Electron Microscopic Study and X-ray Probe Microanalysis of the Liver of LEC Rat, an Animal Model of Wilson Disease

Morphological identification of lysosomes was established by acid phosphatase reaction [1]. Pericanalicular lysosomes exhibited lead deposits after incubating liver slices with a lead and phosphate solution under acidic condition. Transitional elements, copper and iron, are stored in lysosomes as detoxified forms of cuprothioneins and hemosiderins, respectively. Genetic background of adult-onset storage diseases in the liver remained undetermined until 1993 when ATP7B was cloned in patients with Wilson disease (WD) [2]. In 1996, HFE was first cloned in patients with hemochromatosis, followed by TFR2 of non-HFE hemochromatosis. These storage diseases of the liver were named secondary lysosomal diseases due to genetic defects of extralysosomal molecules [3]. Light microscopy is a standard laboratory examination of the modern pathology, but has a drawback in detecting heavy metal toxicoses. Special staining methods such as rhodanine and rubeanic acid for copper, and Berlin blue for iron are needed. It must be kept in mind that negative results do not rule out overloading, namely toxic effect of transition metals. Aggregation of metal-rich lysosomes may be needed in positive histochemical staining, or minimal overloading of metal in lysosomes may be toxic [4]. Electron Microscopy is widely used in clinical diagnosis and pathological investigation. Structural changes of intracellular organelles provided by electron microscopy are important information for understanding disease mechanism of patients and animal models. In addition, X-ray microanalysis, especially energy dispersive X-ray microanalysis (EDX), realizes detection of not only trace element deposits but also compositional pattern of heavy metal complexes [5]. The liver is a central organ for homeostasis of trace elements of iron and copper. Iron absorption in the gut is regulated by hepcidin molecules secreted from the liver, while excess copper is secreted from the liver into bile. The hepatic copper transporter ATP7B is defective in patients with WD whose biliary excretion of copper was completely blocked [6, 7]. Excess copper is first stored in the liver, and then in extrahepatic organs including the central nervous system. In the liver, copper is stored in hepatocellular lysosomes as cuprothioneins. Recent studies indicate that lysosomes are also loaded with iron, and may be replaced by iron during long-term copper cheletion because of more severe hypoceruloplasminemia [8]. Both iron and copper are transition elements and primary sources in radial generation. As a result, multi-organ damage may be inevitable when excess amounts of iron and copper are accumulated in the liver and other organs. Long-Evans cinnamon (LEC) rats are an animal model of WD with an ATP7B mutation and hypoceruloplasminemia [9]. There is evidence to suggest that transition metals of both iron and copper may be involved in variety of fulminant hepatitis and chronic hepatitis of LEC rats [10, 11]. Because these transition elements are finally stored in hepatocellular lysosomes as detoxified forms, ultrastructural element analysis on LEC rats may provide information of cytoprotective response to hepatotoxic elements during chronic hepatitis leading to hepatic fibrosis and malignancy in ATP7B-linked copper toxicosis.