10 Experimental liver tumours in animals

10 Experimental liver tumours in animals P. GRASS0

Anatomically, the liver sits astride the main pathway by which substances absorbed from the intestine gain access to the systemic circulation. Biochemically, it possesses a vast array of enzymes which are capable of metabolizing the majority of foreign compounds and most endogenous ones. It comes as no surprise that the liver is the organ most frequently affected adversely in toxicological studies on rodents. According to Rowe et al (1959) and Weil and McCollister (1963), adverse effects on the liver occurred in one out of every four or five toxicological studies. Although no comparative studies of this nature have been conducted recently, most toxicologists would support the figures obtained by these authors. The liver is also the organ most frequently affected in carcinogenicity studies in rodents. In fact, in a survey of 97 compounds that had been adequately tested for carcinogenicity with a positive result, it was found that liver cancer was produced by 37 of them, cancer of the lung (the organ next most frequently affected) was produced by 17, of the stomach by 9, of the bladder by 7 and of the kidney by 2. In most of these instances the liver was the only organ affected, but in a few instances cancer was found in other organs as well (IARC, 1982). The frequency with which this organ is affected in chemical carcinogenicity studies contrasts sharply with the very few human carcinogens known to affect it. In fact, out of 40 or so compounds and processes that are known to be responsible for producing cancer in man, only three affect the liver - aflatoxin, vinyl chloride and arsenic (IARC, 1979b). The same contrast is observed if one compares the natural incidence of primary hepatic tumours in rodents and man. The natural incidence in rats is in the range of 0 to 6% (Keysser et al, 1980; Grass0 P, personal observation), while that in mice is from 0 to 100% (Grass0 and Hardy, 1975). In man, it is in the order of 0.04% in the Western World and up to 0.12% in some parts of Africa and Asia (Higginson, 1963). Despite these major differences in natural incidence and sensitivity to carcinogens, the induction of hepatic cancer in laboratory rodents is still regarded as a valid indication of cancer risk for man - not only for hepatic cancer but for cancer in other organs as well. For this reason, great attention has been given over the last two decades to the histogenesis and pathogenesis of hepatic tumours, and in particular to the characterization of the early stages BailliPreS

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of tumour development, in the expectation that such studies would provide an insight into the development of hepatic cancer in man and possibly of cancer in other organs as well. HEPATOCARCINOGENIC

CHEMICALS

A wide variety of chemicals are known to be capable of producing tumours in experimental animals. The best documented of these are: 1. 2. 3. 4. 5. 6.

liver

the nitrosamines; the aflatoxins; the chlorinated hydrocarbons; the hypolipidaemic agents (including di-2-ethylhexylphthalate); the pyrrolizidine alkaloids; miscellaneous compounds such as phenobarbitone (Thorpe and Walker, 1973) 4-dimethylaminoazobenzene (Druckrey, 1959), 2-acetylaminofluorene (Edwards and White, 1941), butylated hydroxytoluene (Olsen et al, 1986), cycasin (Laqueur, 1968) and, o-aminoazotoluene (Andervont, 1950).

The nitrosamines are versatile carcinogens and affect virtually every organ of the rodent. They do not occur naturally but are produced readily by chemical interaction between primary or secondary amines and nitrites or nitrates. Over 200 nitroso-compounds have been synthesized and most of them are carcinogenic. The generic formula is R Rl>

N.NO

In general terms the symmetrical dialkyl nitrosamines (e.g., dimethyl- and diethyl-) are hepatocarcinogenic, but many of them induce tumours in other organs as well. The asymmetrical nitrosamines usually spare the liver and produce tumours in other organs, chiefly the oesophagus. The cyclic nitrosamines do not appear to have a predilection for any particular organ but some (e.g., N-nitrosopiperidine and N-nitrosopyrrolidine) may cause liver tumours in rats. There are many other nitrosamines which cannot be placed in either of the classes just mentioned and are grouped chemically as ‘Nitrosamines with functional groups’. The major target organs are limited to the liver, oesophagus and bladder, despite the fact that the chemical structure of the substituent group is very diverse indeed (Magee, Montesano and Preussmann, 1976; Preussmann and Stewart, 1984). The liver tumours produced by the nitrosamines are generally hepatocellular, but a few tumours of endothelial cells or bile ducts have been reported (Peto et al, 1984). Nitrosamines show a remarkable species versatility: hepatic or other tumours have been induced in all of the 15 or so species tested so far (Grasso, 1981). The fungal toxin aflatoxin Bl is the best known of the family of aflatoxins and readily induces hepatocellular tumours in the liver of several strains of rat.

EXPERIMENTAL Table 1. Some hydrocarbons.

LIVER

TUMOURS

examples

of tumour

Liver

Pesticides DDT Dieldrin Chlordane Chlordecone (Kepone) Heptachlor Hexachlorocyclohexane Methoxychlor Mirex Polychlorinated biphenyls Toxaphen Pentachlorophenol Solvents Carbon tetrachloride Chloroform 1,1,2,2-Tetrachloroethane Tetrachloroethylene 1,Zdichloroethane 1,1,2-trichloroethane Organs affected 6 = mononuclear

185

IN ANIMALS production

tumours

in liver

by chlorinated

in

Rats

Mice

-

+ +

fl,+ fl)-

++ + ++ +-

Mutagenicity

--(:,2) +

+ + + +

- (T4) -

+ (WI

other than liver: I= thyroid; phagocyte system.

of rats and mice

+ 2 = kidney;

(IARC

Reference Monographs)

-ve -ve ? -ve --ve -ve -ve -ve -ve 7 -ve

Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol. Vol.

5 5 20 20 20 20 20 20 18 20 20

(1974) (1974) (1979a) (1979a) (1979a) (1979a) (1979a) (1979a) (1978) (1979a) (1979a)

+ve -ve fve --Ye +ve -ve

Vol. Vol. Vol. Vol. Vol. Vol.

20 20 20 20 20 20

(1979a) (1979a) (1979a) (1979a) (1979a) (1979a)

3 = stomach;

4 = spleen;

5 = lung;

The other well-known aflatoxins, B2 and Gl, (Wogan, 1964) and their hydroxy metabolites M 1 and M2 (Wogan and Paglialunga, 1974), which are excreted in the milk, are also carcinogenic to the rat liver, but are 500 to 1000 times less potent than Bl. The mouse is much less susceptible to the carcinogenic activity of aflatoxin Bl than the rat (Barnes, 1970). The other aflatoxins have not been as thoroughly investigated in the mouse as aflatoxin Bl. Aflatoxin Bl is a versatile carcinogen and has produced tumours in the stomach, kidney and colon of rats as well as in the liver (Butler and Barnes, 1968, Ward et al, 1975). The other aflatoxins do not appear to share this versatility. Aflatoxin Bl has induced hepatic tumours in primates and other species in addition to the rat (Busby and Wogan, 1984). The chlorinated hydrocarbons are much more selective in their ‘target’. Most of those that are carcinogenic produce tumours in the liver. A few of them, however, produce tumours in other organs as well, notably in the kidney and thyroid. The liver tumours are hepatocellular in origin. The mouse liver is particularly susceptible to the carcinogenic activity of these chemicals, the rat liver much less so. In fact there are many compounds in this group which are only carcinogenic in the mouse (Table 1). Compounds with a hypolipidaemic action (clofibrate, fenofibrate, nafeno-

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pin, WY-14,643) are also ‘organ-specific’ in their carcinogenic effect, affecting the liver of both rats and mice. Some of these agents have been claimed to produce benign tumours of the exocrine pancreas in rats but not in mice. The evidence for this is, at present, inconclusive (Cohen and Grasso, 1981; Reddy and Lalwani, 1984). As far as can be ascertained, no liver or any other type of tumour has been induced by this class of compound except in rats, mice and hamsters. The pyrrolizidine alkaloids are derived from various species of Senecio and are the active toxic ingredient in aqueous extracts of Senecio, which are used in some parts of the world as a herbal remedy. In man, they produce venoocclusive disease of the liver, characterized by occlusion of the hepatic venous radicles. In animals, this group of alkaloids are potently toxic and hepatocarcinogenic in rats and mice (Schoental, 1968), but do not produce venous occlusion. Although many species of laboratory animals from mouse to primate have been employed in the study of hepatocarcinogenesis, the vast majority of mechanistic studies have been carried out in mice and rats and the following account of the pathogenesis of experimentally produced liver cancer in animals will be confined to these two species. There are two principal stages in the development of experimentally induced cancer: the period prior to the development of tumours - the socalled ‘latent’ period - and the period during which tumours appear and develop. THE LATENT

PERIOD

The latent period embraces the period which commences with the first administration of the carcinogen and ends with the appearance of the first liver tumour in one of the group of treated animals. Conceptually, the first administration of a carcinogen is thought to produce some alteration in target cells which is not enough by itself to produce cancer. Continuous administration of the chemical then produces additional effects, which are thought to be cumulative, and which, in some cells, may become sufficient for malignant transformation to take place. Since the entire organ is exposed to the chemical, the early changes must occur in all or at least the majority of liver cells. On the other hand, only one or at most a few tumours are found in treated animals and these appear to arise from a single or a small group of cells. So far, there is no explanation why only a very few liver cells undergo transformation while the vast majority do not do so. According to current thinking, alteration in the DNA is the most likely cause of cell transformation but it is not certain whether this is sufficient by itself to cause the development of cancer. Early manifestation of hepatic effects The early stages of the latent period have been studied intensively and a considerable mass of data has accumulated on the hepatic changes produced by hepatocarcinogens. Broadly speaking, all hepatocarcinogens produce

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overt hepatic changes if given at high doses. The type of change produced varies enormously. At one end of the scale there is phenobarbitone, which induces hepatomegaly and cytomegaly with proliferation of SER, and at the other end are compounds like the nitrosamines, acetylaminofluorene and aflatoxin, which produce extensive hepatocellular necrosis and a shrinkage of liver mass. In between these two extremes there is a wide variety of compounds which may produce liver enlargement, cell necrosis and/or fatty change, separately or simultaneously, and of different degrees of severity. These morphological changes are dose-related and for each compound there is a threshold dose for the production of histological evidence of damage or for liver enlargement. At appropriately low doses no discernible evidence of damage occurs. These morphological changes are accompanied by a wide variety of biochemical changes. Three types of biochemical changes are currently attracting considerable attention. A: Changes which involve interactions with the DNA. These include alkylation and adduct formation, resulting in damage to the DNA with consequent stimulation of DNA repair. These biochemical changes are produced by a reactive species as a consequence of the metabolism of the compound. (Sir Rudolph Peters coined the phrase ‘lethal synthesis’ to describe this event.) These reactive species interact with RNA and cell proteins as well as with DNA and the adducts formed with all three cell constituents can be detected chemically. In appropriate in-vitro or in-vivo tests these agents induce mutations in a variety of organisms from bacteria to fungi, and from cells in culture to in-vivo tests in mice and rats (Lawley, 1977; Roberts, 1978; Seeberg, 1981). This area has been intensively studied recently because of the currently prevalent view that such changes may induce mutations in the liver cells, one consequence of which is the production of cancer. Because carcinogens that produce adducts with the DNA also produce mutations in vitro and in vivo, this class of compounds are known as ‘genotoxic carcinogens’. One consequence of extensive alkylation of the DNA is the inhibition of replication and transcription, which pathologically is observed as a suppression of mitosis (Farber, 1973). The compounds mentioned above in groups 1,2 and 5 and some of those mentioned in group 6 belong to this class of hepatocarcinogens. They are hepatotoxic at high doses, but the DNA interaction is regarded as the essential lesion which is responsible for their carcinogenic activity. Studies in which the carcinogenic dose levels were correlated with those that produce a toxic effect showed that these agents produce cancer at levels well below the toxic dose (see Grass0 and Gray, 1977). B: Changes in the activity of a variety of enzymes. Examples are mixed function oxidase (MFO) linked to cytochrome P450 and associated cytochromes. These enzymes are usually involved in xenobiotic metabolism and their stimulation is thought to be a manifestation of an enhanced need to eliminate foreign chemicals. An increased activity of MFO is usually but not invariably

188

I’.

GRASS0

accompanied by liver enlargement. Both phenomena are dose-related. Of these two effects, the first to appear in a dose-response study is an increase in MFO activity, liver enlargement appearing at a higher dose-level (SchulteHermann, 1974; Goldsworthy et al, 1986). The chlorinated hydrocarbons, phenobarbitone and butylated hydroxytoluene are examples of hepatocarcinogens which produce changes of this sort. C: Increase in the number of peroxisomes and of enzymes associated with these organelles. Such changes are produced principally by the hypolipidaemic agents and are also associated with liver enlargement (Cohen and Grasso, 1981; Reddy and Lalwani, 1984). With the exception of DNA interactions, the biochemical and morphological changes mentioned in the previous paragraphs are also produced by substances which are not known to be carcinogenic to the rat and mouse liver. Strictly speaking, they are ‘non-specific’ toxic effects. On the other hand, many compounds which produce these toxic effects also produce cancer of the liver, particularly if administered at high dose levels, so they cannot be dismissed as irrelevant in the pathogenesis of hepatocellular carcinoma. At low dose levels, where these effects are absent or marginal, no cancer is produced (Grasso, 1979). As a consequence, there is considerable interest in attempting to define the extent to which they are responsible for the production of tumours and the mechanism by which they operate. Since most of these compounds are nonmutagenic (‘non-genotoxic’) it is unlikely that they cause tumours by a direct interaction with the DNA, so a study of their mechanism of action acquires a greater degree of interest and importance. Intermediate changes There is no specific point on a timescale which divides the early and intermediate changes, Conventionally, the early stage ends at the time of appearance of discrete nodular lesions, but for the purpose of this communication, the early stage has been considered to end and the intermediate stage to commence at the time of appearance of enzyme-altered foci in the liver parenchyma. A variety of enzymes can be demonstrated histochemically in the liver of untreated rats; they are uniformly distributed throughout the liver, although some enzymes may be predominantly present in the centrilobular or in the periportal area. During the process of carcinogenesis a disturbance of this uniform distribution occurs and islands of diminished (e.g., glucose-6-phosphatase, ATPase, alkaline phosphatase) or enhanced enzyme activity (y-glutamyl transpeptidase) appear seemingly scattered in a random fashion in the liver (Farber, 1973; Scherer and Hoffmann, 1971). The first systematic study of this phenomenon was carried out in 1964 by Gossner and Friedrich-Freksa when islands free of glucose-6-phosphatase (G6Pase) were observed in the liver of rats treated orally with diethylnitrosamine. Schauer and Kunze (1968) confirmed this finding and reported that

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similar islands of enzyme-free activity were observed when liver sections were stained for nucleoside-5’-triphosphatase (ATPase). It was subsequently shown that similar islands developed within a few weeks (approximately four to eight) when other hepatocarcinogens, including dibutylnitrosamine, 2acetylaminofluorene, 1,2-dimethylhydrazine and iV-nitrosomorpholine, were administered in the diet or drinking water to rats (Farber, 1973; Scherer and Emmelot, 1975). Islands of enhanced enzyme activity were also observed to develop after treatment with carcinogens. The best example of this group is yglutamyl transpeptidase (y-GT) and islands of increased activity of this enzyme have been reported within a few weeks of commencing the treatment. A single island of disturbed enzyme activity may show changes in several of these enzymes simultaneously. The number and size of the islands correlate quantitatively with the dose of the carcinogen administered and with the duration of treatment. They become more numerous as the experiment progresses and some of the islands become larger (Scherer and Emmelot, 1976; Sirica et al, 1978). In addition to these islands of altered enzyme activity other islands of cells, either rich in glycogen or basiphilic, appear early on in the latent period. The glycogen in these islands is PAS-positive and is digested by salivary diastase just as readily as ordinary glycogen. It differs from ordinary glycogen in that it is resistant to mobilization by starvation. In fact these islands can best be demonstrated in the livers of rats that have been starved overnight. Bannasch has shown that these glycogen-rich islands appear during treatment with several hepatocarcinogens (Bannasch et al, 1979, 1980). The basiphilic islands consist of small cells, the cytoplasm of which stains a reddish-blue. Because the cluster of cells that make up these islands has a different tinctorial appearance from surrounding cells they stand out prominently in paraffin sections stained by haematoxylin-eosin. The number and size of both the islands of altered enzyme activity and the glycogen-rich islands appear to be related to the amount (in terms of dose and time) of the carcinogen administered. It is not yet certain whether they represent the site at which hepatocellular carcinomas eventually develop. According to some authors, each of these islands is a clone of cells derived from a single hepatocyte that has undergone mutation (Emmelot and Scherer, 1980); others regard them as a group of contiguous cells that have undergone a phenotypic change (Pitot et al, 1985). It would seem, however, that these islands of altered enzyme activity represent more than just a phenotypic change in a number of adjacent cells. Scherer and Hoffmann (1971) showed that five weeks after the simultaneous administration of a high dose of dimethylnitrosamine and [3H] thymidine once only to hepatectomized rats, the hepatic parenchyma was heavily labelled, but the label was lost from enzyme-deficient islands, suggesting that the repeated cell division in the focal area caused dilution of the radioactive marker. This is in keeping with the view that these islands are clones of altered cells, but the question of whether the progenitor cell is genotypically or phenotyically altered is unanswered. Emmelot and Scherer (1980) suggested the following sequence of events from their studies on the development of enzyme-altered islands prduced by diethylnitrosamine administration:

20

I’. GRASS0

‘ype IA islands: 200 ,um in diameter and appear in mid-zonal area at about five weeks. ‘ype IB islands: larger, appear a few weeks later than type IA; characterized by internal heterogenicity of marker enzymes. ‘ype II: at least 1 mm in diameter and appear as small nodules. Marked deficiency of G6Pase and ATPase. These islands are glycogen-positive. ‘he data upon which the foregoing account of enzyme-deficient and glycogen llands is based has been obtained by the administration of genotoxic epatocarcinogens. ‘Non-genotoxic’ carcinogens also produce islands defiient in enzyme activity, particularly G6Pase and ATPase, as well as islands ch in enzyme activity, particularly in y-glutamyl transpeptidase. One nportant group of rodent hepatocarcinogens (the hypolipidaemic agents), owever, appear to be an exception since they do not induce islands of ylutamyl transpeptidase. The validity of regarding this change as an essential recursor of neoplasia is open to question. The same question mark, for other :asons, hangs over the other enzyme islands (Farber, 1980; Pitot et al, 1985). Despite these reservations, it would seem sensible to look upon these islands f change as a useful marker for tumour development in view of the diversity f carcinogens that produce them and their dose-dependence. ‘he hyperplastic nodule ‘he islands of altered enzyme activity are usually followed by the appearance f localized proliferative foci, which are generally known as hyperplastic odules (Farber, 1973). These foci were thought to be the precursors of denomas and carcinomas by the early pathologists based on experience with ther tissues, in particular the mammary gland tissue in rats and the skin of lice. Sequential studies of the stages of carcinogenesis in these tissues :vealed that hyperplastic nodular lesions develop several weeks before utonomous growth and that they appeared to be the sites from which amours developed (Foulds, 1969). In these tissues, the development of the :sion could be observed clinically in the live animal as well as histologically; in le liver the early nodular lesions could only be studied histologically, since lch nodular lesions are not detectable clinically unless they are very large. The development of the hepatic nodule and its biological and biochemical haracteristics has been mainly studied by treating rats with single or repeated oses of 2-acetylaminofluorene, diethylnitrosamine, nitrosomorpholine or Aatoxin, followed by hepatectomy, administration of phenobarbitone or Ime other agent that induces liver enlargement, or no further treatment. The iethods employed are reviewed extensively by Emmelot and Scherer (1980), ‘arber (1973, 1980), Pitot et al (1985) and Bannasch et al, (1980). From these studies, it would appear that histologically the nodules appear ithin two to five weeks depending on the dose and on the carcinogen dministered. They are encased in a thin fibroconnective tissue at least for part f their circumference. The cells within the nodule may be indistinguishable .om normal hepatocytes, but they may be larger with a nuclear/cytoplasmic itio less than normal. The larger nodules may contain small islands of

EXPERIMENTAL

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TUMOURS

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191

basiphilic cells within them. In general the nodules contain glycogen which is resistant to mobilization by starvation and to the administration of glucagon. They are deficient in G6Pase and glycogen phosphorylase activity (Farber, 1973). The amount of RNA, DNA and protein in the hyperplastic nodules is less than that in the surrounding liver. The DNA itself shows obvious alterations in structure. This is most clearly shown in the nodules removed from animals treated with 2-acetylaminofluorene. The DNA from such nodules showed clear biochemical and morphological evidence of damage. Although there is no clear evidence of an alteration in the rate of DNA and RNA synthesis in the nodule, there is some evidence that the rate of synthesis of both DNA and RNA is accelerated in the basiphilic areas, suggesting a defective regulation of both the synthesis and breakdown of RNA and DNA (Farber, 1973). One interesting feature of nodular hyperplasia is the absence of haemosiderin in the hepatocytes within the nodule when rats are subjected to a gross iron overload by the administration of high doses of iron-dextran or some similar iron preparation (Williams, 1980). When sections of such livers are stained by the Perles’ Prussian Blue technique these nodules stand out as pale round areas in contrast to the surrounding hepatocytes, which are stained deeply blue because they are loaded with haemosiderin. A constant finding in hepatocytes from hyperplastic nodules is a considerable proliferation of smooth endoplasmic reticulum (SER). This increase is accompanied by a concomitant decrease in G6Pase and may represent what has been called ‘hypoactive hypertrophic smooth endoplasmic reticulum’ (Hutterer et al, 1968). The significance of this finding is at the moment poorly understood. Reversibility studies have shown that a high proportion of the nodules induced by 2-acetylaminofluorene are reversible-very few of the nodules persist and progress to carcinoma. At the moment there do not appear to be any distinguishing biochemical or morphological characteristics to identify those nodules which will progress from those which will reverse (Pitot et al, 1985). Transplantability has been widely employed to study the growth characteristics of tumours and of tumour-like lesions. Several such studies are on record which attempted to assessthe potential of hyperplastic nodules from rats and mice for autonomous growth. These early studies on both spontaneous and induced lesions showed that whereas hepatocellular carcinomas could be successfully transplanted, tissue samples from hyperplastic nodules did not survive for any length of time. This failure of survival led to the conclusion that the technique employed may not have been adequate to allow the graft to survive long enough to demonstrate its growth potential. In a more recent study grafts from normal hepatic tissue, hyperplastic nodules and hepatocellular carcinoma were implanted into the inguinal mammary fat pad of immunologically compatible female rats. A high percentage of grafts from normal liver and from hyperplastic nodules survived for about 40 weeks. Neither showed any increase in size whereas carcinomas increased steadily and rapidly. This carefully conducted study confirmed the results of earlier

192

P. GRASS0

investigators. It showed that the hyperplastic nodule has no more potential for autonomous growth than normal liver (Williams et al, 1977). The growth potential of the hyperplastic nodules was also studied in vitro. These studies, unlike the in-vivo studies, showed that the hepatocytes from nodules are more readily subcultured than normal hepatocytes but the number of subcultures is limited. Under similar conditions, normal hepatocytes do not survive when subcultured while carcinoma cells can be subcultured indefinitely. Thus in-vitro studies give some indication that cells from nodular hyperplastic lesions have some growth advantage over normal cells but do not possess the same growth potential as carcinoma cells (Slifkin et al, 1970).

STAGE

OF TUMOUR

Morphology

DEVELOPMENT

of hepatic tumours in rodents

There are few descriptions of the macroscopic appearance of liver tumours in rats and mice. One of the earliest and most detailed description is that of Opie (1944). He found that in rats approximately two-thirds of primary liver tumours appear in the right upper lobe. Tumours as small as 1 mm in size can often be recognized by a small elevation in the normally smooth surface of the liver. Large tumours appear as round or irregular masses which may be up to 7 cm in diameter. In their early stages, tumours appear as discrete pin-point lesions which on section may be white, grey or yellow in appearance. The larger tumours are more variable in colour and are likely to show haemorrhage, necrosis or degenerated cystic areas. Large tumours often rupture and cause a fatal haemorrhage. Although most of the naturally occurring and experimentally induced tumours develop from the liver parenchymal cell, tumours may develop from virtually every constituent cell of the liver. Thus, haemangiosarcomas have been induced in a dose-related manner by vinyl chloride in rats and mice (Maltoni, 1975; Keplinger, 1975) and bile-duct tumours have been induced by 4-dimethylaminoazobenzene (Price et al, 1952) and diethylnitrosamine in rats (Peto et al, 1984) and azonaphthalene in mice (Turusov and Takayama, 1979). The biological characteristics of these tumours have not been studied in the same depth as tumours that originate from the hepatocyte. The commonly accepted criteria for classifying tumours other than hepatocellular tumours are found in Schauer and Kunze (1976) for the rat and Turusov and Takayama (1979) for the mouse and have not substantially changed since the earliest classification of Stewart and Snell (1957). The criteria for the classification of hepatocellular tumours have, however, undergone substantial change since then. The most recent classification of the rat hepatocellular tumours is found in a publication by Keysser et al (1980). There are at least three well-known attempts at classifying mouse tumours (Jones and Butler, 1975; Turusov and Takayama, 1979; Vesselinovitch and Mihailovic, 1984).

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A considerable overlap exists between the various classifications although they differ in the descriptive terms employed. Basically the following two major categories are recognized - non-proliferative focal lesions and proliferative localized lesions. The former are universally acknowledged to be nonneoplastic, although there is some evidence that they may be connected with the development of tumours. The proliferative localized lesions embrace a range of nodular growths which are non-neoplastic at one end and malignant tumours at the other. Although there is general agreement on the criteria for labelling a particular nodular growth as malignant, the dividing line between the non-neoplastic lesions and the adenomas is extremely difficult to define and much of the controversy on the classification of liver tumours revolves around this issue. The following classification is based on that published by Vesselinovitch for the mouse and that by Keysser et al (1980) for the rat and applies to both species. Focal non-proliferative

lesions

(a) Clear cellfoci. These usually consist of glycogen-rich cells, but sometimes the cells may contain fat. (b) Acidophilic cell foci. Cytoplasm of the cells is acidophilic and has a ground-glass appearance. (c) Basophilic cell foci. The cells are basiphilic and smaller than normal. Proliferative localized lesions (a) Hyperplastic Nodule. An expansive growth which compresses adjacent liver cells. The histological structure is very close to that of the normal liver. Central veins and portal triads are present within the nodule. (b) Hepatocellular Adenoma. Expansive growth which compresses adjacent liver cells. Liver plates are 1-Lcells thick and show a moderate degree of undulation (waviness). Absence of portal triads within the lesion. Normal hepatocellular morphology. Note: both (a) and (b) may be totally or partially composed by glycogen-rich or fatty cells. (c) Hepatocellular Carcinoma (1) Trabecular carcinoma. Liver cords are of irregular thickness and alternate with sinusoidal vessels which are often dilated. Papilliform and adenoid areas are present and accompanied by marked sinusoidal dilation. (2) Adenocarcinoma. Adenoid areas predominate but very rarely do they form the entire neoplasm. There are usually gross architectural and cellular abnormalities. (3) Poorly difSerentiated carcinoma. Cells of various shapes and sizes, mitotic figures common, nuclear pleomorphism and hyperchromasia as well as reduction in nuclear/cytoplasmic ratio. The general architectural pattern is usually grossly distorted but retains some of the features mentioned above.

194 Ultrastructural

P. GRASS0

studies

Extensive ultrastructural studies have been conducted on chemically induced hepatic tumours in rats and mice. Although several changes have been described involving most cellular organelles, there are none that are characteristic of neoplasia (Svoboda and Higginson, 1968).

Dose-response relationships The dependence of the incidence of hepatic tumours on the dose of carcinogen administered was first demonstrated unambiguously by Druckrey in the late 1950s. He looked upon his studies as a pharmacological approach to carcinogenesis since one of the features of pharmacological activity is a relationship between the strength of the response to the magnitude of the dose. Such a relationship exists also between toxic effects and administration of the test chemical. Relationships of this sort are considered to be evidence for a causal relationship between a specific toxic or carcinogenic effect and the test chemical administered. In his studies, Druckrey used principally the hepatocarcinogens 4diethylaminostilbene and 4-dimethylaminoazobenzene. He showed that by increasing the dose of a carcinogen the number of animals affected increased provided the dose was not so high as to shorten the life of the treated animals. Druckrey also showed that, as the incidence of tumours increases, the tumours appeared at a much younger age. This phenomenon is called shortening of the latent period (Druckrey, 1959). The pioneering work of Druckrey has been confirmed by numerous other investigators with a wide variety of hepatocarcinogens (Turusov and Takayama, 1979). The large-scale study on dimethyl- and diethylnitrosamine carcinogenesis in rats, in which 5 120 rodents and 16 dose levels were employed is a recent example (Peto et al, 1984). In this study the dose-response showed three principal features: firstly, as the dose increased there was a gradual increase in the number of tumours; secondly, the mean latent period became progressively shorter; thirdly, a point was reached when most of the animals developed tumours. At this point, no further increase in tumour incidence occurred, but there was a dramatic shortening of the latent period (Table 2). This result was obtained from a large-scale study on two genotoxic carcinogens, and, although no other carcinogens of this class have been studied in the same depth, it would appear that the features observed in the large-scale study are present also in dose-response studies on other genotoxic carcinogens. The dose-response results obtained from the class of non-genotoxic carcinogens are different from those obtained by genotoxic carcinogens. As one proceeds from the lower to the higher dose levels, little or no increase in tumour incidence is observed until a dose is reached at which the incidence of tumours shows a dramatic increase (Table 3). This ‘step-wise’ type of doseresponse appears to be characteristic of non-genotoxic carcinogens and, although the reason for the difference in the type of dose-response cannot be fully accounted for, it would appear that the incidence of liver tumours

EXPERIMENTAL

LIVER

TUMOURS

19.5

IN ANIMALS

increases significantly only when there is overt perturbation of hepatic cell physiology (see paragraphs B and C, pp. 187 and 188). Most ‘genotoxic’ hepatocarcinogens produce tumours after a limited period of treatment (Druckrey, 1967). An important recent example is vinyl chloride (Maltoni and Lefemine, 1975). When administered for only 52 weeks at a series of dose levels, it produced a dose-related increase in tumours of the liver (haemangiosarcomas) and in tumours of the Zymbal gland and kidney (Table 4). These results are consistent with the view that the intracellular changes produced by genotoxic carcinogens are irreversible. It must be remembered, however, that the doses used in experiments of this sort were of the order of several milligrams per kilogram bodyweight and may not be representative of the cellular response to the very low levels of carcinogens such as may occur in the environment. At such low levels, the capabilities of the cell to repair DNA damage may be sufficient to prevent cancer from developing. Very few experiments have been conducted to study the effect of mediumterm administration, say 52 weeks, of a non-genotoxic hepatocarcinogen and then to follow the course of the treated animals for at least another year. In one such study (on Ponceau MX, a non-genotoxic food colour) the test compound was administered for 11 months at which time a laparotomy was performed. Half the animals had hepatic nodules; the other half were without any lesion. The animals were killed 12 months after the laparotomy, during which period no colouring was added to their food. At necropsy, no tumours were found in the animals that had no nodules at laparotomy, but two carcinomas were found among the group that had nodular lesions. All the remaining animals in this group still had nodules at laparotomy. The conclusion reached tentatively from this study was that the biochemical

Table 2. Tumour incidence and median time of appearance in male rats of Colworth Farm strain (Peto et al, 1984). Treatment* (wm) 16.9 8.45 6.34 5.28 422 3.17 2.64 0

Incidence of malignant tumours 93.8 83.4 75.0 64.9 54.1 58.4 37.5 2

(%)

of tumours

Time of death:? days after treatment (median) 222 468 538 545 697 770 750 920

* Dimethylnitrosamine in drinking water. t Tumours were found in animals that died or were moribund. N.B.: similar results were obtained by dimethylnitrosamine and diethylnitrosamine in both sexes.

killed

when

in females

196

P. GRASS0 Table 3. Dose-response relationship between doses of DDT and hepatic tumour incidence in CF-I mice (Tomatis et al, 1982).

Dose (ppm) 0 2 10 50 250 * DDT nogen.

Number of animals ____ M F 125 126 111 135 117

117 110 126 109 105

is an example

Percentage with tumours at weeks O-69

70-99

M

F

0 9.1 0 4.0 26.7

0 0 0 4.2 13.8

M 15 39.2 56.8 46.1 96.8

of a non-genotoxic

F 0 2.4 1.9 4.3 70.9 carci-

lesions are very probably reversible but that some of the nodular lesions are persistent and a few of these may progress to carcinomas (Grass0 and Gray, 1977).

FACTORS HEPATIC

AFFECTING THE NATURAL INCIDENCE TUMOURS IN RATS AND MICE

OF

Both benign and malignant liver tumours occur naturally in rats and mice. The incidence of liver tumours (malignant and benign) in the rat is about O6%, depending on the strain (Keysser et al, 1980; Grasso, personal observation). In the mouse the range is much wider. The incidence in a few strains (e.g., CF-W and Balb/c) is from 0 to 2%, while in other strains the incidence is between 75 and 100% (Grass0 and Hardy, 1975). In most strains, the liver tumour incidence is between 10 and 50%. In both rats and mice, the incidence of liver tumours is higher in males than in females. This sex difference is, in general terms, more marked in mice than in rats. Because of the marked sex difference, the influence of steroid hormones attracted the attention of the early investigators. It was found that orchidectomy reduced the incidence of liver tumours in male mice to a level comparable to that found in females. The implantation of cholesterol pellets containing diethylstilboestrol or oestradiol had a similar, though not quite as marked an effect. However, the administration of testosterone propionate did not increase the incidence of tumours in female or male mice, while oestradiol benzoate did not alter the incidence of tumours in C3H male mice (a highincidence strain). Despite the lack of consistency, these results suggest that sex hormones may play some role in the development of spontaneous tumours in mice. The role of other hormones is much less clear (Grasso, 1984).

60 60 59 59 59 59 58

10 000 6000 2500 500 250 50 0

60 60 59 59 59 59 58

F

to vinyl

(52) (67) (41) (74)

M

6 (50) 4 (59) 1 (26) 1 (97) 0 0 0

F 3 3 6 0 1 0 0 (116)

(59) (68) (77)

M

chloride

Numbers in brackets indicate average latency. * 4 hours a day, 5 days a week for 52 weeks. Survival at appearance of first tumour (a subcutaneous t 13 weeks old at commencement of treatment,

M

Cone (ppm)

related

Zymbal gland turnouts

10 3 1 3 0 0 0

of turnouts

Survival at 26 weeks

Table 4. Incidence al, 1975).

4 10 7 6 2 1 0

(72) (71) (77) (84) (56) (135)

F

angiosarcoma)

Liver

2 2 2 0 1 1 0 (101) (135)

(61) (34) (58)

M

Other F

1 (70) 1 (73) 1 (82) 0 0

M

F 0 1 (84) 2 (98) 5 (96) 0 0 0

Hepatomas

(Experiment

1 (60) 0 0 0 1 (90) 0 0

Ratst

2 (62)

1 (51)

sites

in Sprague-Dawley

Angiosarcoma

exposure*

1 (70) 0 0

2 (8-4

3 (54) 4 (67) 5 (69)

M 2 1 1 4 4 1 0

(67) (59) (99) (83) (80) (135)

F

Brain

2 (76) 0 0 0 0

F

et

5 54) 1 (50) 2 (84) 0 0 0 0

(Maltoni

2 (62)

2 (52)

M

at 135 weeks)

Nephroblast

terminated

E

% 52

2

t:

198

P. GRASS0

As far as can be ascertained there are no systematic studies on the role of hormones in tumour development in rats. Other investigators sought to ascertain the role of the diet in influencing the incidence of hepatic tumours in both mice and rats. It was found that severe restriction of the diet from an average of 6.0 g a day to 3.84.0 g reduced body weight gain and totally abolished the incidence of tumours in C3H mice. Caloric restriction, achieved by reducing the fat content of the diet, produced a similar result. It was further shown that some dietary constituents may increase the natural incidence of tumours when given in excess. Thus casein mixed in the diet in amounts from 9 to 45% produced a concentration-related incidence of hepatic tumours in C3H mice. Groundnut oil given as 5 or 10% in the diet to C57BL mice (a strain with a low incidence of liver tumours) had a similar effect (Grasso, 1984). Diet would appear to be of some importance in influencing hepatic tumour incidence in rats as well as in mice. A 20 to 25% reduction in calorie intake reduced the incidence of liver tumours. It was also found that food restriction, even if only limited to adolescence, has the same beneficial effect (Grasso, 1984; Ross et al, 1982). Deficiency of some dietary constituents would appear to increase the risk of development of hepatic carcinoma. The best-known example is choline deficiency. Rats maintained on a choline-deficient diet develop hepatic cirrhosis within a year and a high proportion develop carcinoma later. Cirrhosis has also been demonstrated to develop in rats fed a purified amino acid diet deficient in choline, methionine, vitamin B12 and folic acid. Carcinoma develops in some of these rats (Salmon and Copeland, 1954; Newberne et al, 1969). The great interstrain variation in hepatic tumour incidence in the mouse led some investigators to search for genetic factors that might be responsible for it. According to Heston (1978) the occurrence of liver tumours in mice is controlled by multiple genetic factors which account for the high incidence of tumours in C3H and for the low incidence in C57BL. Introduction of the Aq gene in C3H mice by careful breeding was found to raise the liver tumour incidence in this strain. There is no indication as to which other genes, if any, are involved in influencing the development of hepatic tumours (Heston and Vlahakis, 1961). Nevertheless, a change in the incidence of hepatic tumours has been observed to occur over several years in the same strain of mice and it is claimed that it is due to a change in the genetic constitution of the animal. This gradual change is known as ‘genetic drift’ (Topham, 1972; Heston, 1978). There are no published studies on the genetic control of hepatic tumour incidence in rats. The possibility that a virus infection may account for the high and variable hepatic tumour incidence in mice was entertained by Andervont in 1950a. He observed that mice with a high incidence of liver tumours had a high incidence of mammary gland tumours as well. Despite the fact that the correlation between the incidences of these tumours was not very good, intensive investigations were carried out to explore a possible aetiological relationship between the mammary tumour agent and hepatic tumours. The results of this work clearly demonstrated that the mammary tumour agent was specific for

EXPERIMENTAL

LIVER

TUMOURS

199

IN ANIMALS

mammary gland tissue and played no role in the causation of hepatic tumours. Unfortunately, there are no published data on any possible relationship between the mouse hepatitis virus, which infects many mouse colonies, and liver tumour development. The recent discovery of oncogenes has opened up a new avenue for investigation. Oncogene activation was studied in spontaneous tumours of the B6C3Fl mouse. This mouse has a moderately high incidence of hepatocellular tumours (approximately 10%) and is the strain most frequently used at the moment for screening chemicals for carcinogenic activity. It was found that 30% of naturally occurring mouse hepatocellular adenomas and 77% of naturally occurring hepatocellular carcinomas scored positive results by DNA transfection. These transforming genes were identified as an activated Ha-ras gene in all the adenomas studied and in most of the carcinomas studied. The oncogenes recovered from two of the carcinomas could not be classified as belonging to the ras gene family (Reynolds et al, 1986). In the rat there is no mention of a possible association between hepatic tumour incidence and a virus infection. Recent observations on oncogene activation revealed that spontaneous hepatic tumours (benign and malignant) from the Fischer 344 rat failed to yield activated oncogenes (Reynolds et al, 1986). ‘OVAL’

CELLS

IN HEPATOCARCINOGENESIS

‘Oval’ cells were first described in the study of the early lesions produced by the hepatocarcinogens acetylaminofluorene and dimethylaminoazobenzene (butter yellow). They were so-called because of their oval shape. The oval appearance of the nucleus accentuated the oval appearance of the cells. The amount of cytoplasm per cell was much less than that of hepatocytes. These oval cells were accompanied by recognizable bile duct hyperplasia (Dalton and Edwards, 1942; Farber, 1956). Oval cells were originally thought to be the precursor cells of the hepatocytes, but careful histological and electron-microscopic studies revealed that they possess some structural similarities to bile-duct cells, and they were considered to be intermediate between ductular cells and hepatocytes (Sirica and Cihila, 1984). Recently it has been shown by immunocytochemistry that oval cells appearing a few days to a few weeks after the administration of acetylaminofluorene are rich in cytochrome P448 but do not contain any cytochrome P450. Bile duct cells, whether quiescent or proliferating (e.g., after bile-duct ligation), are rich in P448 but contain no P450, while hepatocytes, including those newly formed as a result of reactive hyperplasia, contain both types of cytochrome. Thus, oval cells probably represent abnormal newly proliferated bile duct cells (C. Betton, 1986 personal communication; Powell et al, 1986). INITIATION

AND PROMOTION

OF HEPATIC

CARCINOGENESIS

Long before systematic investigations of this aspect of tumour causation in the liver were commenced, some studies of the mechanism of production of

200

P. GRASS0

skin tumours revealed that some carcinogens when applied only once or a few times to the skin of mice failed to elicit tumours even when the mice were allowed to live out their natural lifespan. If, however, substances like croton oil were applied regularly to this area of carcinogen-treated skin, both papillomas and carcinomas developed after several months of treatment. Reversal of treatment by these agents failed to induce tumours. This type of experimental model gave rise to the expressions ‘initiation’ and ‘promotion’. Clearly, the application of the carcinogen in small doses had ‘initiated’ the treated area of skin while the subsequent application of the croton oil ‘promoted’ the development of tumours. Thus, for practical purposes, ‘initiation’ means the production of a change in the treated cell or cells which transforms it or them into potential cancer cells. ‘Promotion’ is meant to signify the process by which the potential of the transformed cell to develop into a cancer is realized. Although histologically no changes in the skin occur from the application of an ‘initiating’ dose of carcinogen, the application of a promoter to mouse skin results in the production of epithelial hyperplasia (Slaga et al, 1980). Although there is no strict correlation between the production of hyperplasia and tumour promotion, it would seem that the cell proliferation involved in hyperplasia plays an important role in the process of ‘promoting’ tumour development. These concepts, developed over many years of experimentation on mouse skin, are being applied to study the mechanism of tumour development in the liver. The stages of initiation and promotion are not as clearly marked as in the skin but there are indications that if any of the classical hepatocarcinogens, e.g., the nitrosamines or acetylaminofluorene, are given for a brief period of a few weeks, few tumours (10 to 20%) will develop if the treated animals are allowed to live out their lifespans. If, however, the brief period of treatment with the carcinogen is followed by prolonged treatment with agents such as phenobarbitone, then the tumour incidence increases up to 80% or over. Promoters not only enhance tumour production after a brief treatment with carcinogens but they also enhance the development of islands of focal change and of hyperplastic nodules. It is of interest to note that the majority of the promoters that have been employed so far produce liver enlargement within a few days when given at high doses. Since this type of liver enlargement involves an increase in the number of cells it would seem that hyperplasia may play some role in promoting liver tumours, as it does in the skin model (Schulte-Hermann, 1985). The importance of hyperplasia in promoting hepatic tumour development was demonstrated clearly by Pound and Lawson (1975), who showed that the yield of tumours of the liver from the administration of dimethylnitrosamine is considerably enhanced by hepatectomy or by hepatotoxic doses of carbon tetrachloride. Both of these procedures induce a marked restorative hyperplasia. Little attention has been given so far to the circulatory disturbances that may accompany the distortion of the liver architecture by the hepatic nodule. The atrophic cells seen at the edge of the nodular growth is probably an indication of sinusoidal compression that might be expected from an expansile lesion. The blood supply to the nodule itself has, surprisingly, attracted little attention. From studies of experimentally induced cirrhosis in the rat, there

EXPERIMENTAL

LIVER

TUMOURS

IN ANIMALS

201

are indications that when nodular growths develop, the hepatic circulation becomes considerably distorted. The flow rate of blood is reduced and the blood supply to the regenerative nodules becomes predominantly arterial. This would suggest that the blood supply to the hyperplastic nodules is very probably arterial too. There are no studies of this sort in the mouse, presumably because of the technical difficulties involved in manipulating the relatively small liver in this species (Koo et al, 1976). CONCLUSION Hepatic tumours in rats and mice can be produced by the administration of a large number of chemicals, which structurally are very diverse indeed. This chemically heterogeneous group of carcinogens can be divided into two broad classes on the basis of their mutagenic potential: those that are mutagenic (also called ‘genotoxic’) and those that are non-mutagenic or only equivocally so (also called ‘non-genotoxic’). The genotoxic hepatocarcinogens are able to form adducts with the DNA and can induce DNA repair in mouse or rat hepatocytes. At high doses these agents are hepatotoxic but they can induce cancer at levels where no morphological evidence of an adverse effect is seen. The non-genotoxic carcinogens do not produce any measurable adverse effect on the DNA but an adverse effect on the liver (ranging from liver enlargement to cell necrosis) is observed at all dose levels which produce cancer. There are major differences in the type of carcinogenic response produced by the two classes of compounds, as exemplified by the ‘step-wise’ dose response result to DTT, a non-genotoxic agent, and the virtually linear response to dimethylnitrosamine. Both types of hepatocarcinogens produce islands of altered enzyme activity and nodular hyperplasia. The role of both these lesions in the production of cancer is still in dispute. There is little firm evidence that islands of altered enzyme activity are a necessary precursor of hyperplastic nodules or of cancer. The evidence that tumours may develop from nodular hyperplasia has a better basis and most workers regard these lesions as precancerous. If this view is correct, then the early appearance of nodular hyperplasia would suggest that the common pathway for the production of tumours by both types of carcinogen is reached at an early stage - after a few weeks or months. Cirrhosis of the liver is difficult to produce in experimental animals and its role in the production of tumours in these species cannot be assessed at the moment. It would appear, however, that nodular hyperplasia when produced by the non-genotoxic hepatocarcinogens is the counterpart of cirrhosis in man in so far as both appear to result in a greatly enhanced risk of cancer development. Dietary factors have a profound influence on the natural occurrence of hepatic tumours in mice and rats. The role of genetics or homones is still imperfectly understood but they seem to play some role in determining spontaneous tumour incidence.

202

P. GRASS0

Initiation and promotion have been studied in a number of models. Some similarities to the mouse skin model have been reported. Despite the considerable amount of experimental investigations on the pathogenesis of chemically-induced cancer of the liver in rats and mice, the reward has been disappointing. There are still many gaps that need to be filled before we can understand and place in perspective the molecular and cellular events that lead to neoplasia.

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Stewart HL, & Snell KC (1957) The histopathology of experimental tumours of the liver of the rat. Acta Unio Int Contra Cancrum 13: 770. Svoboda D & Higginson J (1968) A comparison of ultrastructural changes in rat liver due to chemical carcinogens. Cancer Research 28: 1703. Thorpe E & Walker AIT (1973) The toxicology of dieldrin (HEOD). II Comparative long-term toxicity studies in mice with dieldrin, DDT, phenobarbitone, c(-BHC and p-BHC. Food and Cosmetics Toxicology 11: 433442. Tomatis L, Turusov V, Day N & Charles RT (1972) The effect of long-term exposure to DDT on CF-1 mice. International Journal of Cancer 10: 489. Topham JC, Tucker MJ & McIntosh DAD (1972) Spontaneous liver changes in control rats and mice over an 18-month period. Proceedings of the European Society for Drug Toxicity 13: 314-319. Turusov VS & Takayama S (1979) Tumours of the liver. In Turusov VS (ed.) Pathology of Tumours in Laboratory Animals, Vol. II Tumours of the Mouse. (IARC Scientific Publications 23). Lyons: IARC. Vesselinovitch SD & Mihailovic N (1984) Kinetics of induction and growth of basiphilic foci and development of hepatocellular carcinoma by diethylnitrosamine in the infant mouse. In Popp JA (ed.) Mouse Liver Neoplasia ch. 5, p 61. Washington DC: Hemisphere Publishing Company. Ward JM, Sontag JM, Weisburger EK & Brown CA (1975) Effect of lifetime exposure to aflatoxin Bl in rats. Journal of the National Cancer Institute 55: 107-133. Weil CS & McCollister DD (1963) Safety evaluation of chemicals. Relationship between shortand long-term feeding studies in designing an effective toxicity test, Journal of Agricultural and Food Chemistry 11: 486. Williams GM (1980) The pathogenesis of rat liver cancer caused by chemical carcinogens. Biochimica et Biophysics Acta 605: 167-189. Williams GM, Klaiber M & Farber E (1977) Differences in growth of transplants of liver, liver hyperplastic nodules and hepatocellular carcinomas in the mammary fat pad. American Journal of Pathology 89: 379-390. Wogan GN (1964) Experimental toxicity and carcinogenicity of aflatoxin. In Wogan GN (ed.) Mycotoxins in Foodstuffs, p 163. Cambridge Mass.: MIT Press. Wogan GN & Paglialunga S (1974) Carcinogenicity of synthetic aflatoxin Ml in rats. Food and Cosmetics Toxicology 12: 38 l-384.