**2. The chemical reagent (DEN)-induced liver fibrosis/cirrhosis and carcinogenesis in C57BL/6 inbred mice fed a drinking water diet**

DEN is a nitrosamine-containing chemical reagent, and its toxic properties in mammals are well established [21–23]. Studies have shown that oral administration of the smallest quantities of DEN or dimethylnitrosamine (DMN) results in severe hepatic cell injury in both animals and humans [24–27]. The most prominent chemical reagents are intense neutrophilic infiltration, extensive centrilobular hemorrhagic necrosis, bile duct proliferation, fibrosis, and bridging necrosis that end in hepatocarcinogenesis [28].

DEN was first brought to general attention in 1937 [29] when it was reported to be the causative agent that induces liver injury in men. This assumption was experimentally confirmed in 1954 by Barnes and Magee, who found that a single oral or parental dose (20–40 mg/kg body weight) of the reagent acts primarily as a liver poison producing severe liver necrosis in both small and large animals [30] of which the landmark experiment shows a sharp line of demarcation between the totally destroyed parenchyma and apparently uninjured liver cell areas in the model liver lesions [30]. Furthermore, the animal model shows that chronic application of the chemical agent in rats resulted in a high incidence of hepatic malignancy [31]. The extension of these studies has been subsequently revealed by many other researchers [30, 32, 33]. These findings suggest that the chemical and physical properties of DEN may be of interest for understanding the hepatic pathological characteristics underlying the evolution of liver fibrosis/cirrhosis/ HCC formation [25], although the hepatotoxic and carcinogenic mechanism was not known until 1963 [34].

Moreover, although the application of DEN has become a well-established model in animals for studies of the pathogenetic alterations underlying the formation of liver fibrosis, cirrhosis and cancers, the single diet DEN for analyzing liver fibrosis/cirrhosis in wild-type mice has never been investigated. As such, applied C57BL/6 wild-type inbred mice with a single diet of 0.014% DEN in drinking water; 6 days/week for 15 weeks, we successfully induced a liver fibrosis/cirrhosis/cancer mouse model [19]. The model shows that DEN is extremely effective in inducing hepatic fibrosis [21], cirrhosis and cancers (**Figure 1B**) that are compatible with the deterioration of liver functions [22, 35].
