MAINTENANCE OF PHENOBARBITAL-INDUCIBLE CYP2B GENE-EXPRESSION IN C57BL6 MOUSE HEPATOCYTES IN PRIMARY CULTURE AS SPHEROIDS/

Expression of the Cyp2b-9 and Cyp2b-10 genes was investigated in prima ry cultured adult C57BL/6NCrj mouse hepatocytes in monolayers or durin g the formation of spheroids (multicellular aggregates). Both the cons titutive and phenobarbital-inducible expression of Cyp2b-9 and Cyp2b-1 0 mRNA decreased rapidly after transferring the hepatocytes to monolay er culture. The decrease was dependent on cell-density, and became mor e rapid in more densely seeded cells. However, in spheroid culture, th e Cyp2b-9 and Cyp2b-10 mRNAs were induced by phenobarbital at high lev els for at least 4 days either with continuous exposure from the start of cultivation or for 24 h before harvesting. The expression of both Cyp2b-9 and Cyp2b-10 species was confirmed by either reverse transcrip tase-polymerase chain reaction, or Western blotting. Although more Cyp 2b-9 than Cyp2b-10 was expressed in the liver of control mice, the amo unts of the latter became relatively overwhelming in untreated hepatoc ytes because of a faster decline of Cyp2b-9 species in culture. The le vel of mRNA induced by phenobarbital was concentration-dependent, the highest being at 2 or 4 mM, which was equivalent to in vivo treatment levels. There was more Cyp2b-10 species than those of Cyp2b-9 after ex posure to phenobarbital both in vivo and in vitro. Phenobarbital also induced CYP1A2 mRNA, which was again peculiar to spheroid culture. Alt hough the expression levels of both Cyp2b-9 and Cyp2b-10 species was v ery low in hepatocytes cultured without dexamethasone even in the pres ence of phenobarbital, the addition of 10(-7) or 10(-6) M dexamethason e caused an increase in the mRNA. When given concomitantly with phenob arbital, the expression was greatly enhanced. Nicotinamide or isonicot inamide similarly enhanced the expression of the two mRNA species, but the levels in monolayer cultures were still far lower than those in v ivo. In contrast to the findings for mRNA expression, the protein leve ls in the presence of nicotinamide were similar to those in vivo under both monolayer and spheroid conditions. Since previous efforts to mai ntain expression of the CYP2B1 and CYP2B2 genes in primary cultured ra t hepatocytes, orthologous to mouse Cyp2b-9 and Cyp2b-10 genes, requir ed special cell attachment factors, our spheroid culture system, in wh ich mouse hepatocytes are simply seeded onto noncoated dishes, has adv antages for mechanistic studies. (C) 1995 Academic Press, Inc.