Investigations of dose, age, and duration of administration on the hepatorenal damage induced in mice by cultural products of Penicillium viridicatum

Investigations of Dose, Age, and Duration of Administration on the Hepatorenal Damage Induced in Mice by Cultural Products of Penicillium viridicatuml IWAN T. BUDIARSO,* WILLIAM W. CARLTON, AND JOHN TUITE Departments of Veterinary Microbiology, Pathology and PubIic Health, and Veterinary Physiology and Pharmacology, School of Veterinary Science and Medicine, and Department of Botany and Plant Pathology, School of Agriculture, Purdue University, Lafayette, Indiana 47907 Received January 25, 1911

Investigation of Dose, Age, and Duration of Administration on the HepatorenalDamageInduced in Mice by Cultural Productsof Penicillium viridicatum. BUDIARSO, IWAN T., CARLTON, WILLIAM W., and TLJITE,JOHN F. (1971). Toxicol. Appl. Pharmacol. 20, 357-379.The following studies werecompletedin micefed rice culturesof P. viridicatum: the approximate LD50-14days,agesusceptibility,life span.and pathogenesis of hepatorenal damage. Mice werefed the dried, ground rice culture mixed with a purified diet at concentrationsfrom 2.5 to 30%. Dietsproducing mortalitiesclosestto 50% werethosecontaining 10and 15% concentrationsof the rice culture. In the agesusceptibilitystudy, miceof ages2-18 wk werefed a diet containing 50% rice culture ofP. viridicatum. Although mortality wasnot great in groupsolder than 6 wk of age, weight loss,and lowering of packedcell volume occurred in all groups fed the fungal diets. Older mice did not becomeresistantto the toxic effectsof P. viridicatrrm becausehepatic and renal lesionswere observedin mice of ages8, 10, 14, and 18 wk. These hepatic lesionswerecenteredin the biliary systemand includednecrotizing cholangitis,bile duct proliferation, periductal fibrosis, and ductal ectasia. Hepatic cell changesincludedmarked variation in sizeand shapeof nuclei, bizarre mitotic figures, and multinucleated cells. To provide data on the life spanof test mice, micewerefed rice cultures at a 7.5% concentration in the diet. Clinical signsof toxicity included anemia,jaundice,and high mortality. None survived beyond 9 wk of feeding. Most of the micehad hepaticfocal necrosisand bile duct proliferation. Tubular necrosiswaspresentin most of the kidneys. The developmentof the hepatorenallesionswas followed in mice fed 5 % P. viridicatum dietsandkilled after l-24 wk of feeding.Most of the mice presentedsignsof toxicity suchasroughenedhair coatsearly andmany were anemicafter 7 wk of feeding.Body weightsof test groupswerealwaysless than controls. Hepatic changesprogressedfrom perilobular, hydropic, and fatty degenerationof hepatocytesto necrotizing cholangitis followed by ’ Thisstudywassupportedin part by CooperativeAgreement12-14-lOO-9091(51), Market Quality Research Division,ARS, USDA, Beltsville,Maryland and NIH Grant ES00463,NationalInstitutes of Health,Bethesda, Maryland. Published asJournalPaperNo. 4310,PurdueUniversity,Agricultural ExperimentStation,Lafayette,Indiana47907. ’ Presentaddress:Department of Veterinary Pathology, Faculty of Veterinary Medicine, Bogor Institute of Agricultural

Sciences, Taman Ade Irma No. I, Bogar, Indonesia.

357

358

BUDIARSO,

CARLTON,

AND

TUITE

hepatic cell necrosis. In more chronically fed mice, proliferation of bile duct cells was accompanied by bile duct hyperplasia and periductal fibrosis, and these changes were followed by bile duct ectasia with loss of hepatic parenchyma. The nuclear alterations in hepatocytes were similar to those described in mice of the age susceptibility study.

Carlton et al. (1968, 1970) reported that weanling mice were very sensitive to the toxic effects of P. viridicatum, and cultures of this fungus consistently produced lesions in the liver and kidneys. Pharmacologic and toxicologic studies indicate age-related variation of susceptibility among animals to certain toxicants. A review of the dose-response relationships, in rats of different ages, to aflatoxin has been published (Butler, 1969). Carlton and Tuite (1970a) did not find, based upon mortality, any variation in susceptibility between younger and older guinea pigs fed the same concentration of P. ziridicutum diet. However, in older guinea pigs the loss of body weight was greater than in younger pigs fed the same fungal diet. In previous studies, relatively high dietary concentrations of P. ciridicatum cultures were fed, and acute intoxication was induced. In the present study, the LD50 of cultures was estimated, age susceptibility studied, and the life span of mice fed continuously a low concentration of fungal diet was determined. The morphopathogenesis of the hepatorenal damage in mice induced by the chronic feeding of a P. riridicatum diet was also studied. METHODS

The preparation of the rice cultures of P. viridicatum (isolate 66-68-2) has been described (Budiarso et al., 1971). Trial Z (Determination of LD50). Dried ground rice cultures were mixed with a purified diet at dilution of 2.5, 5.0,7.5, 10.0, 15.0,20.0,25.0, and 30.0%. Six weanling Swiss albino mice were allotted to each diet and 6 mice fed the purified diet only served as the controls. All surviving mice were killed at the end of the second week. Trial ZZ (age susceptibility). Dried ground rice cultures were mixed with a purified diet at a concentration of 50 % (test diet). Ten male, Swiss albino mice of a specified age were fed the test diet for a 2-wk period. Five mice of the same age group were used as controls and fed the purified diet. All surviving mice were killed at the end of the second week. Trial ZZZ(life span). Dried ground rice cultures were mixed with a purified diet at a concentration of 7.5% and fed to 27 weanling, Swiss albino mice. Ten mice fed the purified diet served as controls. The controls were killed when all test mice had died. Blood was collected only from 5 mice killed in extremis. Trial ZV (pathogenesis qf hepatorenal damage). Trial IVa consisted of 100 mice fed a 10 % rice culture diet of P. viridicatum, and trial IVb consisted of 150 mice fed a 5 % fungal diet. Fifty and 75 mice fed the purified diet only served as controls for the 2 trials. Ten test mice and 5 controls from each trial were killed for necropsy at intervals given in Tables 5 and 8. Mice that died during the experimental period were grouped with those which were killed. Because previous studies established that the target organs were the liver and kidney, only these organs were collected at necropsy unless gross abnormalities were

Penicillium viridicatum

MYCOTOXICOSIS

359

found in other tissues. Tissues fixed in 10 % buffered formalin were processed for paraffin sections and stained with hematoxylin and eosin. Blood was collected at necropsy for determination of packed cell volume (PCV) by the microhematocrit method and total serum protein (TSP) by A0 refractometer. RESULTS

Clinical Observations Trial Z (determination of LDSO-14 days). No clinical

signs of toxicity occurred in control mice or test mice fed the fungal diets at 2.5 and S.O’k concentrations. Three mice fed the 7.5 ‘A diet had roughened hair coats and arched backs by day 8. One of these was jaundiced and died on day 12. At the end of week 2, 3 mice were clinically ill. The onset of clinical signs in some mice of the 10 % and 15 % fed groups occurred at about day 5 and included depression, huddling, and yellowish discoloration of the ears. About half of the mice died during week 2 (Table 1). Survivors were clinically ill. TABLE AVERAGE

BODY

WEIGHT

AND

1

MORTALITY

OF MICE Penicilliumviridicatum (TRIAL

Test groups

0

Control

12 13 14 14 14 13 14 13 14

20 17 20 17 14 14 12 11 12

2.5% 5.0% 7.5% 10.0% 15.0% 20.0% 25.0% 30.0%

PV” PV PV PV PV PV PV

PV

CULTURE

OF

Mortality, No. dead/No. started

Average body weight (g) Week 1

FED A RICE 1)

Week 2

1

2

26 2.5 27 19 15 12

016 O/6 016 O/6 l/6 l/6

016 016 O/6 V-5

416

616 616

Dead Dead Dead

616 416

416 316

QP. viridicatum.

Clinical evidence of toxicity in the groups fed the 20.0,25.0, and 30.0% fungal diets began as depression at day 4, and the signs of toxicity were as described above. No mice survived beyond week 1 (Table 1). The average body weights of the 2.5 % and 5 % test mice were comparable to the control group. The weight gains of the 7.5% and 10% groups were markedly reduced as compared to the control, and the other test groups lost weight. The LD50-14 day was determined by estimation from the numbers of deaths given in Table 1 and found to be between 10 and 15 % concentration of the rice culture. Trial ZZ(age susceptibility). The signs of intoxication in test mice of ages 3 and 4 wk were similar to those of the 10 and 15 % test group of trial I. No mice survived beyond week 1 (Table 2). The clinical signs of toxicity in groups of ages 5 and 6 wk were as described in trial I, and some mice developed a yellowish discoloration of the ears. All mice died within 13 days.

360

BUDIARSO,CARLTON,

ANU TUITE

TABLE 2 AVERAGEBODY WEIGHT,~EEKLYAND TOTAL MORTALITYOF CONTAINING 50 % RICE CULTURE OF Penicillium viridicatum

Age (wk) 3

Test groups

Control 50% PV”

4

Control 50% PV

5

Control 50% PV

6 8 10

Control 50% PV Control 50% PV Control 5004 PV

14

Control 50% PV

18

Control 50% PV

MICEFED DIET (TRIAL II)

Average body weights (g)

Mortality, No. dead/No. started

Week

Week

0

1

2

1

2

Total

14 12 22 20 28 26 30 28 33 31 38 38 38 43 46 49

23 9 28 14 31 18 33 22 35 23 40 28 37 33 46 38

26 30 33 18 34 22 36 20 41 27 39 29 46 30

015 lo/lo 015 lo/lo O/5 2/10 O/5 2110 oj5

015

O/5 lo/lo 015 lO/lO O/5 lo/lo 015 lo/lo O/5 Z/l0 015

o/10

OF O/5

w3

O/5

v3 O/5

2110

O/5

O/5

O/5 o/10 015 O/l0

o/5 3jlO 015 o/10

Oil0

2110

2/10

O/5 3110 O/5 o/10

o P. viridicatum.

In mice 8 wk of age, clinical signsbegan at day 5. These were followed in some mice by jaundice and anemia. Posterior limb weaknessand fluffy hair coats were present in somemice at day 9. Two mice died at day 11 and the survivors were clinically sick when killed after 2 wk. The IO-wk-old mice had fluffy hair coats, but other signs of mycotoxicosis were absentin the first week of feeding. At day 9,3 mice were sick, and 2 of thesedied at day 12. The 8 surviving mice were clinically ill at termination. The appearance of the 14-wkold mice was similar. Condition deteriorated rapidly in week 2, and 3 mice died. When killed, 4 mice were icteric and 2 were anemic. The mice of the 18-wk-old group had fluffy hair coats in the first week of feeding. In week 2, the signsof toxicosis were variable but included jaundice and posterior weakness.All mice survived the experimental period. In all test groups there was a loss of body weight indicating that mice did not become completely resistant to the toxic effects of cultures of P. viridicatum. The averagesof PCV of mice in all test groups were markedly lower than those of the control groups, and the ranges of PCV of the test groups were wider (Table 3). The average percentagesof TSP of all test groups were higher and the rangeswere wider than those of the control groups (Table 3). The averagesand ranges of liver weights of the test groups were consistently lower than the controls but, except for the 3-wk-old group, the liver weight asa percentage of the body weight of the test groups were consistently higher than the controls.

Penicillium viridicatum

361

MYCOTOXICOSIS

TABLE

3

AVERAGE PACKED CELL VOLUME (PCV), AVERAGE SERUM PROTEIN, AVERAGE LIVER WEIGHT, AND LIVER WEIGHT AS PERCENT OF BODY WEIGHT OF MICE FED 50% RICE CULTURES OF Penicilliumviridicatum (TRIAL II)

Test groups 3

6

8

Serum protein (%) (range)

Control 50% PV”

4

PCV (%I kinge) (3Z5) -

-

50% PV

-

-

Control

-

-

50% PV

-

-

Control

-

-

50% PV

-

-

Control

43’ (4146)

(6Z.6)

$8)

(7.::;

(4::8)

(7.Z.5)

(243-338)

(5.Z.3)

(424i4)

(6.Z.8)

$44)

(5.G2.2)

(42%)

(6Z.6)

1.71 1.8)

Control

Control 50% PV

18

5.6 5.3 4.4 5.9

6.3

5.1 5.7

(0.93-l .45)

50% PV 14

Liver

wt. % body wt.

4.4 (1.0%65) 1.13 (0.90-1.38) 1.74 (1.39-1.98) 1.23

50% PV 10

wt

cd (range) 1.47 (1.25-1.63) 0.45 (0.25-0.62) 1.31 (1.20-1.37) 0.83 (0.50-I .oo)

(5.2.2) -

Control

Liver

Control 50% PV (5.::

1.8)

(1.47-2.00) 1.21 (0.71-1.52) 1.87 (1.61-2.11) 1.66 (1.32-2.23) 1.60 (1.12-2.11) 1.47 (1.10-2.10) 1.96 (1.76-2.14) 1.69 (1.35-2.00)

4.7 6.1 4.6 6.3 4.1 5.1 4.3 5.6

a P. viridicatum.

Trial ZZZ(life span).In general the signsof intoxication were similar to those described in the 10 and 15% test groups of trial I, but anemia, icterus, and deaths were not observed until week 2 (Table 4). From week 2 to the termination, a few mice were clinically sick, and some had a yellowish discoloration of the ears and occasionally of the tail. No mice survived beyond 9 wk feeding.

Average body weights of the test mice were consistently below those of the controls. The average PCV of the test mice which were killed in extremis was 6.6 %, and for the control mice it was 44%. The % TSP of test groups was 6.5 with a range between 5.7 and 8.7; the average TSP of control groups was 6.9 with a range between 6.6 and 7.2. 14

362

BUDIARSO,

CARLTON,

AND

TUITE

TABLE 4 AVERAGE

Feeding period (Wkj

BODY WEIGHT RICE CULTURE

AND CUNICAL OF Penicillium

MORBIDITY

viridicatum

OF MICE FED 1.5 4; (TRIAL Ill)

Average body weight (g) No. of mice 27 27 20 15 12 12 1

5 2

- Control grow 20” 26 28 31 33 36 38 40 41

No. affected

Test group

Clinical illness

16 17 19 23 24 21 21 20 22

8 17 10 5 3 4 5 3 2

Anemia

lcterus

’ Initial averagebody weightof control and testgroupswas11g.

Trial ZVa (pathogenesis of hepatorenal damage). Sixty percent of the test mice were clinically affected by the end of the first week. A few mice were found dead each day beginning from day 7, and 50 had died by the end of week 3 of feeding. The signs of mycotoxicosis included depression, huddling, and roughened hair coats. These were soon followed by anemia, jaundice, and dehydration, terminated by death. The average body weights of surviving mice of the test groups were markedly lower than those of the controls (Table 5). Except for the values for the first week, the averages of PCV of the controls were consistently higher and the ranges were narrower than those of the test groups. In contrast, the averages of % TSP in the controls were consistently lower than those of the test groups, but the ranges of the latter were wider. For the first 3 wk the averages of liver weights of the controls were higher than those of the test mice. Beginning with week 4, the average liver weights and ranges of the test groups were greater than those of the controls. Except for the first week, liver weights as a y0 of body weights increased with time in the test mice and were numerically greater than those of the controls. TriaZZVb. In general, the signs of toxicosis were similar to those described in trial IVa, except that the onset of the disease was delayed somewhat. The average body weights of the test groups were consistently below those of the controls, but the differences were never very great (Table 8). Significant differences were not found between test and control mice in the average PCV values for the first wk of feeding. The PCV values of the test mice were below those of the control mice at the other determinations. Also, the range of PCV values in the test mice was wider than that of the controls, and as many of the test mice were anemic. During the first 3 wk, the average % TSP of the test mice was slightly above that of controls. However, after week 3, the average % TSP for the test mice dropped below that of the controls, but the difference was never very great. The ranges of % TSP were wider for the test mice than those of the controls (Table 8).

(wk)

Feeding period

’ P. viridicatum.

-____-._-

TABLE

5

11 -

10% PV”

Control

lO%PV

Control

lO%PV

Control

lO%PV

Control

lO%PV

0 11

-

Control

group

Test

__-

-

-

23 13 -

13 -

-

-

16

29

-

-

-

-

18

3

2

1

Week

-

-

22

32

-

-

-

-

-

4

Average body weights (g)

23

33

-

-

-

-

-

-

-

-

5

22

33

-

-

-

-

-

-

-

___ 6 __-

-

$4)

(20?3) 46 (40-48)

(4::8)

(2lzO)

(26)

(6.;%5)

(5.Z.3)

(6.z.0)

(6.ili.5)

(6.Z.6)

(5.Z.4)

(2?50)

(5.;;.8)

(5.E.O)

(0.8Z?15) 0.55 (0.42-0.65) 1.35 (1.18-1.60) 0.88 (0.70-1.17) 1.88 (1.65-2.10) 1.28 (0.88-2.50) 1.66 (1.55-1.90) 1.81 (0.80-3.50) 1.70 (1.60-1.85) 2.28 (0.90-3.33)

bwe)

(range)

(5.E.S)

-

10.4

5.1

8.3

5.3

8.1

6.5

6.7

5.9

4.4

Average liver weight % body weight -.-5.9

WEIGH-~ AND viridicatum

Average liver weight (Et)

AVERAGE LIVER OF Penicillium

Average serum protein (%)

PROTEIN, CULTURE

(4226)

(3zl8)

(3&3)

Average PCV (%I (range)

AVERAGE BODY WEIGHT, AVERAGE PACKED CELL VOLUME (PCV), AVERAGE SERUM AVERAGE LIVER WEIGHT AS PERCENT ot: BODY WEIGHT OF MICE FED 10% RICE (TRIAL IVa)

364

BUDIARSO, CARLTON, AND TUITE

TABLE 6 GROSS LESIONS IN MICE FED lo:‘, RICE CULTURE OF Penicilliumz+idicarwn (TRIAL 1Va)

Interval of feeding (wk)

No. with hepatic lesions No. mice examined

1

13

2 3 4 6

45 17

Necrosisand Cystic hemorrhage bile ducts ___2 22

0 0

No. with green kidneys ~__ __

10

0

10 10

10 10

6 5

1 15 6 3 2

95

54

11

27

The average liver weights of the test mice were generally greater than the controls during the first 5 wk. Subsequently, the liver weights of the test mice dropped below those of the controls for the remainder of the experimental period. The ranges were variable in both test and control groups. Liver weights as a percent of body weight were greater for the test groups than for the controls for most of the observations, but the differences were not great after the first 4 wk. Gross Pathology Trial Z(determination of L 0.50). Gross lesions were not observed in mice fed the 2.5 %

and 5 % fungal diets. One mouse, in the group fed the 7.5 % diet was icteric; its liver had necrotic foci scattered through all lobes, and the kidneys were swollen and pale. Three of the surviving mice killed at the termination had gross lesions of the liver and kidneys. Gross changes were found in one of 4 mice of the 10 % fungal diet group which died. The subcutaneous tissues and ears were discolored yellow, and the kidneys were green. The 2 survivors killed at the termination had small foci of necrosis scattered in the liver (Fig. 1); in one, the bile ducts were cystic and the kidneys were green. One of 3 mice of the group fed the 15 % fungal diet which died was icteric. The 3 survivors killed at the termination had hemorrhagic and necrotic foci in the liver. No renal lesions were noted. In the groups fed the 20,25, and 30 ‘A fungal diets, necropsy lesions included loss of adipose tissue, dehydration, and a mild jaundice. The thoracic and abdominal viscera were normal. Trial ZZ(age susceptibility). Dehydration and loss of adipose tissue occurred in mice of the 3- and 4-wk-old groups. In the 5-wk-old group, pinpoint foci of necrosis occurred in the livers of 5 mice, and in 3 of these the kidneys were green. In the 6-wk-old group, 1 mouse had clay-colored liver and kidneys and 5 mice had foci of necrosis in all hepatic lobes. The urine of 4 mice was green. Gross lesions were not found in the other 4 mice. All mice in the 8-wk-old group had gross hepatic lesions including foci of necrosis and hemorrhage, cystic dilatation of bile ducts, particularly near the hilus, and distention of the gall bladder. The kidney lesions varied from a yellowish to greenish discoloration.

85

10 10

10

4 6 -

35 17

45 17

2 3

10 ._95

13

22

28

8 7

8

4 9

5

1 50

-

35 7

7

-

1

Tubular necrosis ____

Duct cell proliferation

7

I

Bile pigment

No. with hepatic lesions

viridicatum

--

29

11

2 3

16 3

II

2

7 2

5

Bile

-

-

-

Protein

Casts

--

1

5 2

3

Dilated tubules

No. with renal lesions

LIVER AND KIDNEY OF MICE FED 10% RICE CULTURE OF Penicillium (TRIAL IVa)

13

IN

Focal necrosis

LESIONS

No. mice examined

Interval of feeding W) -__ 1

M~CROSCOPK

TABLE 7

8 5

Fl

5 8

P 3

=z f 3 c $ Ia

2.

b 3 =.

366

BUDIARSO,

CARLTON,

TABLE

AND

TUITE

8

AVERAGE PACKED CELL VOLUME (PCV), AVERAGE SERUM PROTEIN, AVERAGE LIVER WEIGHTS, AVERAGE OF LIVER WEIGHT AS “/o OF BODY WEIGHT, AND AVERAGE BODY WEIGHT OF MICE FED 5 0/O RICE CULTURE OF Penicillium viridicatum (TRIAL LVb) -

Feeding period W) 1

Test groups

Average PCV % (range)

Average serum protein %

(range)

Control (3Z5)

(5.::.2)

(3 lY9,

(5.%4,

(394-40)

(6.%

(4OY7, 49 (45-55)

(6. E.0, 6.5 (6.2-7.0)

(2?45) 47 (44-50)

(6.:1:.7) (6.kk3)

(3c47)

(5.:1:.6)

(::8)

(6.::?.2) 6.7 (5.8-8.8)

5% PV” 2

Control 5%PV

3

Control 5%PV

4

Control S%PV

6

Control 5%PV $45,

8

Control (425)

(6.::;.0)

(183:8)

(2l2.4)

(5.G.O) 6.7 (6.5-7.4) 6.6 (6.2-8.3)

(4Z7)

(6.Z.2,

(lcE1,

(5.5hLzs.2, 7.2 (6.5-8.1) 6.4 (5.8-7.0)

5%PV 12

Control (26) 5%PV

16

Control 5%PV

20

Control (384i5, 5%PV (zo,

24

Average liver weight (8) (range) --. __1.43 (1.25-1.63) 1.16 (0.95-I .35) 1.34 (1.15-1.43) 1.49 (1.12-2.05) 1.27 (1.10-1.40) 1.44 (1.28-1.75) 1.24 (1.19-1.30) 1.52 (0.95-2.06) 1.66 (I .40-2.05) 1.28 (0.85-2.20) 1.86 (1.44-2.28) 1.74 (1.24-2.42) (1.7::16) 1.76 (1.24-2.90) 2.05 (1.65-2.30) 1.50 (1.32-2.10) 2.93 (2.25-3.42) 2.10 (1.00-2.90)

Control (3Z5)

(7.::;.6)

5%PV (5.Z.O) LIP. viridicatum.

(2.0:?.70) 2.33 (1.65-3.10)

Average liver weight (% body weight)

Average body weight w

7.3

20

6.2

19

5.2

26

6.4

23

4.5

28

5.7

25

3.9

31

5.3

29

4.6

36

4.4

29

4.7

40

4.9

36

4.6

43

4.9

36

4.6

45

4.0

37

5.9

49

4.9

43

4.5

55

5.2

45

Penicillium

viridicatum MYCOTOXICOSIS

367

FIG. 1. Numerous small hemorrhagic and necrotic foci are scattered throughout the 2 livers (left and right) from mice fed 10% P. viridicatum diet for 2 wk (control liver in center). FIG. 2. Liver from mouse fed 7.5 % P. viridicatum for 2 wk. Several foci of necrosis are present. Hematoxylin and eoxin. x56.

368

BUDIARSO, CARLTON, AND TUITE

The lesions observed in the mice of the IO-, 14-, and 18-wk-old groups were similar to those described above for the 8-wk-old group. However, cystic dilatation of the bile ducts was more frequent and severe. Body fat was markedly reduced, and the carcasses were icteric. Trial III (life span). In general, the lesions were similar, although variable in severity in mice dying after different periods of feeding. Gross lesions were observed after week 2 of feeding. In addition to the changes described in trial II, the gallbladder and common bile duct were distended with either normal or lymphlike bile (“white bile”) and, occasionally, with hemorrhagic or dark green inspissated bile. Other gross changes included yellowish or clay white discoloration of livers which were frequently associated with anemia. Gross change in the kidneys did not occur consistently, but in a few mice the kidneys were green with a few foci of necrosis and some were pitted. Anemia was apparent about week 2 of feeding and was indicated by generalized paleness of the carcasses and of the blood. Trial IVu and b (pathogenesisof hepatorenal damage).Tables 6 and 9 summarize the kinds and incidences of gross lesions. In trial IVa, the lesions appeared in week 1, and in trial IVb in week 3 of feeding. The lesions varied in severity in mice necropsied after different periods. The gross lesions included dilatation of bile ducts and distention of the common bile duct with “white bile” or dark green inspissated bile. In addition to the renal lesions and anemia, some mice in both trials had peritonitis, and a few older mice of trial 1Vb also had splenomegaly. TABLE 9 GROSSLESIONSIN MICE FED5% RICE CULTUREOF Penicilliumviridicatum (TRIAL IVb) No. with hepatic lesions Interval WI -~-__-~~ 1 to4 6to 12 16 to 24

No. mice examined

No. mice anemic

Focal necrosis and hemorrhage

Clay color

Cystic bile ducts

45 61 39

0 22 10

0 18 5

0 7 9

145

32

3 10 7 20

23

16

Microscopic Pathology Trial I (determination of LD50). In 1 of the 6 mice fed the 2.5% fungal diet, fatty

change occurred in the perilobular hepatocytes. Variation in size and shape of the nuclei of hepatocytes was a feature of the livers of the other 5 mice. The nuclei were hyperchromatic and more of the cells were binucleate as compared to controls. The changes in the livers of the mice fed the 5 y0 fungal diet were essentially the same as described above. No renal lesions were observed in either group. The hepatic lesions of 4 of the 6 mice of the 7.5 % group were similar and included disseminated foci of necrosis of hepatocytes (Fig. 2) containing in some foci “ghosts” of portal triads (Fig. 3). Such lesions were accompanied by periductal edema and fibrosis

Penicillium viridicatum

MYCOTOXICOSIS

369

FIG. 3. Focus of necrosis in periportal area of liver from mouse fed 7.5 % P.viridicatum diet for 2 wk. “Ghosts” of portal structures are illustrated. Hematoxylin and eosin. x56. FIG. 4. Dilated tubules of kidney of mouse fed 7.5 % P. viridicatum for 2 wk contains protein and bile pigment casts, Hematoxylin and eosin. x56.

370

BUDIARSO,

CARLTON,

AND

TUITE

and hypertrophy of the epithelium of the bile ducts. A few foci of obliterative cholangitis, necrosis of the gallbladder and mitotic figures in the epithelium of bile ducts occurred in some livers. The renal lesions of these 4 mice consisted of necrosis of a variable number of tubules, dilatation of tubules and presence of proteinaceous casts in some tubules (Fig. 4). Bile pigment in and calcification of cortical tubules occurred in some kidneys. With the exception of 1 mouse, the hepatic lesions observed in the mice fed the 10% fungal diet were similar to those described above, but were more severe. In addition to the renal lesions described for the preceding group, evidence of regeneration occurred in the tubular epithelium. The hepatic lesions of the mice of groups fed the diets containing 15-30 % concentrations of the rice culture were similar in character but variable in severity. The changes included numerous foci of necrosis in the liver (as Fig. 2) and of some larger intra- and extrahepatic bile ducts. Hemorrhage and mild cellular infiltration about the necrotic foci occasionally occurred. Obliterative cholangitis and hypertrophy of the epithelium of some bile ducts occurred in some livers. Two mice from the 15 % group had no renal lesions, but in 4 mice, necrosis of renal tubules was accompanied by regenerative changes and hypertrophy of the epithelium ofother tubules. A few bile pigment and proteinaceous casts occurred in some tubules. Two mice of the 20 % group and 3 of the 25 % group had no renal lesions. In the other mice of these 2 groups, multiple foci of necrosis of cortical tubules occurred along with varying amounts of a proteinaceous material in some tubules. The renal lesions in the mice fed the 30 % fungal diet varied from mouse to mouse, but included foci of hypertrophied epithelial cells and extensive tubular necrosis accompanied by dilatation of tubules with numerous proteinaceous and bile pigment casts (as Fig. 4). Trial II (age susceptibility). The microscopic changes in the livers of the 3- and 4-wkold mice were similar to those described in mice fed the 15 to 30 % fungal diets of trial I. The changes in the livers of 5- and 6-wk-old groups included, in addition to lesions described for younger mice, hypertrophy and hyperplasia of the epithelium of both intra- and extrahepatic bile ducts. Occasionally these bile ducts were also dilated. In some livers the reactions included periductal edema and fibrosis. The renal lesions of the 3- through 6-wk-old mice were essentially similar. The changes varied from mild to extensive tubular necrosis with proteinaceous casts in some tubules. Bile pigment casts and hypertrophied tubules were noted in some kidneys of the 5- and 6-wk-old mice. The liver lesions of mice of ages 8, 10, 14, and 18 wk, varied in severity, but the predominant changes were essentially similar and were centered in the biliary system. The alterations varied from reactive changes in the epithelium of bile ducts to hypertrophy and hyperplasia of the epithelial cells, usually accompanied by cystic dilatation (Fig. 5). In many livers, proliferation of bile ducts (Fig. 6) and bile duct cells occurred. The epithelium of some bile ducts was necrotic, and the lumen of other bile ducts was constricted by proliferated fibrous connective tissue. The extrahepatic bile ducts were surrounded by increased amounts of fibrous connective tissue which was infiltrated with a mixed population of inflammatory cells. Hypertrophy and hyperlasia of the epithelium

Penicillium viridicatum

MYCOTOXICOSIS

371

FIG. 5. Mouse fed 50 % P. viridicatum diet for 2 wk. The epithelium of this dilated bile duct is hypertrophied and hyperplastic. The connective tissue is increased around the duct and contains a few inflammatory cells. Hematoxylin and eosin. x560.

FIG. 6. Mouse fed 50% P. viridicatum diet for 2 wk. Numerous patent proliferated bile ducts are surrounded by admixture of bile ductule cells, fibroblasts and inflammatory cells. Hematoxylin and eosin. x 140.

372

BUDIARSO, CARLTON, AND TUITE

FIG. 7. Hepatocyte with an abnormal nucleus is illustrated and is a feature of the liver lesions of mice fed P. uiridicatum. Hematoxylin and eosin. Y 1400. FIG. 8. Hepatocytes with multilobed nuclei are observed in hypertrophied 50% P. uiridicutum for 2 wk. Hematoxylin and eosin. x 1400.

hepatocytes of mice fed

Penicillium viridicatum

MYCOTOXICOS~S

373

were accompanied by focal necrosis and mucous metaplasia. The lumens were partially filled with protein material mixed with some cellular debris. Some of the intrahepatic bile ducts were so dilated that they displaced the hepatic parenchyma. Nodules of fibroblasts replaced foci of necrotic hepatocytes. The changes in the hepatocytes included variation in size and shape of the nuclei which were enlarged, hyperchromatic and contained prominent nucleoli. Bizarre nuclei occurred (Fig. 7) as well as hepatic cells with multiple nuclei or with multilobed nucleus (Fig. 8). Mitotic figures were more common in the epithelial cells of bile ducts of older mice (Fig. 9). Renal lesions occurred in most of the mice of the last 4 age groups. The severity varied from mouse to mouse of the same group and among the various groups. Tubular necrosis was either focal or extensive and was accompanied by proteinaceous casts. Occasionally tubular regeneration and dilatation occurred along with variable numbers of calcific foci. Trial III (life span). Microscopic lesions were consistently found in the livers of mice which died from week 2 to the termination of the experiment. The early changes were similar to those described in livers of mice fed the fungal diets of trial I. In older mice, the liver lesions, such as changes in the biliary system and the hepatocytes, were similar to those described in mice for ages 8 through 18 wk old in trial II. The changes in the liver in the intermediate stage were essentially a mixture of early and late lesions. The epithelium of a grossly distended common bile duct varied from low cuboidal to 2-layered pseudostratified columnar epithelium in which many cells were vacuolated. The epithelial cells of the submucosal glands had either undergone mucous metaplasia or were flattened. The renal lesions were similar to those in mice of trials I and II. Trial IV(pathogenesis ofhepatorenal damage). The kinds of hepatic and renal lesions and the number of mice affected at the different periods are given in Tables 7 and 10. The changes in the livers followed a fairly orderly fashion, although varying in severity. The early alterations began as either perilobular hydropic or fatty degeneration accompanied by slight hypertrophy of the bile duct epithelium. These changes were followed by a necrotizingcholangitis and disseminated foci of coagulative necrosis of hepatocytes (as Fig. 2) with or without “ghosts” of portal structures (as Fig. 3). Necrotic areas often interconnected. Occasionally hemorrhage occurred into the areas of necrossis. In the livers with more chronic lesions, proliferated bile duct cells occurred along the interlobular septa and were accompanied by patent bile ducts (as in Fig. 6). Also, hypertrophy and hyperplasia of the intra- and extrahepatic bile ducts were usually accompanied by periductal fibrosis (Fig, 10). These changes were followed by dilatation of the ducts (Fig. IO), and the gradual expansion caused pressure atrophy of the surrounding hepatic parenchyma. Sacculations and accessory lobulations of the mucosa of the bile ducts occurred (Fig. 11). These changes were found in both intrahepatic and extrahepatic (Fig. 12) bile ducts as well as the common bile duct. In more chronically fed mice, mucous metaplasia of the epithelium of the common bile duct occurred. The bizarre changes in the hepatocytes were similar to those described in trial II. The renal lesions were similar to those described in kidneys of the preceding trials. Peritonitis found at necropsy was confirmed microscopically and was an extension of a pancreatitis which apparently spread to the pancreas from the affected common bile duct.

374

BUDIARSO, CARLTON. AND TUITE

FIG. 9. Liver of mouse fed 50% P. ciridicatum diet for 2 wk. Hyperplasia of bile duct epithelium is accompanied by mitotic figures. These hypertrophied and hyperplastic ducts are surrounded by proliferated fibrous connective tissue. Hematoxylin and eosin. x350.

FIG. 10. Liver of mouse fed 10% P. ~ividirarum diet for 6 wk. Enlarged bile ducts with small sacculations are cuffed by proliferated fibrous connective tissue. Hematoxylin and eosin. ~56.

LESIONS

45

61

39 145

6to12

16 to 24

AND KIDNEY

OF MICE

15 ss

25

34

9

3

16

12 22

12

5

10

0

Focal Bile Duct cell necrosis pigment proliferation

No. with hepatic lesions

IN LIVER

7

17

1

No. mice Fatty examined degeneration

1 to4

Interval of feeding Wd

MICROSCOPIC

5 % RICE

10 CULTURE

OF

5

11 <

1

10

ii

0

1

SI

4

24

9

16

7

7

2

6 33

22

5

Proteinaceous casts

1%)

7 19

10

2

17

9

7

1

Bile pigment Tubular casts regeneration

No. with renal lesions

Penicilliumviridicatum (TRIAL

Focal Bile duct Mucous tubular Tubular dilatation metaplasia necrosis hypertrophy

FED

TABLE

34 1 $2 8 8 t;

s c F* -. G% “a is 3 sz

P

376

BUDIARSO, CARLTON, AND TUITE

FIG. 11. Liver of mouse fed 5 % P. viridicarum diet for 14 wk. Markedly dilated intrahepatic bile ducts have sacculations and accessory ducts in the proliferated fibrous connective tissue. Hematoxylin and eosin. x35. FIG. 12. Liver of mouse fed 5 % P. viridicatum diet for 14 wk. Markedly dilated extrahepatic duct with sacculations and accessory ducts in surrounding proliferated fibrous connective tissue. Hematoxylin and eosin x35.

Penicillium viridicatum

MYCOTOXICOSIS

377

DISCUSSION In previous reports, the concentrations of fungal cultures fed were great and the mice died early in the feeding period. Thus, the approximate LD50 of the cultures of P. viridicatum was not determined. In this report an attempt was made to determine the LD50-14 days. Several methods of analysis and estimation of the LD50 were considered (Bliss, 1935; Litchfield and Wilcoxon, 1949; Reed and Muench, 1938), but none was considered suitable. Therefore, the LD50 was determined by estimation and supported by the presence of hepatic lesions. Deaths occurred in the first week in groups fed the 15 % or higher concentration of rice culture and in week 2 in groups fed the 10 % or 7.5 y0 fungal diets. The results of this study indicated that while 1%wk-old mice were not killed by a 50 % rice culture diet signs of mycotoxicosis were produced, thus older mice were not entirely resistant. Mice of every age group had obvious lesions of P. viridicatum toxicosis. However, there were rather different tissue changes in the mice from ages of 3 through 6 wk than in the mice of ages 8 through 18 wk. In the former groups the hepatic lesions were not exudative or proliferative and consisted, in general, of hepatic parenchyma necrosis and necrosis of bile duct and associated portal structures. The changes in the older group included, in addition to the lesions described for the younger groups, proliferation of bile duct cells and of bile ducts with hypertrophy and hyperplasia of bile duct epithelium. Formation of inflammatory granulation tissue and cellular exudation around the intra- and extrahepatic bile ducts was more frequent and abundant. Lesions in mice of all trials occurred both in the biliary system and in the liver parenchyma. The latter lesions were, perhaps, the consequence of biliary damage because foci of necrosis were always located in fairly close proximity to interlobular bile ducts and presumably developed about the canals of Herring-that portion of the biliary system joining the bile canaliculi with the smallest intrahepatic bile ducts (cholangioles). Bile may in itself result in necrosis of liver cells as such lesions have been produced by experimental ligation of the common bile ducts in rats (Cameron and Oakley, 1932; Cameron, 1953; Cameron et al., 1957) and in sheep (Leaver and Christie, 1965; Quin, 1933). Thus the hepatic cell changes in the mice of this study may represent reactions to a mixture of bile and toxin(s). Another interesting aspect of the hepatic pathology was the many hypertrophied liver cells, usually located close to portal areas. The nuclei of these cells were large, deeply basophilic and contained coarse granular chromatin, and often the shape was bizarre. “Giant” liver cells containing 4 or more nuclei or multilobulated nuclei were observed and may represent either amitotic cell division or a precancerous change as described in the rats poisoned by cyasin (Plum, 1952) and in aflatoxicosis of guinea pigs (Butler and Barnes, 1964). An interesting observation in this study was that the number of mice with acute renal lesions declined as the feeding time progressed (especially in test mice of trial IV). Possibly the kidneys became more resistant to the toxins as the mice grew older, or the most susceptible mice died leaving a population more resistant to renal damage, or the liver more effectively detoxified the toxins, or all 3 mechanisms were operative. Variation in toxicity with age has been reported in rats in aflatoxicosis (Wogan, 1965) and in cyasin poisoning (Campbell et al., 1966).

378

BUDIARSO, CARLTON, AND TUITE

Hypertrophy of epithelial cells was observed in the kidneys of ducklings and rats fed aflatoxin-contamined peanut meal (Newberne et al., 1964; Newberne, 1965), in mice fed corn cultures of P. cyclopium (Carlton and Tuite, 1970b), and in mice injected SC with filtrates of Czapek cultures of P. cyclopium, P. expansum, and P. roqueforti (Kurata et al., 1968). It seemsunlikely that the hepatorenal lesionsin the test mice of our experiment were induced by aflatoxins, becausemice are very resistant to the toxic effects of aflatoxins (Platonow, 1964)and none of the isolates of P. viridicatum tested by chromatography by Mislivec et al. (1968) produced aflatoxins. A number of antibiotics and other metabolites have been isolated from cultures of P. viridicatum including viridicatin, citrinin, oxalic acid, and ochratoxin A. None of thesehas been establishedasthe causeof the toxicity of the isolates used in this study. The PCV values of sometest mice in thesetrials were consistently lower than those of the control groups. The anemia was apparently due to direct action of the toxic fungal metabolites on the hematopoietic tissues,but bone marrow was not studied. Hemolysis apparently did not occur because evidence of destruction of erythrocytes was not found in the spleenor other viscera, and hemoglobinuria was not observed. Anemia has been described in animals poisoned with Stachybotrys atra, in man affected with alimentary toxic aleukia, and in birds poisoned by mycotoxins (Forgacs and Carll, 1962). In most of these toxicoses, destruction of the bone marrow has been described. REFERENCES BLISS,C. I. (1935).The calculationof the dosage-mortalitycurve. Ann. Appl. Biol. 22, 134-167. BUDIARSO, I. T., CARLTON, W. W., andTUITE,J. (1971).Phototoxic syndromeinducedin mice by rice culturesof Penicillium viridicatum and exposureto sunlight.Pathol. Vet. in press. BUTLER,W. H. (1969).Aflatoxicosis in laboratory animals.In: AJlatoxin (L. A. Goldblatt, ed.), pp. 223-236.M.I.T. Press,Cambridge,Massachusetts. BUTLER, W. H., andBARNES, J. M. (1964).Toxic effectsof groundnut mealcontainingaflatoxin to rats and guineapigs. Brit. J. Cancer 17, 699-710. CAMERON, G. R. (1953). Liver regenerationand biliary obstruction. J. Pathol. Bacterial. 41, 283-288.

CAMERON, G. R., and OAKLEY,D. L. (1932).Ligation of the common bile duct. J. Puthol. Bacterial.

35, 769-798.

CAMERON, G. R., GRIFFITHS, D. B., and HASAN,M. S. (1957).Liver necrosisfollowing obstruction of the common bile duct. J. Pathol. Bacterial. 74, 327-333. CAMPBELL, M. E., MICHELSEN, O., YANG, M. G., LAQUER,G. L., and KERESZTESY, J. C. (1966). Effects of strain, age and diet on the responseof rats to the ingestionof Cycas cirinalis. J. N&r. 88, 115-124. CARLTON, W. W., and TUITE,J. F. (1970a).Mycotoxicosis inducedin guineapigsand rats by corn culturesof Penicillium viridicatum. Toxicol. Appl. Pharmacol. 16, 345-361. CARLTON, W. W., and TUITE,J. F. (1970b).Toxic effectsin miceof corn culturesof Penicillium cyclopium and Penicillium frequentans. Toxicol. Appl. Pharmacol. 17, 289-290. CARLTON, W. W., TIJITE,J., and MISLIVEC,P. (1968).Investigationsof the toxic effectsin mice of certain speciesof Penicillium. Toxicol. Appl. Pharmacol. 13, 372-387. CARLTON, W. W., TUITE,J. F., and MISLIVEC,P. (1970).Pathology of the toxicosisproducedin mice by corn culturesof Penicillium viridicatum Proc. UJNR Conferenceon Toxic Microorganisms,Honolulu, Hawaii, pp. 94-106. FORGACS, J., and CARLL,W. T. (1962).Mycotoxicoses. Advan. Vet. Sci. 7, 273-382. KURATA,H., UDAGAWA,S. C., ICHINOE,M., KAWASAKI,Y., TAZAWA, M., TANABE,H., and ODUDAIRA,M. (1968). Studies on the population of toxigenic fungi in foodstuffs. VI. Histopathologic changesin mice causedby toxic metabolitesof fungal isolated from domesticrice. J. Food Hyg. Sot. Jap. 9, 385-394.

Penicillium viridicatum

MYCOTOXICOSIS

379

D. D., and CHRISTIE, G. S. (1965). Pathological changes following experimental obstruction of the common bile duct, hepatic bile duct and hepatic artery in sheep and their relation to facial eczema. Aust. J. Exp. Biol. Med. Sci. 43, 1-16. LITCHFIELD, J. T., and WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. 96, 99-l 13. MISLIVEC, P. B., HUNTER, J. H., and TUITE, J. F. (1968). Assay for aflatoxin production by the genera Aspergillus and Penicillium. Apple.Microbial. 16, 1053-1055. NEWBERNE, P. M., CARLTON, W. W., andWOGAN,G. N. (1964).Hepatomasin rats andhepatorenalinjury in ducklingsfed peanutmealor AspergillusJ7avus extract. Pathol. Vet. 1,105-l 32. NEWBERNE, P. M. (1965).Carcinogenicityof aflatoxin contaminatedpeanutmeals,In: Wogan, Mycotoxins in Foodstufi (G. N. Wogan, ed.), pp. 187-203. MIT Press, Cambridge, Massachusetts. PLATONOW, N. (1964).Effect of prolongedfeedingof toxic ground nut mealin mice. Vet. Rec. 75, 589-590. PLUM, C. M. (1952). Experimentally produced liver fibrosis with red blood macrocytosisin rabbits. Acta Pathol. Microbial. &and. 31, 90-97. QUIN, F. I. (1933).The effect of surgicalobstruction of the normal bile flow. Onderstqoort J. Vet. Sci. Anim. ind. 1, 505-526. REED, L. G., and MUENCH,H. (1938).A simplemethodof estimatingfifty percentend-points. Amer. J. Hyg. 27, 493-497. WOGAN, G. N. (1965).Chemicalnature and biologicaleffectsof the aflatoxins. Bacterial. Rev. 30,460-470. LEAVER,