Karyotypic changes in a multistage model of chemical hepatocarcinogenesis in the rat

Karyotypic analysis of the stages of rat hepatocarcinogenesis has been facilitated by the development of an initiation-promotion-progression (IPP) protocol that permits separation and characterization of morphologically normal and altered hepatocytes in each of these three stages. The expression of the membrane antigen gamma-glutamyl transpeptidase (GGT) during the promotion and progression stages of rat hepatocarcinogenesis permits the isolation, culture, analysis, and comparison of hepatocytes in the two stages, which express this marker of carcinogenesis. Female rats were administered 10 mg diethylnitrosamine/kg at 5 days of age. One group of initiated rats was maintained on dietary phenobarbital admixed into a laboratory chow diet at 0.05% for 9 months after weaning (promotion protocol). This initiation-promotion (IP) group was compared with one subjected to the complete IPP protocol. The IPP group was initiated with diethylnitrosamine, maintained on phenobarbital for 6 months after weaning, and then subjected to a 70% partial hepatectomy and administered 100 mg ethylnitrosourea/kg 24 h later. These rats on the IPP protocol were then maintained on phenobarbital for an additional 3 months prior to sacrifice. At sacrifice, single hepatocyte suspensions were obtained and separated into populations of cells expressing or not expressing GGT. These hepatocyte populations were cultured separately and subjected to standard cytogenetic analysis. At least five animals per treatment and 100 metaphase spreads of good morphology per animal were examined. Although GGT- cells from the IP protocol were 80% tetraploid and 20% diploid, the GGT+ hepatocytes were greater than 90% diploid. The GGT+ cells from this protocol had a low rate of random aneuploidy (4.0 +/- 1.3%) compared with corresponding cells from the IPP protocol, but a higher level of background aneuploidy compared with GGT- cells from the IP protocol. The GGT+ hepatocytes from animals on the IPP protocol had a 35% incidence of aneuploidy. In addition, the GGT+ population had a 28 +/- 5% incidence of chromosomal breakage and a 17 +/- 5% incidence of chromosomal rearrangements. The primary nonrandom chromosomal changes observed in cells from the IPP protocol included duplication of all or part (1q37-43) of chromosome 1 and the loss of chromosomes 3p and/or 6q. These studies indicate that rat hepatocytes in the stage of promotion are euploid, whereas those in the stage of progression exhibit considerable genetic instability. The presence of multiple copies of chromosome 1 or a duplication of a region of this chromosome indicates that alteration of gene dosage for one or more of the genes present in this region is critical to the neoplastic conversion of rat hepatocytes, whereas the loss of all of 3p and the last light band of 6q may indicate the presence of tumor suppressor genes. Thus, the IP and IPP protocols coupled with the ability to isolate GGT+ and GGT- hepatocytes permit the differential cytogenetic characterization of the stages of promotion and progression in rat hepatocarcinogenesis.