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Penicillium viridicatum mycotoxicosis in the rat. IV. Attempts to modify the tissue responses
Fd Cos;net.Toxi~ol.Vol, 12. pp. 331 340, Pergamon Press 1974. Printed in Great Britain
P E N I C I L L I U M V I R I D I C A T U M M Y C O T O X I C O S I S IN THE RAT. IV. A T T E M P T S TO M O D I F Y THE TISSUE R E S P O N S E S M. D. MCCRACKEN and W. W. CARLTON Department o] Veterinary M icrobiolo~ly and Patholo,qy, School of Veterinary Science and Medicim':
and J.
TU1TE
Department q['Botan) and Phmt Patholoqy, School ql'Agriculture, Purdue Unieersity, West lx{/hyette, Indiana 47907, U S A (Received 24 January 1974) Abstract -Male rats were fed a rice culture o f Penicillium viridicatum a t a dietary concentration of 50°~ and were administered tripelcnnamine hydrochloride, syrosingopine or prednisolone. Other groups fed the fungal diet at 25% dietary concentration were administered prednisolone or tetracycline. One group fed this diet was castrated prior to feeding. Tripelennamine, syrosingopine and tetracycline were not effective in reducing the incidence or severity of the ocular and scrotal lesions that developed in the test rats. Prednisolone was somewhat effective in reducing the incidence and severity of scrotal lesions and decreased the severity of the ocular lesions. Tetracycline, but not tripelennamine or syrosingopine, reduced the severity of the hepatic lesions. Castration effectively prevented the scrotal lesions, but did not altcr those of the eye and liver.
INTRODUCTION
Male rats fed grain cultures or fungal mats of Penicillium vMdicatum developed lesions of the eye (McCracken, Carlton & Tuite, 1974a), and of the scrotum and liver (Carlton & Tuite, 1970; McCracken, Carlton & Tuite, 1974b, c). Because the ocular, and especially the scrotal, lesions were markedly inflammatory in character, studies were completed to ascertain how the development of the lesions was affected by an antihistamine, tripelennamine, the serotonin antagonist, syrosingopine, the corticosteroid, prednisolone, and the antibiotic, tetracycline. In addition, one group of rats was castrated and fed the fungal diet. The results of these studies are presented in this report. EXPERIMENTAL
Experimental animals. Male rats were purchased at a weight of about 250,300 g and were housed and fed as previously described (McCracken et al. 1974a). Rats were examined daily and the incidence and character of ocular and scrotal alterations were recorded. Orchidectomy was performed under ether anaesthesia on one group of rats in Trial II and the rats were allowed 2 wk for recovery. Fungal diets. Rice cultures of P. vMdicatum were prepared as previously described and mixed with a purified diet (McCracken ~'t o/. 1974a). Experimental design and conduct. In Trial I, male rats were fed the fungal grain cultures at a dietary concentration of 50'~f, and groups of these rats were given either tripelennamine 331
332
M . D . MCCRACKt;N, W. W. CARLTON and J. TUITE
(Pyribenzamine, supplied by Ciba Pharmaceutical Co., Summit, N.J.) in a daily sc dose of 5 mg/kg body weight, syrosingopine (Singoserp. supplied by Ciba Pharmaceutical Co.) in a daily sc dose of 25 mg/kg or prednisolone in a daily sc dose of 5 mg/kg. Rats in Trial II were fed the fungal grain culturc at a dietary concentration of 25!'; either alone or together with a daily sc dose of 5 mg prednisolone/kg or a daily intramuscular dose of 5 mg tetracycline/kg. The treatments were continued for up to 6 wk. Autopsy. The eyes, scrotum, liver, stomach and kidneys were removed at autopsy. Eyes were processed as previously described (McCracken et al. 1974a). The other tissues were fixed in 10}~ buffered formalin, processed for paralfin sectioning and stained with haematoxylin and eosin for histopathological examination.
RESULTS
Trial 1 Clinical signs. Signs of toxicity in the groups of rats fed the fungal diet were similar to those previously described and included loss of activity, roughened hair, anorexia, diarrhoea and progressive loss of body weight (Table 1). Administration of tripelennamine hydrochloride, syrosingopine or prednisolonc had no effect on the incidence or severity of the clinical signs. Mortality was high in the test groups given tripelennamine or syrosingopine. Table I. Meall body weights aml im'idencc qf ocuhtr lesions in raz.~.[ed a culture o[ P. viridicatum and treated with tripeh, nnanline, s l'ro.~inHofffne or prednisohme (Trial I) Mean body weight (g) at wk Experimental group
0
1
4
Purified diet~501}~iPV~ Tripelennamine f§ o/ 50,0 PV+4- + tripelennamine§ 50"; PV{ + syrosingopine§ Prednisolonet§ 50'}; PV:{ + prednisolone§
363 367 331 334 339 364 357
387 310 344 283 280 370 306
455 208 407 236 200 405 190
Total gain (g) 92 - 159 76 -98 139 4l -167
Mortality*
Gross ocular lesions*
0/10 2,,"15 0/5 7/'15 10/15 1/10 6/15
0 18 30/30 0/10 30/30 28/'30 0,/'24 30/30
*Mortality is expressed as no. dead/no, in group and gross ocular lesions as no. of eyes affected/no examined. +Control groups. ++Rice culture of P. viridicamm fed at a level of 50°~; in the diet. ¢Tripelennaminc or prednisolone given in daily sc doses of 5 mg/kg body weight and s wosingopme in daily sc doses o1"25 mg/kg.
Ocular effects. Clinically detectable ocular lesions occurred in all rats fed only the fungal diet. Lesions, first noted after 7 10 days of feeding, were similar to those previously described (McCracken et al. 1974a), beginning as a slight haziness of the cornea and increasing in severity so that the cornea became opaque. Hypopyon and corneal vascularization occurred in severely affected eyes. Tripelennamine and syrosingopine had no apparent effect on the development or severity of the clinically detectable ocular changes (Table 1), but ocular disease appeared to be less severe in test rats given prednisolone. The initial microscopic ocular lesions in the test rats consisted of corneal oedema and hydropic degeneration of the corneal epithelium. In mildly affected eyes, accumulations of a mixture of inflammatory cells were present around the limbal vessels and within the
Penicillium viridicatum mycotoxicosis
333
substantia propria. In more severely affected eyes, diffuse interstitial keratitis was accompanied by iridocyclitis, hypopyon and corneal vascularization (Table 2). No difference in the incidence of corneal oedema was observed in test rats given prednisolone. However, the incidence of hypopyon and corneal vascularization was lower in this group than in that fed the fungal diet alone (Table 2). The incidence and severity of ocular lesions in the rats treated with tripelennamine or syrosingopine were comparable to those in the fungal control group (Table 2). F~ffects o~ scrotum. Gross scrotal lesions in test rats were similar to those previously described (McCracken et al. 1974b). Oedematous swelling of the scrotum began after 2 -3 wk of feeding and was followed by necrosis and ulceration of the scrotal epidermis. The administration of tripelennamine or syrosingopine had little observable effect on the incidence or severity of scrotal swelling and necrosis (Table 3). Scrotal necrosis followed sooner upon the initial swelling in test rats given tripelennamine. Scrotal oedema was observed in 7 of 15 rats treated with prednisolone but was severe in only one rat, and epidermal necrosis was observed in only two (Table 3). Microscopic scrotal lesions occurred in almost all test rats and consisted of a severe necrotizing cellulitis characterized by loci and bands of necrotic leucocytes in the epididymal connective tissue and scrotal fascia. Necrosis and ulceration were present in the overlying epidermis. Differences in microscopic lesions between rats given tripelennamine or syrosingopine and those fed the fungal diet alone were a lower incidence of testicular lesions and of rats with lesions in the epididymis (Table 3). Prednisolone decreased the incidence and severity of the scrotal lesions (Table 3); two of the 15 test rats given prednisolone had no lesions. Lesions of the epididymal fat and the scrotal fascia surrounding the tunicae were extremely variable, ranging from no lesions to very severe changes. Changes in the epididymal adipose tissue consisted of focal mononuclear cell accumulations and areas of necrosis small-to-moderate in size, Five rats had lesions of the epididymal fat, but there were no lesions outside the tunicae. Seven of these 15 rats had lesions of severe necrotizing cellulitis similar to that present in the rats given only the fungal diet. Necrosis and ulceration of the epidermis occurred in only three of the 15 prednisolone-treated test rats. Hepatic ehanyes. Gross hepatic lesions in test rats consisted of a variable number of yellow-to-white loci and often the smaller hepatic lobes were the more severely affected. There was no difference in the incidence or appearance of gross hepatic lesions among the various treatment groups (Table 4).
Microscopic hepatic changes in the test rats were characterized by a necrotizing cholangitis with pericholangitis, proliferation of the biliary epithelium and focal areas of hepatic cell necrosis, often adjacent to the bile ducts. Periductal fibrosis resulted in stenosis and obliteration of the bile ducts in the more severely affected livers. Neither the incidence nor severity of hepatic changes in rats given tripelennamine or syrosingopine differed significantly from those in the group fed the fungal diet alone (Table 4). Focal necrosis and periductal fibrosis of the liver were particularly severe in test rats given prednisolone. Necrotic loci were more numerous and larger in these rats, but bile-duct proliferation tended to be less severe. EfJ'ects on other or,qans. Lesions in other organs included gastric erosions, extra-scrotal skin necrosis and biliary pigment in the renal tubular epithelium. There were no apparent differences among the various treatment groups in the development of these lesions.
14 26 9 27 26 24 2~
Experimental group
l-h_lrilied diet* 50", P V I Tripclennamine*:~ 50", PV'~ + tripelennamine.+,. 50"i, PV+ + s_',rosingopinc + Prcdnisolor~c'::~: 500 L PV+ + prednisoJone~ 0 26 0 27
22 0 26
26 0 2S
Keralitis
0 25 0 27
Corneal ocdema
') 0 6
0 12 0 7
Cornea] vessels
20 0 12
0 I8 I) 13
h'itis
No. of eyes with m i c r o s c o p i c lesions
10 15 5 15 15 10 15
0 14 0 10 12 0 7
Sx~clling 0 12 0 9 10 0 2
Necrosis
(h-oss scroml lesions
0 13 0 15 14 0 7
(~ellulitis of scl-olu m
0 10 0 10 10 0 3
Fpidermal necrosis
,
4 0 6
0 6 0 2
Syncchia
9 0 9
0 17 0 S
H}popyon
0 13 {) 2 I 0 0
Tcsticular lesions
0 15 0 7 7 0 5
I~pididvmal lesions
0 11 0 6 9 0 7
Penile lesions
~FY(~.~l'll¢lUpillt, (~" prudJlisol(mc (Ti'lal l)
M i c r o s c o p i c findings
No. of a n i m a l s affected b \
oHd lt'd¢lte'd Ivil/l lrlp(,l(,llllal~liHc.
* ( o n t r o l groups. +Rice culture of P. Hridicatum fed at a level of 50",, m the dieT. !Tripe er +t n nc or prcdn sol< l e g xe 1 in daitx sc doses c,15 mg kg body \xcight and ";5 to~,ingopit'~c in dailx sc doses of 25 mg kg.
Purilied dice :' 50°11 PVi" Tripelennamine*'i50°i, PV'I" + tripelennaminc + 50";. PV+ + syrosingopine} P r e d n i s o l o n e *+ 50°ii PV+ + prednisolone++
No. of *als cx:u~lincd
(;t'¢1~.~ d l i d /Hi(l'(l',('O[~/(' StTtU ii [(',SiO/L~, i'll t'tll~ ~'l [ tl tldtltI'(' 0,1 P, \ i r i d i c a l u l D
Expcrilnental gro ~t~
"]['til31e .)
*Control grotips. ";'Nice culture of P. cu'idic~,,rmn Ibd at a < \~.t ~ " 50" in Ihc diet. i:::lripclcnnamhlcor prcdnisoh~m.',.'ixcn in,!~ , ,L ,h,,.:, ,,I 7 m~ kg h,M\ v, c M h l ; m d 5\lo>n~<,pu~c hldail', ~,cJ~,<',,fi25 m# k~ ~()nl 3 24 <:yes cx;.lmlncd.
No. of eves examined microscopicall 3
'l able 2. O,:.td,w /c:,i<;ns hz ~'a~s /bd a c~drt,,., 0 / P . ~iridicatum a~d rr(,ared ~il/l rHp,.'i
~2
y
t'-
h
~:
i.
+
1I 0 7
15
10
15
15 IO
0 9 (t
5 15 5
Gross lesions
3
12 0
14
0 I4 0
Pericholangitis
9
15 0
14
0 15 0
Bile-duct proliferation
12
15 0
14
0 14 0
Pcriduclal tibrosis
No. of animals affected by
*Control groups. fRice culture ofP. riridicatuut ted at a level of _{) i, m tile diet. ~Tripelennamine or prcdnisolone ~iven m daily sc doses of 5 m g k g hod) weight and s xrosingopinc in dail\ sc doses of 25 mgkg.
syrosingopine$ Prednisolone$ 5()";i PVf + prednisolone{
.S0 o5~ PV
Purified diet* 50",i PVi Tripelennamine*++ 50",, PVt + tripelemmmine++
Experimental group
No. of rats examined
12
15 0
14
0 14 0
Necrotizing cholangitis
12
13 0
13
0 13 0
Scrotal necrosis
Table 4. Gross and microscopic hepatic lesi(ms in raL';./ed a cullure o/'P. viridicatum ~md treated ~,'ilh trilwh, nmmline, s),rosin#opim, or ~rednisolone (Trial I)
'Ji
©
©
@
~2
336
M . D . M('(I',A('Kt N. W. W. CAP,L](IN and J. Tt,I iI
Trial II Signs of toxicity in rats fed the 251}o fungal diet were minimal during the first 3 wk. During 4-6 wk of feeding, signs of toxicity appeared and were similar to those observed in Trial I. Test rats progressively lost weight and those given prednisolone showed the greatest reduction. Castrated rats fed the fungal diet maintained their body weights (Table 5). Table 5. Resl~o#~se ol iulacl aJ~d castrated rats to a cult~t'e 0/P. viridicatum amt to tr~'almctlt with IwedHisohme m" wtracyclille ( 77"ial II1 Mean body weight tit wk Experimental group Purilied diet+ 25<'~; PV{ Prednisolone+ 257. PV{ + prednisolone,~ Tetracycline't 25"~; PV + + tetracycline§ Castrated rats+ 251',, PV + + castration
0
1
5
Total gain (g)
362 367 357 383 363 362 355 341
403 374 367 366 4(ll 370 379 364
422 323 355 286 461 321 417 337
60 44 2 - 97 9", 41 62 - 4
Mortality* 010 0"15 0'5 I 15 I 5 0.15 0'5 015
*No. dead/no, in group. +Control groups. +Rice culture ofP. ~:iridicatum fed at a level ol" 25<'; in the dict. ~Given in daily doses of 5 mg/kg body wright, prednisolonc sc and tetracycline intramuscularly,
Ocular effects. Clinical ocular lesions were similar to those described in Trial I. Castration and treatment with tetracycline had no apparent effect on the incidence or severity of these lesions, but the lesions appeared less severe in test rats given prednisolone. Microscopic ocular lesions were present in most eyes of test rats and were similar to those of Trial I (Table 6). Castration or treatment with tetracycline had no apparent effect on the incidence of ocular lesions. The incidence of iridocyclitis, synechia and hypopyon was less in the test rats given prednisolone (Table 6). EfJbcts on scrotum. Scrotal swelling took a longer time to develop in rats fed the 25'~; fungal culture, usually occurring after 3 5 wk of feeding. Scrotal epidermal necrosis developed within 2 11 days of the initial swelling. The pattern of necrosis was similar to that seen in Trial I. starting approximately l ' 0 c m distal to the prepuce and progressively spreading over most of the scrotum. The incidence of scrotal swelling and epidermal necrosis was less in test rats given prednisolone (Table 7), but treatment with tetracycline had no effect on the incidence or severity of the scrotal alterations. Swelling and necrosis of the scrotum was observed in only three of 15 castrated test rats (Table 7) and. in these rats, the lesions were associated with remnants of the epididymal fat incompletely removed at surgery. Microscopic scrotal lesions in test rats were similar to those observed in Trial 1 and consisted of necrotizing cellulitis of the scrotal fascia with necrosis and ulceration of the epidermis. Diffuse orchitis was observed in about half of thc rats fed the fungal diet alone or in association with tetracycline treatment. Orchitis was characterized by oedema, marked infiltration of a mixture of inflammatory cells and necrosis and calcification of the seminiferous tubules. The incidence of microscopic scrotal lesions was greatly decreascd in the castrated test rats (Table 7) as 11 of 15 had very mild or no lesions. Lesions were generally confined
[9
18
0 24
Corneal oedema
22
18
0 29
Keratitis
16
12
0 18
Vascularization
13
7
0 18
Iritis
9
4
0 13
Synechia
Hypopyon
½
28
21
21
*No. of eyes affected/no, examined. ?Including tetracycline, prednisolone and castrated controls. ~:Rice culture ofP. viridicatum fed at a level of 25% in the diet. §Given in daily doses of 5 mg/kg body weight, prednisolone sc and tetracycline intramuscularly.
26/30
19
14
15
7
O
23
20
0 29
Microscopic lesions
-g
24
22
30 30
No. of eyes examined microscopically
castration
30/30
28/'30
0/30 29/30
Incidence of gross ocular lesions
25% Pv~; +
tetracycline§
25% Pv:~ +
prednisolone§
25% pv~ +
Controlt 25~o PV~
Experimental group
Table 6. Ocular lesions in intact and castrated rats.led a culture ofP. viridicatum and treated with prednisolone or tetracycline (~fi'ial lli
338
M . D . MCCRA('Kt N, W. W, CARl I'ON and .1. T t i ri
Table 7. Microscopic lesions of the scrotum and male relwoduetice organs in illtucl alTd e~lslruted #'tlls ted u eztllltre o/'P. viridicatum and in rats fi_,d the limHal diet and treated with prednisohme or tetracycline (Trial 111 No. of animals aflL'ctcd by' Experimental group Control* 25}o PV+ 253~; PVt + prednisolone++
No. of rats examined
Scrotal cellulitis
Epidermal necrosis
Testicular lesions
Epididymal lesions
Penile lesions
6 15
0 1I
0 S
0 7
0 11
0 0
15
?
6
1
g
~
15
13
7
,',;
10
(I
15
5
3~
257,; Pv+ + tetracycline++ 257o PV* + castration
0
*Anhnals given purified diet. +Rice culture of P. uiridicatum fed at a levcl of 25'~ii in the diet. ~.Givcn in daily' doses of 5 mg/kg bod 3 weight, prcdnisolonc sc alnd telracxclinc inlramuscularl 5. ~Necrosis usually associated with suture abscesses.
to the adipose connective tissue about the cremaster muscle and consisted of infiltration of lymphocytes and numerous pigment-laden macrophagcs. In three rats, the lesions were more severe, and exudate and necrosis was present around the suture material. No lesions were present in the scrotal epidermis in 12 of the 15 castrated test rats. Small loci of necrosis were confined to the distal portion of the scrotum in one rat. Necrosis and ulceration of the epidermis occurred in two rats, with more severe lesions in the subcutis. Test rats given prednisolone showed a slight reduction in the incidence of microscopic scrotal lesions compared with those given the fungal diet alone and testicular lesions were present in only one rat (Table 7). Treatment with tetracycline had no apparent effect on the incidence or severity of microscopic scrotal lesions. Hepatic changes. Gross hepatic changes in the test rats were usually less severe than in Trial I. Mild hepatic changes included mottling, varying from dark red or pale tan to yellow, with occasional small red loci. Yellow foot. 1 2 mm in diameter, were observed in some rats of all the groups given the fungal diet. The most severe gross changes were observed in the livers of test rats given prcdnisolonc. Microscopic hepatic changes in the test rats consisted of necrotizing cholangitis, pericholangitis, periductal fibrosis and bile-duct proliferation, lesions similar to those observed in Trial I. The incidence of focal necrosis of hcpatocytes was low in all the fungal-diet groups except in the group given prednisolone (Table 8). Hepatic lesions appeared slightly less severe in test rats given tetracycline, as the incidence of necrotizing cholangitis was reduced and focal necrosis was not observed. Castration and treatment with prednisolone had no apFarent effect on the severity of the hepatic lesions (Table 8t. DIS('USSION
The observation that treatment with an antihistamine or a catecholamine depressor had no apparent protective effect against the scrotal and ocular lesions described in this study suggests that histamine and serotonin may not play a significant or major role in the pathogenesis of these lesions. However, because only a single dose level was given, it is possible that effective blood and tissue levels of the drugs were not achieved. In the scrotum, the lesions begin in the perivascular areas in the adipose connective tissue and at an early stage
Penicillium eiridicatum mycotoxicosis
339
Table 8. Microscopic hepatic lesions in intact and castrated rats ]ed u culture of P. viridicatum and treated with prednisolone or tetracycline
Experimental group Controls* 25°4, PV+ 25°,£ PV + prednisolone + 25°; PV + tetracycline{ 25~;;, PV + castration
No. of animals affected by Bile-duct Periductal Necrotizing proliferation fibrosis cholangitis
No. of rats examined
Pericholangitis
6 15
0 10
0 11
0 12
0 10
0 3
15
6
9
8
7
9
15
8
12
9
4
0
15
10
13
14
11
1
Focal necrosis
*Animals given purified diet. ?Rice culture of P. viridicatum fed at a level of 25",i; in the diet. §Gix.cn in daily, doses of 5 mg,'kg bod}. weight, prednisolone sc and tetracycline intramuscularly.
are largely confined to these areas, indicating that the toxin(s) may have a specific action especially on the vessels of loose connective tissue. Because of the established role of histamine and serotonin in increasing vascular permeability, the initial scrotal lesions could have originated through a mechanism involving these substances. The later lesion of epidermal necrosis appeared to be secondary to ischaemia. Prednisolone decreased the incidence and reduced the severity of the scrotal lesions and decreased the incidence of some ocular lesions, but not the incidence of corneal oedema. Prednisolone can influence several phases of the inflammatory reaction and the particular phase was not ascertained by the methods of this study, but significant anti-inflammatory properties of glucocorticoids are the effects on vascular permeability and stabilization of lysosomal membranes (Weissmann & Thomas, 1964). Hepatic lesions were generally more severe in prednisolone-treated test rats. The reason for this is unknown, but pathogenic mechanisms of lesion induction apparently differ from organ to organ. It is possible that prednisolone-induced 'stress' activated a latent bacterial infection, as the lesions in some of the livers resembled those produced by Corynebacterium kutscheri (LeMaistre & Tompsett, 1952). An extensive literature points to damage to Descemet's endothelium as most significant to the development of corneal oedema. Our observations are consistent with these data, as oedema preceded any other ocular change. The hepatic lesions appear to begin with damage to the biliary epithelium followed by secondary inflammatory and reparative changes. Because neither the ocular nor hepatic lesions appear to be initially vascular, it would not be unexpected for an antihistamine, a 5-hydroxytryptamine antagonist or a corticosteroid to be ineffective in preventing their development. Prednisolone did appear to reduce the more purely inflammatory ocular lesions, such as iridocyclitis and corneal vascularization. Castration appeared to inhibit the development of scrotal lesions, as alterations were either absent or were very mild and confined to adipose connective-tissue remnants. Further experiments would be necessary to determine whether the protective effects were due only to the removal of androgen secretion. Testosterone is known to increase the blood supply to and stimulate the growth of male accessory organs, including the scrotum (Zarrow, Yockim and McCarthy, 1964). Because the lesions in the scrotum, stomach and skin begin around vessels and appear to be the result of a blood-borne toxin, castration
340
M . D . MCCRACKI'N, W. W. CARLFON and J. TLm~
may have inhibited scrotal necrosis through atrophy of the scrotum and a resulting reduction in the amount of loose connective tissue and blood supply. Acknmvledgements This work, published as paper no. 5392 of the Agricultural Experiment Station, Purdue U niversity, West Lafayette, was supported in part by National Institutes of Health Grant ESCA-00463. U nitcd States Department of Agriculture Cooperative Agreement 12-14-100-909(51), Market Quality Research Division and by National Institutes of Health Postdoctoral Special Fellowship No. F03-GM-50866.
REFERENCES Cahon. W, W. & Tuite, J. (1970). Mycotoxicosis induced in guinea pigs and rats by corn cultures of Penicillium ziridicatum. Toxic. appl. Pharmac. 16, 345. LeMaistre. C. & Tompsett, R. (1952). The emergence of pseudotubcrculosis in rats given cortisone. J. e~p. Med. 95, 393. McCrackcn. M. D.. Carlton, W. W. & Tuite. J. {1974a). Pe~tkillmm t,iridicamm mycotoxicosis in the rat. 1. Ocular lesions. Fd Cosmet. Toxicol. 12, 79. McCracken. M. D., Carlton. W. W. & Tuite..1. {1974b). Pe~icillim, ciridicamm mycotoxicosis in the rat. 11. Scrotal lesions. Fd Cosmet. Toxicol. 12, 89. Mc('rackcn. M. I).. Carlton. W. W. & Tuilc. J. (1974c). P~,Jlicillium riridicutlml lnVCOloxicosis in the ral. lII. Hepatic and gastric lesions. Fd Cosmet. Toxicol. 12, 99. Weissmann, G. & Thomas, L. (I 964). The effects of corticosteroids upon connective tissue and lysosomes. Receipt Pro.q. Horm. Res. 20, 215. Zarrow, M. X., Yockim, J. M. & McCarthy, J. L. (1964). Experimental Endocrim)logy. A Sourcehook o/Basic 7k,chtliques. Academic Press, New York.
Mycotoxicose par Peniciilium viridicatum ehez le rat. IV. Essais de modification de r6actions des tissus R~sum~--On a administre du chlorhydrate de tripelennamine, de la syrosingopine ou de la prednisolone/~ des rats mfiles dont le regime alimentaire comportait/t raison de 50~/o une culture sur riz de Penicillium viridicatum. D'autres groupes, dont le r6gime ne comportait que 25% de cette culture, ont recu de la prednisolone ou de la tetracycline. Les rats d'un des groupes soumis fi ce regime alimentaire avaient etd chfitres au prdalablc. La tripdlennamine, la syrosingopine et la tetracycline n ' o n t pas 6te capables de diminuer la frequence ou la gravit6 des lesions oculaircs et scrotales contractdes par les rats s o u m i s / l ces essais. La prednisolone a fait preuve d'une certaine efficacit6 en diminuant la frequence et la gravite des lesions du scrotum et ia gravite des lesions oculaires. La tetracycline - m a i s non la tripelennamine et la syrosingopine--a attenu6 la gravite des l&ions hepatiques. La castration a eflicacemcnl prdvcnu lcs ]dsions du scrotun~, m~lis n'a pas cntra]nc dc modifications des lesions des yeux et du foie.
Penicillium viridicatum-Mycotoxicose bei der Ratte. IV. Versuchen die Gewerbereaktionen zu ver~indern Zusammenfassung MS.nnlichcn Ratten wurde cine Reiskultur yon Pe~Ticillium viridicutum in emer Konzentration yon 501~; in der Kost verffiltcrt, und es wurde ihncn Tripelenaminhydrochlorid. Syrosingopin oder Prednisolon verabreicht. Anderen Gruppen wurde die Pilzdiiit :,u 25",, in dcr Kost vcrftittert, und cs wurde ihncn Prednisolon oder Tetrucyclin vcrahrcicht, l i n e mit dicser Kost geffitterlc Gruppc wurde vor Beginn kaslricrt. Tripclenamin, S)rosingopm und TetracEclin zeigtcn kcincn Effckt, des Aufh'ctcn odor dic Stfirkc der ocularen und scrotalen L~isionen zu reduzicrcn, die sich in den getesteten Ratten entwickelten. Prcdnisolon zeigtc cinigen Effekt in dcr Rcduktion des Auftrelcns und in der Sfiirke der scrotalen L~isionen und verringcrte die Stiirkc dcr ocularcn L~isionen. Kastrierung verhinderte cfl'cktiv die scrotalcn L/.isionen, abcr verSndcrlc nichl dic der Augcn rind l,cber.
P E N I C I L L I U M V I R I D I C A T U M M Y C O T O X I C O S I S IN THE RAT. IV. A T T E M P T S TO M O D I F Y THE TISSUE R E S P O N S E S M. D. MCCRACKEN and W. W. CARLTON Department o] Veterinary M icrobiolo~ly and Patholo,qy, School of Veterinary Science and Medicim':
and J.
TU1TE
Department q['Botan) and Phmt Patholoqy, School ql'Agriculture, Purdue Unieersity, West lx{/hyette, Indiana 47907, U S A (Received 24 January 1974) Abstract -Male rats were fed a rice culture o f Penicillium viridicatum a t a dietary concentration of 50°~ and were administered tripelcnnamine hydrochloride, syrosingopine or prednisolone. Other groups fed the fungal diet at 25% dietary concentration were administered prednisolone or tetracycline. One group fed this diet was castrated prior to feeding. Tripelennamine, syrosingopine and tetracycline were not effective in reducing the incidence or severity of the ocular and scrotal lesions that developed in the test rats. Prednisolone was somewhat effective in reducing the incidence and severity of scrotal lesions and decreased the severity of the ocular lesions. Tetracycline, but not tripelennamine or syrosingopine, reduced the severity of the hepatic lesions. Castration effectively prevented the scrotal lesions, but did not altcr those of the eye and liver.
INTRODUCTION
Male rats fed grain cultures or fungal mats of Penicillium vMdicatum developed lesions of the eye (McCracken, Carlton & Tuite, 1974a), and of the scrotum and liver (Carlton & Tuite, 1970; McCracken, Carlton & Tuite, 1974b, c). Because the ocular, and especially the scrotal, lesions were markedly inflammatory in character, studies were completed to ascertain how the development of the lesions was affected by an antihistamine, tripelennamine, the serotonin antagonist, syrosingopine, the corticosteroid, prednisolone, and the antibiotic, tetracycline. In addition, one group of rats was castrated and fed the fungal diet. The results of these studies are presented in this report. EXPERIMENTAL
Experimental animals. Male rats were purchased at a weight of about 250,300 g and were housed and fed as previously described (McCracken et al. 1974a). Rats were examined daily and the incidence and character of ocular and scrotal alterations were recorded. Orchidectomy was performed under ether anaesthesia on one group of rats in Trial II and the rats were allowed 2 wk for recovery. Fungal diets. Rice cultures of P. vMdicatum were prepared as previously described and mixed with a purified diet (McCracken ~'t o/. 1974a). Experimental design and conduct. In Trial I, male rats were fed the fungal grain cultures at a dietary concentration of 50'~f, and groups of these rats were given either tripelennamine 331
332
M . D . MCCRACKt;N, W. W. CARLTON and J. TUITE
(Pyribenzamine, supplied by Ciba Pharmaceutical Co., Summit, N.J.) in a daily sc dose of 5 mg/kg body weight, syrosingopine (Singoserp. supplied by Ciba Pharmaceutical Co.) in a daily sc dose of 25 mg/kg or prednisolone in a daily sc dose of 5 mg/kg. Rats in Trial II were fed the fungal grain culturc at a dietary concentration of 25!'; either alone or together with a daily sc dose of 5 mg prednisolone/kg or a daily intramuscular dose of 5 mg tetracycline/kg. The treatments were continued for up to 6 wk. Autopsy. The eyes, scrotum, liver, stomach and kidneys were removed at autopsy. Eyes were processed as previously described (McCracken et al. 1974a). The other tissues were fixed in 10}~ buffered formalin, processed for paralfin sectioning and stained with haematoxylin and eosin for histopathological examination.
RESULTS
Trial 1 Clinical signs. Signs of toxicity in the groups of rats fed the fungal diet were similar to those previously described and included loss of activity, roughened hair, anorexia, diarrhoea and progressive loss of body weight (Table 1). Administration of tripelennamine hydrochloride, syrosingopine or prednisolonc had no effect on the incidence or severity of the clinical signs. Mortality was high in the test groups given tripelennamine or syrosingopine. Table I. Meall body weights aml im'idencc qf ocuhtr lesions in raz.~.[ed a culture o[ P. viridicatum and treated with tripeh, nnanline, s l'ro.~inHofffne or prednisohme (Trial I) Mean body weight (g) at wk Experimental group
0
1
4
Purified diet~501}~iPV~ Tripelennamine f§ o/ 50,0 PV+4- + tripelennamine§ 50"; PV{ + syrosingopine§ Prednisolonet§ 50'}; PV:{ + prednisolone§
363 367 331 334 339 364 357
387 310 344 283 280 370 306
455 208 407 236 200 405 190
Total gain (g) 92 - 159 76 -98 139 4l -167
Mortality*
Gross ocular lesions*
0/10 2,,"15 0/5 7/'15 10/15 1/10 6/15
0 18 30/30 0/10 30/30 28/'30 0,/'24 30/30
*Mortality is expressed as no. dead/no, in group and gross ocular lesions as no. of eyes affected/no examined. +Control groups. ++Rice culture of P. viridicamm fed at a level of 50°~; in the diet. ¢Tripelennaminc or prednisolone given in daily sc doses of 5 mg/kg body weight and s wosingopme in daily sc doses o1"25 mg/kg.
Ocular effects. Clinically detectable ocular lesions occurred in all rats fed only the fungal diet. Lesions, first noted after 7 10 days of feeding, were similar to those previously described (McCracken et al. 1974a), beginning as a slight haziness of the cornea and increasing in severity so that the cornea became opaque. Hypopyon and corneal vascularization occurred in severely affected eyes. Tripelennamine and syrosingopine had no apparent effect on the development or severity of the clinically detectable ocular changes (Table 1), but ocular disease appeared to be less severe in test rats given prednisolone. The initial microscopic ocular lesions in the test rats consisted of corneal oedema and hydropic degeneration of the corneal epithelium. In mildly affected eyes, accumulations of a mixture of inflammatory cells were present around the limbal vessels and within the
Penicillium viridicatum mycotoxicosis
333
substantia propria. In more severely affected eyes, diffuse interstitial keratitis was accompanied by iridocyclitis, hypopyon and corneal vascularization (Table 2). No difference in the incidence of corneal oedema was observed in test rats given prednisolone. However, the incidence of hypopyon and corneal vascularization was lower in this group than in that fed the fungal diet alone (Table 2). The incidence and severity of ocular lesions in the rats treated with tripelennamine or syrosingopine were comparable to those in the fungal control group (Table 2). F~ffects o~ scrotum. Gross scrotal lesions in test rats were similar to those previously described (McCracken et al. 1974b). Oedematous swelling of the scrotum began after 2 -3 wk of feeding and was followed by necrosis and ulceration of the scrotal epidermis. The administration of tripelennamine or syrosingopine had little observable effect on the incidence or severity of scrotal swelling and necrosis (Table 3). Scrotal necrosis followed sooner upon the initial swelling in test rats given tripelennamine. Scrotal oedema was observed in 7 of 15 rats treated with prednisolone but was severe in only one rat, and epidermal necrosis was observed in only two (Table 3). Microscopic scrotal lesions occurred in almost all test rats and consisted of a severe necrotizing cellulitis characterized by loci and bands of necrotic leucocytes in the epididymal connective tissue and scrotal fascia. Necrosis and ulceration were present in the overlying epidermis. Differences in microscopic lesions between rats given tripelennamine or syrosingopine and those fed the fungal diet alone were a lower incidence of testicular lesions and of rats with lesions in the epididymis (Table 3). Prednisolone decreased the incidence and severity of the scrotal lesions (Table 3); two of the 15 test rats given prednisolone had no lesions. Lesions of the epididymal fat and the scrotal fascia surrounding the tunicae were extremely variable, ranging from no lesions to very severe changes. Changes in the epididymal adipose tissue consisted of focal mononuclear cell accumulations and areas of necrosis small-to-moderate in size, Five rats had lesions of the epididymal fat, but there were no lesions outside the tunicae. Seven of these 15 rats had lesions of severe necrotizing cellulitis similar to that present in the rats given only the fungal diet. Necrosis and ulceration of the epidermis occurred in only three of the 15 prednisolone-treated test rats. Hepatic ehanyes. Gross hepatic lesions in test rats consisted of a variable number of yellow-to-white loci and often the smaller hepatic lobes were the more severely affected. There was no difference in the incidence or appearance of gross hepatic lesions among the various treatment groups (Table 4).
Microscopic hepatic changes in the test rats were characterized by a necrotizing cholangitis with pericholangitis, proliferation of the biliary epithelium and focal areas of hepatic cell necrosis, often adjacent to the bile ducts. Periductal fibrosis resulted in stenosis and obliteration of the bile ducts in the more severely affected livers. Neither the incidence nor severity of hepatic changes in rats given tripelennamine or syrosingopine differed significantly from those in the group fed the fungal diet alone (Table 4). Focal necrosis and periductal fibrosis of the liver were particularly severe in test rats given prednisolone. Necrotic loci were more numerous and larger in these rats, but bile-duct proliferation tended to be less severe. EfJ'ects on other or,qans. Lesions in other organs included gastric erosions, extra-scrotal skin necrosis and biliary pigment in the renal tubular epithelium. There were no apparent differences among the various treatment groups in the development of these lesions.
14 26 9 27 26 24 2~
Experimental group
l-h_lrilied diet* 50", P V I Tripclennamine*:~ 50", PV'~ + tripelennamine.+,. 50"i, PV+ + s_',rosingopinc + Prcdnisolor~c'::~: 500 L PV+ + prednisoJone~ 0 26 0 27
22 0 26
26 0 2S
Keralitis
0 25 0 27
Corneal ocdema
') 0 6
0 12 0 7
Cornea] vessels
20 0 12
0 I8 I) 13
h'itis
No. of eyes with m i c r o s c o p i c lesions
10 15 5 15 15 10 15
0 14 0 10 12 0 7
Sx~clling 0 12 0 9 10 0 2
Necrosis
(h-oss scroml lesions
0 13 0 15 14 0 7
(~ellulitis of scl-olu m
0 10 0 10 10 0 3
Fpidermal necrosis
,
4 0 6
0 6 0 2
Syncchia
9 0 9
0 17 0 S
H}popyon
0 13 {) 2 I 0 0
Tcsticular lesions
0 15 0 7 7 0 5
I~pididvmal lesions
0 11 0 6 9 0 7
Penile lesions
~FY(~.~l'll¢lUpillt, (~" prudJlisol(mc (Ti'lal l)
M i c r o s c o p i c findings
No. of a n i m a l s affected b \
oHd lt'd¢lte'd Ivil/l lrlp(,l(,llllal~liHc.
* ( o n t r o l groups. +Rice culture of P. Hridicatum fed at a level of 50",, m the dieT. !Tripe er +t n nc or prcdn sol< l e g xe 1 in daitx sc doses c,15 mg kg body \xcight and ";5 to~,ingopit'~c in dailx sc doses of 25 mg kg.
Purilied dice :' 50°11 PVi" Tripelennamine*'i50°i, PV'I" + tripelennaminc + 50";. PV+ + syrosingopine} P r e d n i s o l o n e *+ 50°ii PV+ + prednisolone++
No. of *als cx:u~lincd
(;t'¢1~.~ d l i d /Hi(l'(l',('O[~/(' StTtU ii [(',SiO/L~, i'll t'tll~ ~'l [ tl tldtltI'(' 0,1 P, \ i r i d i c a l u l D
Expcrilnental gro ~t~
"]['til31e .)
*Control grotips. ";'Nice culture of P. cu'idic~,,rmn Ibd at a < \~.t ~ " 50" in Ihc diet. i:::lripclcnnamhlcor prcdnisoh~m.',.'ixcn in,!~ , ,L ,h,,.:, ,,I 7 m~ kg h,M\ v, c M h l ; m d 5\lo>n~<,pu~c hldail', ~,cJ~,<',,fi25 m# k~ ~()nl 3 24 <:yes cx;.lmlncd.
No. of eves examined microscopicall 3
'l able 2. O,:.td,w /c:,i<;ns hz ~'a~s /bd a c~drt,,., 0 / P . ~iridicatum a~d rr(,ared ~il/l rHp,.'i
~2
y
t'-
h
~:
i.
+
1I 0 7
15
10
15
15 IO
0 9 (t
5 15 5
Gross lesions
3
12 0
14
0 I4 0
Pericholangitis
9
15 0
14
0 15 0
Bile-duct proliferation
12
15 0
14
0 14 0
Pcriduclal tibrosis
No. of animals affected by
*Control groups. fRice culture ofP. riridicatuut ted at a level of _{) i, m tile diet. ~Tripelennamine or prcdnisolone ~iven m daily sc doses of 5 m g k g hod) weight and s xrosingopinc in dail\ sc doses of 25 mgkg.
syrosingopine$ Prednisolone$ 5()";i PVf + prednisolone{
.S0 o5~ PV
Purified diet* 50",i PVi Tripelennamine*++ 50",, PVt + tripelemmmine++
Experimental group
No. of rats examined
12
15 0
14
0 14 0
Necrotizing cholangitis
12
13 0
13
0 13 0
Scrotal necrosis
Table 4. Gross and microscopic hepatic lesi(ms in raL';./ed a cullure o/'P. viridicatum ~md treated ~,'ilh trilwh, nmmline, s),rosin#opim, or ~rednisolone (Trial I)
'Ji
©
©
@
~2
336
M . D . M('(I',A('Kt N. W. W. CAP,L](IN and J. Tt,I iI
Trial II Signs of toxicity in rats fed the 251}o fungal diet were minimal during the first 3 wk. During 4-6 wk of feeding, signs of toxicity appeared and were similar to those observed in Trial I. Test rats progressively lost weight and those given prednisolone showed the greatest reduction. Castrated rats fed the fungal diet maintained their body weights (Table 5). Table 5. Resl~o#~se ol iulacl aJ~d castrated rats to a cult~t'e 0/P. viridicatum amt to tr~'almctlt with IwedHisohme m" wtracyclille ( 77"ial II1 Mean body weight tit wk Experimental group Purilied diet+ 25<'~; PV{ Prednisolone+ 257. PV{ + prednisolone,~ Tetracycline't 25"~; PV + + tetracycline§ Castrated rats+ 251',, PV + + castration
0
1
5
Total gain (g)
362 367 357 383 363 362 355 341
403 374 367 366 4(ll 370 379 364
422 323 355 286 461 321 417 337
60 44 2 - 97 9", 41 62 - 4
Mortality* 010 0"15 0'5 I 15 I 5 0.15 0'5 015
*No. dead/no, in group. +Control groups. +Rice culture ofP. ~:iridicatum fed at a level ol" 25<'; in the dict. ~Given in daily doses of 5 mg/kg body wright, prednisolonc sc and tetracycline intramuscularly,
Ocular effects. Clinical ocular lesions were similar to those described in Trial I. Castration and treatment with tetracycline had no apparent effect on the incidence or severity of these lesions, but the lesions appeared less severe in test rats given prednisolone. Microscopic ocular lesions were present in most eyes of test rats and were similar to those of Trial I (Table 6). Castration or treatment with tetracycline had no apparent effect on the incidence of ocular lesions. The incidence of iridocyclitis, synechia and hypopyon was less in the test rats given prednisolone (Table 6). EfJbcts on scrotum. Scrotal swelling took a longer time to develop in rats fed the 25'~; fungal culture, usually occurring after 3 5 wk of feeding. Scrotal epidermal necrosis developed within 2 11 days of the initial swelling. The pattern of necrosis was similar to that seen in Trial I. starting approximately l ' 0 c m distal to the prepuce and progressively spreading over most of the scrotum. The incidence of scrotal swelling and epidermal necrosis was less in test rats given prednisolone (Table 7), but treatment with tetracycline had no effect on the incidence or severity of the scrotal alterations. Swelling and necrosis of the scrotum was observed in only three of 15 castrated test rats (Table 7) and. in these rats, the lesions were associated with remnants of the epididymal fat incompletely removed at surgery. Microscopic scrotal lesions in test rats were similar to those observed in Trial 1 and consisted of necrotizing cellulitis of the scrotal fascia with necrosis and ulceration of the epidermis. Diffuse orchitis was observed in about half of thc rats fed the fungal diet alone or in association with tetracycline treatment. Orchitis was characterized by oedema, marked infiltration of a mixture of inflammatory cells and necrosis and calcification of the seminiferous tubules. The incidence of microscopic scrotal lesions was greatly decreascd in the castrated test rats (Table 7) as 11 of 15 had very mild or no lesions. Lesions were generally confined
[9
18
0 24
Corneal oedema
22
18
0 29
Keratitis
16
12
0 18
Vascularization
13
7
0 18
Iritis
9
4
0 13
Synechia
Hypopyon
½
28
21
21
*No. of eyes affected/no, examined. ?Including tetracycline, prednisolone and castrated controls. ~:Rice culture ofP. viridicatum fed at a level of 25% in the diet. §Given in daily doses of 5 mg/kg body weight, prednisolone sc and tetracycline intramuscularly.
26/30
19
14
15
7
O
23
20
0 29
Microscopic lesions
-g
24
22
30 30
No. of eyes examined microscopically
castration
30/30
28/'30
0/30 29/30
Incidence of gross ocular lesions
25% Pv~; +
tetracycline§
25% Pv:~ +
prednisolone§
25% pv~ +
Controlt 25~o PV~
Experimental group
Table 6. Ocular lesions in intact and castrated rats.led a culture ofP. viridicatum and treated with prednisolone or tetracycline (~fi'ial lli
338
M . D . MCCRA('Kt N, W. W, CARl I'ON and .1. T t i ri
Table 7. Microscopic lesions of the scrotum and male relwoduetice organs in illtucl alTd e~lslruted #'tlls ted u eztllltre o/'P. viridicatum and in rats fi_,d the limHal diet and treated with prednisohme or tetracycline (Trial 111 No. of animals aflL'ctcd by' Experimental group Control* 25}o PV+ 253~; PVt + prednisolone++
No. of rats examined
Scrotal cellulitis
Epidermal necrosis
Testicular lesions
Epididymal lesions
Penile lesions
6 15
0 1I
0 S
0 7
0 11
0 0
15
?
6
1
g
~
15
13
7
,',;
10
(I
15
5
3~
257,; Pv+ + tetracycline++ 257o PV* + castration
0
*Anhnals given purified diet. +Rice culture of P. uiridicatum fed at a levcl of 25'~ii in the diet. ~.Givcn in daily' doses of 5 mg/kg bod 3 weight, prcdnisolonc sc alnd telracxclinc inlramuscularl 5. ~Necrosis usually associated with suture abscesses.
to the adipose connective tissue about the cremaster muscle and consisted of infiltration of lymphocytes and numerous pigment-laden macrophagcs. In three rats, the lesions were more severe, and exudate and necrosis was present around the suture material. No lesions were present in the scrotal epidermis in 12 of the 15 castrated test rats. Small loci of necrosis were confined to the distal portion of the scrotum in one rat. Necrosis and ulceration of the epidermis occurred in two rats, with more severe lesions in the subcutis. Test rats given prednisolone showed a slight reduction in the incidence of microscopic scrotal lesions compared with those given the fungal diet alone and testicular lesions were present in only one rat (Table 7). Treatment with tetracycline had no apparent effect on the incidence or severity of microscopic scrotal lesions. Hepatic changes. Gross hepatic changes in the test rats were usually less severe than in Trial I. Mild hepatic changes included mottling, varying from dark red or pale tan to yellow, with occasional small red loci. Yellow foot. 1 2 mm in diameter, were observed in some rats of all the groups given the fungal diet. The most severe gross changes were observed in the livers of test rats given prcdnisolonc. Microscopic hepatic changes in the test rats consisted of necrotizing cholangitis, pericholangitis, periductal fibrosis and bile-duct proliferation, lesions similar to those observed in Trial I. The incidence of focal necrosis of hcpatocytes was low in all the fungal-diet groups except in the group given prednisolone (Table 8). Hepatic lesions appeared slightly less severe in test rats given tetracycline, as the incidence of necrotizing cholangitis was reduced and focal necrosis was not observed. Castration and treatment with prednisolone had no apFarent effect on the severity of the hepatic lesions (Table 8t. DIS('USSION
The observation that treatment with an antihistamine or a catecholamine depressor had no apparent protective effect against the scrotal and ocular lesions described in this study suggests that histamine and serotonin may not play a significant or major role in the pathogenesis of these lesions. However, because only a single dose level was given, it is possible that effective blood and tissue levels of the drugs were not achieved. In the scrotum, the lesions begin in the perivascular areas in the adipose connective tissue and at an early stage
Penicillium eiridicatum mycotoxicosis
339
Table 8. Microscopic hepatic lesions in intact and castrated rats ]ed u culture of P. viridicatum and treated with prednisolone or tetracycline
Experimental group Controls* 25°4, PV+ 25°,£ PV + prednisolone + 25°; PV + tetracycline{ 25~;;, PV + castration
No. of animals affected by Bile-duct Periductal Necrotizing proliferation fibrosis cholangitis
No. of rats examined
Pericholangitis
6 15
0 10
0 11
0 12
0 10
0 3
15
6
9
8
7
9
15
8
12
9
4
0
15
10
13
14
11
1
Focal necrosis
*Animals given purified diet. ?Rice culture of P. viridicatum fed at a level of 25",i; in the diet. §Gix.cn in daily, doses of 5 mg,'kg bod}. weight, prednisolone sc and tetracycline intramuscularly.
are largely confined to these areas, indicating that the toxin(s) may have a specific action especially on the vessels of loose connective tissue. Because of the established role of histamine and serotonin in increasing vascular permeability, the initial scrotal lesions could have originated through a mechanism involving these substances. The later lesion of epidermal necrosis appeared to be secondary to ischaemia. Prednisolone decreased the incidence and reduced the severity of the scrotal lesions and decreased the incidence of some ocular lesions, but not the incidence of corneal oedema. Prednisolone can influence several phases of the inflammatory reaction and the particular phase was not ascertained by the methods of this study, but significant anti-inflammatory properties of glucocorticoids are the effects on vascular permeability and stabilization of lysosomal membranes (Weissmann & Thomas, 1964). Hepatic lesions were generally more severe in prednisolone-treated test rats. The reason for this is unknown, but pathogenic mechanisms of lesion induction apparently differ from organ to organ. It is possible that prednisolone-induced 'stress' activated a latent bacterial infection, as the lesions in some of the livers resembled those produced by Corynebacterium kutscheri (LeMaistre & Tompsett, 1952). An extensive literature points to damage to Descemet's endothelium as most significant to the development of corneal oedema. Our observations are consistent with these data, as oedema preceded any other ocular change. The hepatic lesions appear to begin with damage to the biliary epithelium followed by secondary inflammatory and reparative changes. Because neither the ocular nor hepatic lesions appear to be initially vascular, it would not be unexpected for an antihistamine, a 5-hydroxytryptamine antagonist or a corticosteroid to be ineffective in preventing their development. Prednisolone did appear to reduce the more purely inflammatory ocular lesions, such as iridocyclitis and corneal vascularization. Castration appeared to inhibit the development of scrotal lesions, as alterations were either absent or were very mild and confined to adipose connective-tissue remnants. Further experiments would be necessary to determine whether the protective effects were due only to the removal of androgen secretion. Testosterone is known to increase the blood supply to and stimulate the growth of male accessory organs, including the scrotum (Zarrow, Yockim and McCarthy, 1964). Because the lesions in the scrotum, stomach and skin begin around vessels and appear to be the result of a blood-borne toxin, castration
340
M . D . MCCRACKI'N, W. W. CARLFON and J. TLm~
may have inhibited scrotal necrosis through atrophy of the scrotum and a resulting reduction in the amount of loose connective tissue and blood supply. Acknmvledgements This work, published as paper no. 5392 of the Agricultural Experiment Station, Purdue U niversity, West Lafayette, was supported in part by National Institutes of Health Grant ESCA-00463. U nitcd States Department of Agriculture Cooperative Agreement 12-14-100-909(51), Market Quality Research Division and by National Institutes of Health Postdoctoral Special Fellowship No. F03-GM-50866.
REFERENCES Cahon. W, W. & Tuite, J. (1970). Mycotoxicosis induced in guinea pigs and rats by corn cultures of Penicillium ziridicatum. Toxic. appl. Pharmac. 16, 345. LeMaistre. C. & Tompsett, R. (1952). The emergence of pseudotubcrculosis in rats given cortisone. J. e~p. Med. 95, 393. McCrackcn. M. D.. Carlton, W. W. & Tuite. J. {1974a). Pe~tkillmm t,iridicamm mycotoxicosis in the rat. 1. Ocular lesions. Fd Cosmet. Toxicol. 12, 79. McCracken. M. D., Carlton. W. W. & Tuite..1. {1974b). Pe~icillim, ciridicamm mycotoxicosis in the rat. 11. Scrotal lesions. Fd Cosmet. Toxicol. 12, 89. Mc('rackcn. M. I).. Carlton. W. W. & Tuilc. J. (1974c). P~,Jlicillium riridicutlml lnVCOloxicosis in the ral. lII. Hepatic and gastric lesions. Fd Cosmet. Toxicol. 12, 99. Weissmann, G. & Thomas, L. (I 964). The effects of corticosteroids upon connective tissue and lysosomes. Receipt Pro.q. Horm. Res. 20, 215. Zarrow, M. X., Yockim, J. M. & McCarthy, J. L. (1964). Experimental Endocrim)logy. A Sourcehook o/Basic 7k,chtliques. Academic Press, New York.
Mycotoxicose par Peniciilium viridicatum ehez le rat. IV. Essais de modification de r6actions des tissus R~sum~--On a administre du chlorhydrate de tripelennamine, de la syrosingopine ou de la prednisolone/~ des rats mfiles dont le regime alimentaire comportait/t raison de 50~/o une culture sur riz de Penicillium viridicatum. D'autres groupes, dont le r6gime ne comportait que 25% de cette culture, ont recu de la prednisolone ou de la tetracycline. Les rats d'un des groupes soumis fi ce regime alimentaire avaient etd chfitres au prdalablc. La tripdlennamine, la syrosingopine et la tetracycline n ' o n t pas 6te capables de diminuer la frequence ou la gravit6 des lesions oculaircs et scrotales contractdes par les rats s o u m i s / l ces essais. La prednisolone a fait preuve d'une certaine efficacit6 en diminuant la frequence et la gravite des lesions du scrotum et ia gravite des lesions oculaires. La tetracycline - m a i s non la tripelennamine et la syrosingopine--a attenu6 la gravite des l&ions hepatiques. La castration a eflicacemcnl prdvcnu lcs ]dsions du scrotun~, m~lis n'a pas cntra]nc dc modifications des lesions des yeux et du foie.
Penicillium viridicatum-Mycotoxicose bei der Ratte. IV. Versuchen die Gewerbereaktionen zu ver~indern Zusammenfassung MS.nnlichcn Ratten wurde cine Reiskultur yon Pe~Ticillium viridicutum in emer Konzentration yon 501~; in der Kost verffiltcrt, und es wurde ihncn Tripelenaminhydrochlorid. Syrosingopin oder Prednisolon verabreicht. Anderen Gruppen wurde die Pilzdiiit :,u 25",, in dcr Kost vcrftittert, und cs wurde ihncn Prednisolon oder Tetrucyclin vcrahrcicht, l i n e mit dicser Kost geffitterlc Gruppc wurde vor Beginn kaslricrt. Tripclenamin, S)rosingopm und TetracEclin zeigtcn kcincn Effckt, des Aufh'ctcn odor dic Stfirkc der ocularen und scrotalen L~isionen zu reduzicrcn, die sich in den getesteten Ratten entwickelten. Prcdnisolon zeigtc cinigen Effekt in dcr Rcduktion des Auftrelcns und in der Sfiirke der scrotalen L~isionen und verringcrte die Stiirkc dcr ocularcn L~isionen. Kastrierung verhinderte cfl'cktiv die scrotalcn L/.isionen, abcr verSndcrlc nichl dic der Augcn rind l,cber.