Download Preneoplastic lesions as end points in

Carcinogenesis vol.7 no.5 pp.689-695, 1986
COMMENTARY
Preneoplastic lesions as end points in carcinogenicity testing.
I. Hepatic preneoplasia
P.Bannasch
Abteilung filr Cytopathologie, Institut fur Experimentelle Palhologie,
Deutsches Krebsforschungszentrum, Im Neuenbcimer FeW 280. D-6900
Heidelberg, FRG
Introduction
The evaluation of the carcinogenic risk deriving from chemical
compounds depends mainly on conventional histopathology up
to the present. The accepted end point in carcinogenicity testing
is the tumor as defined histologically. A great disadvantage of
this approach is the long lag period in the development of tumors
induced by chemicals. In order to overcome this drawback, many
efforts have been made to detect early biological or morphological lesions which might be specific for carcinogens. A great
number of short-term tests carried out in vitro provided valuable
information about the reactions of cellular constituents and biological macromolecules to the chemicals tested, but they did not
allow an unequivocal prediction of the carcinogenic potential of
the respective compounds in whole animals, not to mention man.
The introduction of modern micromorphological methods such
as electron microscopy and cytochemistry in the evaluation of
whole-animal studies also revealed many new aspects on carcinogen-induced cellular and subcellular alterations which appeared
to be unreliable as indicators of the carcinogenic risk of chemicals.
However, during the past two decades a number of characteristic
cellular changes has been detected in various tissues, especially
in the liver. These changes regularly precede the development
of certain tumor types, and are regarded as preneoplastic lesions
(1,2). The altered cell populations usually form well-defined foci.
They appear prior to the development of tumors in the target
tissue of the carcinogen, and should be duly considered in the
evaluation of the carcinogenic risk from chemicals in bioassays.
Definition of preneoplasia
The definition of preneoplasia has to include prestages of both
benign and malignant neoplasms, especially since many observations suggest that benign tumors are often nothing but intermediate stages in the development of malignant neoplasms.
Hence, 'preneoplasia' is not identical to 'precancer'. At the histological level, preneoplastic lesions may be defined as phenotypically altered cell populations which have no obvious neoplastic
nature, but have a high probability of progressing to a benign
or malignant tumor. Theoretically, such a lesion might consist
of definitive tumor cells which are only prevented from growing
'autonomously' by superordinate mechanisms residing outside
the transformed cells, such as the frequently postulated microenvironmental factors or influences of the immune system. On
the other hand, preneoplastic lesions might also be composed of
cells which are phenotypically altered by the carcinogenic agent
but lack important properties of the final tumor cells. Numerous
recentfindingssuggest that certain types of focal lesions preceding
•Abbreviations: 7-GT, 7-glutamyltranspeptJdase; NNM, Mnitrosomorpholine.
© IRL Press Limited, Oxford, England
the development of tumors do indeed consist of preneoplastic cells
which are only fully transformed after additional intracellular
changes (2). We have to admit, however, that other types of focal
lesions may also contain neoplastic cells which might be mixed
with preneoplastic cells or even occupy the whole lesion. Thus,
the definition of preneoplastic lesions used in this commentary
refers to the histological level and does not necessarily mean that
the lesion is made up of preneoplastic cells. The important question as to whether the conception of preneoplasia is only useful
at the organizational level of the tissue or can also be applied
to single cells will be discussed later on.
In human and experimental pathology, 'hyperplasia' has often
been regarded as an early stage in tumor development (3). However, the use of the term hyperplasia in the context of carcinogenesis entails a dilemma. By definition, hyperplasia is an
increase in the number of tissue-specific cells which depends on
extracellular growth-stimulation factors, such as hormones, while
neoplasia is an 'autonomous' increase in the number of cells
which is independent of such stimuli. A hitherto unsolved problem is that early stages of neoplastic development may be characterized by a proliferation of cells which cannot be clearly
distinguished from normal cells. Although such lesions may have
the potential to progress to tumors without any further exposure
to known carcinogenic or growth-stimulating factors, they are
frequently classified as hyperplasia. It is difficult to avoid this
classification as long as we are unable to identify preneoplastic
or early neoplastic lesions in many tissues. However, this problematical nomenclature by no means implies that hyperplasia in
its strict sense is a prerequisite for neoplasia. At least in chemical
carcinogenesis, it is highly probable that proliferating precursor
lesions of tumors are already composed of irreversibly altered
cells which are not comparable with proliferating normal cells
in true hyperplasia, no matter whether we can detect the irreversible cellular changes or not. Because of the uncertainty of the
biological behaviour of 'hyperplastic lesions', the discussion on
their consideration in the evalution of carcinogenesis bioassays
remains controversial.
Foci of altered hepatocytes
Hepatic preneoplasia has been extensively studied in different
species, especially in rats and mice, by many workers using various experimental models (4-9). In rats, preneoplastic hepatic
foci have been considered end points of carcinogenicity testing
by a number of authors (10-14).
Phenotypic patterns
A landmark for studying early stages of hepatocarcinogenesis
was the finding that treatment of rats with nitrosamines produced
focal liver lesions which were characterized by an excessive storage of glycogen (15) and usually also by a reduction in the activity of the microsomal enzyme glucose-6-phospnatase (16) as
demonstrated by cytochemical methods. From light and electron
microscopical investigations of a series of experiments in which
rats were treated continuously or over short periods (stop experiments) with /V-nitrosomorpholine, a sequence of cellular changes
689
P.Bannasch
Table I. Morphological phenotype and classification of foci of altered hepatocytes
Type of focus
Glycogen
SER
RER/ribosomes
Mitosis
Clear cell foci
Acidophilic cell foci
Intermediate cell foci
Tigroid cell foci
Basophilic cell foci
Mixed cell foci
mixed
mixed
mixed
mixed
has been inferred which leads from clear and acidophilic hepatocytes storing glycogen in excess ('glycogenosis') to basophilic
hepatoma cells poor in glycogen, but rich in free or membranebound ribosomes (15,17). The preneoplastic clear and acidophilic
glycogen storage cells constitute foci which persist after withdrawal of the carcinogen and may progress through intermediate,
mixed and basophilic cell foci to neoplastic nodules (adenomas)
and hepatocellular carcinomas. This developmental sequence has
also been observed during hepatocarcinogenesis induced in rats
by a number of other chemicals, such as diethylnitrosamine,
dimethylaminoazobenzene or thioacetamide (18), and has been
adopted for the classification of specific hepatocellular lesions
in rats among which 'foci of altered hepatocytes' were separated
from 'neoplastic nodules' by two working groups (19,20). Recently a somewhat more sophisticated nomenclature has been proposed (Table I) which takes into consideration that a certain type
of the basophilic foci, called 'tigroid cell focus' (21) apparently
differs in many respects from the basophilic foci described earlier
(22). Preneoplastic foci which exhibit cytomorphological changes
similar to those of the rat have also been detected in other species
including primates (7,18,23).
In addition to changes in the amount of the glycogen, the endoplasmic reticulum and the ribosomes, and alterations in the activity of glucose-6-phosphatase, a large number of other cellular
changes has been described as 'negative' or 'positive' markers,
respectively, for the carcinogen-induced foci (6,9,18,22—29).
Examples of enzymes which frequently show a decreased activity
such as glucose-6-phosphatase are the membrane-bound adenosine triphosphatase (6,30), acid and alkaline nucleases (31,32),
the glycogen phosphorylase (33,34) and adenylate cyclase (35).
An increased activity or content has been found in many (albeit
not all) foci with 7-glutamyltranspeptidase (7-GT*), glucose-6phosphate dehydrogenase (34,38), epoxide hydrolase (39,40),
uridine-diphosphate-glucuronyltransferase (41,42), various isoenzymes of cytochrome P-450 (43,44) and glutathione
transferases (42,44). Moreover, some other functional alterations
have been described in preneoplastic hepatic foci of the rat, namely a resistance to experimental hemosiderosis (45,46), an enhanced glutathione content (47) and a loss of lipid peroxidation (48).
The cytochemical changes appearing in focal liver lesions induced
with chemicals in mice differ in some respects from those of the
rat, but they show also many similarities (7,23).
Observations in different species indicate that the cytochemical
pattern of the preneoplastic foci may be rather heterogenous and
is apparently influenced by many factors, such as the dose and
duration of the carcinogenic treatment, the localization of the focal
lesions within the liver lobule and the age, sex and strain of the
animals (9,18,26,28,49-51). Some observations suggest that an
early reduction of the activity of an enzyme such as glucose-6phosphatase (52,53) or ATPase (54) might be followed by a reappearance of the enzyme activity or, perhaps, the appearance
of an isoenzyme (55) in later stages of hepatocarcinogenesis. Of
690
particular interest is that the activity of 7-GT which has been
considered to be a reliable indicator of preneoplastic changes in
the liver by a number of laboratories (37) may be partly or totally lacking in certain types of preneoplastic foci in rat liver
(21,56—58). On the other hand, a periportal focal increase in
the activity of this enzyme may also occur in rat liver with age
(59) or after partial hepatectomy (60). Two laboratories (61,62)
reported that the hepatocarcinogenic peroxisome-proliferating
hypolipidemic agent nafenopin may 'suppress' histochemical 7GT activity in focal hepatic lesions induced by N-2-fluorenylacetamide. In mice, an increased activity of 7-GT has been observed in focal lesions induced by continuous administration of
safrole (63) or o-aminoazotoluene (64), but not in those produced
by injection of a single dose of diethylnitrosamine in infant animals (23).
In old untreated animals, the various types of foci may develop
'spontaneously', possibly due to a contamination of the food or
the environment with small amounts of carcinogens (65,66). An
exceptionally high incidence of spontaneous foci in certain rat
strains suggests that genetic factors most probably also play an
important role (65,66).
Phenotypic instability
A serious problem for the evaluation of preneoplastic cellular
changes in the liver of rodents has become more and more obvious during the last few years. In various experimental models,
it was found that most phenotypic 'markers' of hepatocarcinogenesis which have been described so far are not stable and may
be mimicked by cellular reactions which are not necessarily related to neoplastic transformation. Thus, under certain experimental conditions, foci (or nodules) have been observed which
resemble in their phenotype the persistent lesions discussed, but
may disappear after cessation of treatment. In addition to this
'reversion-linked' phenotypic instability, a 'progression-linked'
phenotypic instability which is related to metabolic and morphologic changes during transformation of preneoplastic into neoplastic cell populations has to be taken into consideration.
With respect to the reversion-linked phenotypic instability, it
has been known for some time that clear cell areas or foci induced by carcinogens may be partly reversible after withdrawal
(17,67,68). A partial reversibility of cytomorphological and cytochemical changes, including clear, acidophilic and basophilic features which are similar to those in persistent preneoplastic foci,
has also been observed (69) in focal lesions induced in rat liver
by the short-term procedure of Solt and Farber (70). A 'reversion', 'remodeling', 'neodifferentiation' or 'maturation' of carinogen-induced focal liver lesions had earlier been reported by
a number of other authors (25,46,71 -74). An enhanced cytoplasmic basophilia due to ribosomal increase and accompanied
by glycogen reduction as in basophilic hepatic foci may develop
in a reversible manner under various pathological conditions, esecially after high doses of hepatocarcinogens which frequently
lead to so-called megalocytosis (75,76). For an estimation of the
extent of phenotypic instability under the experimental conditions
of the Solt/Farber system, the recent statement by Farber (77)
is of interest: according to his experience, 95-98% of the nodular
lesions are reversible, while only 1-3% persist and may progress to hepatocarcinomas. Even if some observations seem to
indicate that 'remodeled' lesions may reappear after long lag
periods (74,78), the frequently used adjectives 'neoplastic' or
'preneoplastic' for nodules produced in the Solt/Farber system
should be avoided. The term 'hepatocyte nodule' proposed by
Farber (79) or 'proliferating hepatic nodule' appears to be much
Hepatic preneoplasia
more appropriate. The only reliable way to distinguish between
the reversible and the persistent focal lesions, the latter being
considered as preneoplastic, are stop experiments. Thus, it has
been proposed that stop experiments should be conducted whenever foci with a disputed significance develop after application
of certain compounds tested (80).
The progression-linked phenotypic instability was already mentioned when the sequential cytomorphological changes during
hepatocarcinogenesis were discussed. The results of correlative
cytomorphological and cytochemical studies of various enzymes,
especially those of carbohydrate and drug metabolism, support
this concept. Studies of the key enzymes of the alternative pathways of carbohydrate metabolism suggest that the frequently
observed heterogeneous enzyme histochemical patterns do not
emerge at random but are characteristic of certain stages in an
ordered sequence of metabolic and morphologic changes developing during the process of carcinogenesis (4,34,38). Recent findings of Buchmann and colleagues (44), who investigated the
behaviour of a number of enzymes involved in the metabolism
of xenobiotic compounds during hepatocarcinogenesis in rats
using antibodies to the respective enzymes, indicate that such an
ordered pattern of metabolic changes is not only characteristic
of the carbohydrate metabolism but also of other metabolic
pathways of hepatocytes undergoing neoplastic transformation.
The concept of a progression-linked phenotypic instability is
at variance with reports from some other authors who described
a phenotypic stability in focal lesions induced by a single dose
of the carcinogen in newborn or hepatectomized adult animals
followed by phenobarbital treatment (26,81 -83). The reason for
this discrepancy is not clear at present. An important difference
in the evaluation of the experimental results may be that the authors did not determine the histochemical patterns in individual foci
but used a statistical approach which did not allow correlation
of closely associated changes in metabolic pathways. Another
crucial point may be that the additional administration of phenobarbital leads to a rather stable phenotypic expression in the focal
lesion, the cause of which is poorly understood at present (84).
Outset and proliferation kinetics
The current ideas on the cell cycle-dependent primary interaction
of the carcinogen and the possible clonal origin of the preneoplastic cell populations in the liver have been discussed in detail
by Rabes (29). Pereira et al. (85) proposed that initiation of the
preneoplastic foci requires formation of O'-methylguanine, but
Silinskas et al. (86) have recently shown that formation of O6methylguanine in rat liver DNA by nitrosamines does not predict
initiation of preneoplastic hepatic foci.
Various authors investigated the kinetics of cell proliferation
in enzyme-altered foci of the preneoplastic rat liver by autoradiographic determination of the incorporation of [3H]thymidine (29,
30,49,53,87,88). In all of these studies, a considerable increase
in cell proliferation was found in the foci as compared with the
surrounding tissue or the liver parenchyma of untreated controls.
However, most of these studies did not consider the different
cell populations composing the foci. When these cell populations
were investigated separately it turned out that the incorporation
of [3H]thymidine is increased only slightly, if at all, in clear and
acidophilic glycogen storage foci (80). A pronounced and steadily
increasing cell proliferation was only linked with the appearance
of mixed and basophilic cell populations in foci, nodules and carcinomas. These results are in line with the sequence of cellular
changes described earlier, but they do not exclude a minor increase in cell proliferation in the clear and acidophilic cell foci.
This important aspect would need further clarification, especially
since an increase in cell proliferation is a prerequisite for the experimentally well founded hypothesis of a clonal origin of the
preneoplastic hepatic foci (89). In contrast to this hypothesis,
some observations suggest a simultaneous alteration of many
hepatocytes in larger areas of the liver parenchyma (15,17).
Dose-dependence and relation to neoplasia
The dose-dependence of the sequential cellular changes induced
in rat liver by stop experiments with Af-nitrosomorpholine (NNM)
has been investigated with morphometric methods (90). The majority of the foci developed after withdrawal of the carcinogen.
With all dose levels studied, a sequence was established leading
from clear cell and acidophilic cell glycogen storage foci through
mixed cell foci and neoplastic nodules to hepatocellular carcinomas. The first appearance and the frequency of the different
lesions investigated proved to depend on the dose of carcinogen
administered. With increasing dose of NNM, the number of focal
lesions considerably increased, and this was accompanied by an
earlier development of mixed and basophilic cell populations.
There was no indication of any reversibility of the focal lesions
under these experimental conditions. On the contrary, the foci
became larger and acquired phenotypic markers closer to neoplasia without further action of the carcinogen. A progressive
development has also been revealed by morphometric methods
for basophilic foci induced with a single injection of diethylnitrosamine in infant mice (91,92).
Histological transitions between focal hepatic lesions, hepatic
adenomas (neoplastic nodules) and carcinomas have been described by many authors (17,25,27). While these observations
indicate that the adenomas originate from the foci and may progress to carcinomas, it is highly probable that the latter can also
develop directly from the foci without going through a nodular
intermediate stage (93,94).
In addition to these cytomorphological and morphometric results, alterations in a number of functional cellular changes strongly support the concept of a close relation of foci, nodules and
carcinomas. Thus, a similar decrease or increase in the activity
of many enzymes has been demonstrated by enzyme histochemical methods in these lesions (18,24—27). Several authors have
demonstrated in rats or mice that preneoplastic foci, neoplastic
nodules and carcinomas share a common defect in that they do
not accumulate iron in a siderotic liver produced artificially (45,
46,95).
Of particular interest are data concerning the dose-dependence
and development of enzyme-altered foci as published by several
groups (24,96). Although the methods used were somewhat
varied, all the results indicate that quantitative correlations exist
between the size and the number of foci and the dose and duration
of carcinogen treatment. From a statistical point of view there
seems to be a good correlation of dose—time dependence for
the early foci and for the later development of liver tumors.
Although it has not been taken into consideration so far that a
considerable phenotypic instability of enzyme-altered foci and
nodules induced by hepatocarcinogens has been observed under
certain experimental conditions as mentioned earlier (5,69,71,79),
most of these findings are in accord with the concept of a precursor-product relationship between the foci of altered hepatocytes and hepatic tumors. In this context, it should be mentioned,
however, that a direct relationship between focal liver lesions,
nodules and carcinomas has been questioned by some authors
(99). Large discrepancies between the number of foci appearing
early during hepatocarcinogenesis and final tumor yield are in691
P.Bannasch
deed a common finding (6,24,92,100,101). It remains to be clarified whether this is only related to temporal factors or whether
perhaps only a small number of foci have the potential for progression to malignancy. The steady progression in lesion development observed in a number of models appears to support an
important role of time. The possibility that the final tumors may
develop in a multicentric fashion from a number of focal lesions
also deserves further attention (90).
Usefulness in bioassays
There appears to be general agreement that the evaluation of bioassays may be considerably improved by consideration of preneoplastic focal liver lesions. If a test compound produces
significantly more foci of altered hepatocytes in the treated
animals as compared with the concurrent untreated controls, this
suggests a carcinogenic potential of the respective chemical.
Because of a possible reversion-linked phenotypic instability of
the lesions, stop experiments which allow a clear distinction between reversible and persistent preneoplastic foci should be conducted whenever hepatic foci with a disputed significance appear
(80). Although all hepatocarcinogens investigated so far induced some type of focal hepatic lesion prior to the development
of the hepatic tumors, it is not yet clear whether this is always
the case. Hence, the absence of hepatic foci after administration
of a chemical does not preclude a carcinogenic potential of this
compound.
A comparison of the results obtained in a number of experimental models is hampered by both the remarkable variation in the
experimental approaches in different laboratories and the frequent
lack of a clear separation of the (preneoplastic) foci of altered
hepatocytes from the (neoplastic) nodular lesions. Only a few
'clean' models for carcinogenesis bioassays using preneoplastic
hepatic foci have been proposed. The most convincing models
are those in which the test compound is administered for a limited
period (11,80) and the animals are investigated up to several
weeks or months after withdrawal (stop experiment). In an extreme type of stop experiment a single dose is applied to newborn
animals (92,102) or to adult animals after partial hepatectomy
(100,103). Frequently, the additional application of phenobarbital has been used to 'promote' the 'initiated' cells possibly induced
by the test compound, either in young animals (98,104) or in
adult animals after partial hepatectomy (26,105). This approach
has been adopted for the scncalled 'rat liver foci bioassay' (10,
14). Although the additional application of phenobarbital renders
this system more 'sensitive', many problematical interpretations
are based on this 'two-stage model' since most authors imply
that the operational distinction between the 'initiator' and the 'promoter' allows mechanistic explanations. However, a controversial
discussion is still going on as to whether phenobarbital merely
promotes initiated cells or acts in accord with the 'initiator' as
a weak carcinogen (81,106-108). Nevertheless, the 'rat liver
foci bioassay' has been widely used during the past few years
(10-14).
In a recent review, Pereira and Stoner (14) compared the rat
liver foci assay with the strain A mouse lung tumor assay to detect
carcinogens. The authors stated that the rat liver foci assay was
sensitive to 69% of 54 compounds found to be carcinogenic in
long-term bioassays and the strain A lung tumor assay to 54 %
of 93 carcinogens. None of the 10 compounds found to be noncarcinogenic in long-term bioassays were active in the rat liver
foci assay, while seven of 23 non-carcinogens (30%) were active in the lung tumor assay. Ten of the 17 carcinogens negative
in the rat liver foci assay are believed to exhibit tumor-promoting
692
activity, three are direct acting alkylating agents (dimethylsulfate,
epichlorohydrin and /3-propiolactone), and the remaining three
are azobenzene, 1,2-dibromoethane and thioacetamide. Thirtytwo of the 43 carcinogens negative in the lung tumor assay were
active in either (i) the mouse liver only, (ii) the rat and not in
the mouse or (iii) in both the rat and mouse liver but not in other
organs of the mouse.
Extremely complex models have been developed on the basis
of an experiment described by Teebor and Becker (109) and
additional considerations derived from other studies (70,110—
112). From a review taking into consideration 80 compounds
tested, Parodi et al. (113) concluded that the 'preneoplastic nodules' induced by these complex procedures were more predictive than the Ames test as a statistical trend. However, there was
a somewhat better predictivity of the 'preneoplastic nodules' for
liver tumors, as compared with other tumors. It has been emphasized earlier that all of the complex models mentioned should
be interpreted very cautiously (80). The reversibility of many
foci and nodules appearing in these models after cessation of treatment does not seem to be a major problem from a practical point
of view since some foci and nodules persist in all models described. Therefore, any production of foci and nodules might
be sufficient to indicate carcinogenic effect on the liver. However,
the additional application of chemicals, some of which are potent
carcinogens in these test systems, hampers the interpretation of
the results as long as we know little about the different effects
of the various experimental procedures. In any case, stop experiments are to be recommended in order to discriminate clearly
between reversible and persistent foci and nodules, the persisting
lesions being regarded as preneoplastic (foci) or neoplastic
(nodules) in nature (80).
Acknowledgements
I would like to express my appreciation to Dr Heidc Zerban for support in the
preparation of this manuscript and to Ursula Biallas for secretarial help.
References
1. Symposium of Early Lesions and the Development of Epithelial Cancer (1976)
Cancer Res., 36, 2475-2706.
2. Bannasch.P. (1984) Sequential cellular changes during chemical carcinogenesis.
J. Cancer Res. Clin. Oncol., 108, 11 - 2 2 .
3. Farber.E. and Cameron.R. (1980) The sequential analysis of cancer development. Adv. Cancer Res., 31, 125-226.
4. Bannasch.P., Hacker.H.J., Klimek,F. and Mayer,D. (1984) Hepatocellular
glycogenosis and related pattern of enzymatic changes during hepatocarcinogenesis Adv. Enzyme. Regul., 22, 97-121.
5. Farber,E. (1984) Pre-cancerous steps in carcinogenesis. Their physiological
adaptive nature. Biochim. Biophys. Ada, 738, 171-180.
6. Scnerer.E. (1984) Neoplastic progression in experimental hepatocarcinogenesis.
Biochim. Biophys. Ada, 738, 219-236.
7. WardJ.M. (1984) Morphology of potential preneoplastic hepatocyte lesions
and liver tumors in mice and a comparison with other species. In PoppJ.A.
(ed.), Mouse Liver Neoplasia, Hemisphere Publishing Corporation, Washington, New York, London, pp. 1 - 2 6 .
8. Weinbren.K. (1984) Precancerous states in the liver. In Carter,R.L. (ed.),
Precancerous States, Oxford University Press, London, New York, Toronto,
pp. 254-277.
9. Moore,M.A. and Kitagawa.T. (1986) Hepatocarcinogenesis in the rat; the
effect of the promoters and carcinogens in vivo and in vitro. Int. Rev. Cytol.,
in press.
10. Pereira,M.A. (1982) Rat liver foci bioassay. J. Am. Coll. Toxicol., 1, 1 0 1 117.
11. Williams,G.M. (1982) Phenotypic properties of preneoplastic rat liver lesions
and applications to detection of carcinogens and tumor promoters. Taxicol.
Pathol., 10, 3 - 1 0 .
12. Laib.R.J., Kiein.K.P. and Bolt,H.M. (1985) The rat liver foci bioassay: I.
Age-dependence of induction by vinyl chloride of ATPase-dcficienl foci. Car-
cinogenesis, 6, 65-68.
Hepatic preneoplasia
13. Laib.RJ., PeUio.T., Wunschd.U.M., Zimmermann,N. and Boft.H.M. (1985)
The rat liver foci bioassay: D. Investigations on the dose-dependent induction of ATPase-deficient foci by vinyl chloride at very low doses. Carcinogenesis, 6, 69-72.
14. Pereira.M.A. and Stoner.G.D. (1985) Comparison of rat liver foci assay and
strain A mouse lung tumor assay to detect carcinogens: a review. Fund. Appl.
Toxicol.. 5, 688-699.
15. Bannasch.P. and MOller.H.A. (1964) Lichtmikroskopische Untersuchungen
uber die Wirkung von N-Nitrosomorpholin auf die Leber von Ratte und Maus.
Arzneim. Forsch., 14, 805-814.
16. Gossner.W. and Friedrich-Freksa.H. (1964) Histochemische Umersuchungen
Ober die Gtucose-6-Phosphatase in der Rattenlcber wahrend der Cancerisiening
durch Nitrosamine. Z Natwrforscn., 19b, 862-864.
17. Bannasch.P. (1968) The cytoplasm of hepatocytes during carcinogencsis. Light
and electron microscopic investigations of the nkrosomorpholine-intoxicated
rat liver. Rec. Res. Cancer Res., 19, 1-100.
18. Bannasch.P., Mayer.D. and Hacker.H J . (1980) Hepatocellular glycogenosis
and hepatocarcinogenesis. Biochim. Biophys. Acta, 605, 217—245.
19. Squire.R.A. and Levitt.M.H. (1975) Report of a workshop on classification
of specific hepatocellular lesions in rats. Cancer Res., 35, 3214-3223.
20. Stewart.H.L., Williams.G., Keysser.C.H., Lombard.LS. and Montali.R.J.
(1980) Histologic typing of liver tumors of the rat. / . Nail. Cancer Inst.,
65, 179-206.
21. Bannasch.P., Benner.U., Enzmann.H. and Hacker.H.J. (1985) Tigroid cell
foci and noeplastic nodules in the liver of rats treated with a single dose of
aflatoxin B,. Carcinogenesis, 6, 1641-1648.
22. Bannasch.P., Zcrban.H. and Hacker.H.J. (1985) Foci of altered hepatocytes,
rat. In Jones.T.C, Mohr,U. and Hunt,R.D. (eds), Monographs on Pathology
of Laboratory Animals, Digestive System. Springer-Verlag, Berlin, Heidelberg,
New York, pp. 10-30.
23. Vesselinovitch.S.D., Hacker.H.J. and Bannasch.P. (1985) Histochemical
characterization of focal hepatic lesions induced by single diethylnitrosamine
treatment in infant mice. Cancer Res., 45, 2774-2780.
24. Emmelot.P. and Scherer.E. (1980) The first relevant cell stage in rat liver
carcinogenesis. A quantitative approach. Biochim. Biophys. Acta, 605, 2 4 7 304.
25. Farber.E. (1980) The sequential analysis of liver cancer induction. Biochim.
Biophys. Acta, 605, 149-166.
26. Pitot.H.C. and Sirica.A.E. (1980) The stages of initiation and promotion in
hepatocarcinogenesis. Biochim. Biophys. Acta, 605, 191-215.
27. WUliams.G.M. (1980) The pathogenesis of rat liver cancer caused by chemical
carcinogens. Biochim. Biophys. Acta, 605, 167-189.
28. Peraino.C, Richards.W.L. and Stevens.FJ. (1983) Multistage hepatocarcinogenesis. In Slaga.T.J. (ed.), Mechanisms of Tumor Promotion, CRC Press,
Boca Raton, Vol. 1, pp. 1-53.
29. Rabes,H.M. (1983) Development and growth of early preneoplastic lesions
induced in the liver by chemical carcinogens. J. Cancer Res. Clin. Oncol.,
106, 85-92.
30. Schauer^A. and Kunze.E. (1968) Enzymhistochemische und autoradiographische Untersuchungen wihrend der Kanzerisierung der Rattenlebcr durch
Difithylnitrosamin. Z Krebsforsch., 70, 252-266.
31.Taper,H.S., Fort.L. and BrucherJ.-M. (1971) Histochemical activity of
alkaline and acid nucleases in the rat liver parenchyma during A'-nitrosomorpholine carcinogenesis. Cancer Res., 31, 913-916.
32. Taper.H.S., Lans.M., deGerlacheJ., Fort.L. and Roberfroid.M. (1983) Morphological alterations and DNase deficiency in phenobarbital promotion of
A'-nitrosomorpholine initiated rat hepatocarcinogenesis. Carcinogenesis, 4,
231-234.
33. Scherer.E. and Emmelot.P. (1976) Kinetics of induction and growth of
enzyme-deficient islands involved in hepatocarcinogenesis. Cancer Res., 36,
2544-2554.
34. Hacker.H.J., Moore.M.A., Mayer.D. and Bannasch.P. (1982) Correlative
histochemistry of some enzymes of carbohydrate metabolism in preneoplastic
and neoplastic lesions in the rat liver. Carcinogenesis, 3, 1265—1272.
35. Ehemann.V., Mayer.D., Hacker.H.J. and Bannasch.P. (1986) Loss of
adenylate cyclase activity in preneoplastic and neoplastic lesions induced in
rat liver by A'-nitrosomorpholine. Carcinogenesis, 7, 567-573.
36. Kalengayi,M.M.R. and Desmet.V J . (1975) Sapiential histotogical and histochemical study of the rat liver during aflatoxin B,-induced carcinogenesis.
Cancer Res., 35, 2845-2852.
37. Hanigan.M.H. and Pitot.H.C. (1985) Gamma-glutamyl transpeptidase — hs
role in hepatocarcinogenesis. Carcinogenesis, 6, 165-172.
38. KJimek.F., Mayer.D. and Bannasch.P. (1984) Biochemical microanalysis of
glycogen content and glucose-6-phosphate dehydrogenase activity in focal
lesions of rat liver induced by A'-nitrosomorpholine. Carcinogenesis, 5, 265 —
268.
39. Enomoto.K., Ying.T.S., Griffin,M.J. and Farber.E. (1981) Immunohistochemical study of epoxide hydrolase during experimental liver carcinogenesis.
Cancer Res., 41, 3281-3287.
40. Kuhlmann.W.D., Krishan.R., Kunz.W., Guenthner.T.M. and Oesch.F.
(1981) Focal elevation of liver microsomal epoxide hydrolase in early preneoplastic stages and its behaviour in the further course of hepatocarcinogenesis.
Biochem. Biophys. Res. Commun., 98, 417-423.
41. Rscher.G., UUrich.D., Katz,N., Bock,W.K. and Schauer,A. (1983) Immunohistochemical and biochemical detection of uridine-diphosphate-glucuronyltransferase (UDP-GT) activity in putative preneoplastic liver foci. Virchows
Arch. B Cell Pathol, 42, 193-200.
42. Sato.K., Kitahara^i., SatonJC., Ishikawa.T., Tatematsu.M. and Ito.N. (1984)
The placental form of glutathkHie s-transferase as a new marker protein for
preneoplasia in rat chemical carcinogenesis. Gann, 75, 199—202.
43. Schuhe-Hermann.R., Roome.N., Timmermann-Trosiener.I. and Schupptevl.
(1984) uTimunocytochemical demonstration of a phenobarbital-inducibie cytochrome P-450 in putative preneoplastic foci of rat liver. Carcinogenesis, 5,
143-153.
44. Buchmann,A., Kuhlmann.W.D., Schwarz.M., Kunz,H.W., Wolf.C.R., MoU,
E., Friedberg.T. andOesch.F. (1985) Regulation and expression of four cytochrome P-450 isoenzymes, NADPH-cytochrome P-450 reductase, the glutathione transferase B and C and microsomal epoxide hydrolase in preneoplastic
and neoplastic lesions in rat liver. Carcinogenesis, 6, 513-521.
45. Williams.G.M., Klaiber.M., Parker.S.E. and Farber.E. (1976) Nature of carry
appearing, carcinogen-induced liver lesions resistant to iron accumulation.
J. Nail. Cancer Inst., 57, 157-165.
46. Williams.G.M. and Watanabe.K. (1978) Quantitative kinetics of development
of N-2-fluorenylacetamide-induced, altered hyperplastic hepatocellular foci
resistant to iron accumulation and of their reversion or persistence following
removal of carcinogen. /. Natl. Cancer Inst., 61, 113-121.
47. Deml,E. and Oesterle.D. (1980) Histochemical demonstration of enhanced
glutathione content in enzyme-altered islands induced by carcinogens in rat
liver. Cancer Res., 40, 490-491.
48. Benedetti.A., Malvadi.G., Fulcen.R. and Comporti.M. (1984) Loss of lipid
peroxidation as a histochemical marker for preneoplastic hepatocellular foci
of rats. Cancer Res., 44, 5712-5717.
49. Rabes.H.M., Scholze,P. and Jantsch.B. (1972) Growth kinetics of diethylnitrosamine-induced enzyme-deficient 'preneoplastic' liver cell populations
in vivo and in vitro. Cancer Res., 32, 2577-2586.
50. Hirota.N. and Williams.G.M. (1979) The sensitivity and heterogeneity of
histochemical markers for altered foci involved in liver carcinogenesis. Am.
J. Pathol., 95, 317-328.
51. Deml.E., Oesterle.D., Wo)ff,T. and Greim,H. (1981) Age-, sex- and straindependent differences in the induction of enzyme-altered islands in rat liver
by diethylnitrosamine. /. Cancer Res. Clin. Oncol, 100, 125-134.
52. Ruebner.B.H., Michas.C, Kanayama.R. and Bannasch.P. (1976) Sequential
hepatic histologic and histochemical changes produced by diethylnitrosamine
in me Rhesus monkey. J. Natl. Cancer Inst., 57, 1261-1268.
53. Pugh,T.D. and Goldfarb.S. (1978) Quantitative histochemical and autoradiographic studies of hepatocarcinogenesis in rats fed 2-acetylaminofluorene.
Cancer Res., 38, 4450-4457.
54. Daoust.R. (1979) Histochemical comparison of focal losses of RNAse and
ATPase activities in preneoplastic rat livers. J. Histochem. Cytochem., 27,
653-656.
55. Karasaki.S. (1976) Ultrastructural and cytcchemical studies on hyperbasophilic
foci with special reference to the demonstration of cell surface alterations in
hepatocarcinogenesis. Cancer Res., 36, 2567-2572.
56. Butler.W.H., Hempsall.V. and Stewart.M.C. (1981) Histochemical studies
on the early proliferating lesions induced in the rat liver by aflatoxin. J. Pathol,
133, 325-340.
57. Moore.M.A., Hacker.H.J., Kunz.H.W. and Bannasch.P. (1983) Enhancement of NNM-induced carcinogenesis in the rat liver by phenobarbital: a combined morphological and enzyme histochemical approach. Carcinogenesis,
4, 473-479.
58. Rao.M.S., Lalwani.N.D. and ReddyJ.K. (1984) Sequential histologic study
of rat liver during peroxisome proliferator [4-diloro-6<2,3-xylidino)-2-pyrirnidinylthioj-acetic acid (Wy-14,643)-induced carcinogenesis. J. Natl Cancer
Inst., 73,983-990.
59. Khagawa.T., Imai.F. and Sato.K. (1980) Re-evaluation of y-glutamy 1 transpeptidase activity in periportal hepatocytes of rats with age. Gann, 71, 3 6 2 366.
60 Bone UI.S.N., Michalopoulos.G. and Jirtle.R.L. (1985) Ability of partial
hepatectomy to induce y-gtutamyhranspeptidase in regenerated and transplanted
hepatocytes of Fischer 344 and Wistar-Furth rats. Cancer Res., 45, 12221228.
61. StauWi.W., Bentry.P., Bieri.F., Fr61uich,E. and Waechter.F. (1984) Inhibitory
693
P.Bannascfa
effect of nafenopin upon the development of diethylnitrosamine-induced enzyme altered foci within the rat liver. Carcinogenesis, 5, 41-46.
62. Numoto.S., Furukawa.K., Furuya.K. and Williams.G.M. (1984) Effects of
the hepatocaicinogenic peroxisome-proliferating hypolipidemic agents Qofibrate and Nafenopin on the rat liver cell membrane enzymes 7-glutamyltranspeptklase and alkaline phosphatase and on the early stages of liver
carcinogenesis. Carcinogenesis, 5, 1603-1611.
63.Lipsky,M.M., Hinton.D.E., KlaunigJ.E., Goldblatt.P.J. and Tmmp.B.F.
(1981) Biology of hepatoceUular neoplasia in the mouse. II. Sequential enzyme histochemical analysis of BALB/c mouse liver during safrole-induced
carcinogenesis. J. Natl. Cancer lnst., 67, 3T7-392.
64. Jalanko.H. and Ruoslahti.E. (1979) Differential expression of a-feto-protein
and 7-glutamyl-transpeptidase in chemical and spontaneous hepatocarcinogenesis. Cancer Res., 39, 3495-3501.
65. BurekJ.D. (1978) Pathology of aging rats. A morphological and experimental
study of the age-associated lesions in aging BN/Bi, WA6/Rij and (WA6XBN)
Fl rats. CRC Press, West Palm Beach, Florida, pp. 58-68.
66. WardJ.M. (1981) Morphology of foci of altered hepatocytes and naturallyoccurring hepatoceUular tumours in F344 rats. Virchows Arch. PathoL Anat.,
390, 339-345.
67. Newbeme.P.M. (1976) Experimental hepatoceUular carcinogenesis. Cancer
Res., 36, 2573-2578.
68. Flaks.B. and Basley.W.A. (1979) Acute fine structural changes in rat hepatocytes induced by a single large dose of 2-acetylarrdnofluorene. Virchows Arch.
B Cell PathoL, 29, 309-320.
69. Moore.M.A., Hacker.H.J. and Bannasch,P. (1983) Phenotypic instability in
focal and nodular lesions induced in a short term system in the rat liver. Carcinogenesis, 4, 595-603.
70. Solt.D. and Farber.E. (1976) New principle for the analysis of chemical carcinogenesis. Nature, 263, 702-703.
71. Khagawa.T. (1971) Histochemical analysis of hyperplastic lesions and hepatomas of the liver of rats fed 2-fluorenylacetamide. Gann, 62, 207-216.
72. Ito.N., Hananouchi.M., Sugfliara.S., Shirai.T., Tsuda^., Fukushima,S. and
Nagasaki.H. (1976) Reversibility and irreversibility of liver tumor in mice
induced by the a-isomer of 1,2,3,4,5,6-hexachlorocydohexane. Cancer Res.,
36, 2227-2234.
73. Ogawa.K., Medline.A. and Farber.E. (1979) Sequential analysis of hepatic
carcinogenesis. A comparative study of the ultrastructure of preneoplastic,
malignant, prenatal, postnatal, and regenerating liver. Lab. Invest. ,41, 22-35.
74. Tatematsu.M., Nagamine.Y. and Farber.E. (1983) Redifferentiation as a basis
for remodeling of carcinogen-induced hepatccyte nodules to normal appearing liver. Cancer Res., 43, 5049-5058.
75. Theodossiou.A., Bannasch,P. and Reuss.R. (1971) Gh/kogen und endoplasmatisches Retikuhim der Leberzelle nach hohen Dosen des Carcinogens NNitrosomorpholin. Virchows Arch. Abt. B Zellpathol., 7, 126-146.
76. Taper.H.S. and Bannasch.P. (1979) Histochemical differences between socalled megalocytosis and neoplastic or preneoplastic liver lesions induced by
N-nitrosomorpholine. Eur. J. Cancer, 15, 189-196.
77. Farber.E. (1984) Cellular biochemistry of the stepwise development of cancer
•with chemicals: G.H.A.Clowes Memorial Lecture. Cancer Res., 44, 54635474.
78. Williams.G.M., Hirota.N. and RiceJ.M. (1979) The resistance of spontaneous
mouse hepatoceUular neoplasms to iron accumulation during rapid iron loading
by parenteral administration and their transplantability. Am. J. PathoL, 94,
65-74.
79. Farber.E. (1982) The biology of carcinogen-induced hepatocytc nodules and
related liver lesions in the rat. Toxicol. Pathol., 10, 197-205.
80. Bannasch.P., Moore.M.A., Klimek.F. and Zerban.H. (1982) Biological
markers of preneoplastic foci and neoplastic nodules in rodent liver. Toxicol.
Pathol., 10, 19-34.
81. Goldworthy.T., Campbell.H.A. and Pitot.H.C. (1984) The natural history
and dose-response characteristics of enzyme-altered foci in rat liver following phenobarbital and diethylnitrosamine administration. Carcinogenesis, 5,
67-71.
82. Peraino,C, Staffekfc.E.F., Cames.B.A., Ludeman.V.A., BtomquisU.A. and
Vesselinovitch.S.D. (1984) Characterization of histochemically detectable
altered hepatocyte foci and their relationship to hepatic tumorigenesis in rats
treated once with diethylnitrosamine or benzo[a]pyrene within one day after
birth. Cancer Res., 44, 3340-3347.
83. GoMworthy.T.L. and Pitot,H.C. (1985) The quantitative analysis and stability
of histochemical markers of altered hepatic foci in rat liver following initiation by diethylnitrosamine administration and promotion with phenobarbital.
Carcinogenesis, 6, 1261-1269.
84. Moore.M.A., Tsuda.H., Ogiso.T., Mera.Y. and Ito.N. (1984) Enhancement
of phenotypic instability by or-difluoromethylornithine and butylated hyroxy-
694
anisole in rapidly induced rat liver lesions. Cancer Lett., 25, 145-151.
85. Pereira.M.A., lin.L.-H.C. and Herren,S.L. (1983) Role of C-mcthylation
in the initiation of GGTase-positive foci. Chen-BioL Interactions, 43, 313 —
322.
86. SUinskasJC.C, Zucker.P.F. and Archer.M.C. (1985) Formation of C-methylguanine in rat liver DNA by nhrosamines does not predict initiation of preneoplastic foci. Carcinogenesis, 6, 773-775.
87. Barbason.H., Fridman-Manduzo.A., Lelievre.P. and Betz.E.H. (1977) Variations of liver cell control during diethylnitrosamine carcinogenesis. Eur. J.
Cancer, 13, 13-18.
88. Schulte-Hermann.R., Ohde.G., SchupplerJ. and Tunmermann-Trosiener.I.
(1981) Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with the tumor promoters phenobarbital, hexachlorocyclohexane,
steroid compounds and nafenopin. Cancer Res., 41, 2556-2562.
89. Rabes.H.M., Bucher.T., Hartmann.A., Linke.I. and Dunnwald.M. (1982)
Clonal growth of carcinogen-induced enzyme-deficient prcneoplasnc ceU populations in mouse liver. Cancer Res., 42, 3220—3227.
90. Moore.M.A., Mayer.D. and Bannasch.P. (1982) The dose-dependence and
sequential appearance of putative preneoplastic populations induced in the
rat liver by stop experiments with A'-nitrosomorpholine. Carcinogenesis, 3,
1429-1436.
91. Goldfarb.S., Pugh/T.D., Koen.H. and He,Y.-Z. (1983) Preneoplastic and
neoplastic progression during hepatocarcinogenesis in mice injected with diethylnitrosamine in infancy. Environ. Health Perspect., 50, 149-161.
92. Vesselinovitch.S.D. and Mihailovkh.N. (1983) Kinetics of diethylnitrosamine
hepatocarcinogenesis in the infant mouse. Cancer Res., 43, 4253—4259.
93. Bannasch.P. (1976) Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodules. Cancer Res., 36, 2555-2562.
94. Williams.G.M. (1976) Functional markers and growth behaviour of preneoplastic hepatocytes. Cancer Res., 36, 2540-2543.
95. Lipsky,M.M., Hinton.D.E., Goldblatt.P.J., Klaunig.J.E. and Trump.B.F.
(1979) Iron negative foci and nodules in safrole-exposed mouse Uver made
siderotic by iron-dextran injection. Pathol. Res. Pract., 164, 178-185.
96. Kunz.W., Schaude.G., Schwarz.M. and Tennekes.H. (1982) Quantitative
aspects of drug-mediated tumour promotion in liver and its toxicotogical implications. In Hecker.E., Fusenig.N., Marks,F. and Kunz.W. (eds), Carcinogenesis — A Comprehensive Survey, Raven Press, New York, pp. 111 -125.
97. Tatematus.M., Takano,T., Hasegawa.R., Imaida.K., NakanowatariJ. and
Ito.N. (1980) A sequential quantitative study of the reversibility or irreversibility of liver hyperplastic nodules in rats exposed to hepatocarcinogens.
Gann, 71, 843-855.
98. Cater.K.C, Gandolfi.AJ. and Sipes.I.G. (1985) Characterization of diethylnitrosamine-induced focal and nodular lesions in the livers of newborn mice.
ToxicoL PathoL, 13, 3 - 9 .
99. O'Connor.C.A. and Woodward.S.C. (1981) Industry's role in cancer research:
anticipating regulatory problems. Regul. Toxicol. Pharmacol., 1, 316-334.
100. Scherer.E. and Emmelot.P. (1975) Foci of altered liver cells induced by a
single dose of diethylnitrosamine and partial hepatectomy; their contribution
to hepatocarcinogenesis in the rat. Eur. J. Cancer, 11, 145 — 154.
101. Kaufmann,W.K., Mackenzie.S.A. and Kaufrnan.D.G. (1985) Quantitative
relationship between hepatocytic neoplasms and islands of ceUular alterations
during hepatocarcinogenesis in the male F344 rat. Am. J. Pathol., 119, 171 174.
102. Vesselinovitch.S.D., Rao.K.V.N. and Mihailovich.N. (1979) Neoplastic
response of mouse tissues during perinatal age periods and its significance
in chemical carcinogenesis. J. Natl. Cancer lnst. Monogr., 51, 239-250.
103. Scherer.E., Hoffmann.M., Emmelot.P. and Friedrich-Frcska.H. (1972)
Quantitative study on foci of altered liver cells induced in the rat by a single
dose of diethylnitrosamine and partial hepatectomy. J. Natl. Cancer lnst.,
49, 9 3 - 106.
104. Peraino.C, Staffeldt.E.F. and Ludeman.V.A. (1981) Early appearance of
histochcmkaUy altered hepatocyte foci and liver tumors in female rats treated
with carcinogens one day after birth. Carcinogenesis, 2, 463-465.
105. Phot,H.C, BarsnessJL., GoMsworthy,F. and KitagawaJ. (1978) Biochemical
characterisation of stages of hepatocarcinogenesis after a single dose of
diethylnitrosamine. Nature, 271, 456-458.
106. Rossi.L., Ravera.M., Repetn'.G. and Santi.L. (1977) Long-term administration
of DDT or phenobarbital-Na in Wistar rats. Int. J. Cancer, 19, 179-185.
107. Feldman.D., Swarm.R.L. and Becker J . (1981) Ultrastructural study of rat
liver and liver neoplasms after long-term treatment with phenobarbital. Cancer
Res., 41, 2151-2162.
108. WardJ.M. and Ohshima.M. (1985) Evidence for lack of promotion of the
growth of the common naturally occurring basophilic focal hepatoceUular proliferative lesions in aged F344/NCr rats by phenobarbital. Carcinogenesis,
6, 1255-1259.
Hepatic preneoplasia
109. Teebor.G.W. and Becker.F.F. (1971) Regression and persistence of hyperplastic nodules induced by N-2-fluorenylacetamide and their relationship to
hepatocarcinogenesis. Cancer Res., 31, 1-3.
110. Ito.N., Tatematsu.M., Nakanishi.K., Hasegawa,R., Talcano.T., Imaida.K.
and Ogiso.T. (1980) The effects of various chemicals on the development
of hyperplastic liver nodules in hepatectomized rats treated with yV-nitrosodiethylamine or ^-2-fluorenylacetainide. Gann, 71, 832-842.
Ul.Tsuda.H., Lee.G. and Farber.E. (1980) Induction of resistant hepatocytes
as a new principle for a possible short term in vivo test for carcinogens. Cancer
Res., 40, 1157-1164.
Received on 27 January 1986; accepted on 10 February 1986
695