Hepatitis in vervet monkeys caused by Fusarium moniliforme

J. COMP.

PATH.

1987

HEPATITIS

VOL.

97

IN

VERVET

K. JASKIEWICZ,

W.

MONKEYS MONILIFORME

F. 0.

CAUSED

BY MARASAS

andJ.J.

BY FUSARIUM

F. TALJAARD*

National Research Institute for Nutritional Diceam, South Aftican Medical Research Council, P. 0. Box 70, Tygcrberg 7505. South Africa and *Department of Chemical Pathology, Tygerberg Hospital, Tygerberg 7505, South Africa

INTRODUCTION

The fungus Fusarium moniliforrne Sheldon is one of the most prevalent seed-borne fungi associated with maize (
AND

METHODS

Fungus Cultures Fusarium monilifrme MRC 826 was isolated as a single-conidial culture from home-grown maize produced in an area of Transkei, southern Africa, where there is a high incidence of oesophagealcancer in man (Kriek et al. 1981b). Stock cultures were maintained by lyophihzation and used to inoculate whole yellow maize kernels. 0021-9975/87/030281+

11 $03.00/O

0

1987

Academic

Press

Inc.

(London)

Limited

282

K.

JASK’EWICZ

et al.

Cultures in 2 1 flasks were incubated in the dark at 25°C for 14 days, followed by 14 days at 15°C (Gelderblom, Thiel, Marasas and van der Merwe, 1984). The culture material was then lyophilized, ground to a fine meal and stored in the dark at 0°C until used (Marasas et al., 1984b).

Animals Twelve young vervet monkeys weighing approximately 1 kg each were fed a standard primate diet (Table 1). Ten monkeys were fed this diet containing different concentrations of lyophilized culture material of F. monilifrme MRC 826, while two monkeys served as controls. The diets were monitored for aflatoxin with negative results. In a pilot experiment, one monkey was fed a diet containing 5 per cent of culture material. After 21 days of treatment and following liver biopsy, the dose was lowered to 1 per cent and during the next 14 days to 0.25 per cent. The remaining 11 monkeys were divided into treatment groups and a control group as follows: One group of 5 monkeys received 1 per cent of culture material for 14 days after which the dose was lowered to 0.25 per cent for the remainder of the experiment due to high toxicity. A second treatment group of 4 monkeys received 0.25 per cent of culture material for 160 days and the dose was lowered to 0.1 per cent thereafter. The 2 control monkeys received the standard diet without culture material. The monkeys were caged individually in a controlled environment at 24 f 1.5”C and 50 per cent humidity with 15 to 20 air changes per hour and a 12 h artificial lighting cycle. They were immobilized at regular intervals with ketamine hydrochloride (Ketalar, Parker-Davis) for weighing and taking blood samples. Liver biopsies were performed by laparotomy under halothane general anaesthesia on one monkey from each treatment group on days 2 and 6 of the experiment and all the monkeys were biopsied on day 12 and again after 180 days.

Ingredient

Amount

Commerciai pre-cooked maize meal Protein-vitamin-mineral supplement (PVM, Dreyer & du Bruyn, 1968) Vitamin mix* Whole wheat brown bread Fruit * I;itamin Mix Vitamin A Vitamin D3 Vitamin E Vitamin K Vitamin B,, Folic acid Riboflavin Niacin Thiamin Pantothenic acid Biotin Inositol Choline chloride Pyridoxin Maize meal

per monkey per day 58 g 10 87 lg + slice f apple

2g 5 mg

8g 5 mg 70 mg 200 mg 500 mg 5g 150 mg 5g 2g 50 g 150 g 500 mg 776.57 g 1000~00

g

FUNGAL

HEPATITIS

IN

VERVETS

283

Light Microscop_y Liver biopsy samples were fixed in neutral buffered formalin, processed to paraffin wax in the conventional way and sections were stained with haematoxylin and eosin (HE) for histopathological examination. Some sections were also stained by the periodic acid Schiff (PAS), Masson’s trichrome and Gordon and Sweet’s silver reticulin techniques. Cryostat-cut frozen sections were tested for fatty changes and immunoglobulin deposits by the direct immuno-fluorescence method (Bancroft and Stevens, 1977). Electron Microscofiy

(EM)

Immediately after biopsy, the liver tissues were cut into cubes measuring 1 mm or less, fixed in 4 per cent cold buffered glutaraldehyde overnight and subsequently treated in the conventional way. Biochemical Studies Serum samples were analysed by routine chemical pathology methods including the (ALT), lactic following: aspartate aminotransferase (AST), a 1anine aminotransferase dehydrogenase (LD), alkaline phosphatase (ALP), g amma glutamyltranspeptidase (GGT), bilirubin, cholesterol, albumin and globulin (Lynch, Raphael, Mellar, Spare and Inwood, 1969). Immunological

Tests

Complete blood counts, serum protein electrophoresis and analysis of serum anti-mitochondrial, anti-smooth muscle and anti-nuclear were performed routinely (Sherlock, 1970).

for the presence autoantibodies

RESULTS

Macroscopic

Changes

The clinical condition of all monkeys was satisfactory throughout the experiment. The liver df the monkey used in the pilot experiment and fed 5 per cent of F. monilifrme MRC 826 culture material for 21 days was slightly reduced in size, soft, yellowish, and bled easily. Scattered petechiae were present on mucosal and serosal membranes and the bleeding time was prolonged. Monkeys treated with 1 per cent of culture material and biopsied after 12 days presented similar but milder macroscopic changes. No macroscopic changes were observed in animals initially treated with 0.25 per cent of culture material or in the controls. Microscopical

Changes

The lobular structure of the liver of the pilot monkey biopsied at 2 1 days was disturbed and the trabecular structure was replaced by a tangled mass of abnormal hepatocytes in various forms and stages of degeneration and necrosis (Fig. 1). I n d’ 1v1‘d ua 1 ce 11s or small groups of cells were necrotic and sometimes bridging necrosis linked necrotic foci in pericentral and periportai areas. The

284

K.

JASKIEWICZ

et

d.

collapsed reticular architecture made this clearly visible. Some individual cells either disappeared and were replaced by a few inflammatory cells, or underwent coagulation and produced hyaline (Councilman) bodies. Other cells showed various stages of degeneration including vacuolar, balloon-like and feathery. No fatty changes occurred in the hepatocytes. The infiltrate that accompanied resorption and necrosis was mainly mononuclear and non-specific in nature with an increased number of Kupffer cells. Intralobular and intracellular cholestasis was mild and irregularly distributed. On the second day of exposure to either 1 or 0.25 per cent of culture material, there were no significant differences between the two fungal dietary treatments. The trabecular architecture was preserved. Sinusoids were either irregularly dilated or collapsed due to swollen hepatocytes with pale, slightly granular, glycogen-rich cytoplasm and prominent nuclei. After 6 days, more advanced degenerative changes occurred in hepatocytes with scattered hyaline bodies and isolated cell necrosis in some of them. These changes were more common after 12 days and focal accumulations of mononuclear cells and Kupffer cell proliferation became distinct. These changes were more common in the monkeys treated with the greater concentration of culture material and, in some cases, the hepatic lesions were similar to those in the pilot monkey, but less advanced. Eosinophilic degeneration and scattered necrosis were the predominant changes in the hepatocytes at this stage. The reticular structure of the liver was preserved for the most part and collapse and shrinkage occurred only in small foci. Cellular infiltration and cholestasis were also mild. Liver biopsies of both treatment groups of monkeys fed culture material for 180 days revealed that the lobular structure was mostly preserved but the trabecular structure was focally disturbed. In all cases, scattered mononuclear infiltration occurred, particularly in association with necrotic or acidophilic hepatocytes. Some infiltrations tended to accumulate in periportal and pericentral areas. In most cases, the border plate was invaded by mononuclear cells. In one case, the chronic inflammatory response was limited to periportal areas and caused granulomas. The intensity of the inflammatory response and the degenerative or necrotic changes in the hepatocytes were similar in both treatments, but individual differences were noted. All cases suggested active chronic hepatitis (Fig. 2) which was either mild or moderate and in two cases severe. In these two cases, the fibroblastic activity and disturbed lobular structure resulted in active cirrhosis and direct immunofluorescence revealed IgG deposits in sinusoid walls. No abnormal changes were observed by light microscopy in the two control monkeys. EM Changes On the second day of exposure to either 1 or 0.25 per cent of culture material, the most prominent ultrastructural changes in the liver were in the configuration of the endoplasmic reticulum of hepatocytes. In some cells, an

FUNGAL

HEPATITIS

IN

VERVETS

285

increase of smooth membranes only occurred, while, in others, this was accompanied by dilatation of smooth endoplasmic reticulum (SER) and loss of ribosomes in the reticular structure. Some cells contained vacuolated or deformed mitochondria with a tendency to accumulate close to the cell membrane. Dilated bile canaliculi contained reduced, short or flattened microvilli (Fig. 3). Characteristic nucleolar segregation, resulting in the rearrangement of nucleolar constituents into granular and fibrillar areas, was noted. After 6 and 12 days of exposure, hepatocytes showed more severe degenerative changes such as the presence of polysomes, numerous lysosomes, bile deposits and lamellar structures. Some dilated sinusoids contained necrotic debris or dying hepatocytes (Fig. 4). as well as nuclear changes were After 180 days of exposure, cytoplasmic observed in the hepatocytes of both treatments. Cytoplasmic changes included increased SER, vesiculated membranes, disorganized rough endoplasmic reticulum (RER), polysomes, lysosomes, lamellar bodies and electron-dense granules or needle-like structures in mitochondria. Nuclear changes in most hepatocytes were characterized by nucleolar hypertrophy, with rearrangement of constituents in the nucleolar cap pattern or the less common ring-like shape and chromatin clumping (Fig. 5). Distended sinusoids showed “ghost hepatocytes” or early collagen deposits and thick basal membranes (Fig. 6). No abnormal EM changes were seen in the livers of the two control monkeys. Biochemical Changes The serum of the pilot monkey treated with 5 per cent of culture material had normal or slightly increased activities of the enzyme indicators of hepatocellular damage (AST, ALT, LD) during the first few days of the experiment, but activity increased IO- to 20-fold at the end of the second week. Monkeys treated with 1 per cent of culture material showed 6- to IO-fold increases in activity of these enzymes compared to the controls after 14 days and decreased activity during the 2 to 3 weeks after the lowering of the dose to 0.25 per cent (Fig. 7). The activities then increased gradually up to 18-fold in one high response animal. During the first 21 days of the experiment, gradual increases of ALP and GGT activity occurred in the serum and activity remained high up to 180 days. In monkeys which received 0.25 per cent culture material, the increase in hepatocellular enzyme activity was much slower during the first 6 to 7 weeks of the experiment, but increased steadily during the following 8 to 9 weeks. No dramatic decreases occurred when the dose was lowered to 0.1 per cent after 160 days. In fact, some monkeys showed an unexpected increase of AST, ALT and LD activities after 180 days (Fig. 7). Some monkeys in both treatment groups .sh@wed moderate increases in concentration of conjugated and unconjugated bilirubin in the serum during the first few weeks of the experiment and gradual increases in globulin and cholesterol (Fig. 7). Albumin concentration remained unchanged.

Fig.

1. Trabectilar structure of the liver replaced by a tangled mass of hepatocytes presenting various forms and stages of degeneration and necrosis. Note also focal mononuclear infiltration (arrows). Monkey fed 5 per cent culture material for 21 days. HE X 504.

Fig. 2. Hepatitis acidophilic Fig. 3. Distended dilatation material Fig. 4. Dilated reticulum 12 days.

with disturbed lobular structure and mononuclear bodies (arrow). Monkey fed 0.25 per cent culture intercellular bile canaliculus with shortened of rough endoplasmic reticulum and dense for 2 days. EM x 11 880. sinusoid contains and mitochondria EM X 7200.

and irregular mitochondria.

for

Some 180

microvilli Monkey

hepatocytes days. HE (arrow). fed 1 per

remnants of necrotic hepatocytes. Changes in the rough in the hepatocyte on the left. Monkey fed 1 per cent culture

Fig. 5. An unusual pattern of segregation of nucleolar constituents. component with four satellite-like foci in the dense ring-shaped 0.25 per cent culture material for 180 days. EM X 18 900. Fig. 6.

infiltration. material

In the centre filamentous

contain X 315. Note cystic cent culture endoplasmic material

for

is the enlarged granular component. Monkey fed

Early collagen deposits and part of fibroblastic cell in the space of Disse, separated from (lower part of the picture) by endothelial lining with thick basal membrane (arrow). 0.25 per cent followed by 0.1 per cent culture material for 180 days. EM x 7800. page 287

the sinusoid Monkey fed

FUNGAL

I I

I

HEPATITIS

IN

I

287

VERVETS

,

I

I

I 0

1600

-

1400

$ .-2

1200

5 .z= .’

1000

i t: E : c%

800

l-

600 400 2oc

0

612

30

I 90

60

Time

Fig.

---

I

I

120

150

_-

180

(days)

7. Changes in serum AST activity and cholesterol concentrations in High mat&al of Puusarium monilt$wme MRC 826, [Group 1: O--O: Mean value of remaining monkeys; 0 - - 0: High response monkeys value of remaining monkeys; O---O: controls; +=Time of change 0 = Time of change in dose (0.25 to 0.1 per cent)].

vervet monkeys fed culture response monkey; O--O: Group II:- 0 - - 0: Mean in dose (1 to 0.25 per cent)

lTmmunologicaL Findings During the sixth month of the experiment, increased concentrations of both IgG and IgM up to 10 g per 1, occurred in some monkeys fed culture material. Corresponding maxima in the controls were 5.8 g per I, and 0.85 g per I respectively. Serum anti-mitochondrial, anti-smooth muscle and anti-nuclear autoantibodies in these cases remained negative. No abnormal haematological changes were noted. DISCUSSION

A wide variety of pathological changes in the liver of man is caused by a large number of known and unknown toxins. Liver biopsy assists in establishing whether drug therapy or accidental intoxication is responsible for a lesion, but is rarely conclusive because absolute criteria for this type of hepatic lesion have not been established (Read, 1979). Some therapeutic drugs and toxins cause characteristic hepatic lesions that can be reproduced in laboratory animals, but many others cause non-specific histological lesions that mimic some common liver diseases (George and Watt, 1979). This presents

288

K.

JASKIEW’CZ

t?t al.

considerable problems in the pathological diagnosis of liver lesions in man. Amongst hundreds of hepatotoxic substances that have to be considered is the naturally occurring mycotoxin, aflatoxin. The present finding that very low dietary concentrations of culture material of F. monilifrme MRC 826 cause a toxic hepatitis in primates, adds the unidentified hepatotoxin produced by this fungus to this list. This finding is considered to be very important in view of the widespread occurrence of F. monilifrme in a human staple food and the possibility of exposure of large population groups in Africa and elsewhere. The morphological picture of acute liver injury in vervet monkeys caused by F. moniliforme simulates acute viral hepatitis with intrahepatic cholestasis. Subacute and chronic stages simulate viral chronic active hepatitis. The lesions have some similarities to those induced in primates by aflatoxin (Svoboda, Grady and Higginson, 1966; Alpert, Serck-Hanssen and Rajagopolan, 1970; Wogan, 1973), and to the hepatitis due to aflatoxicosis in man (Tandon, Tandon and Ramalingaswami, 1978), but differ in several respects. The most significant similarities in the liver pathology caused by aflatoxin and F. monilaforme are the nuclear and nucleolar changes, i. e. marginal clumping of nuclear chromatin and large nucleoli with segregation of fibrillar and granular components. These characteristic changes caused by both agents may be the results of impaired RNA synthesis due to the inhibition of RNA polymerase activity (Svoboda et al., 1966; Jezequel, 1976). Nucleolar segregation occurs after the administration of some chemical carcinogens which are inhibitors of DNA and RNA synthesis (Monneron, 197 1; Jezequel, 1976) and is also known to be caused by actinomycin Duezequel, 1976). It remains to be seen whether the segregation of nucleolar components observed in the present experiment is also an early indicator of the hepatocarcinogenicity of F. moniliforme in vervet monkeys. Biochemical changes such as increased activities of AST, ALT and LD reflect hepatocellular damage and raised ALP, GGT and cholesterol activities confirm intrahepatic cholestasis (Popper and Schaffner, 1970) noted in light examinations. Chronic intoxication leads to and electron microscopic mononuclear infiltration, fibroblastic activity, focal condensation or collapse of the reticular structure and ultimately to liver cirrhosis (Read, 1979). Biotransformation of the toxic material by enzymatic processes such as oxidative hydrolysis or reduction in SER causes early cellular changes such as dilatation and glycogen accumulation in the cellular cytoplasm (Stenger, 1970). Cellular lesions during the first few days of the experiment were mild. The formation of whorls, lamellar structures, polysomes and lysosomes following further toxin administration can be interpreted as evidence of degeneration or regeneration (Trump, Dees and Shelburne, 1983). The increase in surface density of the SER may be related to hepatic cholesterol synthesis (Jones and Schmucker, 1977; Trump et al., 1983). Electron-dense granules or needle-like structures present in mitochondria were the result of liver cell injury. Some dense intramitochondrial inclusions and curled cristae may be an effect of cholestasis (Perez, Gorodisch and Demartire, 1969; Popper and Schaffner, 1970).

FUNGAL

HEPATITIS

IN

VERVETS

289

Increase in globulin, together with unchanged albumin concentration in serum, represents a reaction of the reticuloendothelial system in general and does not reflect liver cell damage (George and Watt, 1979). It can reflect increased activity of the immune system. Other observations such as increased concentrations of IgG and IgM in serum, IgG deposits, thick basal membranes in the liver sinusoids and increased severity of liver injury in response to a lower dose (0.1 per cent) of toxic material after 6 months of intoxication, tend to support the concept of a hypersensitivity reaction (Read, 1979). The importance of this reaction which can lead to liver cirrhosis, even if there is irregular exposure to low doses of a toxin as has been reported in chlorpromazine, halothane, alpha-methyldopa and isoniazid treatment (Read, 1979), warrants further investigation. A chronic feeding experiment of vervet monkeys with low dietary concentrations of F. monilifrme MRC 826 and attempts to isolate and chemically characterise the hepatotoxic metabolite(s) produced by this fungus are being continued. SUMMARY

The fungus Fusarium monilforme Sheldon is a common contaminant of maize (
We thank Dr P. G. Thiel for aflatoxin analyses and Mr J. W. Seier, Mr M. J. van Wyk, Mr F. S. Venter, Mr C. W. Woodroof and Mrs C. L. Schreuder for competent technical assistance.

290

K.

JASK’EWICZ

et d.

REFERENCES

i2lpert, E., Serck-Hanssen, A. and Rajagopolan, B. (1970). hflatoxin-induced hepatic injury in the African monkey. Archives of Environmental Health, 20, 7233728. Bancroft, J. D. and Stevens, A., Eds. (1977). Theory and Practice of Histological ‘Techniques. Churchill Livingstone, Edinburgh. Dreyer, J. J. and du Bruyn, D. B. (1968). Composition of a food mixture developed at the NNRI for supplementation of predominantly cereal diets. South i?frican Medical

Journal,

42,

600-604.

Gelderblom, (1984).

W. C. A., Thiel, P. G., Marasas, W. F. 0. and van der Merwe, K. J. Natural occurrence of fusarin C, a mutagen produced by Fusarium monitifrme. Journal of Agricultural and Food Chemistry, 32, 1064-1067. George, C. F. and Watt, P. J. (1979). The liver and response to drugs. In Liver and Biliary Disease. Pathophysiology, Diagnosis, Management. R. Wright, K. G. M. VI. Alberti, S. Karran, and G. H. Millward-Sadler, Eds, W. B. Saunders Company, London, pp. 344-377. Jezequel, A. M. (1976). Ultrastructural changes induced by drugs in the liver. In The Hepatobiliary System. W. Taylor, Ed, Plenum, New York. pp. 179-203. Jones, A. L. and Schmucker, D. L. (1977). C UI.rent concept of liver structure as related to function. Gastroenterology, 73, 833-85 1. Kriek, N. P. J., Kellerman, T. S. and Marasas, W. F. 0. (198laj. A comparative study of the toxicity of Fusarium verticillioides ( = F. monilzforme) to horses, primates, pigs, sheep and rats. Onderstepoort journal of I’eterinary Research, 48, 129913 1. Kriek, N. P. J., ?\/larasas, W. F. 0. and Thiel, P. G. (1981 b). Hepatoand cardiotoxicity of Fusarium verticillioides (F. monilzjorme) isolated from southern African maize. Food and Cosmetics Toxicology, 19, 447-456. Lynch, M. G., Raphael, S. S., Mellar, L. D., Spare, P. D. and Inwood, M. G. H., Eds, ( 1969). Medical I,aboratory Technology and Clinical Pathology. W. B. Saunders Company, London. Marasas, W. F. 0. (1982) Mycotoxicological investigations on corn produced in esophageal cancer areas in Transkei. In Cancer of the Esophagus. C. J. Pfeiffer, Ed., Volume 1. CRC Press, Boca, Raton, pp. 29940. Marasas, W. F. O., Kriek, N. P. J., Fincham, J. E. and van Renshurg, S. J. (1984b). Primary liver cancer and oesophageal basal cell hyperplasia in rats caused by Fusarium

moniliforme.

International

Journal

of Cancer,

34,

3833387.

Marasas, W. F. O., Kriek, N. P. J.? Wiggens, V. M., Steyn, P. S., Towers, D. J. and Hastie, T. J. (1979). Incidence, geographic distribution, and toxigenicity of Fusar-ium species in South African corn. Phytopathology, 69, 1181-l 185. Marasas, W. F. O., Nelson, P. E. and Toussoun, T. t1. ( 1984aj. Toxigenic Fusarium Species., Identity and Mycotoxicology. Pennsylvania State University Press, University Park, pp 216-221. Marasas, W. F. O., Wehner, F. C., van Rensburg, S. J. and van Schalkwyk D. J. ( 1981). Mycoflora of corn produced in human esophageal cancer areas in Transkei, southern Africa. Phytopathology. 71, 792-796. Monneron, A. ( 197 1). Action of some drugs on liver nuclei and polysomes. In Advances in Cytopharmacology, Volume 1. Raven Press, New York, pp. 131-138. Perez, V., Gorodisch, S. and Demartire, J. (1969). Oral contraceptives: Long-term use produces fine structural changes in liver mitochondria. Science, 165, 805-811. Popper, H. and Schaffner, F. (1970). Pathophysiology of cholestasis. Human Pathology, 1, l-25. Read, A. E. (1979). The liver and drugs. In Liver and Biliary Disease. Pathophysiology, Diagnosis, Management. R. Wright, K. G. M. M. Albertini, S. Karran, and G. H. Millward-Sadler, Eds, W. B. Saunders Company, London, pp. 823-847. Sherlock, S. ( 1970). The immunology of liver diseases. American Journal of Medicine, 49, 693-698.

Stenger,

R. J. (1970).

Organelle

pathology

of the liver.

Gastroenterology,

58,

555-574.

FUNGAL

HEPATITIS

IN

VERVETS

291

Svoboda, D., Grady, H. J. and Higginson, J. (1966). Matoxin B, injury in rat and monkey liver. .4merican Journal oj Pathology, 49, 1023-I 05 1. Tandon, H. D., Tandon, B. N. and Ramalingaswami, V. (1978). Epidemic of tosic hepatitis in India of possible mycotoxic origin. Arch&s oj‘Patholo,glr and Laboratoy Medicine, 102, 373-376. Trump, B. F., Dees, J. H. and Shelburne, J. D. (1983). The ultrastructure of the human liver cell and its common patterns of reaction to injury. In The Liver. E. A. Gall and F. IL. Mostofi, Eds, LVilliam and Wilkins, Baltimore, pp. 80-120. \l:ogan, G. N. #:1973). Mycotoxins and lilrer injury. In The Liver. E. A. Gall and F. Ii. Mostofi, Eds, \Yilliams and Wilkins, Baltimore, pp. 161-181. [Received-f& publication,

Januagl

13th, 1986i