THE IN OVO CARCINOGENEICITY ASSAY (IOCA): A REVIEW OF AN EXPERIMENTAL APPROACH FOR RESEARCH ON CARCINOGENESIS AND CARCINOGENICITY TESTING

Harald Enzmann¹ and Klaus D. Brunnemann²

1Bayer AG, Institute of Toxicology, 42096 Wuppertal, Germany and ²American Health Foundation, Valhalla, NY 10595, USA

Received 7/21/97 Accepted 11/14/97

TABLE OF CONTENTS

1. Abstract

2. Introduction: avian models of carcinogenicity

3. Carcinogen-induced cellular changes in embryonic avian liver

3.1 Induction of adenoid formations by carcinogens
3.2 Carcinogen-induced hepatocellular nuclear enlargement
3.3 Quantification of carcinogen-induced nuclear enlargement
3.4 Significance of the carcinogen-induced nuclear enlargement

4. Carcinogen induced focal hepatic lesions in the in ovo carcinogenicity assay (IOCA)

4.1 Induction of focal hepatic lesions in avian embryonic liver
4.2 Experimental induction of focal hepatic lesions in rodents
4.3 Focal hepatic lesions as end points in carcinogenicity testing

5. Focal hepatic lesions as endpoints in carcinogenicity testing
Induction of damage to mitochondrial DNA in ovo

5.1 Mitochondrial DNA as target for genotoxic chemicals
5.2 Studies of mitochondrial DNA in carcinogen-exposed embryonic turkey liver

6. Perspectives of the IOCA

7. Acknowledgements
8. References

1. ABSTRACT

Up to now, the carcinogenicity of a substance, i.e., its cancer-causing effect, has been usually determined with the help of extensive animal testing. The in ovo carcinogenicity assay (IOCA) has been suggested as a rapid and inexpensive, non-animal, method for carcinogenicity testing and for experimental studies on mechanisms of carcinogenesis. The substance to be tested is injected into a fertilized ovum. No later than four days before the hatching date, the embryos are released, and the liver is removed for identifying the effect of hepatocarcinogens.

Histological, cytological and molecular biological alterations of the embryonic liver have been induced with a variety of chemical carcinogens. After sufficiently high doses of hepatocarcinogens, tubular structures predominate in the liver and replace the normal trabecular pattern. The cell and nuclear size of the hepatocytes in embryonic liver is severely increased after exposure to chemical carcinogens over a wide dose range including doses that fail to elicit cytotoxicity in the embryonic liver.

The in ovo exposure of avian embryos to hepatocarcinogens has also been shown to induced focal lesions in the embryonic livers. In embryonic turkey liver, in ovo exposure to carcinogens mostly induced basophilic hepatocellular altered foci (foci of altered hepatocytes) that were similar to carcinogen-induced lesions in rodent liver. In embryonic quail livers, most of the focal lesions were composed of cells that morphologically appeared more similar to cholangiocytes than to hepatocytes.

The in ovo exposure of embryonic liver to hepatocarcinogens resulted in damage to the mitochondrial DNA (mtDNA). Electrophoresis on agarose gels revealed a carcinogen-induced effect on the molecular size of the mtDNA. The content of mtDNA of the regular size of 16 kilobases (kb) dose-dependently decreased. At the same time, the amount of fragmented mtDNA of various sizes increased.

The advantages of bird embryos for carcinogenicity testing are considerable: The IOCA is rapid, since the in ovo phase does not exceed 24 days. Since no facilities for animal housing are required, it is less expensive than whole animal experiments. There is less potential of human exposure during the experiment than in animal experiments and studies in a non-rodent system may help to discriminate between chemicals that are rodent specific carcinogens and chemicals that may constitute a potential cancer hazard to man.

2. INTRODUCTION: AVIAN MODELS OF CARCINOGENICITY

The use of birds in experimental cancer research is even older than the experimental induction of tumors by chemicals. Four years before the first chemical induction of an experimental tumor was described in rabbits, the viral induction of chicken sarcomas was published. Since then, the experimental induction of carcinogenesis in birds has been predominantly used for studies on viral carcinogenesis. The sensitivity of birds to chemical carcinogens became obvious with the elucidation of the turkey X disease which resulted in the discovery of the strongest known carcinogens, the aflatoxins.

More recently, liver cell cancer in birds has emerged as a potential model for the synergistic effects between viral hepatitis and exposure to chemical carcinogens Bird embryos have been known to be more sensitive to viral transformation than the hatched birds. The

use of bird embryos has also been suggested for tests for genotoxicity demonstrating chemically induced damage to nuclear DNA.

The disadvantage of this experimental approach are the potential metabolic differences between this non-mammalian system and the rodents usually used for carcinogenicity studies. Depending on the test chemical used, activation or inactivation of the administered chemicals in the embryonic tissue may be a crucial determinant. Drug-metabolizing enzyme activities in turkey and chick liver have been shown to be similar to the activities in rat liver. In the embryonic liver, however, the activation of xenobiotics is less effective than in adult liver or at hatching. Nevertheless, measurable enzyme activities are found as early as on day 5 of development. Since the mixed function oxidase enzyme system can be induced even at this early stage, the sequential administration of an enzyme inducing agent and of the potentially carcinogenic test substance might be advantageous.

3. CARCINOGEN-INDUCED CELLULAR CHANGES IN EMBRYONIC AVIAN LIVER

3.1 Induction of adenoid formations by carcinogens

Exposure of avian embryonic liver to the hepatocarcinogenic nitrosamines N-nitrosomorpholine (NNM) and diethylnitrosamine (DEN) resulted in the occurrence of tubular (adenoid, ductular) formations instead of the physiological trabecular organization of the embryonic liver (10, 11). Extracellular deposits of a brownish substance (bile pigment) were occasionally observed in these cell populations. Neither basal membranes nor fibrillary structures were detectable at the luminar cell pole. After high doses of the nitrosamines, the whole liver was composed of a mixture of small basophilic hepatocytes and large acidophilic hepatocytes including cells with a ground glass appearance. A similar mixture of large acidophilic hepatocytes and small basophilic hepatocytes has long been shown in the liver after exposure of rats to the very same nitrosamines (12). The occurrence of tubular structures is a typical histological feature of adenoid hepatocellular tumors (13), but may also be observed in severely damaged human liver (14,15).

3.2 Carcinogen-induced hepatocellular nuclear enlargement

The cell and nuclear size of the hepatocytes in embryonic liver is severely increased after exposure to chemical carcinogens. This effect on the nuclear size is dose dependent (table 1) and occurs at doses lower than the doses required for the induction of the tubular formations or preneoplastic focal lesions (see below). Single hepatocytes with unusually large nuclei (figure 1) are observed after exposure to low doses that fail to elicit cytotoxicity in the embryonic liver. At low doses, only a small minority of hepatocytes exhibit enlarged nuclei after exposure to low doses of carcinogens whereas the vast majority of nuclei are apparently normal. Therefore, the 95% quantile or the 99% quantile rather than the arithmetic mean or the median of a sample is the most appropriate means of assessing the exposure-induced nuclear size differences.

Figure 1: Effect of diethylnitrosamine on nuclear size: obviously enlarged hepatocyte nucleus (center) after exposure to 0.4 mg of diethylnitrosamine. Hematoxylin eosin stain, objective magnification 40-fold.

3.3 Quantification of carcinogen-induced nuclear enlargement

The measurement of cell and nuclear size may be performed using flow cytometry (16). Most hepatotoxins, however, cause qualitatively and quantitatively different damage to periportal and perivenous hepatocytes (17,18) and cell suspensions are not suitable for the discrimination of periportal and perivenous hepatocytes. In addition, cell suspensions do not allow an exact distinction between cells of the carcinogen-induced focal lesions (see below) and the surrounding extrafocal tissue. Therefore, we preferred to measure nuclear size in the tissue sections. There were no differences in nuclear size between periportal and perivenous hepatocytes in untreated control embryos (table 2).

It is worth to consider that thickness of the section may be a crucial point in the planimetric measurement of nuclear areas. The measured areas of peripherally cut nuclei (small areas) strongly depends on the thickness of the section. Focusing the microscope for the maximal nuclear area will have the consequence that with an infinite increase in section thickness no peripherally cut nuclei, but exclusively the maximal (central) area of nuclei is measured. On the other hand, the ideally central profiles will give the maximal area values both in infinitely thin and in infinitely thick sections. Therefore, whereas small profiles may be underrepresented in the measured data and their number may be biased by the thickness of the section, the maximal area values will hardly be biased by variations in thickness of the section, if enough profiles per sample are measured.

3.4 Significance of the carcinogen-induced nuclear enlargement

The size of liver cell nuclei may reflect various physiological and pathological processes. In rodents, distinct alterations in the ploidy pattern are characteristic during postnatal development (19). Alterations of the nuclear size of parenchymal cells have frequently been observed during various stages of hepatocarcinogenesis. Various authors reported the occurrence of enlarged or hyperploid nuclei at early stages of the carcinogenic process (20-24), effects of the carcinogenic treatment on the number of binucleated hepatocytes (24-27) or proposed in vitro test systems based on the carcinogen-induced nuclear enlargement (28).

At least some of these carcinogen-induced phenomena seemed also to be linked to liver regeneration. Regeneration after partial hepatectomy (29-31) or after toxic injury (32) results in a shift towards higher ploidy levels and a reduced number of binucleated hepatocytes. Consequently, the occurrence of enlarged nuclei during chemically induced hepatocarcinogenesis was regarded as to be due to the combination of regenerative and toxic effects (33). More recent experiments, however, showed that enlarged nuclei occurred after low doses of carcinogens that failed to induce other signs of nonspecific toxic effects (34). Nuclei with unusually high ploidy level were observed in LEC rats with hereditary hepatitis (35) before these rats developed preneoplastic liver cell foci and hepatocellular carcinomas without any deliberate exposure to carcinogens (36). According to Clawson and coworkers an enlargement of nuclei was also observed after, low, and essentially non-toxic doses of mutagenic carcinogens (37). Similarly, effects on the nuclear size were observed in the IOCA even after small doses that did not induce foci of altered hepatocytes and did not induce nonspecific toxic effects, such as cell death, loss of glycogen or fatty change. This observation is not compatible with the assumption, that the enlarged nuclei merely reflect a compensatory regeneration due to toxic cell loss. Both the occurrence of aneuploid or hyperploid nuclei caused by nonspecific effects on the mitotic spindle (38) and a carcinogen-mediated acceleration of the naturally occurring polyploidization program (39) may account for the observed effects.

These findings suggest that the occurrence of enlarged nuclei at early stages of experimental carcinogenesis, induced by low doses of chemical carcinogens indicate cellular changes, that may be involved in the carcinogenic process.

4. CARCINOGEN INDUCED FOCAL HEPATIC LESIONS

4.1 Experimental induction of focal hepatic lesions in rodents

In rodent liver, the induction of altered foci is widely used as a rapid model for hepatocarcinogenesis (40-42). Sasaki and Yoshida (43) were the first to notice that foci of altered hepatocytes, consisting of clear and basophilic cells, precede the occurrence of chemically-induced liver tumors. During the last few years, further subtypes of altered hepatocellular foci showing

characteristic morphological or metabolic alterations were found (44,45). Several morphometric studies support a predominant developmental sequence from clear cell foci through mixed and basophilic cell foci to hepatomas (12,46-50). However, different types of foci not related to this sequence have also been observed (45,51-54). This concept is further supported by the demonstration of a similar developmental sequence of metabolic alterations (55,56) and increasing cell proliferation (57).

Since the occurrence of these foci is perceivable only in tissue not in cell culture systems, experiments in whole animals are required. Nevertheless immortalized cell lines have been described which are phenotypically similar to these foci observed in situ (58).

4.2 Induction of focal hepatic lesions in avian embryonic liver

The heterogeneity of focal lesions observed in avian embryonic liver corresponds to a similar heterogeneity in rodent liver (10-12,45,59). Turkey and quail have been preferred over chicken eggs because of the longer incubation period of turkey eggs (28 days) and quail eggs (24 days) as compared with 21 days in chicken. Assuming, that the development of phenotypically altered preneoplastic hepatic foci requires some time, a longer hatching period may be more suitable. The features of these experimental models are outlined in table 3.

In the turkey embryos foci of altered hepatocytes can be demonstrated. They are similar to preneoplastic liver cell foci that have been observed in the liver of rodents after exposure to carcinogens. In HE stained sections, in ovo exposure to carcinogens results in the occurrence of mostly basophilic cell foci. Basophilic cell foci were solid (figure 2) or showed an adenoid structure (figure 3). Less frequently, clear cell foci were observed.

Figure 2: Solid basophilic cell focus in embryonic turkey liver after exposure to 1 mg of diethylnitrosamine. Hematoxylin eosin stain, objective magnification 10-fold.

Figure 3: Adenoid basophilic cell focus in embryonic turkey liver after exposure to 2 mg of diethylnitrosamine. Hematoxylin eosin stain, objective magnification 20-fold.

Quail eggs have also been used for the exposure of avian embryos to chemical carcinogens (60). Similar to the findings in embryonic turkey liver, high doses of diethylnitrosamine or N-nitrosomorpholine induced foci of altered hepatocytes in embryonic quail liver (61). However, after lower doses of the nitrosamines, the focal lesions in quail were mostly composed of cells, morphologically more similar to cholangiocytes than to hepatocytes. These cells were arranged in distinct glandular patterns.

After high doses of chemical carcinogens, reversibility of focal and nodular hepatocellular lesions rather than progression to fully transformed neoplastic lesions has been described (61-63,64,65). Similar phenomena cannot be exclude for foci induced by high doses of carcinogens that elicit significant liver damage. However, there is no evidence for the reversibility of foci of altered hepatocytes induced by low doses of hepatocarcinogens that do not appreciably alter the surrounding liver (figure 3).

The demonstration of decreased activity of glycogen phosphorylase was the most reliable enzyme histochemical marker in ovo. Similar to findings in rodents (56,66), the majority of the foci of altered hepatocytes showed a decreased activity of glycogen phosphorylase. In addition some foci showed a more violet tinged staining of the reaction product. A similar phenomenon was described in aged control rats (45) and in rats fed a choline-deficient diet. N-nitrosomorpholine induced glycogen storage and increased glucose-6-phosphate dehydrogenase activity in foci of altered hepatocytes in a dose dependent manner. However there was no increase in foci with elevated activity of glycogen phosphorylase (68).

4.3 Focal hepatic lesions as endpoints in carcinogenicity testing

In rodents, foci of altered hepatocytes are regarded as preneoplastic lesions. These lesions may progress to benign and malignant liver cell tumors without any additional exposure. Therefore in rodents foci of altered hepatocytes have frequently been used as end points in carcinogenicity testing ( (5, 17, 18, 32)...41,42). Testing based on the induction of tumors in whole animals usually requires more than two years. Using preneoplastic lesions as end points in carcinogenicity testing may shorten the required time to a few months. Since these experiments are terminated before tumors occur, the animals do not suffer from tumor growth or metastasis. However, some frequently used protocols include partial hepatectomy for the enhancement of the carcinogenic effect by regenerative proliferation, thus requiring abdominal surgery in animals already exposed to the test chemical (75).

Since foci of altered hepatocytes induced in animal experiments have been successfully used as endpoints in carcinogenicity testing, the occurrence of similar lesions in the IOCA may offer an even more accelerated and convenient approach to carcinogenicity testing. It is generally believed that the long term experiments in carcinogenicity testing are necessary for a sufficiently high sensitivity. However, at least for the detection of the carcinogenic effects of small doses of potent carcinogens, the IOCA based on the induction of focal liver lesions seems to be as sensitive as the chronic rodent bioassay which is based on the occurrence of tumors.

5. Induction of mtDNA damage in ovo

5.1 mtDNA as target for genotoxic chemicals

It is becoming clear that alterations of mitochondrial DNA (mtDNA) may be an alternative and perhaps a superior indicator for genotoxic effects (76). DNA damage may be the result of a direct reaction between a genotoxic chemical and the target DNA molecule. It may also be mediated by free radicals (77). Consequently, mtDNA seems more likely to be a target for the effect of these chemicals than the nuclear DNA (nDNA). In addition, several differences between nDNA and mtDNA may contribute to the higher sensitivity of mtDNA to genotoxic effects. This may, to some extent be due to the close association of nDNA with histone and non-histone proteins that may offer some protection, whereas the mtDNA appears much more exposed to genotoxic effects (78). Damage to nuclear DNA may be efficiently removed by repair systems, but mtDNA has a significantly lower DNA repair capacity (79). Furthermore, the D-loop structure of mtDNA is unique insofar as it contains a single-stranded DNA structure which controls both transcription and replication (80). Since single-stranded DNA is more easily damaged by various genotoxic mechanisms, the D-loop may be a natural hot spot for experimentally induced DNA damage (81). In addition, damage to multiple sites on nDNA may, to some extent, be self-limiting, since the enzymes for DNA replication are coded by nDNA. Severe damage to these genes may result in inactivation of enzymes and the inability of damaged cells to replicate. A similar mechanism may not occur after damage to the mtDNA since the enzymes required for the replication of mtDNA are coded in the nuclear genome (78). In contrast, mutated mitochondria may accumulate in cells and eventually dominate in certain daughter cells of a dividing tissue due to the non-mendelian inheritance of mtDNA (82,83). Another advantage of examining the effect of genotoxic chemicals on mtDNA is its invariability from 16 kb. The supercoiled, relaxed or linearized forms of mtDNA are all detectable by gel electrophoresis (84). Presence of DNA fragments of different sizes most likely reflect the induction of DNA damage. In contrast, during preparation the nDNA (one DNA molecule per chromosome!) usually break, unless very sophisticated methods are used. Consequently, the molecular size of nDNA observed depends on the isolation procedure rather than being reflective of DNA damage.

5.2 Studies of mtDNA using carcinogen-exposed embryonic turkey liver

A disadvantage of mtDNA is that only 0.1% to 1% of total cellular DNA is mtDNA. If methods established for investigations on nuclear DNA are to be used, DNA of about 100 to 1000 fold greater samples are required for experiments with mtDNA as compared with experiments with nuclear DNA. Tissue samples from the IOCA offer a sample size much greater than samples from cell culture systems. The purification of mtDNA from embryonic turkey liver according to the protocol of Welter and coworkers (85) gave mtDNA yields of about 0.2 to 0.4 ng per mg tissue wet weight or up to 0.5 µg mtDNA per liver. The physiologically supercoiled structure of mtDNA was well preserved during the isolation procedure and formed a distinct band in gel electrophoresis. Only small amounts of the mtDNA were of the relaxed form (86).

Separation of native as well as ribonuclease treated samples of mtDNA by gel electrophoresis showed a discrete smear starting at the band of the relaxed form of mtDNA (86).

The electrophoretic separation of mtDNA from turkey embryos previously exposed to hepatocarcinogenic nitrosamines revealed smaller size fragments that could be quantitated by densitometric measurement as smear-band-quotient (SBQ). In experiments with diethylnitrosamine, clear-cut damage to mtDNA was induced over a dose range from 1.24 to 12.4 mmol/kg (86) similar to the concentration range in other in vitro tests for genotoxicity (87,88). Table 4 illustrates the effect of the hepatocarcinogenic nitrosamine N-nitrosomorpholine (NNM) on mtDNA in embryonic turkey liver.

6. PERSPECTIVE OF THE IN OVO MODEL OF CARCINOGENESIS

In ovo experiments may fill the gap between experiments with whole animals and cell culture systems, combining some advantages of both approaches. These advantages are summarized below: The IOCA is rapid, since focal and nodular preneoplastic liver lesions can be induced in 24 days and damage to mitochondrial DNA in 4 days. 1. The IOCA is less expensive than whole animal experiments. Since the experiments are terminated several days before hatching and fertilized eggs are commercially available, facilities for animal housing are not required. 2. The IOCA is logistically simple: It does not require highly sophisticated methods or expensive equipment since routine histology is sufficient for the detection of the induced nuclear alterations and focal hepatic lesions. 3. Quantification of the induced effects can be easily achieved by simple morphometric methods both for the nuclear enlargement and for the occurrence of focal hepatic lesions. 4. Proliferation-mediated effects on carcinogenesis are unlikely to affect the induction of foci in the IOCA. Similar to the partial hepatectomy in the Ito-model, the rapid growth of the embryonic liver provides such a high cell proliferation that a stimulating effect of the test substance is of little or no effect. Therefore, merely cytotoxic effects are less likely to mimic carcinogenicity. 5. The pronounced proliferation of the embryonic tissue also contributes to the high sensitivity of the assay. The induction of regenerative proliferation in potential target tissues frequently introduced in animal experiments (36, 39) is not necessary in ovo due to the high proliferation rate of the embryonic tissue. 6. An additional non-rodent species strengthens the extrapolation of animal studies to humans. 7. Finally, there is less potential of human exposure during the experiment: There is no excretion of the injected carcinogens from the eggs. The egg shell may be regarded as a barrier, at least, for the non-volatile chemicals. The amounts of the carcinogenic chemicals that are needed for the IOCA are relatively small as compared to animal experiments.

It may be possible to study carcinogenic effects of chemicals in tissues other than the liver by the IOCA. However, the liver has been the favorite target organ studied in chemically-induced carcinogenesis (17). As more experience with different chemicals and different target tissues is gained the IOCA may become an increasingly valuable tool both for toxicologic carcinogenicity testing and for research on the mechanisms of chemical carcinogenesis.

7. ACKNOWLEDGEMENTS

The authors would like to express their appreciation to Martina Wingenroth and Sigrid Weber for secretarial help.

8. REFERENCES

1. K. Yamagiwa & K. Ichikawa: Experimentelle Studie über die Pathogenese der Epithelialgeschwülste. Mitteilungen Med Fakultät Kaiserl Univ Tokyo 15, 295-344 (1915)

2. P. Rous: A sarcoma of the fowl transmissible by an agent separable from the tumor cells. J Exp Med 13, 397-411 (1911)

3. M.C. Lancaster, F.P. Jenkins, J. Philip: Toxicity associated with certain samples of groundnuts. Nature 192, 1095-1096 (1961)

4. L. Cova, A. Duflot, M. Prave & C. Trepo: Duck hepatitis B virus infection aflatoxin B1 and liver cancer in ducks. [Review] Arch of Virology - Supplementum 8, 81-7 (1993)

5. A.E. al Moustafa, B. Quatannens, F. Dieterlen-Lievre & S. Saule: Tumorigenic effects mediated in the avian embryo by one or more oncogenes associated with v-myc. Oncogene 7, 1667-70 (1992)

6. KH. Tempel, A. Ignatius & I. Stammberger: A short-term test for nucleotoxicity that uses chick embryo cells treated in vitro and in vivo - physico-chemical and biochemical investigations. Comp Biochem Physiol 103C, 73-78 (1992)

7. C.R. Short, W. Flory, L.C. Hsieh, T. Aranas, S.P. Ou & J. Weissinger : Comparison of hepatic drug metabolizing enzyme activities in several agricultural species. Comp Biochem Physiol 91, 419-422 (1988)

8. B. Brunström: Activities in chick embryos of 7-ethoxycoumarin O-deethylase and acryl hydrocarbon (benzo[a]pyrene) hydrolase and their induction by 3,3',4,4'- tetrachlorobiphenyl in early embryos. Xenobiotics 16, 865-872 (1986)

9. JW. Hamilton & S.E. Bloom: Correlation between mixed-function oxidase enzyme induction and aflatoxin B1-induced unscheduled DNA synthesis in the chick embryo in vivo. Environ Mutagenesis 6, 41-48 (1984)

10. H. Enzmann, G. Kaliner, B. Watta-Gebert & E. Löser: Foci of altered hepatocytes induced in embryonal turkey liver. Carcinogenesis 13, 943-946 (1992)

11. H. Enzmann, C. Kühlem, G. Kaliner, E. Löser & P. Bannasch: Rapid induction of preneoplastic liver foci in embryonal turkey liver by diethylnitrosamine. Toxicol Pathol 23, 60-569 (1995)

12. P.Bannasch : The cytoplasm of hepatocytes during carcinogenesis. Electron and light microscopical investigations of the nitrosomorpholine-intoxicated rat liver. Rec Res Cancer Res 19, 1-100 (1968)

13. Y. Ruan, H.J. Hacker, H. Zerban & P. Bannasch: Ultrastructural and immunocytochemical characterization of the cellular phenotype in primary adenoid liver tumours of the rat. Path Res Pract 184, 223-233 (1989)

14. S.N Thung,: The development of proliferating ductular structures in liver disease. Arch Pathol Lab Med 114, 407-411 (1990)

15. A.M. Vandersteenhoven, J Burchette & G Michalopoulos: Characterization of ductular hepatocytes in end-stage cirrhosis. Arch Pathol Lab Med 114, 403-406 (1990)

16. P. Gerlyng, T.Stokke, H.S: Huitfeldt, T. Stenersen, H.E. Danielsen, T. Grotmol & P.O. Seglen: Analytical methods for the study of liver cell proliferation. Cytometry 13, 404 - 415 (1992)

17. A.M. Rappaport: Physioanatomical basis of toxic liver in jury. In: Toxic injury of the liver. Eds: E. Farber & M.M. Fischer, Marcel Dekker, New York, USA, 1-55 (1979)

18. J.J. Gumucio & D.L. Miller: Functional implications of liver cell heterogeneity. Gasteroenterology 80, 393-403 (1981)

19. J. James, J. Tas. K.S. Bosch. A.J.P. de Meere & H.C. Schuyt: Growth patterns of rat hepatocytes during postnatal development. Eur J Cell Biol 19, 222-226 (1979)

20. H.W. Kunz, H.A. Tennekes, R.E. Port, M. Schwartz, D. Lorke,& G. Schaude: Quantitative aspects of chemical carcinogenesis and tumor promotion in liver. Environ Health Perspect 50, 113-122 (1983)

21. J. Carlson & R. Abraham: Nuclear ploidy of neonatal rat livers: Effects of two hepatic carcinogens (mirex and dimethylnitrosamine) J Toxicol Environ Health 15, 551-559 (1985)

22. A.J. Ingram & P.Grasso: Nuclear Enlargement - An early change produced in mouse epidermis by carcinogenic chemicals applied topically in the presence of a promoter. J Appl Toxicol 5, 53-60 (1985)

23. A.J. Ingram & P. Grasso: Nuclear enlargement produced in mouse skin by carcinogenic mineral oils. J Appl Toxicol 7, 289-295 (1987)

24. H. Enzmann, & P. Bannasch: Morphometric study of alterations of extrafocal hepatocytes of rat liver treated with N-nitrosomorpholine. Virchows Archiv B, 53, 218-226 (1987)

25. J.A. Styles, M. Kelly & C.R. Elcombe: A cytological comparison between regeneration, hyperplasia and early neoplasia in the rat liver. Carcinogenesis 8, 391-399 (1987)

26. J.A. Styles, A. Bybee,N.R. Pritchard & M.D. Kelly: Studies on the hyperplastic responsiveness of binucleated rat hepatocytes. Carcinogenesis 11, 1149-1152 (1990)

27. P. Gerlyng, T. Grotmol, T. Stokke, B. Erikstein& P.O. Seglen: Flow cytometric investigation of a possible precursor-product relationship between oval cells and parenchymal cells in the rat liver. Carcinogenesis 15, 53-59 (1994)

28. R.A. Finch, I.M.Evans, & H.B. Bosmann: Chemical carcinogen in vitro testing: A method for sizing cell nuclei in the nuclear enlargement assay. Toxicology 15, 145-154(1980)

29. P. Gerlyng,, A. Abyholm,T. Grotmol, B. Erikstein, H.S. Huitfeldt, T. Stokke & P.O. Seglen: Binucleation and polyploidization patterns in developmental regenerative rat liver growth. Cell Proli 26, 557-565 (1993)

30. J. James, M. Schopman & P. Delfgaauw: The nuclear pattern or the parenchymal cells of the liver after partial hepatectomy. Exp Cell Res., 42, 375-379 (1966)

31. C. Melchiorri, P. Chieco, A.I: Zedda, P. Coni, G.M. Ledda-Columbano & A. Columbano: Ploidy and nuclearity of rat hepatocytes after compensatory regeneration or mitogen-induced liver growth. Carcinogenesis 14, 1825-1830 (1993)

32. O. Klinge: Cytologic and histologic aspects of toxicologically induced liver reactions. Cur Top Pathol 85, 91-116 (1973)

33. H.S. Taper & P. Bannasch: Histochemical differences between so-called megalocytosis and neoplastic or preneoplastic liver lesions induced by N-nitrosomorpholine. Eur J Cancer 15, 189-196 (1979)

34. G.A. Clawson, L.J. Blankenship, J.G. Rhame, & D.S. Wilkinson: Nuclear enlargement induced by hepatocarcinogens alters ploidy. Cancer Res 52, 1304-1308 (1992)

35. Y. Fujimoto, M. Oyamada, A. Hattori, H. Takahashi, M. Sawaki, K. Dempo,M. Mori, & M. Nagao: Accumulation of abnormally high ploid nuclei in the liver of LEC rats developing spontaneous hepatitis. Jpn J Cancer Res 80, 45-50 (1989)

36. M. Sawaki, K. Enomoto, H. Takahashi, Y. Nakajima & M. Mori: Phenotype of preneoplastic and neoplastic liver lesions during spontaneous liver carcinogenesis of LEC rats. Carcinogenesis 11, 1857-1861 (1990)

37. H. Enzmann, C. Kühlem, E. Löser & P. Bannasch: Dose dependence of diethylnitrosamine-induced nuclear enlargment in embryonal turkey liver. Carcinogenesis 16, 1351-1355 (1995)

38. A. Oenfelt: Spindle disturbances in mammalian cells. III. Toxicity, c-mitoses and aneuploidy with 22 different compounds. Specific and unspecific mechanisms. Mutat Res 182, 135-154 (1987)

39. L. Wiest: The effect of diethylnitrosamine on the distribution of cell classes in the parenchyma of the liver of newborn rats. Eur J Cancer 8, 121-125 (1972)

40. G.M. Williams: Phenotypic properties of preneoplastic rat liver lesions and applications to detection of carcinogens and tumor promoters. Toxicol. Pathol. 10, 3-10 (1982)

41. P. Bannasch: Preneoplastic lesions as end points in carcinogenicity testing. I. Hepatic preneoplasia. Carcinogenesis 7, 689-695(1986)

42. N. Ito, K. Imaida, R. Hasegawa & H. Tsuda: Rapid bioassay methods for carcinogens and modifiers of hepatocarcinogenesis CRC. Crit Rev Toxicol 19, 386-415 (1989)

43. T. Sasaki & T. Yoshida: Experimentelle Erzeugung des Lebercarcinoms durch Fütterung mit o-Amidoazotoluol. Virchows Arch Path Anat 295. 175-200 (1935)

44. P. Bannasch,H. Enzmann, F. Klimek, E. Weber & H. Zerban: Significance of sequential cellular changes inside and outside foci of altered hepatocytes during hepatocarcinogenesis. Toxicol Pathol 17, 617-629 (1989)

45. H. Enzmann, H. Zerban, E. Löser & P. Bannasch: Glycogen phosphorylase hyperactive foci of altered hepatocytes in aged rats. Virchows Arch B 62, 3-8 (1992)

46. H. Enzmann & P. Bannasch: Potential significance of phenotypic heterogeneity of focal lesions at different stages in hepatocarcinogenesis. Carcinogenesis 8, 1607-1612 (1987)

47. M.A. Moore, D. Mayer, & P. Bannasch: The dose dependence and sequential appearance of putative preneoplastic populations induced in the rat liver by stop experiments with N-nitrosomorpholine. Carcinogenesis 3, 1429-1436 (1982)

48. E. Weber & P. Bannasch: Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia induced by single oral exposures to N-nitrosomorpholine. Carcinogenesis 15, 1219-1226 (1994a)

49. E. Weber & P. Bannasch: Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia and neoplasia induced in stop experiments by oral exposure to N-nitrosomorpholine. Carcinogenesis 15, 1227-1234 (1994b)

50. E. Weber & P. Bannasch: Dose and time dependence of the cellular phenotype in rat hepatic preneoplasia induced by continuous oral exposures to N-nitrosomorpholine. Carcinogenesis 15, 1235-1242 (1994c)

51. P. Bannasch, U. Benner, H. Enzmann & H.J. Hacker: Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B1. Carcinogenesis 6, 1641-1648 (1985)

52. H. Enzmann, P. Bannasch: Non-persisting early foci of altered hepatocytes induced in rats by N-nitrosomorpholine. J Cancer Res Clin Oncol 114, 30-34 (1988)

53. E. Weber, MA. Moore, P. Bannasch: Enzyme histochemical and morphological phenotype of amphophilic foci and amphophilic/tigroid cell adenomas in rat liver after combined treatment with dehydroepiandosteron and N-nitrosomorpholine. Carcinogenesis 9, 1049-1054 (1988)

54. H. Enzmann, D. Ohlhauser, T. Dettler, U. Benner, H.J. Hacker & P. Bannasch: Unusual histochemical pattern in preneoplastic hepatic foci characterized by hyperactivity of several enzymes. Virchows Arch B 57, 99-108 (1989)

55. A. Buchmann,W. Kuhlmann, M. Schwarz, W. Kunz, C.R. Wolf, E. Moll, T. Friedberg, & F. Oesch: Regulation and expression of four cytochrome P-450 isoenzymes, NADPH-cytochrome P-450 reductase, the glutathione transferases B and C and microsomal epoxide hydrolase in preneoplastic and neoplastic lesions in rat liver. Carcinogenesis 6, 513-521 (1985)

56. H.J. Hacker, M.A. Moore, D. Mayer & P.Bannasch: Correlative histochemistry of some enzymes of carbohydrate metabolism in preneoplastic and neoplastic lesions in the rat liver. Carcinogenesis 3, 1265-1272(1982)

57. H. Zerban, S. Radig, A. Kopp-Schneider & P. Bannasch: Cell proliferation and cell death (apoptosis) in hepatic preneoplasia and neoplasia are closely related to phenotype cellular diversity and instability. Carcinogenesis 15, 2467-2473 (1994)

58. D. Mayer & B. Schäfer: Biochemical and morphological characterization of glycogen-storing epithelial liver cell lines. Exp Cell Res 138, 1-14 (1982)

59. C. Peraino, E.F. Staffeldt, B.A. Carnes, V.A. Ludeman, J.A. Blomquist & S.D. Vesselinovitch: 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 (1984)

60. G.G. Hatch, F.W. Edens, L.C. Rogers & B. Most: Embryotoxic, tumor, and other post-hatch effects of chemical carcinogens and noncarcinogens following in ovo yolk sac inoculation of Japanies quail. In: Evaluation of Short-term Tests for Carcinogens. Eds: Ashby, J., de Serres, F.J., Shelby, M.D., Margolin, B.H., Ishidate, M. and Becking, G.C., Cambridge University Press, Cambridge, UK, 2.35-2.42 (1988)

61. G.M. Williams & K. Watanabe: Quantitative kinetics of development of N-2-flourenylacetamide-induced altered hyperplastic hepatocellular foci resistant to iron accumulation and their reversion or persistence following removal of carcinogen. J Natl Cancer Inst 61, 113-121 (1978)

62. M.A. Moore, H.J. Hacker & P. Bannasch: Phenotypic instability in focal and nodular lesions induced in a short-term system in the rat liver. Carcinogenesis 4, 595-603 (1983)

63. M. Tatematsu, Y. Nagamine & E. Farber: Redifferentiation as a basis for remodeling of carcinogen-induced hepatocyte nodules to normal appearing liver. Cancer Res 43, 5049-5058 (1983)

64. R. Schulte-Hermann, W. Bursch, B. Krauppgrasl, F. Oberhammer, A. Wagner & R. Jirtle: Cell-proliferation and Apoptosis in normal liver and preneoplastic foci. Environ Health Perspect 101, 87-90 (1993)

65. B. Grasl-Kraupp, B. Ruttkay-Nedecky, L. Mullauer, H. Taper, W. Huber, W. Bursch & R. Schulte-Hermann: Inherent increase of apoptosis in liver tumors: Implications for carcinogenesis and tumor regression. Hepatology 25, 906-912 (1997)

66. G. Seelmann-Eggebert, D. Mayer, D. Mecke & P. Bannasch: Expression and regulation of glycogen phosphorylase in preneoplastic and neoplastic hepatic lesions in rats. Virchows Arch B 53, 44-51(1987)

67. H.J. Hacker, P. Steinberg, I. Toshkov, F. Oesch & P. Bannasch: Persistence of the cholangiocellular and hepatocellular lesions observed in rats fed a choline-deficient/DL-ethionine-supplemented diet. Carcinogenesis 13, 271-276 (1992)

68. H. Enzmann, H. Zerban, A. Kopp-Schneider, E. Löser & P. Bannasch: Effects of low doses of N-nitrosomorpholine on the development of early stages of hepatocarcinogesis. Carcinogenesis 16, 1513-1518 (1995)

69. H.C. Pitot, T.L. Goldsworthy, S. Moran, W. Kennan, H.P. Glauert, R.R: Maronpot & H.A. Campbell: A method to quantitate the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationship to altered hepatic foci. Carcinogenesis 8, 1491-1499 (1987)

70. M.A. Pereira & S.L. Herren-Freund: Liver initiation assay: the rat liver foci bioassay of carcinogens and noncarcinogens. In: Evaluation of short-term tests for carcinogens. Eds: J. Ashby, de Serres F.J., Shelby M.D., Margolin B.H., Ishidate M., Becking G.C., Cambridge University Press, Cambridge, UK, 1.325-1.328 (1988)

71. R.R. Maronpot, H.C., Pitot & C. Peraino: Use of rat liver altered focus models for testing chemicals that have completed two-year carcinogenicity studies. Toxicol Pathol 17, 651-662 (1989)

72. D. Oesterle, U. Gerbracht, E. Deml, B. Schlatterer & E. Eigenbrodt: Comparison of three rat liver foci bioassays-incidence of preneoplastic foci initiated by diethylnitrosamine. Carcinogenesis 10, 1891-1895 (1989)

73. P. Bannasch & H. Zerban:. Predictive value of hepatic preneoplastic lesions as indicators of carcinogenic response. In: Mechanisms of carcinogenesis in risk identification. Eds: Vainio, H., Magee, P.N., McGregor, D.B., and McMichel, A.J., IARC, Lyon, France, 389-427 (1992)

74. G.M. Williams: Chemicals with carcinogenic activity in the rodent liver; mechanistic evaluation of human risk. Cancer Letters 117, 175-188 (1997)

75. N. Ito, H. Tsuda, M. Tatematsu, T. Inoue, Y. Tagawa, T. Aoki, S. Uwagawa, M. Kagawa, T. Ogiso, T. Masui, K. Imaida, S. Fukushima & M. Asamoto: Enhancing effect of various hepatocarcinogens on induction of preneoplastic glutathione S-transferase placental form positive foci in rats - an approach for a new medium-term bioassay system. Carcinogenesis 9, 387-394 (1988)

76. J.A. Allen & M.M. Coombs: Covalent binding of polycyclic aromatic compounds to mitochondrial and nuclear DNA. Nature 287, 244-245 (1980)

77. L.G. Baggetto: Role of mitochondria in carcinogenesis. Eur J Cancer 29A, 156-159 (1993)

78. D.A. Clayton: Replication of animal mitochondrial DNA. Cell 28, 638-705 (1982)

79. G. Singh & E. Maniccia-Bozzo: Evidence for lack of mitochondrial DNA repair following cis-dichlorodiammineplatinum treatment. Cancer Chemother Pharmacol 26, 97-100 (1990)

80. D.D. Chang, R.P.Fisher & D.A. Clayton: Roles for a promotor and RNA processing in the synthesis of mitochondrial displacement-loop strands. Biochim and Biophys Acta 909, 85-91 (1987)

81. G. Singh, S.M. Sharkey & R. Moorehead: Mitochondrial DNA damage by anticancer agents. Pharmac Ther 54, 217-230 (1992)

82. B. Bandy & AJ Davison: Mitochondrial mutations may increase oxidative stress: Implications for carcinogenesis and aging? Free Radic Biol Med 8, 523-539 (1990)

83. D.C. Wallace: Mitochondrial DNA in aging and disease. Scientific American 277 (1997)

84. G. Singh, W.W. Hauswirth, W.E. Ross & A:H. Neims: A method for assessing damage to mitochondrial DNA caused by radiation and epichlorohydrin. Mol Pharmacol 27, 167-170 (1985)

85. C. Welter, D. Dooley & N. Blin: A rapid protocol for the purification of mitochondrial DNA suitable for studying restriction fragment length polymorphisms. Gene 83, 169-172 (1989)

86. H. Enzmann, C. Kühlem, E. Löser & P. Bannasch: Damage to mitrochondrial DNA induced by the hepatocarcinogen diethylnitrosamine in ovo. Mutation Res 329, 113-120 (1995)

87. A. Martelli, L. Robbiano, G.M. Gazzaniga & G. Brambilla: Comparative study of DNA damage and repair induced by ten N-nitroso compounds in primary cultures of human and rat hepatocytes. Cancer Res 48, 4144-4152 (1988)

88. M. Zielenska & J.B. Guttenplan: Effects of UV repair, error-prone repair and critical site of mutation on mutagenesis induced by N-Nitrosamines. Mutation Res 180, 11-20 (1987)