Yanhua Li
Dr. sc.hum.
Demonstration of Viral DNA, Viral Antigens, Hepatocellular Ultrastructure,
Proliferation Kinetics and Enzyme Activity during Hepadnaviral
Hepatocarcinogenesis in Woodchucks
Geboren am 01.09.1963 in Changchun, VR China
Studiengang der Medizin vom SS/WS 1981 bis SS/WS 1986
Magisterarbeit vom SS/WS 1986 bis SS/WS 1989 an der Norman Bethune medizinische
Universität
Promotionsfach: Deutsches Krebsforschungszentrum (DKFZ)
Doktorvater: Prof. Dr. med. P. Bannasch
Sequential hepatocellular changes were studied during carcinogenesis in woodchucks
inoculated as newborns with woodchuck hepatitis virus (WHV), which is closely related
to the human hepatitis B virus. When the woodchucks reached 12 months of age,
aflatoxin B1 (AFB1) was administered to one group of WHV carriers in the diet at dose
levels of 40 µg/kg body weight/day for 4 months and subsequently 20 µg/kg body
weight/day (5 days/week) for lifetime. The histo- and cytomorphology of the liver were
investigated by light and electron microscopy. The WHV DNA and the viral antigens
(WHsAg and WHcAg) were demonstrated in the liver tissue by in situ hybridization and
immunohistochemical approaches. In addition, the cell proliferation kinetics were
investigated using a monoclonal antibody against Ki67. The activities of several enzymes
involved in carbohydrate metabolism and in mitochondrial function were analysed by
enzyme histochemistry. The cytomorphological and cytochemical analysis permitted the
identification of five different types of focal lesions in chronic WHV carriers, namely
APF/GCH, APF/GGH, APF, GSF, and GSF/BCF which seem to be integrated into two
separate cell lineages leading to hepatocellular neoplasms; the amphophilic and
glycogenotic/basophilic cell lineage apparently derived from MDA. The cellular and
subcellular alterations were similar in WHV-infected animals with and without AFB1-
treatment.
MDA constitute a mosaic of glycogen-rich cells, amphophilic cells and ground glass
cells. Well demarcated APF/GCH comprised glycogen-containing cells and glycogen-
poor amphophilic cells, but no ground glass cells. The striking difference between
APF/GGH and APF/GCH was the presence of a few scattered ground glass cells in
APF/GGH. Cells forming APF were poor in glycogen and were characterized by a
homogeneous, granular acidophilic cytoplasm due to pronounced mitochondrial
proliferation, associated with a distinct diffuse or randomly scattered basophilia. GSF
consisted of considerably enlarged hepatocytes excessively storing glycogen and were
well demarcated from the surrounding parenchyma. GSF/BCF were made up of
glycogenotic cells, glycogen-poor basophilic cells, and various types of intermediate
cells.
High levels of WHV DNA replication and WHsAg expression were observed in most
MDA, but the expression of WHcAg was limited to a lower number of hepatocytes in
MDA under the experimental conditions chosen. Cell proliferation was significantly
elevated in MDA in comparison with the liver parenchyma of WHV-free woodchucks.
Three different types of proliferative foci of altered hepatocytes (FAH) representing early
morphological and metabolic alterations emerged from MDA, namely 1) APF/GGH
initially maintaining the viral DNA replication and the expression of WHsAg and
WHcAg in many cells, 2) APF/GCH resembling APF/GGH but being free of ground
glass cells, WHsAg and WHcAg and 3) GSF largely losing the WHV DNA and the
expression of both antigens early during the development. The enzyme patterns of these
two different cell lineages are in many respects opposite and mimic different hormonal
effects. The amphophilic cell lineage shows a reduced activity of glycogen synthase but
increased activities of gluconeogenic and mitochondrial enzymes, reflecting a
thyromimetic effect. In contrast, the glycogenosis is associated with a reduction in the
activity of enzymes involved in glycogen breakdown and gluconeogenesis, resembling a
response of hepatocytes to insulin. Despite the phenotypic diversity in these two
hepatocellular lineages, early changes in energy metabolism due to a disturbance in
signal transduction pathways appear to be a common denominator. FAH and
hepatocellular neoplasms are apparently elicited by a direct interaction of WHV with the
hepatocytes. Although progression of FAH to hepatocellular neoplasms may become
independent from viral replication and antigen expression at an early time point, chronic
active WHV infection is a prerequisite for the development of a high incidence and
number of preneoplastic and neoplastic hepatocellular lesions.
