We modified Ito's medium-term liver foci bioassay by incorporating time-course sacrifices to study pharmacokinetics/pharmacodynamics related to the initiation-promotion process. In our earlier clonal growth modeling studies on a series of chlorobenzenes, the probable existence of two cell types in glutathione-S-trasferase (GSTP) foci became apparent. As suggested by other researchers, these two cell types are either susceptible or resistant to mitoinhibitory effects as a response to external chemical insults, and those resistant to mitoinhibitory effect would have a growth advantage serving as basis for malignant transformation. Thus, as preneo-plastic GSTP foci form, isolation of GSTP positive (GSTP+) cells from liver across time allows capturing changing characteristics of an emerging malignant population. This study describes an attempt to isolate primary GSTP+ hepatocytes using Ito's bioassay protocol with diethylnitrosamine (DEN) as an initiating agent and a 2/3 partial hepatectomy with concomitant dosing of hexachlorobenzene (HCB; 28.4 mg/kg/day) with and without coexposure to PCB 126 (9.8 u/kg/day) as promoting agents. Male F344 rats were sacrificed at 20, 24, 28, 47 and 56 days, and 3, 6, and 9 months following DEN injection. By day 56, a significant increase in liver weight was observed in rats coexposed to HCB and PCB 126. Either primary hepatocytes or liver sections were taken to analyze formation of GSTP+ foci. By treating cell suspension and/or culture with ethacrynic acid (EA) (Stenius et al., Carcinogenesis 15:1561-1566, 1994), GSTP+ hepatocytes were enriched by inducing toxicity to non-GSTP+ cells. In so doing, progressively more malignant cell populations can be subjected to cell cycle kinetic studies, genomic analyses, and clonal growth modeling to aid our insight into the carcinogenic process.
The Toxicologist. Society of Toxicology 43nd Annual Meeting and ToxExpo, March 21-25, 2004, Baltimore, Maryland