The formation of ethoxyfluorescein and fluorescein from diethoxyfluorescein by isolated rat hepatocytes has been used as a basis for separating such cells dependent on their mixed function oxidase activities by fluorescence-activated flow cytometry. Five equal fractions defined by computer-generated regions were isolated. Non-viable cells with low fluorescence (region 1) represented 10-15% of the population, while the remainder with higher mixed function oxidase activities (regions 2-5), were greater than 95% viable by Trypan Blue exclusion. In region 1, 30% of the viable cells were binucleate, 67% diploid while in region 5, 13% were binucleate and 69% tetraploid. At 3 h after sorting, following attachment to glass coverslips, exposure of cells to methyl methanesulphonate, retrorsine or norethindrone resulted in unscheduled DNA synthesis which was 2-fold higher in the tetraploid-rich region 5, while aflatoxin B1, benzo[a]pyrene or 2-acetylaminofluorene caused a 5-fold increase in unscheduled DNA synthesis in these cells, relative to the diploid-rich hepatocytes in region 2.

Incubation of rat liver microsomes with norethindrone and a NADPH-generating system leads to the formation of one N-alkylated porphyrin (green pigment, GP1). Administration of this steroid to male rats in vivo results in the formation of three more-polar green pigments (GP2, 3 and 4). A cytosolic protein (green-pigment converting protein) has been purified from rat liver that, when added to liver microsomal mixtures containing norethindrone (0.03 mM) and a NADPH-generating system, results in the formation of all four green pigments (GP1, 2, 3 and 4). Field-desorption mass spectrometry of the purified green pigments gave protonated molecules, [M + H]+, at m/z 905 for GP1, m/z 909 for GP2, m/z 925 for GP3 and 4. The Mr of the purified cytosolic protein on SDS/polyacrylamide-gel electrophoresis or gel filtration was 37000. Polyacrylamide-gel isoelectric focusing gave a pI value of 5.9. Antibodies raised in rabbits against this protein, after preincubation with rat liver cytosol, subsequently prevented the formation of the more-polar norethindrone-induced green pigments (GP2, 3 and 4). The purified protein in the presence of either NADH or NADPH catalysed the reduction of delta 4-ring-reduced norethindrone, 5 alpha-oestran-17 alpha-ethynyl-17 beta-ol-3-one and, with the appropriate cofactor, the oxidation and reduction of steroids lacking the ethynyl function, e.g. androsterone or dihydrotestosterone. Indomethacin inhibited the reduction of dihydrotestosterone by this protein with an I50 (concn. causing 50% inhibition) value of 4.9 microM. From its physical and enzymic properties it is concluded that green-pigment converting protein is the same as 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50).