CRUDE LIVER MEMBRANE-FRACTIONS AND EXTRACELLULAR-MATRIX COMPONENTS AS SUBSTRATE 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 basal or in-vivo-induced microsomal cytochrome P-450 isoenzyme contents in cultured rat hepatocytes and on the adaptive responses after exposure to phenobarbital or 3-methylcholanthrene in vitro, was investigated. Hepatocytes from untreated or phenobarbital-treated rats were cultured in serum-free, aprotinin-supplemented culture medium in 96-well microtiter plates coated with collagen type I (COL), laminin, fibronectin or crude liver membrane fractions/collagen type I (CMF/COL). Basal cell functions were characterized by measuring the total protein content and lactate dehydrogenase release. The relative contributions of CYP1A1/2, CYP2B1/2, CYP2C6, CYP2C11, CYP3A and CYP4A isoenzymes were determined with ELISA using monoclonal antibodies raised against purified cytochromes P-450 from rat liver microsomes. The characterization of the CMF revealed that contaminations with mitochondria, nuclei and lysosomes are relatively low. Among these, membranes derived from the endoplasmic reticulum appeared to be the major organelle contaminant of the CME The matrix components laminin, fibronectin and collagen type IV were found in appreciable amounts. Hepatocytes from untreated rats, cultured for up to nine days on CMF/COL-coated plates, retained their relative cytochrome P-450 contents at 1.5 - 3-fold higher levels when compared to cells cultured on COL, fibronectin or laminin. Similarly, hepatocytes from phenobarbital-treated rats preserved the contents of barbiturate-inducible CYP2B1/2 and CYP3A proteins best when cultured on CMF/COL. After exposure of hepatocytes cultured on CMF/COL to phenobarbital from days 3-6, CYP3A proteins were enhanced more than twofold and CYP2B1/2, depending on the exposure level, increased 1.3-6-fold. After exposure to 3-methylcholanthrene, a threefold increase of CYP1A proteins was found in CMF/COL and laminin cultures. These results indicate that CMF/COL, as a substratum in rat hepatocyte cultures, regulates gene expression of cytochromes P-450 isoenzymes for up to 9 days and provides a matrix which enables the cells to respond qualitatively similar to the response observed in different zones of the liver. This activity cannot be replaced by single-matrix components.