S-phase In Response Research Articles

Species differences in response to peroxisome proliferators correlate in vitro with induction of DNA synthesis rather than suppression of apoptosis.

Tumorigenesis caused by the peroxisome proliferator (PP) class of non-genotoxic hepatocarcinogens is species restricted; rat and mouse are considered responsive whereas the available evidence suggests that humans, non-human primates, dogs, hamsters and guinea pigs are non-responsive. We have demonstrated previously that the PP, nafenopin can suppress rat hepatocyte apoptosis both in vitro and in vivo. Here we describe the ability of nafenopin to suppress apoptosis in mouse, hamster, guinea pig and rat hepatocytes and induce S-phase in mouse and rat hepatocytes. Hepatocyte monolayers from all species examined degenerated rapidly in culture. However, nafenopin (50 microM) reversibly maintained the viability of both rat and mouse hepatocytes. This maintenance was associated with a decrease (P < or = 0.01) in the number of hepatocytes displaying chromatin condensation patterns characteristic of apoptosis. Treatment of rat and mouse monolayers with 5 ng/ml transforming growth factor-beta 1 (TGF beta 1) induced high levels of apoptosis (P < or = 0.01); co-addition of nafenopin suppressed this induced apoptosis (P < or = 0.01). TGF beta 1 also induced apoptosis in hamster and guinea pig hepatocytes (P < or = 0.01) and unexpectedly nafenopin was able to suppress this induced apoptosis (P < or = 0.01) as well as reversibly maintaining the viability of hamster and guinea pig hepatocyte monolayers. Thus, all the species examined responded to nafenopin by a suppression of both spontaneous and TGF beta 1-induced apoptosis. In contrast, only rat and mouse hepatocytes showed an induction of S-phase in response to nafenopin (P < or = 0.01). Certain key experiments were repeated using the PPs methyl clofenapate (MCP) (100 microM) and Wy-14, 643 (10 microM). Both were able to suppress spontaneous and TGF beta 1-induced apoptosis in rat and guinea pig hepatocytes although the effects of MCP were weak (P < or = 0.05) compared with nafenopin or Wy-14 643 (P < or = 0.01). The rat and mouse liver tumour promoter, phenobarbitone (PB) was assessed also. Rat hepatocytes responded to PB with a suppression of apoptosis and an induction of S-phase (P < or = 0.01). Hamster and guinea pig cells gave no response in the S-phase assay and exhibited no suppression of either spontaneous or TGF beta 1-induced apoptosis. Interestingly, nafenopin suppressed the apoptosis induced by the DNA damaging drugs, etoposide and hydroxyurea (P < or = 0.01) suggesting that PPs can impact on diverse apoptosis signalling pathways. Overall, species differences in response to the non-genotoxic hepatocarcinogens studied, correlate with induction of DNA synthesis rather than with suppression of apoptosis. The data extend our knowledge of the mechanisms of species differences in non-genotoxic hepatocarcinogenesis, posing interesting questions on the relative roles of apoptosis and DNA synthesis in carcinogenesis.

Quantitation of an Epithelial S-Phase Response in the Rat Forestomach and Glandular Stomach Following Gavage Dosing with Ethyl Acrylate

Gavage dosing of the irritant, ethyl acrylate (EA), has been found to induce hyperplasia in the rat forestomach, but no signs of toxicity in the glandular stomach or in organs remote from the site of dosing. To quantitatively describe this effect as a background for subsequent modeling studies, pulse measurements of the number of S-phase cells were made following a single gavage dose of EA. The time-course of the S-phase response in the forestomach epithelium following a high dose (200 mg/kg or a 4% solution in corn oil) indicated that the number of S-phase nuclei was decreased relative to control animals immediately following gavage dosing with a minimum at 6 hr, but that the number of S-phase nuclei increased significantly above control values by 20 hr and remained significantly elevated until at least 48 hr following the gavage dose. A single-dose dose-response study with gavage doses of 0, 2, 10, 20, 50, 100, or 200 mg/kg EA and S-phase analysis at 24 hr following gavage dosing indicated that a significant increase in S-phase nuclei was evident at doses of 20 mg/kg or higher. Dosing with EA for 2 weeks at dose levels of 0, 10, 50, or 200 mg/kg caused a prolonged elevation of S-phase nuclei only at the 200 mg/kg dose level during the 24 hr following the last gavage dose. Lower doses did not induce a significant increase in the S-phase nuclei. In contrast to the forestomach, the S-phase response of the glandular stomach was transient following a single 200 mg/kg gavage dose, and only a marginal response was observed following multiple 200 mg/kg doses. No effects were observed at lower doses. Comparison of these results to prior determinations of the effect of EA on the concentration of nonprotein sulfhydryls (primarily glutathione) in the forestomach and glandular stomach indicate a correlation of the stimulation in S-phase activity in the forestomach with the repletion and overshoot of tissue nonprotein sulfhydryl levels

Mitosis and histopathology in rat liver during methylclofenapate-induced hyperplasia

Liver hyperplasia was induced in rats by daily administration of methylclofenapate (25 mg/kg by gavage). An increase in the incidence of colchicine-arrested metaphases was observed with peaks occurring at 40 h (1.3%), 64 h (6.4%) and 84 h (6.8%) after the start of treatment. This response contrasted with the much larger (21.3%) peak in arrested metaphases at 36 h after partial hepatectomy, but was still unexpectedly large in comparison with the S-phase response to methylclofenapate reported in a previous study. Progressive hypertrophic histopathological changes were apparent during the whole course of treatment.

Age-related cell cycle kinetics in the stimulated mouse vaginal epithelium

The cell cycle of the vaginal epithelium of castrated mice after an estrogenic stimulation has been investigated in adult and in aged animals. In aed animals, some cells (at least the first entering S-phase in response to the stimulus) remain able to react and to complete the prereplicative phase as fast as in younger animals. The dose of the stimulus has an all-or-nothing effect on the triggering of the response, and the critical dose value does ot change with age. The only age effect suggested here is a lower proportion of cells synthesizing DNA in response to the stimulus, indicating an enhanced variability in the responding times of the individual cells.

Potent mitogenic activity of 4-acetylaminofluorene to the rat liver

Administration of 4-acetylaminofluorene (4AAF) to rats by oral gavage (1000 mg/kg) produces a wave of S-phase activity in the liver 36 h later, followed by a wave of mitoses at 48 h. These events were monitored by autoradiography of isolated hepatocytes and by histopathology, respectively. DNA-labelling was shown to occur following both in vivo and in vitro radiolabelling. The level of S-phases observed approached that reported following partial hepatectomy. These effects were not accompanied by unscheduled DNA synthesis (UDS) nor was any frank histopathological damage to the liver evident. 2-Acetylaminofluorene (2AAF) elicited a very weak S-phase response at a dose level of 50 mg/kg, but gave marked UDS between 12 and 48 h.