CRUDE LIVER MEMBRANE-FRACTIONS AND EXTRACELLULAR-MATRIX COMPONENTS ASSUBSTRATE REGULATE DIFFERENTIALLY THE PRESERVATION AND INDUCIBILITY OF CYTOCHROME-P-450 ISOENZYMES IN CULTURED RAT HEPATOCYTES

The influence of cell-substrata interactions on the preservation of ba sal or in-vivo-induced microsomal cytochrome P-450 isoenzyme contents in cultured rat hepatocytes and on the adaptive responses after exposu re to phenobarbital or 3-methylcholanthrene in vitro, was investigated . Hepatocytes from untreated or phenobarbital-treated rats were cultur ed in serum-free, aprotinin-supplemented culture medium in 96-well mic rotiter plates coated with collagen type I (COL), laminin, fibronectin or crude liver membrane fractions/collagen type I (CMF/COL). Basal ce ll functions were characterized by measuring the total protein content and lactate dehydrogenase release. The relative contributions of CYP1 A1/2, CYP2B1/2, CYP2C6, CYP2C11, CYP3A and CYP4A isoenzymes were deter mined with ELISA using monoclonal antibodies raised against purified c ytochromes P-450 from rat liver microsomes. The characterization of th e CMF revealed that contaminations with mitochondria, nuclei and lysos omes are relatively low. Among these, membranes derived from the endop lasmic reticulum appeared to be the major organelle contaminant of the CME The matrix components laminin, fibronectin and collagen type IV w ere found in appreciable amounts. Hepatocytes from untreated rats, cul tured for up to nine days on CMF/COL-coated plates, retained their rel ative cytochrome P-450 contents at 1.5 - 3-fold higher levels when com pared to cells cultured on COL, fibronectin or laminin. Similarly, hep atocytes from phenobarbital-treated rats preserved the contents of bar biturate-inducible CYP2B1/2 and CYP3A proteins best when cultured on C MF/COL. After exposure of hepatocytes cultured on CMF/COL to phenobarb ital from days 3-6, CYP3A proteins were enhanced more than twofold and CYP2B1/2, depending on the exposure level, increased 1.3-6-fold. Afte r exposure to 3-methylcholanthrene, a threefold increase of CYP1A prot eins was found in CMF/COL and laminin cultures. These results indicate that CMF/COL, as a substratum in rat hepatocyte cultures, regulates g ene expression of cytochromes P-450 isoenzymes for up to 9 days and pr ovides a matrix which enables the cells to respond qualitatively simil ar to the response observed in different zones of the liver. This acti vity cannot be replaced by single-matrix components.