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

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.