Changes in hepatocyte ploidy in response to chromium, analyzed by computer-assisted microscopy.

BDF1 mice were given single injections of sodium dichromate (25 mg/kg) on an acute (6 hr to 7 days) or intermediate (2-4 weeks) basis, or multiple injections (12.5 mg/kg) on a chronic (4.5 months) basis. Observed hepatic changes included programmed cell death (apoptosis) in the periportal region with acute exposure and fusion of liver lobes with chronic exposure. Response to chromate exposure was measured by change in hepatocyte nuclear ploidy state (e.g., the proportion of diploid, tetraploid, and octaploid nuclei) based on computer-assisted imaging from histological sections. The computer-assisted imaging system used in this study was superior to traditional methods because it (1) allows rapid ploidy determinations from histological material and (2) can be used to collect regional information. Regional differences in ploidy were seen to occur in a consistent fashion among both control and treated animals. Nuclei adjacent to the portal triad had the lowest ploidy value (highest proportion of diploid nuclei), an intermediate value was found adjacent to the central vein, and the highest ploidy was found in the midzone. These three ploidy-based zones roughly correspond to the three functional zones of A. M. Rappaport (1973, Microvasc. Res. 6, 212-228) and W. H. Lamers et al. W. H. Lamers, A. Hilberts, E. Furt, J. Smith, G. N. Jonges, J. F. Van Noorden, J. W. G. Janzen, R. Charles, and A. F. M. Moorman, 1989, Hepatology, 10, 72-76. Temporal changes in ploidy were seen among control animals (all zones), with young animals (56 days) displaying relatively low ploidy values compared to older animals (184 days). Chromate exposure caused increased ploidy (all zones) among animals treated on an acute basis (the youngest animals). Chromate had no apparent effect on ploidy among animals treated for longer periods of time, probably because of age-related factors.

Suiciding of lymphocytic precursor cells by tritiated nucleosides, in vitro.

Differences in suiciding by various tritiated nucleosides were observed between two functional assays for in vitro lymphocytic precursor cell development, the hemolysin plaque-forming cell (PFC) assay and the B lymphocytic colony-forming cell (CFC-L) assay, using BDF1 mouse spleen cells. PFC growth was markedly reduced by an early (days 0-1) pulse of tritiated deoxyadenosine ([3H]dAdo), but relatively unaffected by a pulse of tritiated thymidine ([3H]dThd) during the same interval. In contrast, CFC-L formation significantly dropped after an early (day 0) [3H]dThd pulse, as well as after pulses of [3H]dAdo and the corresponding tritiated ribosides, uridine and adenosine. This implied a cycling state in an early lymphocytic precursor cell, as opposed to the PFC insensitivity to an early [3H]dThd pulse. The response pattern of colonies and clusters to [3H]dThd supported our notion of a delayed suiciding of CFC contributing to the increase in cluster numbers.

Role of cellular density, in vitro, in anti-tumor activity of CFA-treated and immunized cells.

Incorporation of tritiated deoxythymidine (3HdT) into DNA was used to measure growth, in vitro, of P815 tumor cells admixed with spleen and peritoneal effector cells. At a high tumor cell density ((1x10(5) cells per dish), using anti-theta and anti-macrophage sera, T-cells and macrophages from the peritoneum of immunized mice could be identified as cells possessing anti-tumor activity. A nonspecific inhibition by normal effector cells, which occurred at the high tumor cell density, did not occur at a lower tumor cell density (1x10(4) cells per dish). Therefore, the effects of immunization and Freund's adjuvant treatment on the anti-tumor activity of effector cells were determined more accurately when normal cells were no longer inhibitory. Thus, experimental variables dealing with cellular density (cells/mm2 of the culture vessel surface) and effector:tumor cell ratios play an important role in the anti-proliferative capacity of effector cells.