the lectin-binding characteristics of aflatoxin b1 induced lesions in the rat liver

JOURNAL OF PATHOLOGY, VOL. 154: 269-276 (1 988)

THE LECTIN-BINDING CHARACTERISTICS OF AFLATOXIN B, INDUCED LESIONS IN THE

RAT LIVER DEBORAH J. PRITCHARD AND WIL,LIAM H. BUTLER

Department of' Pathology, British Industrial Biological Research Association, Woodmansterne Road, Carshalton SM4 4DS, U.K.

Received 13 May 1987 Accepted 29 July 1987

SUMMARY Hepatocellular carcinoma was induced in rats by administering aflatoxin B, (AFB,) for 6 weeks. Malignant

tumours were preceded by foci and nodules of altered hepatocytes of three histological types, composed of basophilic, eosinophilic, and vacuolated cells. In addition, there were areas of altered hepatocytes that were considered as hyperplastic.

Lectins were used as histochemical markers to compare the expression of membrane glycoproteins in hepatocellular carcinomas and hepatic nodules with non-nodular or control hepatocytes. There were marked changes in the lectin-binding patterns of the hepatocellular carcinoma cells and the eosinophilic nodules. The lectin- binding patterns of basophilic nodules, vacuolated nodules, and hyperplastic areas were similar to non-nodular or untreated hepatocytes. The similarity in the lectin-binding changes of the eosinophilic nodules and hepatocellular carcinomas suggests that the eosinophilic nodules may be an early stage in the development of carcinoma.

KEY woms-Lectins, aflatoxin, hepatocellular carcinoma, rat.

INTRODUCTION

The mechanisms of rat hepatocarcinogenesis have been extensively studied but there is often uncertainty about the histological criteria used for

In particular, there is debate about the relationship between the presumed 'precursor' nodular lesions and the development of hepatic neoplasia. The nodules have been described as hyperplastic, pre-neoplastic, or neoplastic, which illustrates the uncertainty of their biological n a t ~ r e . ~ - ~ The rat is widely used in toxicity bio- assays, and in some systems the lesions are used as indicators of carcin~genici ty .~,~ It is critical therefore that the biological nature of the hepatic nodules is determined if such assays are to play an important part in carcinogenicity testing pro- grammes. Several model systems have been developed in the rat to study the sequential events

Addressee for correspondence: Dr D. J. Pritchard, Depart- ment of Toxicology, Smith, Kline and French Research Ltd., The Frythe, Welwyn, Herts. AL6 9AR, U.K.

leading to carcinoma. Some of these models are based on a brief exposure to an initiating carcinogen followed by a period of liver cell proliferation induced either by partial hepate~tomy',~ or by the administration of necrotizing agents."- l 2 A number of hepatocarcinogens are also highly toxic to the liver, and it is often difficult to dissociate toxic injury from changes related to neoplasia. Inter- mittent feeding regimes have been developed to reduce the hepatotoxicity of compounds such as 2- acetylaminofluorene (2AAF)13,14 and ethionine. These model systems result in a large number of nodules some of which persist and others do not. While the persistent nodules are clearly more relevant to the development of neoplasia in the liver, their precursor role is still presumptive rather than e~tabl ished. '~ . '~ The hyperplastic or persistent nodules are often considered together as if they were a homogeneous group, but clearly this is not the case as most hepatic carcinogens give rise to a diversity of lesions." Biochemical, biological, and morphological studies have identified some

0022-341 7/88/03026949 $05.00 011988 by John Wiley & Sons, Ltd.

270 D. J . PRITCHARD AND W. H. BUTLER

Table I-Lectin abbreviations and sugar specificity ~

+ / - neuraminidase Sugar Lectin (source) Abbreviation pretreatment specificity

Triticum vulgaris WGA

A rachnis hypogea PNA

Glycine max SBA

Ricinus cornmz4nis RCA

Limux,flavus LFA

(wheat germ)

(peanut)

(soya bean)

(castor bean)

(slug)

- b-N-Acetyl glucosamine

+ P-D-galactosamine

+ a-N-Acetyl galactosamine

+ B-11-galactosarnine, U-D-gahCtoSamine

- N-Acetyl-neuraminic acid

characteristics common to hepatic nodules and carcinomas: for example, gamma-glutamyl transpeptidase, a-fetoprotein, alkaline phosphatase, glucose-6-phosphatase, and glucose-6-phosphate dehydrogenase have all been studied as poten- tial pre-neoplastic marker^.'^-'^ Fr om all these studies it is apparent that the histochemical phenotypes show heterogeneity in the nodular lesions and hepatocellular carcinomas, and although the histochemical phenotypes of the nodules are different from those of the carcinomas, the significance of these differences is not established.

There is evidence that malignancy is associated with abnormalities in gene regulation which may be expressed on the cell surface of hepatocytes in v i t ~ o . ' ~ The cell surface is considered to be an important site for the expression of neoplastic change, and in particular, membrane glycoproteins appear to be altered during neoplastic transform- a t i ~ n . ~ ~ , ~ ~ Small alterations in glycoprotein structure may result in the altered growth patterns and loss of cellular organization that are features of neoplastic development. These observations have encouraged the study of neoplastic cell membranes in an attempt to elucidate further the mechanisms of carcinogenesis. ' s Z 8

It has been established that there are significant changes in the ganglioside patterns of tumour cells, and in the rat there are reports that the ganglioside structure is simplified in hepatocellular carcinoma cells and some hyperplastic nodular cell^.^^.^' These changes in the glycoprotein structure appear to be an early event in neoplastic transformation and may be used to indicate the acquisition of characteristics which enable hyperplastic cells to be

distinguished from malignant ones. Lectins are sugar-binding proteins of non-immune origin that bind specifically to glyc~proteins~ ' and we report here their use as histochemical markers to compare the expression of plasma membrane glycoproteins in aflatoxin B, induced hepatocellular carcinomas and hepatic nodules with non-nodular or control hepatocytes.

Aflatoxin B, has been widely used as a model carcinogen to study the mechanisms of hepatocarcinogenesis in rat, and the histological changes induced by limited feeding regimes are well docu- mented.32-34 In this study we report changes in the membrane glycoprotein patterns of hepatocellular carcinoma and of some aflatoxin-induced hepatic nodules.

MATERIALS AND METHODS

Male weanling F344 rats were obtained from Charles River Laboratories and acclimatized for 7 days prior to the commencement of treatment. Rats were housed in groups of three in plastic cages with wire-mesh bases, fed a standard SDS No. 1 diet (Special Diet Services), and given water ad libitum. The animal room was maintained at constant temperature and humidity with a 12 h light and dark cycle. Treated rats were given crystalline aflatoxin B, dissolved in dimethyl sulphoxide orally three times each week for 6 weeks, and control rats were given equivalent volumes of the carrier solution. Treated rats each received a total of 1.35 mg of aflatoxin B, .

A minimum of four control and four test rats were killed at 4, 6, 10, 14, 24, 32, and 66 weeks after the

AFLATOXIN-INDUCED LIVER LESIONS 27 1

commencement of treatment. (The first two time points occurred during the treatment period.) At each time interval, rats were either perfused with 4 per cent gluteraldehyde or their livers were immersed in 10 per cent buffered formalin. Slices of formalin-fixed tissue were processed into paraffin wax and 5 pm sections were serially stained with haematoxylin and eosin for routine histological examination, and with a range of peroxidase- conjugated lectins for the histochemical study. Prior to lectin incubation, some sections were treated with neuraminidase to remove terminal sialic acid residues.35 The sections were then incubated with 2 : 1 methanol-hydrogen peroxide solution to block endogenous tissue peroxidase. This was followed by washing the sections in phosphate-buffered saline (PBS), pH 7.4 (Oxoid; Dulbeco ‘A’). Slides were then incubated with a range of peroxidase-labelled lectins at concentrations between 10 and 50 pg/ml for between 20 and 120min at room temperature (Table I). The peroxidase label was visualized by incubating the slides in a buffered solution of 3,3’- diaminobenzidine (0.5 mg/ml) and 0.015 per cent hydrogen peroxide for 15 min to produce a brown precipitate at the site of lectin binding.36 Sections were lightly counterstained with haematoxylin, dehydrated in alcohol, and mounted in DPX for examination at the light microscope. The specificity of lectin binding was demonstrated by co- incubating the sections with the lectins and either appropriate or inappropriate inhibitory sugprs.

RESULTS

Mucroscopic, findings At the interim kills the livers of treated rats

had mottled surfaces and often there were numerous pale raised areas or foci scattered over all lobes, At later stages there were adhesions between liver lobes, or between the liver and adipose tissue or the small intestine. Gross nodules were seen only at the end of the study when the livers of all treated rats bore large multiple cream-coloured masses.

Hist o logicai,findings

After 4 weeks of treatment there was a mild proliferation of bile ducts from the portal tracts to the sinusoids in the form of ‘oval’ cells. Peri-portal

hepatocytes were numerous with small nuclei and a basophilic cytoplasm. These were considered to be areas of hepatocyte hyperplasia, which in some cases extended throughout the liver lobe. By 6 weeks, in addition to these changes there were several small islands of altered cells distinct from the diffuse hyperplasia of peri-portal hepatocytes in that they formed discrete foci often compressing the surrounding parenchyma. These lesions were of two general types: one was composed of small cells with a basophilic cytoplasm and small regular nuclei, and the other was composed of larger, often atypical cells with an eosinophilic cytoplasm (Figs 1 and 2).

From 10 weeks the bile duct proliferation extended in some cases throughout the liver lobule. Individual cells throughout the liver had vacuolated cytoplasm and pyknotic nuclei and were recognized to be undergoing individual cell necrosis. The basophilic and eosinophilic foci had often developed into large discrete nodules and they compressed the surrounding hepatic tissue. The cellular atypia was often extensive in the eosinophilic nodules and some cells showed cytoplasmic vacuolation. In some cases, the larger nodules were of a mixed population containing both eosinophilic and basophilic cells. A third type of focal lesion was seen, consisting of cells with large cytoplasmic vacuoles, and this gave rise to pale staining foci (Fig. 1). In addition to the well- defined foci or nodules, there were areas of small hepatocytes with regular staining characteristics (Fig. 3) and although the lobular architecture was disrupted they did not compress the surrounding tissue and were therefore not true nodules. These apparent hyperplastic areas were seen in the liver of most treated rats from 10 weeks.

The nodular and hyperplastic lesions developed and persisted throughout the study. By 32 weeks, the eosinophilic nodules with more extensive nuclear and cellular pleomorphism were larger than the basophilic nodules.

At the termination of the study, the remaining seven treated rats had developed hepatocellular carcinoma, in addition to the range of simple nodular lesions previously described. There was minimal bile duct and peri-portal hepatocyte hyperplasia in most of the treated rats, and although most had both basophilic and eosinophilic nodules the livers of these animals were dominated by masses of hepatocellular carcinoma. The carcinomas were classified into three types according to the classifi- cation of Stewart and Sne11.37 The first type was an ‘adenomatous’ hepatocellular carcinoma with cells

212 D. J. PRITCHARD AND W. H. BUTLER

forming duct-like structures; the second, a ‘trabecular’ type carcinoma with thickened cords of hepatocytes; and the third, a more anaplastic ‘solid’ tumour devoid of trabecular structures with closely packed cells showing moderate amounts of cellular and nuclear pleomorphism. 3 2 These histological variants often occurred in the same animal. Only one rat from the study showed metastasis in the lung. Two of the rats killed at the end of the study had an extensive leukaemic infiltration throughout the liver.

Fig. 3-Liver 4 weeks after the end of AFB, treatment showing a hyperplastic area, demarcated by arrows, composed of small regular staining cells not compressing the surrounding tissue

Lectin-binding studies

and are summarized in Tables IT and 111. The results of these studies were semi-quantitated

Control liver-In the absence of enzyme digestion there was no SBA, PNA, or RCA staining on hepatocytes of control or non-nodular livers. When sialic acid was removed, the binding sites for these lectins

of hepatocytes. In some cases, there was also binding to the sinusoidal membrane although at the light microscope level it was not always possible to dis- tinguish this from staining of endothelial cells. Binding sites for WGA were not masked by sialic acid and staining was seen on the bile canalicular membrane and on the endothelial lining cells of control liver sections (Fig. 4).

Fig. I-Liver 4 weeks after the end of AFB, treatment showing a were then exposed on the bile canalicular membrane small basophilic focus (B) composed of small regular cells, and a small focus (V) composed of cells with vacuolated cytoplasm

Hepatocellular carcinoma-There were marked alterations in the lectin-binding patterns of the hepatocellular carcinomas compared with normal liver, although within a single tumour mass there was often heterogeneity in the binding patterns. In the ‘adenomatous’ and ‘trabecular’ tumours, the lectin-binding sites were heavily localized along the membrane or within the cytoplasm of most cells (Fig. 5). Other tumour cells were either negative or else lectin-binding sites were located in the cytoplasm. In the adenomatous tumours, there was some membrane staining with RCA and PNA in the

also changes in the lectin-binding patterns of the

Fig. 2-Liver at the end of a 6-week treatment with AFB,, showing a small eosinophilic nodule composed of pale staining atypical cells, with some cytoplasmic vacuolation

absence of neuraminidase digestion. There were

AFLATOXIN-INDUCED LIVER LESIONS 273

‘solid’ type, anaplastic hepatocellular carcinomas. In some cells the reaction product was clustered along the tumour cell membrane or clumped within the tumour cell cytoplasm (Fig. 6).

Eosinophilic nodules-There were considerable differences in the lectin-binding patterns of the eosinophilic foci and nodules compared with non- nodular tissue. There was either a total loss or reduction in lectin staining or a clustering of staining product on the bile canalicular membranes of nodular cells. In the larger nodules, there was often a localization of binding sites within the cytoplasm of some nodular cells (Figs 7 and S), in addition to the changes in membrane staining.

Basophilic nodules, vacuolated nodules, and hyperplastic areas-There were no alterations in the lectin-binding patterns of the basophilic foci and nodules, vacuolated foci, and hyperplastic areas compared with the control or non-nodular areas. The basophilic cells retained the even distribution of binding sites along the bile canalicular membrane and could therefore not be distinguished from non- nodular areas on the basis of their binding patterns (Fig. 9).

There was no staining with Limaxjavus (LFA) on non-nodular or any nodular hepatocytes that preceded carcinoma development. There was slight membrane and cytoplasmic staining on the luminal border cells of the ‘adenomatous’ hepatocellular carcinomas and in the cytoplasm of some solid, anaplastic carcinoma cells.

DISCUSSION

The histological changes induced by a limited feeding experiment with aflatoxin B, are similar to those reported by other worker^.^*.^^ Hepatotox- icity, as determined by single-cell hepatocyte necrosis and subsequent hepatocyte proliferation, has been proposed to be a prerequisite in the early stages of liver carcinogenesis. 12840 However, in the AFB, system the toxic damage was minimal, sug- gesting that this did not play a significant role in the development of malignancy. Other more toxic carcinogens such as 2AAF,I3 3-methyl- diamin~benzidine,~’ and ethionine when adminis- tered continuously give rise to oval cell and bile duct proliferation which may then lead to fibrosis and cholangiofibrosis. However, the role of oval cell and bile duct proliferation in carcinogenicity remains

unclear since, as in this study, neoplasia may develop in the absence of marked ductular cell proliferation.

There was a 100 per cent incidence of hepatocellular carcinoma after 66 weeks, preceded by a variety of hepatic foci and nodules. The basophilic and eosinophilic lesions occurred with a similar frequency in treated rats and it was not possible to identify a premalignant lesion based on their occurrence and incidence. There was a long latent period between the appearance of these nodular lesions and the development of overt hepatocellular carcinoma. It is clear that there were fewer carcinomas than there were foci and nodules, suggest- ing either that not all the foci and nodules progressed to carcinoma, or that within the period of study these lesions had not time to develop into carcinoma.

Changes in membrane glycoproteins are thought to result in the altered growth patterns and loss of cellular organization of neoplastic cells and therefore the lectin histochemistry provided information about the nature of hepatic nodules and their association with malignant development.

While there was variation and heterogeneity in the membrane glycoprotein changes of the hepatocellular carcinoma cells, there were often similarities with the distribution of membrane glycoproteins in the eosinophilic nodules. The basophilic, vacuolated, and hyperplastic lesions were similar to control or non-nodular tissue. In the carcinoma and eosinophilic nodular cells, the clustering of the lectin staining may be due to a redistribution of binding sites or to conformational changes in the membrane structure leading to an exposure or masking of specific sugar residues, rather than to de novo syn- thesis of glycoproteins. It has been shown that the number of glycoprotein residues is similar in transformed and control cells, and that the differential expression is due to a redistribution of existing binding sites within the membrane.42s43

Sialic acid is frequently the terminal residue on galactosamine sites, giving rise to the negative surface charge of cells, and changes in the expression of membrane-bound sialic acid are thought to be important in the manifestation of malignant behav- i o ~ r . ’ ~ , ~ ~ Sialic acid levels have been shown to be elevated in the glycoproteins and glycolipids of hyperplastic nodules and carcinomas induced by 2AAF,30 and the authors suggested that the increased expression of sialic acid levels in the nodules is evidence of the premalignant nature of these lesions.


Table 11-Summary of lectin binding in AFB,-induced hepatic nodules (A) Lectins with prior neuraminidase digestion

- __

SBA PNA RCA

Non-nodular Bile canalicular membrane Sinusoidal membrane Hepatocyte cytoplasm Portal tract vessels Bile ducts Endothelial cell

Basophilic nodule Bile canalicular membrane Sinusoidal membrane Hepatocyte cytoplasm Endothelial cell

Eosinophilic nodule* Hepatocyte membrane

Hepatocyte cytoplasm

Hyperplastic areas Bile canalicular membrane Sinusoidal membrane Hepatocyte cytoplasm

Vacuolated nodules Bile canalicular membrane Sinusoidal membrane Hepatocyte cytoplasm Endothelial cell

+ + + + + + + + +

- +/- +/-

+ + + + + + +I- +/-

[ + + I [ + + I

+/- [ + + I or + or +/-

+ + + + +/- +/ -

+ + + + + + +/ - +/-

+ -

+/- + + + + + + -

[+ + I or + -

+/- -

-

+ + + +/-

Key to semi-quantitative data: -, negative; + / - , minimal staining; +, mild staining; + +, moderate staining; [ ], clustering of binding sites.

*There was variation and heterogeneity in the lectin- binding patterns of some eosinophilic nodules, and this is represented by two values in the table.

Lirnux ftavus lectin has been demonstrated to bind specifically to sialic acid residues on hepato- ~ y t e s , ~ ’ but in this study there was no LFA binding on control or nodular tissue. This may be because on the rat hepatocyte membrane sialic acid is in a masked form, due either to its position within the glycocalyx or to the effects of fixation that prevent it binding to LFA. Sialic acid, however, is undoubt- edly present on the cell surface because its removal by neuraminidase unmasks pre-terminal galactosyl residues.

In the present study, sialic acid residues were visualized on the cell membrane of luminal border

Table 11-Summary of lectin binding in AFB,-induced hepatic nodules (B) Lectins without prior neuraminidase digestion

WGA LFA

Non-nodular Bile canalicular membrane Sinusoidal membrane Hepatocyte cytoplasm Portal tract vessels Bile ducts Endothelial cell

Basophilic nodule Bile canalicular membrane Sinusoidal membrane Hepatocyte cytoplasm Endothelial cell

Eosinophilic nodule* Hepatocyte membrane

Hepatocyte cytoplasm

Hyperplastic areas Bile canalicular membrane Sinusoidal membrane Hepatocyte cytoplasm

Vacuolated nodules Bile canalicular membrane Sinusoidal membrane Hepatocyte cytoplasm Endothelial cells

+ + + + + +

+/-

++ + + +l-

[+ + I or + [+ + I

+ + +/-

++ + + +/-

Key to semi-quantitative data: - , negative; + / - , minimal staining; + , mild staining; + +, moderate staining; [ ], clustering of binding sites.


cells and in the cytoplasm of some tumour cells but not on well-differentiated carcinomas, nodular, or hyperplastic lesions. Therefore there was no evidence in this study that the expression of sialic acid can be used as an early indicator of malignant behaviour .

Alterations in the expression of glycoproteins may be associated with a simple hyperplastic response of the cells following acute injury. If this were so, one would expect both the basophilic and eosinophilic nodules, which by definition are proliferative, to express similar changes in membrane glycoproteins.

Fig. 4 - The WGA-PO binding patterns in an area of control liver. Binding sites are located along the hepatocyte bile canalicular membrane and on the endothelial lining cells. Counterstained with haematoxylin

Fig. 5 - Adenomatous hepatocellular carcinoma showing the clustering of WGA-PO staining on the membrane or within the cytoplasm of tumour cells. Counterstained with haematoxylin

Fig. 6 - An anaplastic hepatocellular carcinoma showing the redistribution of WGA-PO staining on the membrane and within the cytoplasm of tumour cells. Counterstained with haematoxylin

Fig. 7 - Eosinophilic nodule showing the redistribution of WGA-PO staining in the nodular cells. Counterstained with haematoxylin

Fig. 8-A high power of the previous eosinophilic focus showing the clustering of WGA-PO staining on the membrane and within the cytoplasm of nodular cells. Counterstained with haematoxylin

Fig. 9 - A small basophilic nodule showing no difference in the distribution of WGA staining on the nodular cells compared with adjacent non-nodular tissue. Counterstained with haematoxylin

AFLATOXIN-INDUCED LIVER LESIONS 275

Table 111-Summary of lectin binding in AFB,-induced hepatocellular carcinomas (A) Lectins with prior neuraminidase digestion

SBA PNA RCA

‘Solid’ type anaplastic carcinomas* Hepatocyte membrane +/- [ + + I [ + + I

Hepatocyte cytoplasm +/- [ + + I -

Endothelial cell + o r + / - + Adenomatous and trabecular carcinomas* Hepatocyte membrane + + + + + + Hepatocyte cytoplasm +/- + -

Endothelial cell + + +

or - or +/-

or +/- or [+ + I or [+ + I

or [+ + I or [+ + I or +

Key to semi-quantitative data: -, negative; +/-, minimal staining; +, mild staining; + + , moderate staining; [ ], clustering of binding sites.

Therc was variation and heterogeneity in the lectin- binding patterns of carcinomas, and this is represented by two values in the table.

Table 111-Summary of lectin binding in AFB ,-induced hepatocellular carcinomas (B) Lectins without prior neuraminidase digestion

WGA LFA

‘Solid’ type anaplastic carcinomas* Hcpatocyte membrane + + +

or [+ + I Hepatocyte cytoplasm +/-

or + + Endothelial cell + Adenomatous and trabeculur carcinoma* Hepatocyte membrane ++ Hepatocyte cytoplasm + + Endothelial cell +

or [+ + I

or +/-

+ +

[ + + I or - + + or - +

Key to semi-quantitative data: - , negative; + / - , minimal staining; +, mild staining; + +, moderate staining; + + +, marked staining; [ ], clustering of binding sites.


Previous histochemical studies have shown that both the basophilic and eosinophilic foci and nodules have a heterogeneous pattern of enzyme histochemical reaction^.'^ However, in the present study this was clearly not the case since the alterations in glycoprotein expression were specific to the eosinophilic nodule and were similar to the glycoprotein expression of hepatocellular carcinomas. Thus, the similarity in the lectin-binding changes of the eosinophilic nodules and hepatocellular carcinomas suggests that the eosinophilic nodules may be an early stage in the development of carcinoma.

ACKNOWLEDGEMENT

This work was supported by the Ministry of Agriculture, Food and Fisheries, to whom our thanks are due,

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the lectin-binding characteristics of aflatoxin b1 induced lesions in the rat liver

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