**2.3 Tumor and preneoplastic lesion induction and assessment**

Tumors were induced by a single intraperitoneal injection of DEN (Eastman Kodak Co., Rochester NY; 0.1 mol/g body weight) dissolved in tricaprylin (a.k.a. trioctanoin; Sigma, St. Louis, MO) 12 ± 1 days after birth. Mice, all male, were sacrificed by CO2 asphyxiation. For prenoplastic lesion volume analysis, mice were sacrificed at 16 or 24 weeks of age (Table 2) and representative sections of the liver were frozen immediately on dry ice and stained for glucose-6-phosphatase activity as described (Bugni et al., 2001). Glucose-6-phosphatase deficiency is a hallmark of the majority of preneoplastic lesions in DEN-treated mice (Bugni et al., 2001). For tumor analysis, mice were sacrificed at 32 weeks of age (Figure 1, Figure 3). Livers were removed and weighed, and all tumors larger than 1 mm in diameter were counted. Liver tumors were sampled at random, fixed in RNAlater (Qiagen, Valencia, CA) at 4°C, then drained of RNAlater and transferred to -80°C. Spleens were collected as a source of DNA and frozen on dry ice. All tumors were scored by a single observer blind to genotype. To assess spontaneous tumors (Table 1), mice were allowed to age to 15 months without undergoing any procedures. Tumors larger than 1 mm in diameter were counted.

To assess apoptosis, mitosis, and RNA expression immediately following DEN injection, male mice were either left untreated or injected with DEN (0.1 mol/g body weight), at 12 days of age between 11 a.m. and 3 p.m.. Mice were sacrificed one, two, or three days later between 3 and 6 p.m.. One half of each left lobe and half of each half of the medial lobe were placed in formalin at 4°C. Half of the remaining parts of the left and medial lobes were placed in RNAlater at 4°C. The remaining portions of the left and medial lobes were frozen in pre-chilled tubes on dry ice and transferred to -80°C.

Formalin-fixed tissue was sectioned and stained with hematoxylin and eosin (H&E). Hepatocytes, apoptoses, and mitoses were counted under 600× magnification.
