerous resorptions. Ten of 10 dams given 0.50 mg/kg and 9 of 10 dams given either 0.75 or 0.25 mg/kg had live fetuses at necropsy. Eight of 10 control dams were pregnant and carried normal litters to term, The number of resorptions in dams given 0.25 or 0.50 mg/kg was comparable to the control value, but a sharp increase in resorption rate occurred at the 1 mg/kg level (Table 1). Fetuses from dams given 0.75 or 1 mg/kg were smaller than the controls (Fig. 2) and the average number of fetuses per litter was significantly decreased in dams given 1
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PURMALIS
TABLE
1 SURVIVALANDREPRODUCTIVEDATAFOR RATSGIVENOCHRATOXINABY GASTRIC~NTUBATIONON DAYS~ THROUGH 15 OFGESTATION
Doseb-dkg) Observations
0
0.25
0.50
0.75
1
2
4”
8b
S/l0
s/10
lO/lO
s/10
s/10
s/10
S/l0
s/10
0 150 78 3 3.5 9.4 0 4.3 0.1193
0 163 93 4 4.3 10.0 0 4.1 0.0770
0 166 130 3 2.3 12.8 0 4.0 0.0669
0 168 113 16 14 10.8 0 3.1’ 0.2067
0 91’ 106 78 74 3.5 0 2.4’ 0.3158
0 74” 110 110 100 0 0 -
1 44” 120 120 100 0 0 -
No. dams pregnant
No. of dams bred No. dams killed or dead with ochratoxicosis Average body wt. gain (g) Total no. implantation sites Total no. resorptions Percentage resorbedd Average no. live fetuses per litter Total no dead fetuses Average body wt. live fetuses (g) Variance body wt. live fetuses (g)
10 80 80 100 0 0 -
a Ochratoxicosis developed, dosing discontinued on gestation Day 12. ’ Ochratoxicosis developed, dosing discontinued on gestation Day 10. Only one rat survived. ’ Significantly different from control value (p < 0.01) by Dunnett Multiple Comparison procedure (Ostle, 1973). d Significant (p < 0.05) monotonic increasing dose-response relationship by modified Jonckheere test (Lin and Haseman, 1976).
FIG. 1. Uterus from control dam (left) compared to uterus from dam given 1.O mg/kg of ochratoxin A. All fetuses are resorbed in uterus from treated dam.
mg/kg. Although dams given 0.75 mg/kg had as many live fetuses as control dams, the fetuses weighed significantly less. Teratologic Jindings. Ochratoxin A induced various gross, visceral, and skeletal anomaliesin rat fetusesfrom dams given the smaller dosesof toxin on Days 6 through 15 of gestation (Table 2).
OCHRATOXIN
FIG.
A : A TERATOGEN
335
IN RATS
2. Stunted fetus on left is from a dam given 1.0 mg/kg of ochratoxin A. Thereisa slightdepres-
sionat the bridgeof the snout.Control fetusis on theright. TABLE 2 MALFORMATIONS
IN FETUSES
FROM
RATS GIVEN
OCHRATOXIN
A
BY GASTRIC
INTUBATION
Ochratoxin A (mg/kg) Observations Gross Edema,subcutaneous“ Snout, short, bridge depressed” Eyes, open Body, foreshortened Visceral Kidney, hychonephrosis Kidney, polycystic Heart, left atrium small”
Historical controls
0
0.25
0.50
0.75
o/75 o/75 o/75
o/90 o/90 o/90 o/90
5/128 O/128 O/128 l/128
9196 5196 O/96
511,304 l/24 O/l ,304 O/24 o/1,304 O/24
o/29 o/29 o/29
o/45 o/45 o/45
2133 l/33 o/33
82/83 l/83 17/83 3/83 25183 l/83 O/63 O/63 33183
63163 O/63 lo/63 10163 21/63 3163 O/63 O/63 48163
212,788 o/75 O/2,788 012,788 012,788
Skeletal Ribs, 13 pair 2,284/2,318 51/51 61/61 Ribs, 13 pair, 14th unilateral 6/2,318 o/51 O/61 Ribs, wavy“ 5/2,318 o/51 5/61 Sternebrae,agenesis’ l/61 137/2,318 l/51 Sternebrae,asymmetry 191/2,318 7151 17/61 o/51 O/61 Long bones,major malformation l/2,318 Metacarpal bone, 5th, agenesis” 73/2,318 s/51 O/61 Metatarsal bone, 5th, agenesiP O/2,318 o/51 O/61 All bones,grossimmaturity’ 212,318 o/51 O/61
l/96
1.0
2128 16/28 28128 O/28
019 o/g g/g 18119 l/19 3/19 14/19 2119 o/19 9119 3/19 12/19
’ Significant(p < 0.05)monotonicincreasingdose-response relationshipby modifiedJonckheere test(Lin andHaseman,1976).
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PURMALIS
Alterations observed at necropsy included small fetal size, subcutaneous edema, multiple hematomas, open eyes, and a shortened snout with a characteristic depression on the bridge of the nose (Fig. 2). Only one visceral anomaly was consistently observed in fetuses from ochratoxin A-treated rats; fetuses from dams given 1 mg/kg had very small left atria, but this alteration was not quantitated. Table 2 summarizes the skeletal anomalites found in control fetuses and in fetuses from dams given ochratoxin A. Wavy ribs occurred in several litters from dams given 0.25, 0.5, 0.75, or 1.0 mg/kg. There was a high incidence of agenesis and asymmetry of sternebrae in many fetuses from treated dams. There were major malformations of long bones in one fetus from a dam given 0.5 mg/kg and in three fetuses from dams given 0.75 mg/kg. In addition, two fetuses from treated dams had multiple bizarre anomalies. The fetus from a dam given 0.5 mg/kg had agenesis of all lumbar and sacral vertebrae, agenesis of adrenal glands, diaphragm, ribs, spinal cord, and gonads plus bilateral talipes, stenosis of the rectum, a constriction at the base of the tail, and caudal displacement of the kidneys. A fetus from a dam given 0.75 mg/kg had anterior displacement of the uterus, adrenals, and ovaries, and its kidneys were displaced caudally. That fetus also had major malformations involving most of the long bones. There was prominent skeletal immaturity in most fetuses from dams given 0.5 mg/kg or larger doses of ochratoxin A. DISCUSSION
Although the LD50 (PO) for ochratoxin A in female rats is 22 mg/kg (Purchase and Theron, 1968), we observed signs of toxicosis at dose levels as low as 1 mg/kg given for 10 consecutive days. Two unusual lesions, a large hematoma of the tongue and an infarcted tail, occurred in dams given the largest doses of ochratoxin A and were considered to be related to treatment. The larger doses of ochratoxin A also caused enteritis, renal failure, and necrosis of lymphoid tissue, all of which have been reported in other laboratory species. It is probable that lymphoid necrosis is a characteristic of ochratoxicosis in most species since, when looked for, it has been found in several animal species. As tested in this study, 0.75 mg/kg of ochratoxin A proved to be a very critical dose. There were no signs of toxicosis or pathoanatomic alterations in dams given 0.75 mg/kg, but there were signs of fetotoxicity. Dams given 0.75 mg/kg gained as much weight as control dams, but they had more resorptions and produced fetuses that weighed significantly less than controls. Still et al. (1971) previously reported that ochratoxin A caused a marked increasein the number of dead and resorbed fetuses when given to pregnant rats by gastric intubation on Day 10 ofpregnancy at doses of 6.25,12.5, or 25 mg/kg. Our results also confirm the report by More and Galtier (1974) that ochratoxin A caused an increase in the number of resorptions and a decrease in fetal body weights when given (ip) to pregnant rats at 4 or 5 mg/kg over a 2-, 4-, or 8-day period. These workers described extensive hemorrhage and edema in some fetuses and concluded that ochratoxin A was a teratogen. Since neither edema nor hemorrhage are signs of teratogenicity, they apparently made this judgment on the basis of coelosomy, which occurred in fetuses from treated
OCHRATOXIN A : A TERATOGEN IN RATS
337
dams but not in controls. They looked for, but did not find, skeletal or visceral anomalies. Hood et al. (1975) also noted that although ochratoxin A caused a variety of gross anomalies in fetuses from treated hamsters, there were no skeletal anomalies. We consider ochratoxin A to be a teratogen in rats because multiple gross, visceral, and skeletal anomalies were related to treatment and dose. Hayes et al. (1974) reported that ochratoxin A was teratogenic when given to mice at 5 mg/kg (ip) on one of gestations Days 7 through 12. They found a variety of skeletal anomalies, mostly in ribs and vertebrae. In their study, regardless of the day of exposure, treated dams had significant numbers of dead or resorbed fetuses and the body weights of live fetuses from treated dams were decreased. We conclude that multiple exposure of rats to doses of ochratoxin A larger than 1 mg/kg results in ochratoxicosis and the loss of litters approaches 100%. Multiple doses of 1 mg/kg are embryocidal, doses of 0.75 mg/kg are embryotoxic and teratogenie, and doses of 0.25 to 0.50 mg/kg are principally teratogenic. These low doses suggest that ochratoxin A is a rather potent teratogen; however, the mechanism is unknown. The teratogenic effect involved many organ systems in the three species studied (mouse, rat, and hamster), but embryolethality and embryotoxicity were common to all studies. We did not find any fetuses that had died near term. It was apparent that ochratoxin A killed the embryos soon after initial exposure. Hood et al. (1975) suggest that ochratoxin A may have a direct effect on the fetus. More and Galtier (1974) proposed that since ochratoxin A inactivates phosphorylaseP-kinase (Pitout, 1968) and impairs glycolysis in maternal liver, many of the effects may be due to decreased concentrations of glucose available to developing fetuses. However, Suzuki et al. (1975) recently reported that ochratoxin A increased serum glucose in ochratoxin A-treated rats and caused marked depletion of hepatic glycogen stores. They attributed the glycogen depletion to inhibition of active transport of glucose into the liver, suppression of glycogenesis, and acceleration of glycogenolysis. It is also known that ochratoxin A inhibits mitochondrial respiration (Moore and Truelove, 1970) and that it causes necrosis in lymphoid tissue. There may be more than one mechanism to explain its teratogenic activity. ACKNOWLEDGMENTS
Mr. William Burr is thanked for assistancewith illustrations in this paper, Mr. Marshall Brunden for the statisticalanalysis,and Ms. Kay Franz for typing. REFERENCES CHU,F. S.(1975).Studieson ochratoxins. CRC Critical Reviews in Toxicology, 2,499-524. HAYES,W. A., Hook, R. D., ANDHUMPHREY, L. L. (1974).Teratogeniceffectsof ochratoxin A in mice. Teratology 9,93-97. HOOD,R. D., NAUGHTON,M. J., ANDHAYES,A. W. (1975).Prenataleffects of ochratoxin A in hamsters.Teratology 13, 1l-14. LIN, F. O., ANDHASEMAN,J. K. (1976).A modifiedJonckheeretestagainstorderedalternatives whenties are presentat a singleextremevalue. Biometrische Zeitschrift (in press). MOORE,J. H., ANDTRUELOVE, B. (1970). Ochratoxin A: Inhibition of mitochondrial respiration. Science 168, 1102-l103. MoRB, J., AND GALTIER,P. (1974). Toxicite de l’ochratoxine A. I. effect embryotoxique et teratogenechezle rat. Ann. Rech. Veter. 5, 167-178.
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OSTLE, B. (1963).Statistics In Research. Iowa State University Press,Ames,Iowa. PITOUT,M. J. (1968).The effect of ochratoxin A on glycogenstoragein the rat liver. Toxicol. Appl. Pharmacol. 13,299-306. PURCHASE, I. F. H., AND THERON, J. J. (1968).The acute toxicity of ochratoxin A to rats. Fd. Cosmet. Toxicol. 6,479-483. STILL, P. E., MACKLIN, A. W., R.IBELIN, W. E., AND SMALLEY, E. B. (1971).Relationship of ochratoxin A to fetal death in laboratory and domesticanimals.Nature (London) 234,563-
564. SUZUKI, S., SATOH, T., AND YAMAZAKI, M. (1975).Effect of ochratoxin A on carbohydrate metabolismin rat liver. Toxicol. Appl. Pharmacol. 32, 116-122. VAN DER MERWE, IL J., STEYN, P. S., FOURIE, L., DE B. SCOTT,AND THERON, J. J. (1965). Ochratoxin A, a toxic metabolite produced by Aspergillus ochraceus Wilh. Nature, 205:
1112-1113. WILSON, J. G., AND WARKANY, J. (1965).Teratology:
University of ChicagoPress,Chicago, Illinois.
Principles
and Techniques, pp. 271-217.