title: Biotransformation in carcinogen-induced diploid and polyploid hepatocytes separated by centrifugal elutriation.
abstract: Biotransformation in carcinogen-induced diploid and polyploid hepatocytes was studied using isozyme-selective substrates for several enzyme pathways. Diploid hepatocytes were induced by partial hepatectomy, a single injection of diethylnitrosamine, and 4 weeks of 2-acetylaminofluorene (2-AAF) feeding. Then, after an additional 3-5 weeks on the control diet, diploid and polyploid hepatocytes were separated from freshly isolated hepatocytes by centrifugal elutriation. Benzo(a)pyrene hydroxylase, ethoxyresorufin O-deethylase, and methoxycoumarin O-demethylase activities were approximately 15-40% lower in the diploid hepatocyte fraction than in the polyploid cell fraction. Activities of 1-chloro-2,4-dinitrobenzene, glutathione S-transferase, 3-hydroxy-benzo(a)pyrene or 4-hydroxybiphenyl UDP-glucuronosyltransferase, and DT-diaphorase were not different in the two cell fractions. Determination of activity during the 2-AAF treatment indicated that 2-AAF increased 7-ethoxyresorufin O-deethylase and 3-hydroxybenzo(a)pyrene glucuronosyltransferase activities by 300 and 200%, respectively, in both the diploid and polyploid hepatocyte fractions. Administration of phenobarbital for 4 days at the end of the control diet period increased ethoxyresorufin and methoxycoumarin dealkylations by 2- and 4-fold, and 3-hydroxybenzo(a)pyrene glucuronidation and 1-chloro-2,4-dinitrobenzene conjugation with glutathione by 1.5- to 2-fold in both hepatocyte fractions. Slight increases in benzo(a)pyrene hydroxylation and 4-hydroxybiphenyl glucuronidation were also evident in diploid cells. Although there is a slight decrease in cytochrome P-450-dependent monooxygenase activities, these data indicate that carcinogen-induced diploid hepatocytes do not show the typical toxicant-resistant phenotype observed in preneoplastic hepatocytes of altered liver foci, which are characterized by large decreases in monooxygenase biotransformations as well as increased activities of several phase II enzymes. This finding is compatible with the hypothesis that 2-AAF-induced nonploidizing growth of diploid hepatocytes is caused by nontoxic mechanisms in the present experimental paradigm. In addition, carcinogen-induced diploid cells respond to phenobarbital in a manner similar to that of polyploid hepatocytes.
authors.affiliation: Indiana University School of Medicine, Medical Sciences Program, Bloomington 47405.
authors.forename: J B
authors.initials: JB
authors.lastname: Watkins
authors.forename: D
authors.initials: D
authors.lastname: Thierau
authors.forename: L R
authors.initials: LR
authors.lastname: Schwarz
mesh_terms: D015073:2-Acetylaminofluorene; D000818:Animals; D002469:Cell Separation; D004052:Diethylnitrosamine; D004171:Diploidy; D004790:Enzyme Induction / Q000187:drug effects; D006498:Hepatectomy; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000473:pathology*; D008297:Male; D011123:Polyploidy; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D004052:Diethylnitrosamine; D015073:2-Acetylaminofluorene