Virchows Arch. B Cell. Path. 17,149--157 (1974) �9 by Springer-Verlag 1974

The Ultrastructural Effects of Aflatoxin B1 in the Rat Pancreas

M. S. Rao, D. J. Svoboda, and J. K. Reddy

Department of Pathology and Oncology, University of Kansas Medical Center, College of Health Sciences and Hospital, Kansas City, Kansas

l~eceived September 3, 1974

Summary. The acute effects of aflatoxin B 1 (AFB1) on the morphology of the acinar cells of the exocrine pancreas were studied in rats. By light microscopy, the changes were unremark- able except for slight decrease in number of zymogen granules. By electron microscopy, the changes observed were: (a) decrease in the number of zymogen granules and irregularity of the limiting membrane, (b) dilatation of Golgi vesicles, (c) dilatation of endoplasmic reticulum (ER) and disaggregation of ribosomes, and (d) areas of focal cytoplasmic degradation. These changes, although not specific, clearly suggest that aflatoxin B 1 is a pancreaticotoxic agent.

Introduction

Aflatoxins are a group of related toxic agents produced by the mold Asper- gillus flavus which has wide geographic distribution. The carcinogenic action of peanut meal contaminated with aflatoxins was first described by Lancaster etal. (1961) and, since then, various chemical forms of aflatoxins have been identified (Asao et al., 1963). The most toxic member of the group is aflatoxin B 1 (AFB1) (Nesbitt et al., 1962), a potent hepatocarcinogen in several species. The acute and chronic effects of AFB 1 on liver have been studied, in a wide variety of species such as, rats (Butler, 1966; Svoboda et al., 1966; Newberne and Wogan, 1968; Reddy and Svoboda, 1972), ducks (Carnaghan 1965), monkeys (Cuthbertson et al., ] 967 ; Svoboda et al., 1966 ; Alpert et al., 1970), marmosets (Linet al., 1974), and tree shrews (Reddy and Svoboda, unpublished data).

Biochemical studies in liver revealed that AFB 1 binds with DNA and inhibits DNA-dependent RNA polymerase activity (Clifford and Rees, 1966). In the pancreas AFB 1 caused activation of deoxyribonuclease, through interaction with DNA (Schabort and Pitout, 1971). Curiously, however, the morphologic effects of aflatoxin on the pancreas have not been reported. In view of the report that protein deficiency potentiates the aflatoxin toxicity (Madhavan and GopMan, 1965) and since AFB 1 contamination in foodstuffs is widespread (Marth, 1967) particularly in developing countries where protein-calorie malnutrition is prevalent, the present study was undertaken to ascertain the acute effects of AFB 1 on the morphology of pancreatic acinar cells.

Materials and Methods Inbred, male F-344 rats weighing 200-250grams (Simonsen Laboratory, Gilroy, Cali-

fornia) were used in these experiments. The animals were fed standard diet (Purina laboratory chow) ad libitum and had free access to water. The animals were divided into various groups and were injected intraperitoneally with aflatoxin B 1 (AFB1), dissolved in dimethylformamide

150 M. S. Rao et al.

Table 1. Dose and time schedule of administration of aflatoxin B 1

Group No. Dose No. of Sacrifice animals (kg/b.wt) doses

1 Aflatoxin a 4 1 mg 1 12, 24 hrs 2 Aflatoxin 12 250 (zg 1 12, 24, 48, 72 hrs 3 Aflatoxin 4 250 ~tg 2 b 72,96 hrs 4 Control c 2 2 72 hrs

2 96 hrs

a Aflatoxin was dissolved in dimethylformamide. b Aflatoxin, two doses at 0 and 48 hrs, two animals were sacrificed 24 hrs after second dose, and the remaining two animals 48 hrs after the second dose. e Controls received identical volume of dimethylformamide.

(DMF) as shown in Table 1. A similar volume of DMF alone was injected into control groups. Rats from each group were sacrified at various intervals (Table 1) under ether anesthesia. Portions of the pancreas were prepared for light microscopy by fixation in 10 % neutral buffered formalin. Sections 5 t~ thick of paraffin embedded tissues were cut and stained with hematoxy- lin and eosin. For electron microscopy, the tissues were fixed in S-collidine-buffered osmium After fixation for 2 hours at 0-4~ tissues were dehydrated in graded series of alcohol and embedded in Epon 812 (Luft, 1961). Ultra-thin sections were cut with an LKB ultra microtome using glass knives. Sections were stained with lead hydroxide and examined in an electron microscope. Pancreatic amylase activities were determined by radial diffusion method des- cribed by Belanger et al. (1970). Pancreatic homogenates (0.1%) were prepared with 0-01 M phosphate buffer, pi t 7.0. The homogenate was sonicated for one minute using sonifier-cel[ disruptor (Heat Systems-Ultrasonics, Inc., Plainview, Long Island, New York) and centri- fuged for 10 rain at 6000/rpm. The supernatant was used for amylase assay by the radial diffusion method by applying 25 ~tl of sample in a well on the agar-starch substrate (Belanger et al., 1970). A standard curve was obtained as described previously (Reddy et al., 1974, in press) by using known concentrations of 2x crystallized alpha-amylase-AA (hog pancreas; activity 717 units/rag; Schwarz/Mann Research Laboratories, Orangeburg, New York).

Results

W i t h a dose of 1 mg/kg body weight the animals did not survive beyond 24 hours. Therefore, the following descript ions of l ight and electron microscopic

changes are concerned with animals g iven one or two inject ions of af la toxin ]31 in

a dose of 250 ~g/kg b.wt. unless otherwise indicated. The food intake was normal

in animals t r ea t ed with 250 (zg/kg of AFB.

Light Microscopy . In all animals g iven a single dose of AFB1, the acinar cells

of the pancreas appeared unremarkable except for slight decrease in the number of

zymogen granules. I n animals g iven two doses of AFB1, however, the zymogen

granules were marked ly reduced in number . There was no acinar or isolated cell

necrosis and there was no increase in mitot ic act ivi ty . At no stage, in any group,

was an in f l ammato ry react ion seen.

Fig. 1. The pancreatic acinar cell of rat 24 hrs after a single dose of AFB 1 (250 ~g/kg b. wt.). Numerous free ribosomes are seen in the hyaloplasm. Endoplasmie retieulum (er) shows dissociation of ribosomes. Areas of focal cytoplasmic degradation (]cd) are also seen. (n)

Nucleus. • 15600

The Ultrastructural Effects of Aflatoxin B~ in the Rat Pancreas 151

m , ' ""~. "' . "': 4 ~'~,'.* ~ . . . . > "\ ~,.~l "" "," ~

. , ~ _ ::,I ~': ~" ~ ' ~ ' " .. ~ ' , ~ - - ~. * ~

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Fig. 2. Another pancreatic acinar cell 24 hrs after injection of AFB 1 (250 t~g/kg b.wt.) showing vesicular dilatation of the endoplasmic reticulum ( e r ) with few ribosomes attached. Zymogen

granules show peripheral areas of electron luceny ( p z ) , • 21600

1 5 2

m t ~ - - - - . . . . . . . .

, - , ~ -

M. S. Rao et al.

P

.a'?

Fig. 3. Areas of focal cytoplasmic degradation (fcd) in pancreatic acinar cells 48 hours after AFB x injection (250 tzg/kg b. wt.) showing endoplasmic reticulum (er) and degenerative mito-

chondria (m). x 24000

The Ultrastructural Effects of Aflatoxin B 1 in the Rat Pancreas 153

Ultrastructure

Control Group. Ultrastructure of exocrine pancreas in control rats was essen- tially similar to that of normal rats described by Ekholm et al. (1962). The acinar cell cytoplasm consisted of numerous mature zymogen granules, oval to elongated mitochondria, parallel arrays of abundant R E R and several Golgi complexes. The nuclei in many cells contained one to three nucleoli showing an admixture of granules and fibrils.

Experimental Groups : Cytoplasmic Changes : 12 hrs : The parallel arrays of R E R showed moderate segmental dilatation. However, the ribosomes appeared un- altered at this interval. The Golgi complexes were dilated in many acinar cells. Majority of the mitochondria were unremarkable except for an occasional one showing swelling and distortion of cristae. Mitochondrial changes appeared severe at this interval in animals given AFB 1 in a dose of 1 mg/kg. Zymogen granules appeared normal both in number and morphology. Occasional examples of focal cytoplasmic degradation were present in several ceils.

24 hrs. At this interval, the cytoplasm showed extensive changes. R E R was markedly dilated and several vesicles were devoid of ribosomes. Numerous aggregates of free ribosomes were seen in the cytoplasm (Fig. 1). Mitochondrial changes at this interval were similar to those observed at 12 hr. The Golgi complex was dilated and associated with numerous smooth-walled vacuoles. Reduction in the number of mature zymogen granules was a prominent feature at this stage. Several zymogen granules showed structural abnormalities such as variation in size, shape and electron density of the zymogen granule content and discontinu- ities in the limiting membrane (Fig. 2). In addition, several large empty vesicles with a diameter equal to or slightly larger than that of mature zymogen granules were seen in the cytoplasm of several acinar cells. Several acinar cells at this interval contained many areas of focal cytoplasmic degradations.

48, 72 and 96 hrs. During these intervals, the cytoplasmic alterations appeared progressive. Focal cytoplasmic degradations were predominant at 48 and 72 hr, consisting of single membrane limited areas containing either recognizable cyto- plasmic organelles, such as mitochondria and segments of endoplasmic reticulum or dense lamellar bodies (Fig. 3). Majority of acinar cells were almost totally devoid of mature zymogen granules by 48 hr (Figs. 4 and 5). Several single membrane bound vesicles with no electron opaque secretory material were frequently observed. The limitihg membrane appeared irregular and broken (Fig. 4). After two doses, in animals killed at 72 and 96 hrs, the changes in the R E R and Golgi complex persisted. Mitochondria were swollen and showed irregularities of the outer membrane. Several mitochondria were seen in various stages of degeneration leading to myelin body formation (Fig. 5).

Nuclear Changes. Except for slight to moderate enlargement of the nucleoli, no other alterations were noted in the nuclei in any of the groups at the intervals studied.

Pancreatic Amylase Levels. There was a significant difference in the amylase

Fig. 4. Apical cytoplasm of the pancreatic acinar cells 48 hours after AFB 1 injection (250 ~g/kg b. wt.) showing many membrane bound vacuoles (v). Membrane of some of the vacuoles is

broken (arrows). (L) lumen • 14400

154 M.S. Rao et al.

The Ultrastructural Effects of Aflatoxin B 1 in the Rat Pancreas 155

concentration in pancreatic tissue homogenate at 24 hr in animals t reated with AFB~ when compared to controls. At 48 and 72 hr, the pancreatic enzyme concen- trat ion in rats given two doses of AFB 1 was appreciably lower.

Discussion

The observations made during the present investigation indicate a series of alterations in cytoplasmic organelles of pancreatic acinar cells in animals t reated with AFB1.

The RER, the organelle concerned with protein synthesis (Siekevitz and Pala- de, 1953; Weiss, 1958), showed marked vesicular dilatation with disaggregation of ribosomes which probably indicates decreased protein synthesis. The progres- sive decrease in the number of zymogen granules and decrease in the pancreatic amylase content between 24-72 hr after AFB 1 t reatment also suggests that AFB 1 may inhibit protein syntehsis in the exocrine pancreas. The cause of vesicular dilation of the endoplasmic reticulum is not clear; it probably represents a non- specific response to AFB 1 injury since such changes are known to occur in a variety of cells as a response to injuries of various types (Trump and Ericsson, 1965) and in pancreas with various chemicals such as ethionine (Ekholm et al., 1962) and acetyl aminofluorine (Flaks and Lucas, 1973).

The single membrane bound vesicles which made their appearance at 24 hr and persisted at 48 and 72 hr in the cytoplasm of acinar cells, were abundant in rats t reated with two doses of AFB r Some of these vacuoles contained central electron dense area and some were completely electron lucid. These changes probably represent defective formation of zymogen granules because of decreased protein synthesis. The changes in RER, Golgi complex and mitochondria also indicate alteration in protein synthesis.

Mitochondrial changes were marked in animals which were given two doses of aflatoxin B r These changes resembled those seen in livers of animals treated with AFB 1 (Theron, 1965) or carbon tetrachloride (Christie and Judah, 1954). Similar changes were described by Scott and Vermillion (1966) in acinar cells of pancreas of rats deprived of lysine. These authors suggested that the mitochondrial changes were reflections of cellular inactivity.

Hyperplasia and dilatation of the Golgi complex (associated with an increase in smooth walled vesicles) is probably due to an a t tempt at compensation for low output (as a result of decreased protein synthesis), since Golgi complex is known to participate in packaging zymogen proteins.

In control animals the areas of focal cytoplasmic degradation in pancreatic acinar cell cytoplasm were noted infrequently and when present they appeared as very small foci. In animals t reated with one or two doses of AFB1, the size and number of focal cytoplasmic degradations increased greatly. Focal cytoplasmic degradation does not imply any specific response, but represents a common pat tern of cellular reaction to various toxic agents such as B-3-thienyl-Dl-alanine,

Fig. 5. Pancreatic acinar cell after 2 doses of AFB 1 (250 izg/kg b.wt.) showing pleomorphic mitoehondria (m) in various stages of degeneration. Membrane bound vacuoles (v) are also

markedly increased. X 21600

156 M.S. Rao et al.

ethionine , and puromyc in (Hruban et al., 1962; H e r m a n and Fi tzgera ld , P. J . , 1962; Ekho lm et al., 1962; Longnecker et al., 1968), or to any o ther condi t ion in which p ro te in synthes is is decreased (Scot t and Vermill ion, 1966). Various cyto- p lasmic organelles, such as endoplasmic re t icu lum and mi tochondr ia , were common componen t s of the loci of degrada t ion . I n these s tudies we d id not f ind zymogen granules in the lesions. The mechanism by which focal cy toplasmic degrada t ion occurs is uncer ta in . H r u b a n et al. (1962) sugges ted t h a t pinocytosis , phagocy tos i s and seques t ra t ion m a y cont r ibu te to the deve lopmen t of such a l te ra t ions .

Segregat ion of the f ibr i l lar and granu la r components of nucleoli as seen in the hepa tocy te s wi th af la toxin and with var ious o ther carcinogens (Svoboda et al., 1966 a n d 1967) and also in pancreas with ac t inomyc in D in mice (Rodriguez, 1967), was not observed in pancrea t i c ac inar cells wi th A F B 1 t r e a tme n t . I t was sugges ted t h a t a f la toxin B 1 and o ther hepa tocarc inogens inhibi t the synthes is of D N A - d e p e n d e n t R N A (Sporn et al., 1966; R e d d y and Svoboda, 1968) and t h e r e b y decrease p ro te in synthesis . But le r (1966) descr ibed cy toplasmic changes independ- ent of nucleolar changes in hepa tocy te s of ra t s t r e a t e d with af la toxin. J6z~quel and Be rnha rd (1964) descr ibed segregat ion of nucleolus in the ra t pancreas wi th normal R E R and zymogen granules a f te r admin i s t r a t i on of ac t inomyc in D.

I t is ev iden t from our expe r imen ta l s t u d y t h a t A F B 1 is pancrea t ico tox ic in add i t i on to i ts well known hepa to tox ic effects.

This work was supported by Public Health Service Contract CP-3271 from the Division of Cancer Cause and Prevention, National Cancer Institute.

We thank J. D. Prasad for the excellent technical assistance and Miss Juanita Stika for help in preparing this manuscript.

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Janardan K. Reddy, M. B., B. S., M. D. Department of Pathology and Oncology University of Kansas Medical Center College of Health Sciences and Hospital Rainbow Boulevard at 39th Street Kansas City, Kansas 66103, USA