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Introduction: The aim of study was to evaluate impact of long-term dietary cholesterol overload on the cholesterol homeostasis and liver regeneration., Material and Methods: Serum lipid parameters, 14C-cholesterol incorporation, liver DNA synthesis and protein expression was determined in partially hepatectomized (PH) rats fed with a standard (SLD) or hypercholesterolemic (CHOL) diet., Results: 29-day intake of CHOL diet before PH produced increase in serum total cholesterol, LDL lipoprotein, and triglyceride concentration. PH provoked decrease in serum total cholesterol and triglyceride concentration in both groups. PH was associated with increase in serum ALT activity more pronounced in CHOL animals. Hepatic DNA synthesis was increased after PH in both groups, but lower in CHOL. Hypercholesterolemic diet reduced the absorption of radiolabelled cholesterol in intestine and then activity in blood and liver. The 14C-cholesterol hepatic activities tend to increase after PH in both groups. CHOL diet produced up-regulation of Acyl-CoA:cholesterol acyltransferase-2 protein expression. PH was associated with increase of LDL receptor and Acyl-CoA:cholesterol acyltransferase-2 protein expression in both dietary groups., Discussion: Liver regeneration after PH is negatively influenced by CHOL diet. The increased uptake of cholesterol in the liver after PH associated with up-regulation of LDL receptor protein expression suggests preferential use of extrahepatic cholesterol by the liver.

The 3'-azido-3'-deoxythymidine or Zidovudine (AZT) was the first antiretroviral drug used in the treatment of HIV patients, which has good effectiveness but also hepatotoxic side effects that include cell cycle arrest and oxidative/nitrative mitochondrial damage. Whether such an oxidative damage may affect the proliferative-regenerative capacity of liver remains to be clearly specified at doses commonly used in the clinical practice. In this study, we described the oxidative-proliferative effect of AZT administered at a common clinical dose in rat liver submitted to 70% partial hepatectomy (PH). The results indicate that AZT significantly decreased DNA synthesis and the number of mitosis in liver subjected to PH in a synchronized way with the promotion of organelle-selective lipid peroxidation events (especially those observed in plasma membrane and cytosolic fractions) and with liver enzyme release to the bloodstream. Then at the dose used in clinical practice AZT decreased liver regeneration but stimulates oxidative events involved during the proliferation process in a way that each membrane system inside the cell preserves its integrity in order to maintain the cell proliferative process. Here, the induction of large amounts of free ammonia in the systemic circulation could become a factor capable of mediating the deleterious effects of AZT on PH-induced rat liver regeneration.

Nuclear sphingomyelin is a key molecule for cell proliferation. This molecule is organized with cholesterol and proteins to form specific lipid microdomains bound to the inner nuclear membrane where RNA is synthesized. Here, we have reported the ability of the sphingomyelin present in the nuclear microdomain to bind DNA and regulate its synthesis, and to highlight its role in cell proliferation induced by partial hepatectomy. During G1/S transition of the cell cycle, sphingomyelin and DNA content is very high and it is strongly reduced after exogenous sphingomyelinase treatment. During the S-phase of the cell cycle, the stimulation of sphingomyelinase and inhibition of sphingomyelin-synthase are accompanied by the DNA synthesis start. To assess the specificity of the results, experiments were repeated with trifluoperazine, a drug known to affect the synthesis of lipids and DNA and to stimulate sphingomyelinase activity. The activity of sphingomyelinase is stimulated in the first hour after hepatectomy and sphingomyelin-DNA synthesis is strongly attenuated. It may be hypothesized that the nuclear microdomain represents a specific area of the inner nuclear membrane that acts as an active site of chromatin anchorage thanks to the stabilizing action of sphingomyelin. Thus, sphingomyelin metabolism in nuclear lipid microdomains is suggested to regulate cell proliferation.

Rapamycin is a potent antiproliferative agent that arrests cells in the G1 phase of the cell cycle through a variety of mechanisms involving the inhibition of the mammalian target of rapamycin (mTOR) pathway. The majority of normal cells in culture are sensitive to the cytostatic effects of rapamycin, whereas the growth of many malignant cells and tumors is rapamycin resistant. We had shown previously that hepatic DNA synthesis in the late gestation rat fetus is rapamycin resistant even though signaling through the mTOR/S6 kinase (S6K) pathway is attenuated. On the basis of this finding, we went on to characterize the response to rapamycin in a spectrum of tissues during late gestation and the early postnatal period in the rat. We found that rapamycin had no effect on DNA synthesis in major organs such as heart, intestine, and kidney in the fetal and early postnatal rat despite a marked attenuation in the phosphorylation of ribosomal protein S6. In contrast, the proliferation of mature hepatocytes during liver regeneration was highly sensitive to rapamycin. These data indicate that basal cellular proliferation in a wide variety of tissues is rapamycin resistant and occurs independently of mTOR/S6K signaling. Furthermore, the well-characterized effects of rapamycin in tissue culture systems are not recapitulated in the asynchronous cell proliferation that accompanies normal growth and tissue remodeling.

The role of endogenous regucalcin in the regulation of deoxyribonucleic acid (DNA) synthesis activity in the nuclei of regenerating rat liver after partial hepatectomy was investigated. The addition of regucalcin (0.25 and 0.5 microM) in the reaction mixture caused a significant decrease in the nuclear DNA synthesis activity of normal rat liver. This decrease was also seen in the presence of Ca2+-chelator EGTA (0.4 mM), indicating that the effect of regucalcin is not related to nuclear Ca2+. Nuclear DNA activity was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect was completely abolished by the addition of regucalcin (0.5 microM). Nuclear DNA synthesis activity was significantly increased at 24, 48, and 72 h after partial heptectomy. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing nuclear DNA synthesis activity was significantly enhanced at 24 and 48 h after partial hepatectomy. The presence of staurospone (10(-6) M), trifluoperazine (2 x 10(-5) M), or PD98059 (10(-5) M) in the reaction mixture caused a significant decrease in DNA synthesis activity in the nuclei obtained at 24 after partial hepateactomy. The effect of these inhibitors in the presence of anti-regucalcin monoclonal antibody (25 ng/ml) was greater than that in the absence of the antibody. The present study suggests that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis activity in regenerating liver with cell proliferation after partial hepatectomy in rats., (Copyright 2002 Wiley-Liss, Inc.)

Polyamines are key factors in macromolecule synthesis during liver regeneration. It has been postulated that interferon-alpha (IFNalpha) decreases putrescine levels in regenerating liver by inhibiting ornithine decarboxylase (ODC) activity, the main enzyme in polyamine biosynthesis. In the present study, we analysed the effects of a pharmacological dose of IFNalpha on polyamine and ODC levels during the regenerative process following partial hepatectomy in rats. Synthesis of ODC by isolated hepatocytes from IFN-treated rats with regenerating livers was also assessed. Furthermore, we investigated the effect of IFNalpha-2b on DNA and total protein synthesis in 24-hr regenerating livers. No effect on DNA synthesis was observed at the dose of IFNalpha-2b used, but total protein synthesis decreased significantly in IFNalpha-2b-treated rats undergoing liver regeneration (7.0 +/- 2.0 and 12.1 +/- 1.7%. min(-1) in hepatectomized rats treated with IFNalpha-2b and saline, respectively). ODC levels were also reduced significantly (by 50%) in hepatectomized rats treated with IFNalpha-2b versus saline. In parallel with the ODC decrease, the concentrations of putrescine and spermidine (63 +/- 25 vs 101 +/- 15 nmol/g liver and 1.08 +/- 0.35 vs 2.14 +/- 0.22 micromol/g liver, respectively, in IFNalpha-2b- and saline-treated hepatectomized rats) showed similar, significant diminutions. Moreover, the incorporation of [35S]methionine into ODC was decreased dramatically in isolated hepatocytes from IFNalpha-2b-treated hepatectomized rats 12 hr after surgery. In conclusion, the protein synthesis rate in regenerating liver was impaired by therapeutic doses of IFNalpha-2b. In addition, the results presented in this study suggest that IFNalpha-2b negatively regulates ODC synthesis, causing a reduction in polyamine levels during liver regeneration.

The expression of asialoglycoprotein receptor (ASGP-R) on human hepatocarcinoma cells might be exploited to reduce the extrahepatic toxicity of DNA synthesis inhibitors by their conjugation with galactosyl- terminating peptides. We conjugated 2',2'-difluorodeoxycytidine (dFdC), an inhibitor of DNA synthesis active on solid tumors, with lactosaminated poly-L-lysine (L-poly(LYS)). In experiments in vitro, L-poly(LYS)-dFdC inhibited proliferation of Hep G2 cells, a human hepatocarcinoma cell line which maintains the ASGP-R. Inhibition was rescued by asialofetuin. To study the pharmacological action of the conjugate in vivo, we used rats 18-24 hr after 2/3 hepatectomy and observed that regenerating hepatocytes expressed ASGP-R on their surface and internalized L-poly(LYS)-dFdC. Conjugate uptake by bone marrow, spleen, and intestine was negligible. We also found that L-poly(LYS)-dFdC inhibited [3H]thymidine incorporation into DNA of regenerating liver. These results indicated that hepatectomized rats were a suitable animal model to study the pharmacological action, on DNA-synthesizing hepatocytes, of conjugates binding to ASGP-R and carrying inhibitors of DNA synthesis. L-poly(LYS)-dFdC also inhibited [3H]thymidine incorporation in bone marrow, spleen, and intestine. Evidence was obtained that inhibition of DNA synthesis in extrahepatic tissues was a consequence of drug release from hepatocytes into blood-stream after the bond with the carrier has been broken down within liver cells. Possible ways of reducing the exit of dFdC from liver cells, thereby obtaining an inhibition of DNA synthesis restricted to dividing hepatocytes, were discussed.

Quercetin, a widely distributed bioflavonoid, inhibited DNA synthesis in regenerating liver after partial hepatectomy. This inhibition was accompanied by apoptosis, evidenced by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was detected as early as 2 h after injection. Northern blot analysis revealed that quercetin induced the increases in c-fos and p21WAF1CIP1 mRNA levels within 2 h. The expression of p21 protein was also enhanced, while p53 mRNA and protein levels were not affected by quercetin. These results suggest that quercetin-induced apoptosis is associated with the increase in c-fos mRNA level and the upregulation of p21 mRNA and protein expression, probably in a p53-independent pathway.

Background/aim: Follistatin is an antagonist of activins and is effective in promoting liver regeneration after 70% hepatectomy. To examine its efficacy under more critical conditions, we studied the effect of follistatin on liver regeneration in 90% hepatectomized rat., Methods: Human recombinant follistatin was infused into the portal vein immediately after 90%, hepatectomy in 24-h-starved rats, and changes in the liver regeneration rate and nuclear bromodeoxyuridine labeling were measured., Results: In control rats, nuclear labeling was first detected at 11 h of hepatectomy. In follistatin-treated rats, nuclear labeling was markedly increased at 3 h, and was significantly higher than that in control rats at 24, 72, 96, 120 and 144 h. The liver regeneration rate was significantly higher in follistatin-treated rats at 48, 72, 96, 120, 144 and 168 h. To determine the reason for the accelerated growth in starved rats, we compared the expression of mRNA for c-myc, p53, p21CIP1, p15INK4B, p27KIP1, and subunits of activins in fed and starved rats. mRNA for p21CIP1 and p15INK4B, but not p27KIP1 were decreased in 24 h-starved rats compared to the fed rats. mRNA for betaA subunit of activin was not detected in either fed or 24-h-starved rats, whereas that for betaC subunit was increased in starved rats., Conclusion: These results indicate that follistatin induces immediate onset of DNA synthesis in 90% hepatectomized rats and is quite effective in promoting liver regeneration.

Prevention of postoperative hepatic failure is important after hepatic resection. In patients with cirrhosis, impaired liver function and regenerative capacity after major hepatic resection are associated with increased morbidity and mortality. In this study, a combination of epidermal growth factor (EGF) and insulin were used as hepatotrophic factors in an attempt to stimulate DNA synthesis after 70% hepatectomy (HTX). Regenerative capacity was evaluated in normal and cirrhotic rat liver by measuring DNA synthesis in vivo. Micronodular liver cirrhosis was established by the simultaneous oral administration of CCl4 and phenobarbital. Epidermal growth factor plus insulin was injected subcutaneously immediately after and 12 h after HTX or sham operation was performed. Rats were killed 24 h after the operation and liver regeneration was estimated by [3H]-thymidine incorporation into DNA as well as an autoradiographic nuclear labelling index. Hepatectomy increased [3H]-thymidine incorporation significantly in both normal and cirrhotic rats. In cirrhotic rats, [3H]-thymidine incorporation after HTX was significantly lower than in normal rats and administration of a combination of EGF and insulin after HTX enhanced [3H]-thymidine incorporation. In conclusion, DNA synthesis 24 h after HTX is decreased in cirrhotic rats compared with normal rats and EGF supplementation with insulin accelerates DNA synthesis in hepatectomized cirrhotic rats. The data suggest that administration of combinations of exogenous hepatotrophic factors may play a useful role in the treatment of cirrhotic patients undergoing major hepatic resection.

The effects of pHGF on the changes of hepatocyte proliferative cycle and liver regenerative capacity after partial hepatectomized rats were observed by flow cytometry (FCM). The results were as follows: 1) S phase fraction (SPF) in group of normal rats (group A) accounted for 9.89% and increased gradually within 6 h, following a peak at 12th h or 36th h after operation, but in the group of pHGF-treated rats (group B) the peak appeared at 24th h after operation; 2) Proliferation index (PI) of group A was 19.6% before partial hepatectomy, increased to 34.91% within 6 h and reached a peaks at 12th or 36th h after operation, and in group B the peak appeared at 48th h after operation. There were significant differences between two groups in SPF and PI (P < 0.01). The weight of liver began to increase 12 h after operation, and almost reached the preoperative weight 5 days after operation. These findings suggest that pHGF can promote the DNA synthesis and segmentation of hepatocyte.

Liver regeneration is significantly impaired in rats with both alpha-adrenergic hepatic denervation and hereditary vasopressin deficiency. This may implicate a direct role for these agonists in the process of compensatory hyperplasia. The mitogenic capacities of norepinephrine, vasopressin and hepatocyte growth factor (HGF), either alone or in combination were investigated by [3H]thymidine incorporation into hepatocyte cultures prepared from normal and regenerating rat livers. The results show that normal hepatocytes incorporate less [3H]thymidine in response to HGF than do regenerating hepatocytes. In addition, physiological concentrations of vasopressin cause a synergistic stimulation of [3H]thymidine uptake in rat liver cells in the presence of HGF.

Quinolone antibiotics inhibit eukaryotic as well as prokaryotic cell growth and protein synthesis. To determine whether these properties adversely affect hepatic growth and recovery following surgical resection, five groups of healthy, adult male rats (n = 7-8/group) were treated for 10 days with equal volumes of either ofloxacin (50 mg/kg), fleroxacin (25 mg/kg), ciprofloxacin (25 mg/kg), norfloxacin (15 mg/kg) or sterile saline (controls) prior to 70% partial hepatectomy (PH) and daily thereafter until death. Restituted liver mass, DNA and protein synthesis rates were determined at 24, 48 and 72 h PH. The results of the study revealed that all parameters of hepatic regeneration were similar in the five study groups at each time interval. To ensure that an effect on hepatic regeneration was not dose-dependent, additional experiments were performed where 1, 10 and 100 mg/kg ciprofloxacin was administered and DNA synthesis was measured 24 h post-PH. Once again, the results were similar to sterile saline-treated controls. These findings suggest that the quinolone antibiotics are unlikely to have an adverse effect on hepatic recovery following surgical resection of the liver and are safe to use in that setting.

Liver cell proliferation is a complex process that can be affected by a large number of factors such as bile acids, which have been reported to be associated to the pathogenesis of liver cancer. In this work, bile acid-induced modifications in DNA synthesis by regenerating perfused rat liver were investigated. Two-thirds hepatectomy was carried out 24 hr before perfusion of liver with recirculating, erythrocyte-free Krebs-Henseleit solution. The viability of the preparations was maintained under all experimental conditions, as indicated by bile flow, oxygen uptake, perfusion pressure, perfusion flow and release of lactate dehydrogenase and potassium into the perfusate. Livers received (min 10 to min 60) bile acid infusion at a rate of 25 nmol/min/gm liver (i.e., maximal secretion rate/2) in regenerating livers as calculated for taurocholate in separate experiments). Trace amounts of [methyl-14C]thymidine were added to the perfusate at min 30. At the end of the experiments (min 60) the livers were washed, removed, weighed and homogenized to determine radioactivity in whole tissue, in DNA and in non-DNA-related fractions. Taurocholate and, to a lesser extent, taurodeoxycholate and dehydrocholate (but not ursodeoxycholate) were found to reduce 14C incorporation into DNA. This was not due to changes in the content of 14C in whole, regenerating liver tissue. Taurocholate, taurodeoxycholate, dehydrocholate and ursodeoxycholate had no effect on thymidine uptake; moreover, the proportion of 14C found in bile was negligible. However, bile acid-induced modification in the fate of intracellular thymidine was observed. In regenerating livers receiving no bile acid, the 14C carried by thymidine metabolites accounted for about 60% of 14C in whole liver tissue. Taurocholate markedly increased this proportion to about 80%. Reverse-phase high-pressure liquid chromatography revealed that most of this 14C (about 80%) was recovered at the elution time, corresponding to thymidine catabolites rather than to DNA precursors. These results suggest that bile acids induce enhancement of thymidine catabolism that reduces its incorporation into DNA; inhibition in the process of DNA synthesis itself, leading to a subsequent increase in the metabolism of DNA precursors; or both. Moreover, from the diversity in this property for bile acid species it might be inferred that changes in the composition and size of the bile acid pool during liver carcinogenesis or regeneration play a role in the modulation of the proliferative process.

Thioacetamide proved to be a potent necrogenic agent when a single dose of 6.6 mmol/kg was administered intraperitoneally to rats. Its necrogenic ability was assessed on the basis of morphological and biochemical changes. The injury of centrilobular hepatocytes showed a peak of cell death 24 hr after thioacetamide administration; it was followed immediately by the regenerative response. Parallel increases of serum aminotransferases, isocitrate dehydrogenase and gamma-glutamyl transferase activities were observed. Severe liver damage was also evident at 24 hr on the basis of glutathione depletion (29% of control), malondialdehyde production (169%), cytochrome P-450 level decrease (26%) and increased activity of glutathione S-transferase (160%). We checked the regenerative response by determining nuclear DNA content in isolated hepatocytes 0, 6, 12, 18, 24, 36, 48 and 72 hr after thioacetamide administration. Changes in DNA cell distribution between G0-G1, S and G2 + M phases of the cell cycle were observed. The sharp decrease in the percentage of the tetraploid cell population (G2 + M phases) and the abrupt increase of the S-phase cells at 36 and 48 hr suggest transition from adult to fetal in hepatocyte populations obtained 24 and 36 hr after thioacetamide treatment. At 72 hr of treatment, hepatocyte populations showed recovery to adult state. In the shift from the adult to fetal, registered at 24, 36 and 48 hr after thioacetamide administration, mitosis seemed to precede the synthesis of DNA.

Impaired liver regeneration in cirrhosis complicates the surgical treatment of liver tumors which arise in this setting. We developed a rat model to investigate the regenerative response of cirrhotic liver after hepatectomy and studied the effect of exogenous transforming growth factor-alpha (TGF-alpha), a potent liver mitogen. Micronodular cirrhosis was established by the simultaneous administration of CCl4 and phenobarbital. Hepatic DNA synthesis ([3H]thymidine incorporation into DNA) 24 hr after partial hepatectomy in cirrhotic rats was 15.6 +/- 3.4 cpm/micrograms DNA (means +/- SEM), which was significantly lower than in normal rats (37.3 +/- 3.4 cpm/micrograms DNA, P less than 0.05). Exogenous TGF-alpha (30 nmol/kg, sc every 12 hr) significantly improved [3H]thymidine incorporation (35.6 +/- 8.2 cpm/micrograms DNA, P less than 0.05). An autoradiographic nuclear labeling index also confirmed increased DNA synthesis (6.7% vs 13.4%). TGF-alpha had no effect on normal regenerating liver (42.5 +/- 8.8 cpm/micrograms DNA, NS). Although the significance of TGF-alpha-enhanced liver regeneration in cirrhosis has yet to be assessed, this model may be useful for the study of mechanisms which control hepatic proliferation.

Tumor necrosis factor-alpha (TNF) has been reported to increase DNA synthesis in normal rat liver. Therefore, we examined the effects of TNF on rat liver regeneration. TNF, 1.5 micrograms ip every 4 h for 5 d, significantly increased hepatic DNA and RNA contents of regenerating and sham operated livers by up to 45%. Mitotic figures in sham operated liver, usually rare, were increased substantially by TNF. ODC mRNA content and enzyme activity were increased in regenerating liver, and were further increased by TNF. These data indicate that TNF, although not specific for regenerating liver, is a potent stimulus for hepatocyte DNA synthesis and mitosis.

Transforming growth factor beta (TGFB) is a potent inhibitor of DNA synthesis in adult rat hepatocytes in vitro. In the present study, the response of hepatocytes from normal or regenerating rat liver to TGFB was determined. TGFB inhibited DNA synthesis uniformly in hepatocytes from both groups in the absence of EGF. However, hepatocytes from 3 h regenerating liver maintained for 3 days in the presence of EGF were less sensitive to the growth-inhibitory action of TGFB. [3H]-thymidine incorporation was inhibited at 20 pM TGFB by only 7% in hepatocytes from 3 h regenerating liver compared with 70% in normal hepatocytes. By increasing the dose of TGFB to 100 pM, however, the full inhibitory response was restored. Reduced sensitivity was also found when the nuclear labelling index was determined, but no change was observed in cells from rats 3 h following sham hepatectomy. The change in sensitivity to TGFB required the presence of 5 ng/ml EGF or greater. Within a further 24-48 h in culture, the response to lower doses of TGFB was at least partially restored. While the present experimental design cannot directly confirm the role of TGFB as a paracrine inhibitor of liver growth in vivo, the data are compatible with this hypothesis.

Rates of DNA and RNA synthesis as well as transport of RNA into cytoplasmic structures of rat liver cells were studied after administration of amniocen preparation. The drug did not affect the rate of DNA and RNA synthesis in intact rat liver cells, while specific radioactivity of DNA was decreased in regenerating liver tissue 24 hrs after hepatectomy. Amniocen inhibited the rate of rRNA, mRNA and tRNA transport into cytoplasm but contributed to elevated penetration of nucleic acid precursors into rat liver cells. Effect of the drug on cell membrane system appears to be responsible for its biological action related to realization of genetic information.

The presence of a human hepatocyte growth factor (hHGF)-like DNA-synthesis promoter in platelet-poor serum of mice with liver injury was examined. Activity of the serum for stimulating DNA synthesis in cultured rat hepatocytes was low in untreated or vehicle-treated mice, but markedly increased 24 h after carbon tetrachloride administration and then dropped to normal levels prior to the peak of liver DNA synthesis. The effect of the serum was additive with the maximal effects of mouse and human epidermal growth factors, but not with that of hHGF. The growth-stimulating factor in the mouse serum, like hHGF, had affinity for heparin and was heat-labile. These results indicate that the level of a serum hHGF-like hepatocyte growth factor increased in mice treated with carbon tetrachloride prior to liver regeneration.

Tritium-labeled 3-methyldibenzo(c,g)carbazole and 5,9-dimethyldibenzo(c,g)-carbazole, 2 organ-specific hepatocarcinogens in mice, were given intraperitoneally to partially hepatectomized animals at various times during the first cell division cycle following surgery. The former compound, more potent and cytotoxic, gave more striking results showing that the maximum number of adducts per unit weight of DNA in liver were formed when the carcinogen was given at the beginning of the S phase, which is evidence for the role of DNA replication in the initiation step of carcinogenesis. Elimination of these adducts led after several days to a residual number of lesions which was of the same order whatever the time of carcinogen administration. In the case of 5,9-dimethyldibenzo-carbazole there were fewer adducts formed after hepatectomy and their removal was much slower.

A single injection of diaminohexane (DAH) causes an abrupt but reversible decrease in the putrescine concentration in the regenerating rat liver. Under experimental conditions hepatic spermidine and spermin content remains invariable while the synthesis of these compounds temporarily decreases. Incorporation of the radioactive precursors into proteins and RNA of the regenerating liver does not essentially change after DAH injection. However, DNA synthesis is sharply reduced. Incorporation of 3H-thymidine into DNA directly correlates with the putrescine concentration and spermidine synthesis. The results suggest that putrescine is essential for maintenance of normal DNA replication in the regenerating rat liver.

We studied liver regeneration after partial hepatectomy and the effects of inhibition of ornithine decarboxylase (ODC) and putrescine administration. The specific ODC inhibitor, alpha-difluoromethylornithine was given as a 3% oral solution (5.4 g/kg X day intake) to hepatectomized rats as well as sham-operated controls. alpha-Difluoromethylornithine had no effect other than inhibition of the low basal levels of ODC in sham-operated rats, but it markedly inhibited increases in ODC by 85% in hepatectomized rats. alpha-Difluoromethylornithine reduced hepatic deoxyribonucleic acid synthesis by 61%, protein synthesis by 46%, and liver weight increased by 83%, showing that alpha-difluoromethylornithine inhibition of ODC inhibits liver regeneration. Putrescine (2 mmol/kg X day) was then given intraperitoneally to hepatectomized rats and controls. Putrescine had no effect in rats not given alpha-difluoromethylornithine. In rats given alpha-difluoromethylornithine, putrescine markedly reversed the inhibitory effect of alpha-difluoromethylornithine on ODC (83%), deoxyribonucleic acid synthesis (94%), protein synthesis (95%), and liver regeneration (85%). These results document that the increases in ODC are important in hepatic regeneration and that polyamine metabolism plays an important role in the increased deoxyribonucleic acid and protein synthesis in this hepatic proliferative response.

Degenerative and regenerative changes induced in rat liver by single exposure to diethylnitrosamine (DEN) were examined by morphological and biochemical approaches. Apoptotic changes were observed in livers of rats exposed to a 'subnecrogenic' dose of DEN (10 mg/kg) as well as in liver parenchyma of those receiving a necrogenic dose (100 mg/kg). Zonal centrilobular necrosis was observed exclusively in the latter group. Regenerative changes, i.e., increases in DNA synthesis, labeling index and mitotic activity, occurred only in animals exposed to the higher dose. The mitogenic effect obtained in these conditions was about half that induced by two-thirds hepatectomy and the maximum response occurred about 24 h later than in partially hepatectomized rats.

A system for the study of DNA synthesis in isolated nuclei is described for sham and regenerating rat liver. The system has been characterized with respect to nuclear purity, conditions for optimum incorporation of [5-methyl-3H]thymidine triphosphate, time course of incorporation, product analysis by neutral and alkaline sucrose gradients, and the effect of exogenously added DNA. No difference in the basal level of activity was detected between nuclei prepared from normal or regenerating liver when isolated 24 hr after operation. However, exogenous activated DNA preferentially stimulated [5-methyl-3H]thymidine triphosphate incorporation in nuclei from regenerating liver. Activated DNA caused to react with the carcinogen N-acetoxy-2-acetylaminofluorene was a less effective primer-template in this system and decreased in a dose-dependent fashion the incorporation of [5-methyl-3H]thymidine triphosphate to below basal levels in nuclei from both normal and regenerating liver. The carcinogen N-methyl-N'-nitro-N-nitrosoguanidine had no inhibitory effect when assayed in this fashion.

The appearance of a liver DNA synthesis promoter (HP) in rat plasma after dimethylnitrosamine (DMNA) or thioacetamide injection was investigated. After 48 h, DMNA (30 mg kg-1 body weight) produced liver (centrilobular) necrosis and intense hepatic regeneration, as assessed by microscopic observations of liver slices, as well as augmented transaminase levels; HP was detectable under these conditions. After 5 days, transaminases and HP returned to normal values (the latter undetectable), coinciding with a lack of necrotic zones. At 60 mg DMNA kg-1 body weight, necrotic areas were more marked and transaminases and HP levels higher after 48 h than with the lower dose; these increases were even more pronounced at 90 mg DMNA kg-1 body weight. After thioacetamide injection (200 mg kg-1 body wt) the situation at 48 h was very similar, with focal, centrilobular necrosis, frequent regenerative signs, high transaminases and detectable HP. Rats recovered after 7 days in a similar fashion as with DMNA. At 400 mg thioacetamide kg-1 body weight, necrotic areas and regeneration zones were more widespread and transaminases and HP higher after 48 h than with the lower dose. On account of the differing modes of action of DMNA and thioacetamide in rat liver, it is proposed that the appearance of HP activity in plasma could be related to the regenerative process that follows hepatotoxic damage.

Studies on endogenous inhibitors obtained from supernatant fluids of rat hepatocytes indicated that the supernatant arrests the normal cell cycle at two points, namely, before the beginning of the S phase and at the onset of metaphase when assayed in regenerating liver cells in situ. The specificity of this (these) inhibitory substance(s) was evidenced by the finding that no inhibition of cell division was observed in situ in tongue, intestine, or kidneys of animals injected with concentrations of the hepatocyte supernatant that inhibited cell division in regenerating liver. When higher concentrations of the inhibitor-containing supernatant were used, only the tongue epithelial cells were affected in addition to the regenerating liver cells.

The nuclei of cells from regenerating rat liver were incubated with benzo(a)pyrene and the concentrations of the metabolites that covalently bound to DNA of different nuclear fractions were compared. It appeared that DNA associated with nuclear matrix (containing replicating DNA) is modified most intensively. The synchronized mouse embryo cells were incubated with benzo(a)pyrene during S phase and the levels of modifications in short and long single-stranded DNA fragments were compared. It has been observed that replicating DNA is represented in short fragments. These short DNA fragments were found to be modified by benzo(a)pyrene 4-9 times more intensively than total DNA. The possible mechanisms of both the increase in the number of DNA modifications in proliferating cells and the reason for the enhancement of carcinogenic effect on dividing cells are being discussed.

The effect of N-hydroxyurethan (HUR) on DNA synthesis has been tested both in vivo on various tissues and in vitro on concanavalin A (ConA)-stimulated rat thymocytes and compared with the action of urethan and hydroxyurea. HUR suppresses scheduled DNA synthesis, except that of non-stimulated spleen cells in vitro. The inhibition is efficient and rapid and takes place immediately if the drug is administered at the peak of the S-phase. UR inhibits DNA synthesis in vitro only at much higher doses and with different time course. It is effective or slightly effective if it is administered at the peak of the S-phase. A conversion of urethan into HUR the latter depressing DNA synthesis could partly explain the differences observed. No toxicity was found after treatment with drugs at the concentration employed. Finally, the relationships between drug doses and cell responses have been particularly observed in vivo.

The alkylating drug dipin was injected to mice 2 hours before a partial hepatectomy. The duration of DNA synthesis of proliferating hepatocytes was determined by means of cytophotometry of DNA mass in the nuclei, labeled with 3H-thymidine 30 hours after the operation. The DNA mass was doubled simultaneously in diploid and tetraploid nuclei within 18-26 hours. The DNA in labeled nuclei of the control mice was doubled in 8 hours. The kinetics of 3H-thymidine incorporation at different stages of S-period was similar in alkylated and normal cells.

Present investigation documents the influence of 2-mercaptopropionylglycine, a hepatropic detoxicant, on glucose-6-phosphate dehydrogenase, malic enzyme, DNA, and total protein contents in regenerating mouse liver exposed to a chemical carcinogen--DMBA. DMBA, when given alone, decreases the levels of these macromolecular constituents in the regenerating liver. When MPG is given with DMBA, the levels of these, except that of G6P-DH, are maintained at control levels.

Synchronized regenerating rat liver after partial hepatectomy was used to study cell cycle-related DNA base alkylation and liver carcinogenesis. A continuous iv infusion of [14C]N-nitrosodimethylamine (DMN) at a dose of 0.5 mg/kg/hour was given to inbred male Wistar Af/Han rats over a period of 8 hours either during the G1 phase, hydroxyurea-synchronized DNA synthesis, or the G2+M-phase of regenerating liver or to untreated rats (G0-phase liver--carcinogen dose, 1.5 mg/kg/hour). Two hours after the end of the infusion, the amount of 7-methylguanine was highest in the G0-phase liver, with a decrease in the G1 phase, the S-phase, and the G2+M-phase. After continuous DMN exposure, the O6-methylguanine:7-methylguanine ratio was lower in the S-phase and G2+M-phase livers than in the G0-phase and G1-phase livers, indicating an increased O6-methylguanine repair during DNA synthesis and the G2+M-phase. Liver tumors in rats treated by continuous DMN infusion either during the G0 phase or the S-phase developed only after carcinogen exposure during DNA synthesis.

The administration of 5-azacytidine to rats resulted in enhancement of thymidine incorporation into liver DNA. Repeated doses of the drug caused a greater than 10-fold increase of thymidine incorporation. In 5-azacytidine-treated intact rats no changes in the activity of thymidine and thymidylate kinases were observed, whereas a marked depression of thymidine phosphorylase activity occurred. In intact animals the mitotic activity in the drug-treated livers changed slightly; however, 5-azacytidine administration before partial hepatectomy resulted in a dramatic increase of mitotic activity in 24-hr regenerating livers. In this case the observed increase in incorporation of thymidine was paralleled both by increased activity of thymidine and thymidylate kinases and by a decline in thymidine phosphorylase activity. The higher incorporation of radioactivity into liver DNA in intact rats pretreated with the drug can be accounted for, at least partially, by the lower cellular degradation of the injected radioactive thymidine used for labeling. In addition, alterations in the labeling and in the mitotic activity of hepatocytes and nonparenchymal cells caused by 5-azacytidine should be taken into consideration.

Hydrocortisone sodium succinate (65 mg/kg body weight), Zn-ACTH (40 U/kg) and the intraperitoneal injection of Celite (200 mg/kg) decrease the incorporation of 14C-thymidine into the DNA of regenerating rat liver by about 60%. This decrease is not followed by a corresponding inhibition of cell division. The agents applied at the end of G1 phase or in the S phase of the cell cycle probably change thymidine metabolism and the observed decrease of thymidine incorporation does not represent true inhibition of DNA synthesis. The experiment with regenerating liver slices has shown that this disproportion is partly caused by decreased 14C-thymidine transport into the cells.

Dynamics of distribution in subcellular organelles as well as effects on DNA synthesis of heparin, polycanidine (ChAS oligomer of 25 canidine) and their complexes were studied in regenerating rat liver tissue after partial hepatectomy. Polycanidine and heparin penetrated from circulation into hepatocyte cells, nuclei and mitochondria. After consecutive administration of polyelectrolytes polycanidine increased 2-2.5-fold the amount of heparin entering into cells. Polycanidine formed stable complexes with DNP in nuclei. Heparin and its complexes with polycanidine decreased the DNA synthesis within first day. Heparin appears to be responsible for the inhibitory effect, whereas administration of polycanidine only into animals caused a slight increase in 3H-thymidine incorporation into DNA as compared with controls.

Ethanol reversed the ethionine-induced inhibition of 3H-thymidine incoroporation into hepatic DNA in the male and female rat after partial hepatectomy.