Staining also reveals the typically circular shape of the lesion in histologic section, representing a cross section of a three-dimensional spherical entity of several hundred microns diameter [4]. No upper limits are placed upon the size of the foci, however smaller foci are far more common than those extending to a diameter greater than that of a normal hepatic lobule [4].
Foci are typically associated with a variety of abnormalities such as aberrations in metabolic pathways such as carbohydrate and drug metabolism. This has been observed in rats and in other species including humans [3]. Such perturbations may be due in part to abnormalities in enzymes such as glucose-6-phosphatase and ATPase. In addition, other biochemical abnormalities are characteristic of foci including altered glycogen storage, demonstrated first by Bannasch [5], in the case of nitrosamine-induced foci and a deficiency in iron storage, possibly due to ferritin abnormalities.
It is possible to classify foci into various groups based on their phenotypic and histochemical characteristics (Table 1.). This classification is extremely important from a prognostic point of view in carcinogenesis studies.
Table 1. Classification of foci of altered hepatocytes based on their phenotypic and biochemical characteristics.
Species differences.
Foci of altered hepatocytes have been identified in a number of species such as rats, mice (including transgenic mice prone to developing hepatic tumours), hamsters and monkeys [1]. They have also been observed in humans, although their significance in human hepatocarcinogenesis has not been fully investigated [3]. Interestingly, there are a significant number of reports of patients suffering from Von Gierke’s disease or inborn hepatic glycogenolysis type 1, of whom more than 90% develop hepatic tumours during adolescence [3]. These observations support the idea that metabolic changes characteristic of hepatocarcinogenesis are causally related to neoplastic changes in liver cells.
The issue whether foci of altered hepatocytes represent a pre-carcinogenic state has been relentlessly pursued over recent years. Foci are frequently found in the livers of rats administered with hepatocarcinogens in comparison to control rats of equal age [4]. In fact almost all potent hepatocarcinogens have been demonstrated to cause an increased size and number of foci in the pre-carcinogenic state. Evidence for the theory that foci are precursors to liver tumours is derived from morphological and histological observations following experimental studies.
Foci of altered hepatocytes have been observed to increase in size and to acquire characteristic phenotypic markers close to neoplasia [1]. In recent years, mounting evidence has emerged regarding the progression of foci in the carcinogenic process. The series of phenotypic changes often observed in these lesions may represent the evolution of glycogen rich “clear cell foci” into later “mixed cell” populations, basophilic foci and finally to hepatic tumours. In addition to alterations in glycogen storage, there are also increased levels of enzymes such as glucose-6-phosphate dehydrogenase which is associated with cell proliferation [2]. Similar observations have been made for enzymes of xenobiotic metabolism such as cytochrome p450 [2], whilst activities of other enzymes such as succinate dehydrogenase were reduced. Overall, these enzymatic changes may represent an adaptive metabolic mechanism for example enabling enhanced xenobiotic detoxification.
Regression of foci has been observed under various conditions following the cessation of the administration of inducing stimuli [1]. Regression has been observed in so called “stop” experiments in which a carcinogen is administered for a short duration and then stopped. It has also been observed in short term studies whereby a carcinogen is given following treatment with another initiating carcinogen and a stimuli administered to induce liver regression. It has been suggested that some of the reverted cells that undergo regression may maintain some degree of alteration that could render them susceptible to carcinogenesis. In conclusion, regression of foci of altered hepatocytes has been observed under a variety of conditions and is therefore certainly plausible. This observation provides evidence against the theory that foci are a definitive pre-carcinogenic state.
The use of foci of altered hepatocytes in risk assessment.
The incidence of foci of altered hepatocytes may be used as an end point in the detection of chemicals with the potential to induce promotion or initiation of carcinogenesis [6]. If treatment with a suspected carcinogenic substance gives rise to significantly more foci in treated versus control animals, this could confirm the carcinogenic potential of that compound. So far, the majority of hepatocarcinogens investigated show an induction of some types of foci.
One example of a bioassay for the identification of carcinogens by this method is the “rat liver focal bioassay” whereby the test compound is administered for a specified time following which the progress of the animals is followed for several weeks or months. The addition of phenobarbitone has also been used, to render the system more sensitive, but it has been questioned whether phenobarbitone merely promotes initiated cells as intended or whether it acts as a weak carcinogen itself.
The specificity of the utilisation of foci as a bioassay for carcinogenesis is not clear. It is important to note that the organ of interest here is the liver which has a unique capacity to metabolise xenobiotics compared with other organs. It is clear that many carcinogenic substances are specific for a certain organ, for example saccharin which induces bladder cancer [6]. It could therefore be said that the use of foci to identify carcinogenic potential is only useful for carcinogens with the liver as their primary target.
In addition there is some variability in the appearance of foci in different regions of the liver itself, however, results of carcinogenic studies are often assumed to be representative of the entire liver. Differences in the distribution of foci have been observed, for example in rats treated with 2-acetylaminofluorene, which was shown to induce right lobed foci.
In conclusion, the use of foci of altered hepatocytes as an endpoint in carcinogenicity testing serves to decrease the time and expense of such testing and to increase statistical power as the number of induced foci follow a normal (continuous) distribution [6]. In addition the determination of foci may enable risk assessment at very low doses [3].
Conclusion.
In conclusion, foci of altered hepatocytes can be easily identified by the use of various stains. It is now widely accepted that they preceed the development of hepatic tumours by weeks or months in many species including primates [3]. The phenotypes of foci are varied and often unstable and phenotypic instability is often associated with the reversion or progression of foci.
Despite the compelling evidence that foci represent a pre-carcinogenic state, it is obvious that they are not definitively precursors to liver tumours. The lesions arise spontaneously in older, control rats and their incidence can also vary with the strain of the rat used and other factors for example diet. There is some evidence that the carcinogenic potential varies with the type of focus as basophilic and mixed cell foci in particular represent phenotypes that are closer to neoplasia than other foci. Finally, it is important to note that despite their use in predicting carcinogenesis, the absence of such lesions does not exclude carcinogenicity [3].
References.
-
Rinde, E., Hill, R., Chiu, A. and Haberman, B. Toxicology and Industrial Health, 3(1), 145-162, (1987).
- Bannasch, P., Zerban, H. and Hacker, H.J. Pathology of the Liver. McSween. (Human).
- Bannasch, P. and Zerban, H. Mechanisms of Carcinogenesis in Risk Estimation. Ed.Vainio, H., Magee, D.B.and Mc Michael, A.J. 389-427.
-
Popp, J.A. and Goldsworthy, T.L. Toxicologic Pathology, 17(4), 561-568, (1989).
-
Williams, G.M. Toxicologic pathology, 17(4), 663-668, (1989).
-
Glauert, H.P. Progress in Histo- and Cytochemistry, 23, 34-39, (1991).