Exp. Path., Bd. 12, S. 329-335 (1976)

Institute of Pathology, Wilhelm-Pieck-University Rostock (German Democratic Republic)and Department of Anatomy, University of Manitoba, Winnipeg, Manitoba (Canada)

Cytotoxicity of the pyrrolizidine alkaloid fulvineto pancreatic acinar cells in the rat

Light microscopic, histochemical and ultrastructural studies

By H.-P. PUTZKE and T. V. N. PERSAUD

With 6 figures

(Received June 16, 1976)

Key-words: pyrrolizidine alkaloid; fulvine; cytotoxicity; exocrine pancreas; pancreopathy; rat

SummaryVeno-occlusive disease of the liver is caused by the hepatotoxic pyrrolizidine alkaloid fulvine

which is found in the plant Crotola'ria fulva. Histological, histochemical and electronmicroscopicstudies carried out in Wistar rats showed that an aqueous solution of fulvine produced an acutepancreatopathy with interstitial oedema and secondary hypoxic dystrophy of the acinar cells, whenadministered at doses of 10 and 20 mg/kg body weight.

The pyrrolizidine alkaloids are naturally occurring plant toxins which possess a widerange of biological activities and are encountered in different parts of the world. Thesesubstances are considered important in the aetiology of certain diseases, including hepatichaemorrhagic necrosis, megalohepatocytosis, hepatic veno-occlusive lesion, liver cirrhosisand liver neoplasm. It is now established that veno-occlusive disease of the liver is causedby the hepatotoxic pyrrolizidine alkaloid fulvine derived from the leaves and flowers of theplant Crotolaria fulva (BRAS et al. 1954, McLEAN et aJ. 1964, DAVIS and PERSAUD 1970).

Hepatocellular degeneration, damage to the sinusoidal Rupffer cells, and obliterativechanges in the perisinusoidal spaces of Disse in the central region of the hepatic lobule werethe earliest changes observed in the liver following administration of fulvine to rats (PUTZKEet al. 1972). The changes present in the kidneys were less pronounced; hyaline degenerationof the proximal convoluted tubules concomitantly induced by the alkaloid may be accountedfor by a functional adaptation of the tubular epithelium to proteinuria (PERSAUD et al.1970). In order to determine whether the direct toxic action of fulvine was restricted to theliver, we also examined the exocrine pancreas of the treated animals, since the acinar cellsare known to be a highly sensitive indicator of drug toxicity.

Material and methodsFifty inbred male Wistar rats (130-150 g), of which ten served as controls, were used in this

investigation. The animals were fed a standard rat diet ad libitum and had free access to water.Fulvine was administered as an aqueous suspension to the animals by means of a stomach tube asfollows:a) Single doses of 5, 10 and 20 mg fulvine kg/body weight. Each treatment group consisted of ten

animals, and from each two were killed at intervals of 1, 2, 3, 7 and 14 days following treat­ment.

b) Fulvine was administered daily at a dose level of 1 mg/kg for 3 and 5 days respectively to tW(}groups of five animals each. The animals were killed 24 hours following the last treatment. Smallpieces of the pancreas were excised and prepared for light microscopic (HE and Goldner stainingtechniques), histochemical (tryptophan reaction), enzyme-histochemical (alkaline phosphatase,.acid phosphatase, non-specific esterase, adenosine triphosphatase, cytochrome oxidase, succinate·dehydrogenase) and electron microscopic studies (fixed in osmium tetroxide and embedded inVestopal) as described previously (PERSAUD et al. 1970, PUTZKE 1972).

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Fig. 1. Extensive interstitial oedema of the pancreas, 48 hrs. after treatment with fulvine at a dose­level of 10 mg/kg body weight. HE, X 90.Fig. 2. Moderately increased (a) and markedly reduced (b) zymogen granules in the acinar cellsafter fulvine treatment at dose-levels of 5 X 1 mg/kg and 1 x 20 mg/kg respectively. Tryptophanreaction, x 240.

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Results

Light MicroscopyAcute inter- and intra-lobular oedema of varying degrees and extent was detected in the

exocrine pancreas (fig. 1), particularly with higher dose levels of fulvine (5, 10 and 20 mg/kgbody weight). These changes were maximal, two to three days following treatment, andthereafter the severity declined. Acinar or isolated cell necrosis was not observed, the bloodvessels appeared unaffected, and there was no sign of an inflammatory reaction.

The number of zymogen granules in the acinar cells were increased following treatmentat lower dose levels (fig. 2a). Secretory products were scanty in the intercalated ducts. Incontrast, zymogen granules in the acinar cells were markedly reduced (fig. 2b) in animalsreceiving high doses of fulvine (10 and 20 mg/kg body weight). Nevertheless, these granulesappeared as increasingly conspicuous clusters in the acinar cells up to the third day of treat­ment, giving rise to a marked asynchronous state of secretory activity.

Electron Microscopy

Ultrastructurally, there was evidence of marked interstitial oedema and the intercellularspaces between acinar cells, as well as those between acinar cells and capillaries, were severelydilated (fig. 3). In addition, individual histiocytes appeared activated.

Deposits of lipid droplets were localized in the rough endoplasmic reticulum of manyacinar cells (fig. 3). In other acinar cells, the tubular elements of the granular endoplasmicreticulum were moderately distended and there were numerous small cytolysosomes, com­bined with evidence of recent focal cytoplasmic degeneration (fig. 4).

:fFig. 3. Marked oedema between acinar cells and capillaries after fulvine treatment (1 x 10 mg/kg).The basal cytoplasm of surrounding acinar cells shows clusters of lipid droplets. x 20,000.

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Fig. 4. Acinar cells with distended granular endoplasmic reticulum showing numerous small cytolyso­somes and moderate hydropic swelling of the nndeus. x 20,000.

In some cases, the acinar cells were slightly hypertrophied and showed swelling of themitochondria with shortening of the cristae. Occasionally, the mitochondria revealed smallinvaginations. Other acinar cells displayed loss of cytoplasmic ground substance, dilatationof the rough endoplasmic reticulum (fig. 5), r.,nd vacuolisation of the Golgi :1pparatus.

In areas free of oedema, the endoplasmic reticulum of the acinar cells was constrictedand the elongated mitochondria appeared dense with numerous granules in the matrix.The Golgi apparatus was enlarged on account of an enhanced formation of Golgi vacuolesand amorphous secretory precursor gmnules. However, the zymogen grr.,nules themselveswere markedly reduced in both size and quantity (fig. 6).

Fig. 5. Portion of dystrophic acinar cell (left) with oedema of cytoplasmic ground substance andvacuolisation as well as extensive reduction of the rough endoplasmic reticulum. x 20,000.

Fig. 6. Supranuclear region of acinar cell two days after treatment with fulvine (20 mg/kg). Modera­tely enlarged Golgi apparatus showing increl1sed formation of vacuoles and amorphous secretoryprecursor droplets. The mature zymogen granules are reduced in both size and quantity. Note theseverely constricted endoplasmic reticulum and the elongated and electrondense mitochondria.x 9,600.

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Enzyme HistochemistryAcid phosphatase activity was slightly increased between one to three days following

fulvine treatment at dose levels of 5, 10 and/or 20 mg/kg body weight. A moderate increasein the activity of non-specific esterase was likewise observed following treatment with fulvineat a dose level of 1 mg/kg body weight. However, the activity of the esterase was distinctlyreduced one day after treatment with fulvine at dose levels of 5 and 10 mg/kg body weight.The decline in activity was only slight on the second and third days of treatment.

The activity of cytochrome oxidase was moderately reduced up to three days after treat­ment with the alkaloid (5, 10 and 20 mg/kg body weight).

Significant changes in the activity of alkaline phosphatase, adenosine triphosphatase,and succinic dehydrogenase were not detected following any of the treatment schedules.

Discussion

Evidence of a pronounced cytotoxic effect on the rat exocrine pancreas was not observedfollowing treatment with fulvine at low dose levels of 1 mg/kg body weight, administeredover a period of 3-5 days or after a single injection of 5 mg/kg body weight. The secretoryactivity of the acinar cells was disturbed slightly as evident from the accumulation of enzymegranules in the cells, and a concomitant increase of the tryptophan and non-specific esterasereactions. Such changes could be due primarily to a lack of the stimuli essential for the ini­tiation of secretory activity.

On the other hand, treatment of the animals with fulvine at higher dose-levels (10 and20 mg/kg body weight) produced significant morphological alterations in the exocrinepancreatic cells. A marked localized inter- and intralobular oedema was evident with dila­tation of the acinar and acino-capillary interstitial spaces, as it is frequently observed alsoin more advanced forms of Flexner's dysenteric endotoxin (LETTERER 1950).

Two severe levels of changes were evident in the acinar cells adjacent to the oedematousregions, although not all cells of the acinus were involved.

1. Acinar cells with reversible changes, showing dilatation of the cisternae of the roughendoplasmic reticulum, lipid accumulation in the basal portion of the cytoplasm, mitochon­drial swelling, and moderate hydropic swelling of the nucleus. The cytoplasmic degradation,formation of small cytolysosomes, and the enhanced activity of acid phosphatase, wereindicative of a progressive separation process.

2. Irreversible damage of the acinar cells with vesiculation and vacuolization of the roughendoplasmic reticulum, hydropic swelling of the ground substance, vacuolization of theGolgi apparatus, and severe mitochondrial swelling.

It appears that the acinar cells lesions observed after fulvine administration were prima­rily due to circulatory disturbances, and that the subsequent hypoxia was caused by oedema.Evidence in support of this assumption was the lipid accumulation, mitochondrial swelling,and the decrease in cytochrome oxidase activity. The reason for the in.terstitial oedema isnot readily recognizable. Although capillary wall lesions were not evident, it still seemsappropriate to consider the oedema as due to either vascular lesions or haemodynamicchanges, particularly in view of the accompanying severe hepatic lesions (parenchymalnecrosis, dilatation of the sinusoids, hyperaemia, and haemorrhage) which were also present(PERSAUD et 301. 1970). Whether acute cardiac insufficiency was a contributing factor remainsstill to be determined.

Treatment with fulvine at high dose-levels produced secretory disturbances in the acinarcells, which themselves did not reveal any significant structural changes nor any associationwith the interstitial oedema. The ultrastructural observations (engorged endoplasmic reti­culum, elongated and dense mitochondria, enlarged Golgi apparatus with the formation ofvacuoles which were either empty or containing homogenous precursor secretory material,and the presence of relatively fewer and smaller zymogen granules in the apical region of theacinar cells) indicated that the secretory activity of these glandular cells was generally

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reduced. This may be attributed to either a reduction in the humoral stimulus for secretionor to a deficiency in essential protein resulting from the hepatic damage. In other experi­mental series, malnutrition and hunger produced similar changes in the exocrine pancreas,where in extreme cases a cytolysosomal reaction was also induced (RICHTER et 21. 1971,LAZARUS and YOLK 1965, NEVALAINEN and JANIGAN 1974).

It would appear from the observations of the present investigation that high doses offulvine (10 and 20 mg/kg body weight) caused some damage to the exocrine pancreas, yetan immediate toxic effect of the substance on the parenchymal cells can be largely ruled out.The interstitial oedema of the pancreas, induced at the same time as the hepatic damage,caused hypoxic dystrophy and focal necrosis of the acinar secretory cells in the form of anacute pancreatopathy which is likely reversible.

LiteratureBRAS, G., D. B. JELLIFFE and K. L. STUART, Veno-occlusive disease of the liver with non-portal

type of cirrhosis, occurring in Jamaica. Arch. Pathol. 1i7, 285-300 (1954).DAVIS, W. G., and T. V. N. PERS<\UD, Recent studies on the active principles of Jamaican medicinal

plants. W. I. Med. J. 19, 101-110 (1970).LAZARUS, S. S., and B. W. YOLK, Ultrastructure and acid phosphatase distribution in the pancreas

of rabbits. A comparison of alterations following protein deficient and semistarvation diets.Arch. Pathol. 80, 135-147 (1965),

McLEAN, E. K., G. BRAS and P. GYORGY, Veno-occlusive lesions in livers of rats fed Crotolaria fulva.Brit. J. Exp. Pathol. 04, 242-247 (1964\.

NEVALAINEN, T. J., and D. T. JANIGAN, Degeneration of mouse pancreatic acinar cells during fasting.Virchows Arch. Abt. B. Zellpath. Hi, 107-118 (1974).

PERSAUD, T. V. N., H.-P. PUTZKE, D. TESSMANN and A. BIENENGRABER, Microscopic and enzymehistochemical studies on the liver and kidney of rats treated with fulvine, the toxic alkaloidalconstituent of Crotolaria fulva. Acta Histochem. 37, 369-378 (1970).

PUTZKE, H.-P., T. V. N. PERSAUD and A. BIENENGRABER, Fine structure of the liver in experimentalveno-occlusive disease. Exp. Path. 6, 325-332 (1972).

RICHTER, K., H.-P. PUTZKE and A. BIENENGRABER, Histochemische und elektronenmikroskopischeUntersuchungen zur Sekretionskinetik der Azinuszelle des Rattenpankreas im Hungerzustand.Exp. Path. 3, 192-199 (1971).

SCHOENTAL, R., Alkaloidal constituents of Crotolaria fulva Roxb., fulvine and its N-oxide. Aust. J.Chern. 16, 233-238. (1963).

Authors' addresses:Dr. sc. med. H.-P. PUTZKE, Pathologisches Institut der Wilhelm-Pieck-Universitat,

DDR - 25 Rostock, StrempelstraBe 14;Prof. Dr. sc. med. T. V. N. PERSAUD, Department of Anatomy, University of Manitoba, 730 WilliamAvenue, Winnipeg, Manitoba (Canada R3E OW3).

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