Studies on the nephrotoxicity of ochratoxin A in rats

Studies

on the

SHIGETOSHI Research

SUZUKI,

Institute

for

Nephrotoxicity

YOSHIMICHI

KOZUKA,

Chemobiodynamics, Received

Chiba April

30,197s;

of Ochratoxin TETSUO SATOH,' University, accepfed

Izumi-rho,

A in Rats AND

MIKIO

Narashino,

YAMAZAKI

Chiba,

275, Japan

Juiy 15, I975

Studies on the Nephrotoxicity

of Ochratoxin A in Rats. SUZUKI, S., M. (1975).Toxicol.Appl.Pharmacol. 34, 479-490. Administration of ochratoxin A (5 or 15 mg/kg/day, po) to rats daily for 3 days decreasedthe accumulationof p-aminohippuric acid (PAH) in renal cortical slices.PAH clearance(C,,,) was more depressed than inulin clearance(C,,) in ocbratoxin A-treated rats (5 mg/kg/day, PO). The filtration fraction was increasedby ochratoxin A treatment. Ochratoxin A, ochratoxin LX,and citrinin inhibited the accumulation of PAH by KOZUKA,Y.,SATOH,T.ANDYAMAZAKI,

renal tissue in vitro. These results reflected, at least in part, a cytotoxic action of ochratoxin A on renal cortical tissue. Ochratoxin A (OCT A) is a toxic dihydroisocoumarin metabolite produced by several species of Aspergillus (Scott, 1965; Hesseltineet al., 1972)and Penicillium (van Walbeek et al., 1969; Ciegler et al., 1972). This mycotoxin has been reported to have a high acute toxicity for 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), rainbow trout (Doster et al., 1972), beagle dogs (Szczech et al., 1973a, b), and swine (Szczech et al., 1973c), and it may be responsible for kidney disease of swine in Denmark (Krogh et al., 1973). In studies with various species, the pathologic changes produced by OCT A are commonly characterized by renal and hepatic damage and alterations in the intestinal and lymphoid tissues. OCT A is known to cause impairment of and lesions in the proximal convoluted tubules in rats (Purchase

and Theron, 1968; Munro et al., 1974), beagle dogs (Szczech et al., 1973a, b), and swine (Szczech et al., 1973~). Engelbrecht and Purchase (1969) described nonspecific degenerative changes of nuclei with decreasein normal mitosis in kidney cell culture

treated with OCT A. Since little information has been reported on renal function alterations in OCT Ainduced nephrotoxicity, the present study was undertaken to determine the effects of

OCT A on renal function by using in vitro experiments and renal clearance tests. METHODS

OCT A was isolated from the culture medium of Aspergillus ochraceus Wilh. (IFM 4443) and purified and quantified by the method of Davis et al. (1972). The toxin, mp ca. 90°C after crystallization from benzene, was heated at 80°C under reduced pressure to remove the solvent of crystallization. Ochratoxin CI(OCT IX)was obtained by hydrolysis of OCT A in concentrated HCl according to the method of van der Merwe et al. i Presentaddress : Departmentof BiochemicalPharmacology, Faculty of Pharmaceutical Sciences, ChibaUniversity,Yayoi-cho,Chiba,Chiba,280,Japan. Copyright 0 1975 by Academic Press, Inc. 479 All rights of reproduction in any form reserved.

Printed

in Great

Britain

480

SUZUKI

ET AL.

(1965). Citrinin was recrystallized from ethyl alcohol (EtOH) (Krogh et al., 1970) before use. The purity of OCT A, OCT a, and citrinin used was verified by melting point, thin-layer chromatography, and spectrometric analysis. All animals were male Wistar rats weighing 180-220 g for the clearance test or 200-300 g for the other experiments. They were maintained on a standard commercial diet and water ad libitum. Renal function was studied by measurement of p-aminohippuric acid (PAH) accumulation in cortical slices in vitro and standard clearances of inulin and PAH. OCT A dissolved in 0.1 M sodium bicarbonate solution was given po; NaHCO,treated animals were given an equivalent volume of the vehicle, and control animals. were given 0.9% NaCl solution. The volume administered was adjusted to 0.4 ml/ 100 g body weight. Five or more animals were used for each treatment. Accumulation of PAH in the renal cortical slice was made essentially according to, the method of Cross and Taggart (1950). The animals were sacrificed by decapitation 24 hr after the last administration of OCT A (5 or 15 mg/kg) and the kidneys were removed quickly and placed in ice-cold 0.9% NaCl solution. Three or four slices, 0.4 mm thick, were cut from the cortex of each kidney using a Stadie-Riggs microtome. The tissue, weighing about 100 mg, was then incubated in 2.7 ml of a medium containing PAH, 1.32 x 10e4 M (pH 7.4), as described by Watrous and Plaa (1972a). In the experiment in which a determined concentration of OCT A, OCT u, and Lphenylalanine or citrinin was added, an equivalent volume (0.1 ml) of 0.1 M NaHCO, or 99.5 % EtOH was added to the control medium. The slices were initially equilibrated for 15 min and were incubated at 30°C in an atmosphere of 95 % 02-5 % CO, in a Dubnoff metabolic shaker operating at 100 oscillations/min, for 60 or 75 min. At the end of the incubation period, the slices were removed from the medium, blotted on filter paper, and homogenized in 3 ml of 10% trichloroacetic acid (TCA). The homogenate of slices was diluted to 5 ml with distilled water. One milliliter of the residual incubation medium was diluted to 5 ml with 3 ml of 10 % TCA and with an appropriate volume of distilled water. Both slice homogenate and incubation medium were centrifuged at 3000 rpm for 10 min, and the resulting supernates were taken for analysis of PAH by the method of Bratton and Marshall (1939). Accumulation of PAH by renal tissue was expressed as the ratio between the concentration of PAH in the tissue (milligrams per gram of wet tissue) and the final concentration in the medium (milligrams per milliliter) and referred to as S/M. The variation was observed in the S/M values of the control groups. Therefore, the ratios from each experiment were transformed by the formula of Watrous and Plaa (1972b). The formula for this transformation was : Decrease (% of maximum)

=

control (S/M) - treated (S/M) x loo control (S/M)

Clearance procedure was the method described by Yamamoto et al. (1962) with a minor modification. The animals were fasted about 24 hr after final administration of OCT A (5 mg/kg), although water was given ad libitum. Immediately before the experiments, the urinary bladder was emptied by pressing the pubic region, then the animals. were intubed with 50 ml of water/kg. An emulsion of 5 % inulin and 10 % PAH in.

OCHRATOXIN

A AND

NEPHROTOXICITY

481

cottonseed oil was given SCin a dose of 50 ml/kg. The bladder urine was removed by pressing the pubic area 30 min after inulin and PAH injection, then the animal was placed in a cage with a sample tube for urine collection. Urine was collected for 40 min. Blood was collected from the abdominal aorta under ether anesthesia at the end of the clearance period (40 min). Inulin was determined by the anthrone method of Davidson and Sacker (1963). PAH was determined by the diazotization method of Bratton and Marshall (1939). The kidney separated rapidly from OCT A-treated rats (5 or 15 mg/kg, po) was washed with 1.15 % KC1 and weighed. A 20 % (w/v) homogenate was prepared in 1.15 “,(; KC1 using a Potter-Elvehjem homogenizer. The OCT A in the homogenate was removed by extracting four times with 25 ml of chloroform (CHCI,). Each preparation was shaken for 30 min, and these emulsions were centrifuged for 20 min at 3000 rpm. The CHCl, solution in the bottom layers was pooled and evaporated to dryness under vacuum. The residue was dissolved in 1 or 2 ml of CHCIJ, then 4 or 8 ~1 of the resultant CHCl, solution and OCT A standard solution (0.5 and 0.1 /ig/,ul) were cochromatographed on a thin-layer chromatoplate of silica gel G (Merck) with benzene-acetic acid (9: 1) as the mobile phase. The fluorescence intensity of OCT A on the plate was determined in a Hitachi spectrofluorometer (MPF-2A) equipped with a thin-layer chromatogram scanner and automatic recorder (excitation wavelength, 340 nm: emission wavelength, 466 nm). Throughout the experiments including extraction procedure, about 70 % of OCT A was recovered. Differences between the treatment and control groups were compared by Student’s t test. Freshly sliced kidney tissues for electron microscopy were fixed in 2.5 % glutaldehyde in 0.05 M s-collidine buffer (pH 7.2) for 2.5 hr in an ice-water bath (Bennet and Luft, 1959). After washing, tissues were postfixed in 1% osmium tetroxide with s-collidine buffer (pH 7.2) for 1.5 hr in an ice-water bath. The fixed tissues were transferred into 50% EtOH and subsequently dehydrated in graded, ascending, concentrations of EtOH and embedded in Epon 812 (Luft, 1961). Ultrathin sections, pale gold to silver interference colors, were cut on a Porter-Blum MT-2B ultramicrotome with glass knives and mounted on collodion-coated copper grids. These sections were doubly stained with saturated aqueous solutions of uranyl acetate and lead citrate (Reynolds, 1963) and were examined with a Hitachi HU-12 electron microscope. RESULTS

The effects of repeated pretreatment of rats with OCT A on PAH accumulation by rat renal cortical slices are given in Table 1. In each experiment, tissues of OCT Atreated rats were compared to control tissues from rats receiving 0.1 M NaHCO, solution. The results are given as an S/M ratio and as a percentage of the maximum theoretical response for each experiment (i.e., a reduction of S/M to unity). The S/M values of the OCT A-treated tissues were not different from control values 24 hr after FIG. 1. Electron micrograph of control (A) and OCT A-treated (B) proximal convoluted tubules of the rat renal cortical slice. Notice the thickening and increased density of the basement membrane (BM). Magnification ~12,500; line indicates 1 pm.

482

SUZUKI

ET AL.

OCHRATOXIN

A AND

NEPHROTOXICITY

483

484

SUZUKI ETAL.

TABLE 1 EFFECTS OF OCHRATOXIN A PRETREATMENT ON PAH ACCUMULATION BY RAT RENAL CORTICAL SLICES'

Total dose of OCT A

S/MC (mean f SE)

Decrease in S/Md (% of max)

5 15 -

5.0 i- 0.3 6.0 -t 0.3

18 Increasee

OCT A

10 30 -

4.6 k 0.5 5.3 f 0.2 5.0 + 0.1

10 Increasee -

OCT A OCT A

15 45 -

3.3 + 0.3 3.5 + 0.3 5.5 5 0.3

499 44* -

Treatmentb OCT A OCTA Control

OCT A Control

Control

Days of treatment 1

@g/b,

PO)

5.9 f 0.2

-

@Rats were sacrificed24 hr after final administrationof OCT A (5 or 15mg/kg, po) or control (0.1M NaHCO,). bThe sliceswereincubatedfor 60min.All mediacontained0.01M acetate. c Eachvaluerepresents themean+-SEof a groupof five animals. dIn eachexperiment,a maximumdecrease in S/M wouldbea reductionfrom the controlvalueto unity, i.e., control (S/M) - treated(S/M) x loo decrease (% of max)= control(S/M) - 1 oIncreaseovercontrolvalue. f Significantlydifferentfrom controlsatp < 0.001.

single or two successive dosings (5 or 15 mg/kg/day, PO). A significant impairment of PAH accumulation by renal cortical slices in OCT A-treated rats became evident 24 hr after three successive doses (p < 0.001). A dose-response correlation was not observed. Electron micrographs of control tissue and renal tissue 24 hr after three daily doses of OCT A (5 mg/kg/day) are presented in Fig. 1. The proximal convoluted tubules in a control kidney slice had the characteristic .apical brush order and interdigitation of the basal cell membrane. The mitochondria between the interdigitations were slender and well-developed cristae. Thickening and increased density of the basement membrane and oval-shaped mitochondria were observed after OCT A administration. These morphological changes paralleled somewhat in severity the dose of OCT A. Three successivelyadministered dosesof 0.4 ml (per 100 g) of 0.1 M NaHCO, daily did not significantly change the renal function measured 24 hr later, as judged by a comparison with control tissue with 0.9% NaCl solution (Table 2). OCT A-treated rats showed a lower clearance of inulin (C,,) and PAH (C,,,) (p < O.OOl), though C PAH was depressed more than GIN. The ratio of CIN/CPAH was elevated in the OCT A group as compared with the control or 0.1 M NaHCO,-treated rats. The urine volume of rats treated with OCT A was decreased (p < 0.02). The increase in blood PAH concentrations was statistically significant, but changes in blood inulin concentrations were not different.

OCHRATOXIN

A AND

485

NEPHROTOXICITY

TABLE 2 RENAL FUNCTION IN CONTROL AND OCHRATOXIN A-TREATED RAW Parameter

Control

0.1 M NaHCOa __---

Urine volume (ml/hr/lOO g) Cm (ml/min/lOO g) GAH (ml/mid100 8) GdGA” Inulin in blood (mg/lOO ml) PAH in blood (mg/lOO ml)

2.16 0.24 1.03 0.24 16.66 6.29

+ f f. L* +

0.18 0.02 0.05 0.01 0.40 0.65

2.55 0.30 1.17 0.28 16.92 7.38

+ 0.26 + 0.04 + 0.15 -I 0.01 -I: 0.92 + 0.80

OCT A ~.

1.80 0.15 0.40 0.37 17.10

+ + * + +

0.12’ 0.02’ 0.05” 0.01’ 1.07

12.58+ 0.95'

’ Animals were used for clearance tests 24 hr after three successive administrations (5 mg/kg/day, PO), 0.9% NaCl solution or 0.1 M NaHC03 solution. * Significantly different from 0.1 M NaHC03 group at p < 0.02. ’ Significantly different from 0.1 M NaHCO, group at p < 0.001.

of OCT A

The effects of 0.1 M NaHCO, and 0.01 M acetate on the PAH transport system were determined (Table 3). Acetate was found to stimulate significantly the accumulation of PAH by renal cortical slices (p < 0.001) but in 0.1 M NaHCO, did not alter the PAH transport system. TABLE 3 EFFECTS OF NaHCO, AND ACETATE ON PAH ACCUMULATION BY RAT RENAL CORTICAL SLICESONTHE BASE MEDIA" S/M

Expt

NaHCO,”

Acetatec

(mean rf: SE)d

p’

Pf

1 2

+

-

4.7 3.920.2 + 0.4

NS -

NS

3 4

-

+ +

7.8 +0.4 8.2 + 0.6

0.001

0.001 NS

+

0.001

’ The slices were incubated for 75 min. * 0.1 ml of 0.1 M NaHCO, was added to the base medium (2.7 ml). ’ 0.1 ml of 0.01 M acetate was added to the base medium (2.7 ml). d See footnote c, Table 1. e Significantly different from the base medium; NS, not significant. f Significantly different from the medium to the medium used.

The effects of OCT A on PAH accumulation by rat renal cortical slices with and without addition of 0.01 M acetate in vitro are summarized in Table 4. Accumulation of PAH was significantly inhibited by 5 x 10W4-5 x 10m6 M OCT A with and without acetate. The results of this experiment show that the rate of inhibition on PAH accumulation in the medium without 0.01 M acetate may be in proportion to OCT A concentration in the medium. No clear relationship between the rate of inhibition on PAH accumulation and OCT A concentration in the medium was observed in the medium containing 0.01 M acetate. OCT A at a concentration of 5 x 10m4 M had a complete inhibitory effect on PAH accumulation in a medium containing 0.01 M acetate.

486

SUZUKI

ETAL.

TABLE 4 EFFECTSOF~CHRATOXIN AoNPAH ACCUMLJLATIONBYRAT RENALCORTICAL SLICES' Acetate (0.01 M) Without

With

OCT Ab (M)

S/MC

Decrease in S/M (% of max)d

5x104 5 x 10” 5x104 5 x 10-7 0 5x104 5 x 1o-5 5 x 106 5 x 10-7 0

3.3 + 0.3 (6) 5.9 f 0.3 (6) 7.3 zk 0.4 (5) 7.9 + 0.3 (6) 9.2 +_0.6 (4) 1.0 f. 0.1 (6) 2.9 k 0.2 (6) 8.7 k 0.7 (5) 9.7 k 0.8 (5) 11.2 + 0.5 (5)

72” 40” 23” 14 100’ 81’ 25f 15 -

a The slices were incubated for 75 min. b OCT A was dissolved 0.1 ml of 0.1 M NaHC03. An equivalent volume (0.1 ml) of 0.1M NaHC03 was addedto the control medium. c Each value represents the mean + SE. Figures in parentheses indicate numbers of slice experiments. d Seenote d, Table 1. e Significantly different from controls (p < 0.001). f Significantly different from controls (p < 0.05). In order to determine the type of inhibition of PAH accumulation by OCT A in a medium containing 0.01 M acetate, the substrate concentration was varied in the absence and presence of two concentrations of OCT A (5 x 10e5 and 5 x 10e6 M). The results, plotted according to the method of Lineweaver and Burk (1934), are shown in Fig. 2. The plots indicate that OCT A inhibited PAH accumulation non-

-2

0

2 IIS

4

0

2

10

mM

Fro. 2. Lineweaver-Burk plots for PAH. The slices were incubated for 75 min. PAH concentrations ranging from 0.101 x 10m3to 0.254 x 10T3M were used.

OCHRATOXIN

A AND

487

NEPHROTOXICITY

competitively; the Michaelis constant (K,,,) and inhibition constant (KJ for the effect of OCT A on PAH accumulation were 1.17 x 10e3 M for PAH. The effects of OCT A, OCT a, L-phenylalanine, and citrinin on PAH accumulation by rat renal cortical slices are shown in Table 5. OCT A, OCT CI,and citrinin, but not L-phenylalanine had a marked inhibitory effect on active PAH transport in vitro (p < 0.001). TABLE 5 EFFECTS OF OCHRATOXIN

A, OCHRATOXIN

a, PHENYLALANINE,

ANDCITRININONPAH ACCUMULATIONBYRATCORTICALSLICES~ Decrease in

Expt

Substrate*

S/M’

S/M (% of max)”

5

OCT A 0 OCT a

1.0 + 0.1 (6) 11.2 + 0.5 (5)

100’ -

1.5 + 0.2 (5) 10.7 * 1 .o (5)

95’ -

6

0

7 8

L-Phenylalanine 0 Citrinin 0

7.9 + 0.8 8.3 0.7 (5)

2

2.1 *+ 0.5 7.1 0.1 (5) (4)

Fe

’ The slices were incubated for 75 min. All media contained 0.01 M acetate. All substrate was added at 5 x lOA Mto the medium. b OCT A, OCT a, and L-phenylalanine were dissolved in 0.1 ml of 0.1 MNaHCO,. Citrinin was dissolved in 0.1 ml of 99.5”/, EtOH. An equivalent volume (0.1 ml) of the vehicle used for dissolving each substrate was added to the control medium. c See footnote c, Table 4. d See footnote d, Table 1. e See footnote e, Table 4.

DISCUSSION

With regard to the nephrotoxicity of mycotoxins, aflatoxin is known to cause, besides preeminent hepatic damage, acute renal damage in birds, dogs, monkeys, guinea pigs, and rats, consisting of dilated tubules, tubular necrosis, and regeneration (Newberne, 1965). Citrinin caused renal damage in experimental animals (Ambrose and DeEds, 1946). In 1955, Sakai found that citrinin severely depressed the clearance of inulin as well as PAH and elevated the filtration fraction in rats. Most of the renal tubular necrosis produced by citrinin in dogs was located in the distal convoluted tubules and the loop of Henle (Carlton et al., 1974). This serves to differentiate further the renal damage induced in dogs by citrinin from that caused by OCT A, as the latter affected primarily the proximal convoluted tubules (Szczech et al., 1973b). Rats fed 24 ppm of OCT A for a period of 2 weeks had elevated serum blood urea nitrogen (BUN) and significantly increased kidney weights accompanied by marked degenerative changes involving the entire tubular system (Munro et al., 1974). Purchase and Theron (1968) reported that the principal pathological change associated with fatal doses (40 mg/kg, po) of OCT A was necrosis of the renal tubules; the glomeruli were considered normal.

488

SUZUKI

ET AL.

In agreement with the earlier work described above, our results demonstrated an acute toxic effect of OCT A on renal function in rats with changes in transport of PAH and in clearances of glomerular and tubular markers. Repeated administration of nonlethal doses of OCT A (5 or 15 mg/kg, po) daily for 3 days may be required for inhibition of PAH accumulation in medium containing acetate. As described earlier (Weiner and Mudge, 1964; Despopoulos, 1965), OCT A is acidic and has a R-CO-NH-(CHR’),-COOH, R and R’ are an aliphatic and aromatic group respectively, it is therefore unlikely that PAH and OCT A compete for the same transport system. Our results show that inhibition of PAH accumulation by OCT A in vitro may be considered as competitive in medium without added acetate and as noncompetitive in medium containing acetate. OCT A may be transported more actively than PAH and has more affinity for the active transport system. Since PAH transport is accomplished in the proximal tubule by an energy-requiring system susceptible to metabolic inhibition (Cross and Taggart, 1950), the greater sensitivity of renal tissue to OCT A in medium containing 0.01 M acetate is to be expected. Analysis of extractable OCT A from the kidneys of rats given repeated dosing (5 or 15 mg/kg daily, po) indicated that OCT A did not accumulate in the kidney (unpublished data). The range of OCT A content in the kidneys was 2-10 pg/g wet weight of tissue in the 5-mg/kg treatment group and 4-22 ,ug/g wet weight of tissue in the Ifi-mg/kg treatment group. OCT A interacts with proteins in the kidney homogenate (Chu, 1974). Therefore, OCT A bound to protein in the kidney may accumulate resulting in its toxic effect in vivo. in The GIN, CP,W, and the filtration fraction values (FF, calculated as C&&J OCT A-treated rats as compared to controls and NaHCO,-treated rats indicate a toxic effect on tubular transport rather than complete renal shut-down. The elevated PAH concentrations in blood suggest also damage to tubular function (Table 2). The decrease of C,,, may be explained by factors such as reduction of total plasma flow and inhibition of PAH secretion in proximal tubules. After successive doses of OCT A, degeneration of convoluted tubules was present, but the glomeruli appeared normal. In electron micrographs, the basement membranes were thickened and increased in density and the mitochondria were oval shaped. The thickening of the basement membrane may be responsible for tubular malfunction, as the basement membrane is known to act as a barrier for the macromolecular transport system in kidney (Caulfield and Farquhar, 1974). Thickening of the basement membrane in renal tubules was observed after administration of OCT A in rats (Munro et al., 1974). Abnormalities of renal function may reside, at least in part, in the thickening of the basement membrane. ACKNOWLEDGMENTS We thank Dr. M. Kanisawa, Department of Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, who helped in the morphological portions of this study. We also thank Dr. Y. Ueno, Department of Microbial Chemistry, Faculty of Pharmaceutical Science, Tokyo University of Science, Tokyo, for supplying us with citrinin. REFERENCES F. (1946). Some toxicological and pharmacological properties of citrinin. J. Pharmacol. Exp. Ther. 88, 173-186.

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LUFT, J. H. (1961). Improvements in epoxyresin embeddingmethod. J. Biophys. Biochem. Cytol. 9,409-414.

I. C., MOODIE, C. A., KUIPER-GOODMAN, T., Scorr, P. M. AND GRICE, H. C. (1974). Toxicologic changesin rats fed gradeddietary levelsof ochratoxin A. Toxicol. Appl. Pharmacol. 28,18~188. NEWBERNE, P. M. (1965). Carcinogenicity of aflatoxin-contaminated peanut meals. In Mycotoxins in Foodstufi (G. N. Wogan, Ed.), pp. 187-208. M.T.T. Press,Cambridge, Mass. PECKHAM, J. C., DOUPNIK, B., JR. ANDJONES, 0. H., JR. (1971).Acute toxicity of ochratoxin A and B in chicks. Appl. Microbial. 21, 492-494. PURCHASE, I. F. H. AND THERON, J. J. (1968). The acute toxicity of ochratoxin A to rats. MUNRO,

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J. J. (1971).The long term toxicity of ochratoxin A to rats. Food Cosmet. Toxicol. 9,681-682. REYNOLDS, E. S. (1963).The useof leadcitrate at high pH asan electronopaquestainin electron microscopy.J. Cell Biol. 17, 208-212. SAKAI,F. (1955).An experimentalstudy on the toxic effectespeciallyon the kidney of “yellowed rice” polluted by Penicillium citrinum Thorn, aswell asof citrinin, a pigmentisolatedfrom the mould. Folia Pharmacol. Jap. 51,413-442 (in Japanese). SCOTT, DE B. (1965).Toxigenic fungi isolatedfrom cerealand legumeproducts. Mycopathol. PURCHASE,

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