Effects of phenobarbital and 3-methylcholanthrene pretreatment on size, sedimentation velocity, and mixed function oxygenase activity of rat hepatocytes

SVA was used to separate liver cells from rats pretreated for 3 days with PB (40 mg/kg intraperitoneally, twice daily) or 3-MC (50 MG/KG AS A SINGLE INJECTIOn). Twelve fractions of cells were collected with s's ranging from 97 mm/hr (fraction1) to 25 mm/hr (fraction 12). Cells in each fraction were sized and counted electronically. PB caused the average volume of the largest cells recovered (fraction 1) to increase to 16, 725 micrometer3 from 10,500 micrometer3 previously reported in untreated animals. The number of cells recovered in the fast-sedimenting fractions (1 to 6) also increased, but there was only a small rise in the average model volume of hepatocytes determined prior to SVA. In cell suspensions analyzed after PB treatment no evidence was found for a discontinuity in the distribution of density-volume characteristics as previously described in hepatocytes from untreated rats. As expected, prior to sedimentation analysis, hepatocyte suspensions from rats treated with PB contained increased cytochrome P-450. The average ratio (n = 4) of P-450 in separated to unseparated cells ranged from 2.75 (fractions 1 + 2) to 0.38 (fractions 11 + 12), giving a 6.8-fold range in quantity of cytochrome per cell. Over this range, cell volume increased 4.2-fold, indicating a gradient in P-450 concentration similar to that previously reported to exist in cells from untreated rat liver. The gradient for AHH activity was 6.7-fold, suggesting that activity of MFO's paralleled the increase in cytochrome concentration. 3-MC pretreatment caused no significant increase in either size or number of cells in the fast-sedimenting fractions, but the discontinuity in density-volume characteristics which distinguished fast and slow sedimenting cells of untreated rats became marked. Furthermore, both the size and number of cells recovered in fractions 7 to 11 (which include the modal peal volume of unseparated hepatocytes) were increased. The gradient of P-450 (OR P1-450) was less steep in 3-MC-treated cells with pooled fractions 1 and 2 containing an average of 4.62 times as much cytochrome as fractions 11 and 12. The gradient for AHH was 4.29 times. The range of cell volume was 3.3-fold over this range. Additional experimental work was performed to determine whether P-450, AHH, or NADPH cytochrome c reductase exhibited differences in activity or concentration per unit of cell volume between cells on either side of fraction 7 where discontinuity had been noted. Each variable was expressed per unit of cell volume in fractions 5 and 9; ratios were compared but were indistinguishable from unity. It was concluded that induction of MFO activity had occurred equally in both populations of cells. Densities were calculated from s and cell volume. Noticeable loss of density followed PB treatment in cells of all sizes. 3-MC had less of an effect on density but enhanced the increment in density observed at fraction 7, which corresponds to the division between cells with distinct sedimentation characteristics...