Franny Jongbloed

54 CHAPTER 3 administration of either vehicle or irinotecan, all mice were fasted for two hours, after which they were given AL access to food for 10 hours until the moment of sacrifice. Food intake during this period was measured by weighing the food before and afterwards. The mice in both AL groups, AL vehicle and AL irinotecan, ate around three grams per mouse in this 10-hour period. The fasting vehicle group had the highest food intake, 4.3 grams per mouse, whereas the fasted irinotecan mice had an average intake of 3.5 grams per mouse. The systemic toxic effects of irinotecan and the effects of fasting were analyzed via markers of common (LDH) and liver specific (ALT and AST) cellular injury, and kidney function (urea and creatinine). Levels of these markers in the serum increase upon cell damage and death. Leukocyte numbers were determined as markers of bone marrow toxicity. Administration of irinotecan increased markers of both liver (Figure 1A), and kidney injury (Figure 1B). These increases were suppressed in the fasted animals that received irinotecan. A common side effect of irinotecan therapy is depletion of white blood cells from the blood. Irinotecan treatment caused a significant depletion of white blood cells, which was also ameliorated by fasting (Figure 1C). Collectively, these results demonstrate successful replication of the fasting induced chemo-protective phenotype in the liver as described before 10 . Liver transcriptome analysis Principal component analysis To investigate variability between and within our experimental groups, we performed an unbiased principal component analysis (PCA) including all probe sets in the microarray of liver and tumor tissue samples. In the PCA plot of the liver samples, 50.77% of the variance was explained by principal component (PC) 1 and 16.81% by PC2 (Figure 2A). A pattern of distinct clustering of the four individual groups was seen, with the smallest intragroup variability in the fasting vehicle group. Using the AL vehicle group as reference, the fasting vehicle and fasting irinotecan groups diverged mainly by PC1, while the difference with the AL irinotecan group was for the most part by PC2. The fasting irinotecan group differed almost exclusively by PC1 from AL vehicle, in direction and distance similar to that of fasting vehicle, making fasting vehicle and fasting irinotecan the two groups in closest proximity. These data point towards a more differential regulated and homogeneous response to irinotecan in the liver due to fasting.