THE EFFECT OF SHORT-TERM FASTING, PHENOBARBITAL AND REFEEDING ON APOPTOTIC LOSS, CELL REPLICATION AND GENE-EXPRESSION IN RAT-LIVER DURING THE PROMOTION STAGE

Previous work from this laboratory has reported on the effects of two sequential 5 day periods of fasting and subsequent refeeding on tumor promotion in multistage hepatocarcinogenesis in the rat (Carcinogenesi s, 18, 159-166, 1997). In the present extension of the earlier study, the sequential fasting-refeeding regimen was begun at later time point s (28 and 54 days post-initiation) than the first study. This was done to determine whether larger-sized altered hepatic foci (AHF) exhibite d a depletion similar to that of the relatively small AHF in the publi shed experiment and to study concomitant molecular changes during the fasting periods. Groups of animals were fasted in the presence and abs ence of 0.05% phenobarbital (PB) in the drinking water. During the fas ting periods, both body and liver weights decreased dramatically, less in the fast begun at 54 days. This change was accompanied by a signif icant decrease in the bromodeoxyuridine (BrdU) labeling indices of hep atocytes within AHF, Apoptotic bodies increased dramatically in the no n-focal (surrounding the AHF) hepatocytes during the fasting periods. These parameters were slightly lower in hepatocytes of rats administer ed PB during the fasting periods, most notably during the 54-66 day pe riod. With the nick end-labeling method, the proportion of hepatocytes undergoing apoptosis was significantly higher in cells within AHF at the end of each of the fasting periods in all but one group. Concomita ntly, the number of AHF and percentage of liver volume occupied by AHF decreased dramatically during the fasting periods, Refeeding caused a marked increase in BrdU labeling in hepatocytes within and surroundin g AHF during the first week or two, most notably in animals not receiv ing PB during the fasting period. Both the number and volume percentag e of liver AHF returned to control values within similar to 2 weeks of the refeeding regimen. Assays of nuclear DNA fragmentation with sampl es of whole liver indicated that a 'laddering' effect was most noticea ble in livers of animals subjected to the fasting-refeeding regimen wh en phenobarbital was not present during the fasting period. Studies of the levels of mRNA of several genes in the total liver revealed that the expression of c-myc increased 3- to 9-fold during the fasting peri ods but rapidly returned to normal levels after refeeding, Levels of a lbumin and insulin-like growth factor I mRNAs decreased significantly during the fasting period, but rapidly reappeared on refeeding, These results indicate that the extensive loss of AHF during the short-term fasting periods occurs even when the number and volume of AHF are 10- to 50-fold greater at the beginning of the fast than the values publis hed previously. Both the decrease in insulin growth factor I and the e levation of c-myc expression during the fasting period may indicate th e role of these genes in the transcriptional regulation of hepatocyte apoptosis in both normal and preneoplastic hepatocytes in the rat.