Effect of the activation of macrophages on the course of regeneration of rat liver following partial hepatectomy

Stimulation of the Kupffer cells with E. coli endotoxin (the purified lipopolysaccharide) or with prodigiosan (a polysaccharide from Serratia marcescens) 24 h before partial hepatectomy (resection of 65-70% of the liver) stimulated and intensified the onset of liver regenerative activity (evaluated from changes in liver DNA synthesis, the H5 labelling index and the mitotic activity of the hepatocytes). Liver DNA synthesis increased together with the dose of endotoxin (i.v., from 25 to 1000 micrograms/kg body weight). If E. coli endotoxin was injected during or 3 h after partial hepatectomy, partial inhibition of liver DNA synthesis was observed. In mice stimulated with zymosan (a polysaccharide isolated from yeast), administered 5 days before performing partial hepatectomy, proliferation of the hepatocytes (evaluated from changes in the 3H labelling index and in the mitotic activity of the hepatocytes) was evaluated. The results confirm that proliferation is correlated to the state of reactivity of the Kupffer cells.     

Different epidermal cell kinetic effects of hydroxyurea when injected at two different times of the day

Circadian rhythms in epidermal basal cell-cycle progression in hairless mouse skin have been repeatedly demonstrated. A dose of 10 mg/animal hydroxyurea (HU), given to inhibit DNA synthesis was injected intraperitoneally to two groups of hairless mice. One group was injected at 10.00 hours MET, when the cell-cycle progression and cell division rate are relatively high, and another group was injected at 20.00 hours, when the same variables are at minimum values. Various cell kinetic methods--[3H]TdR autoradiography, DNA flow cytometry and the stathmokinetic method (Colcemid)--were used to study HU-induced alterations in cell kinetics. Hydroxyurea (HU) immediately reduced the labelling index (LI) to less than 10% of controls when injected at both times of the day, and higher then normal values were observed 8 hr later. A subsequent decrease towards normal values was steeper in the 20.00 hours injected group. The proportion of cells with S-phase DNA content was transiently reduced in both series, but the reduction was less pronounced and control values were reached earlier in the series injected at 10.00 hours. The observed alterations in LI and fraction of cells in S phase were followed by comparable alterations in the fraction of cells in G2 and in the mitotic rate. Hence the changes in G2 and mitotic rate are easily explained as consequences of the previous perturbations in the S phase. The time-dependent differences in the cell kinetic perturbations caused by HU in the S phase may be explained by a circadian-phase-dependent action of HU on the influx and efflux of cells to and from the S phase, respectively. At 10.00 hours the efflux of cells from S is most heavily inhibited; at 20.00 hours the influx is predominantly blocked. Hence, when physiological flux is high HU mainly blocks the efflux from S, but when flux normally is low, HU mainly blocks the entrance to S. Within 20 hours after the HU injection, the cell kinetic variables had approached the unperturbed circadian pattern.     

Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication

Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.     

Effect of α-difluoromethylornithine on polyamine and DNA synthesis in regenerating rat liver: Reversal of inhibition of DNA synthesis by putrescine

The possibility that α-difluoromethylornithine, a specific, irreversible inhibitor of ornithine decarboxylase could be used to prevent the rise in hepatic putrescine and spermidine content following partial hepatectomy was tested. Administration of α-difluoromethylornithine at a dose of 400 mg/kg every 4 h reduced hepatic putrescine to <2 nmol/g, but had only a small effect on the rise in spermidine seen at 28 h after partial hepatectomy. Such treatment also reduced the rise in DNA synthesis produced by partial hepatectomy by up to 70%. The inhibitory effect towards DNA synthesis could be reversed by administration of putrescine which increased the hepatic putrescine content to about 30–40% of that in the regenerating control livers. These results suggest that accumulation of putrescine rather than spermidine is needed for DNA synthesis after partial hepatectomy. They also suggest that part, but not all of the rise in putrescine normally seen in the liver after partial hepatectomy is needed for the enhanced DNA synthesis associated with liver regeneration. Experiments with lower doses of α-difluoromethylornithine showed that a substantial part of the rise in hepatic ornithine decarboxylase activity could be abolished without affecting either the rise in spermidine content or the increase in DNA synthesis after partial hepatectomy.     

Different Epidermal Cell Kinetic Effects of Hydroxyurea When Injected At Two Different Times of the Day

Circadian rhythms in epidermal basal cell-cycle progression in hairless mouse skin have been repeatedly demonstrated. A dose of 10 mg/animal hydroxyurea (HU), given to inhibit DNA synthesis was injected intraperitoneally to two groups of hairless mice. One group was injected at 10.00 hours MET, when the cell-cycle progression and cell division rate are relatively high, and another group was injected at 20.00 hours, when the same variables are at minimum values. Various cell kinetic methods—[³H]TdR autoradiography, DNA flow cytometry and the stathmokinetic method (Colcemid)—were used to study HU-induced alterations in cell kinetics. Hydroxyurea (HU) immediately reduced the labelling index (LI) to less than 10% of controls when injected at both times of the day, and higher then normal values were observed 8 hr later. A subsequent decrease towards normal values was steeper in the 20.00 hours injected group. the proportion of cells with S-phase DNA content was transiently reduced in both series, but the reduction was less pronounced and control values were reached earlier in the series injected at 10.00 hours. the observed alterations in LI and fraction of cells in S phase were followed by comparable alterations in the fraction of cells in G₂ and in the mitotic rate. Hence the changes in G₂ and mitotic rate are easily explained as consequences of the previous perturbations in the S phase. The time-dependent differences in the cell kinetic perturbations caused by HU in the S phase may be explained by a circadian-phase-dependent action of HU on the influx and efflux of cells to and from the S phase, respectively. At 10.00 hours the efflux of cells from S is most heavily inhibited; at 20.00 hours the influx is predominantly blocked. Hence, when physiological flux is high HU mainly blocks the efflux from S, but when flux normally is low, HU mainly blocks the entrance to S. Within 20 hours after the HU injection, the cell kinetic variables had approached the unperturbed circadian pattern.     

Effect of the parenteral administration of energy substrates in different postoperation phases on the initiation and development of liver regeneration in rats subjected to partial hepatectomy

DNA specific activity in the liver, the total DNA content of the liver and the mitotic index of the hepatocytes were studied after the infusion of glucose or lipid emulsions in female laboratory rats with a mean pre-operation weight of 250 +/- 30 g after partial (65-70%) hepatectomy (PH). The infusions were administered in the early prereplication phase (the 1st to 6th hour after the operation), in the late prereplication phase (the 7th to 12th hour after the operation), or continuously from the 1st to the 12th, or the 1st to the 24th, hour after partial hepatectomy. The effect of these parenterally administered energy substrates on the initiation of liver regeneration was evaluated 18 and 24 hours after partial hepatectomy. The results indicate that the infusion of glucose, in any interval after the operation, inhibited the initial phases of liver DNA synthesis (18 h after PH), but not its further development (24 h after PH). Neither the mitotic index of the hepatocytes, nor the total DNA content of the liver differed from the control groups in the case of rats given a glucose infusion. In the experimental groups given lipid emulsions, inhibition of liver DNA synthesis was recorded 18 h after PH only when the infusions were given from the 1st to the 12th or the 1st to the 18th hour after PH. The total DNA content of the liver 18 h after PH was raised in all the experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA synthesis,mitotic index,drug-metabolising systems and cytogenetic analysis in regenerating rat liver: Comparison with bone marrow test after ‘in vivo’ treatment with cyclophosphamide

Rat-liver cells can be used to reveal “in vivo” clastogenic activity of indirect mutagens, provided that they are stimulated to divide by partial hepatectomy. In order to characterize the rat-liver metabolic capacity in such experimental conditions, several biochemical parameters were measured during the first 54–66 h of liver regeneration in Sprague-Dawley male rats, subjected to a partial hepatectomy. The levels of cytochrome P-450, the activities of styrene monooxygenase, epoxide hydrolase and glutathione-S-epoxide transferase were chosen as markers.All the enzymatic activities and the level of cytochrome P-450 decreased during the first 12 h after the hepatectomy to about 50% of the activities of the sham-operated rats considered as controls. Subsequent recovery of the metabolic capacity was not observed.DNA synthesis and the mitotic index were measured to find the most suitable time for metaphase analysis. DNA synthesis and the number of metaphases were maximal at, respectively, 22–25 and 28–31 h after partial removal of the liver.The sensitivity to clastogenic damage induced by “in vivo” treatment with cyclophosphamide (CPA) was assayed in regenerating liver cells by chromosome-aberration analysis. Different doses, ranging from 5 to 30 mg/kg b.w., were given i.p. to the rats 17 h before or 7 h after partial hepatectomy. Liver cells were collected 31 h after surgery. Clastogenic damage was greater when the drug was administered to the animals after the hepatectomy (24 h of exposure) than before (48 h of exposure).The sensitivity to CPA-induced damage was compared with a bone marrow cell test carried out on non-hepatectomized rats treated in the same way. The results indicated that in these conditions regenerating liver cells are more sensitive than bone marrow cells to the induction of chromosome aberrations by CPA.     

DNA synthesis, mitotic index, drug-metabolising systems and cytogenetic analysis in regenerating rat liver. Comparison with bone marrow test after 'in vivo' treatment with cyclophosphamide

Rat-liver cells can be used to reveal "in vivo" clastogenic activity of indirect mutagens, provided that they are stimulated to divide by partial hepatectomy. In order to characterize the rat-liver metabolic capacity in such experimental conditions, several biochemical parameters were measured during the first 54-66 h of liver regeneration in Sprague-Dawley male rats, subjected to a partial hepatectomy. The levels of cytochrome P-450, the activities of styrene monooxygenase, epoxide hydrolase and glutathione-S-epoxide transferase were chosen as markers. All the enzymatic activities and the level of cytochrome P-450 decreased during the first 12 h after the hepatectomy to about 50% of the activities of the sham-operated rats considered as controls. Subsequent recovery of the metabolic capacity was not observed. DNA synthesis and the mitotic index were measured to find the most suitable time for metaphase analysis. DNA synthesis and the number of metaphases were maximal at, respectively, 22-25 and 28-31 h after partial removal of the liver. The sensitivity to clastogenic damage induced by "in vivo" treatment with cyclophosphamide (CPA) was assayed in regenerating liver cells by chromosome-aberration analysis. Different doses, ranging from 5 to 30 mg/kg b.w., were given i.p. to the rats 17 h before or 7 h after partial hepatectomy. Liver cells were collected 31 h after surgery. Clastogenic damage was greater when the drug was administered to the animals after the hepatectomy (24 h of exposure) than before (48 h of exposure). The sensitivity to CPA-induced damage was compared with a bone marrow cell test carried out on non-hepatectomized rats treated in the same way. The results indicated that in these conditions regenerating liver cells are more sensitive than bone marrow cells to the induction of chromosome aberrations by CPA.     

Effects of partial hepatectomy on the growth characteristics and hypoxic fractions of xenografted DLD-2 human colon cancers.

A recent publication by Leith et al. (Cancer Res. 51, 4111-4113, 1991) showed that administration of epidermal growth factor (EGF) (0.25 mg/kg; q.i.d. x 7) to mice bearing xenografted A431 human epidermoid cancers produced increased tumor growth and a reduction of the percentage of hypoxic cells within neoplasms. In contrast, sialoadenectomy, which removes the majority of circulating EGF in the mouse, resulted in slower tumor growth and increased hypoxic percentages. To investigate such homeostatic mechanisms further, we investigated the growth characteristics and hypoxic fractions of xenografted DLD-2 human colon tumors when tumor-bearing mice received partial hepatectomy (about 65%), unilateral nephrectomy, or nonspecific surgical trauma (laparotomy). Surgical procedures were performed when tumor volumes were about 375 mm3, and hypoxic percentages within tumors were measured by clonogenic excision assay procedures 72 h later. We found that partial hepatectomy increased the growth rates of the transplanted DLD-2 cancers by about a factor of 2.4. This increased growth rate was accompanied by an increase in the mitotic index from about 1.5 to 3.0%. Also, hypoxic fractions were decreased from 0.561 in control tumors to 0.235 in tumors from mice receiving partial hepatectomies. Unilateral nephrectomy and nonspecific surgery manipulations did not alter tumor growth rates, mitotic indices, or hypoxic percentages. These results indicate that partial hepatectomy produces significant alterations in tumor physiology. Results are consistent with a growth factor-mediated mechanism.     

Early nuclear cytochemical changes in regenerating mammalian liver

Various cytochemical parameters were studied cytophotometrically in parenchymal cell nuclei isolated from rat liver at different times following partial hepatectomy. The study was confined to the early period following operation, before DNA synthesis and before mitotic activity ensued. Binding of acridine orange was found to increase approx. 70% over normal controls at 3 h post-hepatectomy followed by a sharp decrease to 60% below controls by 12 h and a return to near control levels at 24 h. These changes were found in both diploid and tetraploid cell nuclei. The initial two-fold difference in AO binding observed between diploid and tetraploid nuclei in normal liver persisted at 3 h but decreased markedly from 6 to 24 h after operation. Chromatin thermal stability, determined by acridine orange microfluorimetry, showed a rapid decrease at 3 h in both diploid and tetraploid cell nuclei followed by an increase above control levels at 6 h which persisted up to 24 h after partial hepatectomy. Parallel measurements of Feulgen dye binding showed no change in the relative DNA content of diploid and tetraploid cells throughout the experimental period. Ratios of specific picric acid and alkaline bromphenol blue dye binding by histone arginine and lysine side chains were found to rise significantly over normal controls at 3 h post-hepatectomy and return to normal levels by 6 h.     

DNA synthesis and mitotic activity in cells of regenerating rat liver following x-irradiation in stages GO and Gl]

Effect of X-rays on DNA synthesis and mitotic activity of regenerating liver cells has been studied. The irradiation was performed at a dose of 630 rad before hepatectomy and 2.5 and 6 hours after the stimulation of liver. With the stimulated liver being irradiated, the number of cells synthetizing DNA and entering into mitosis was seen reduced almost twice, whereas DNA synthesis and entering into mitosis were delayed, resp., by 4 and 6 hours. Irradiation of liver before the stimulation brings about a delay in DNA synthesis and in start of mitosis by 2 and 4 hours, resp., without reducing the numbers of cells capable to synthesize DNA and to enter mitosis.     

Topographic distribution of dividing hepatocytes in the regenerating liver lobule in the period of maximum mitotic activity

Topographic distribution of dividing hepatocytes was studied in the liver lobule after hepatectomy performed at 10-11.30 a. m. The studies were conducted 20-32 h after surgery during the first increase in mitotic activity. It has been established that dividing hepatocytes appear simultaneously in all lobule areas, i. e. that all the hepatocytes are capable of responding to regenerating stimuli. However, further behaviour of dividing hepatocytes becomes different. It is concluded that hepatocytes from outer and intermediate lobule areas possess a more pronounced proliferative potential during peak mitotic activity after partial hepatectomy.     

Biliverdin initiates the liver regeneration in the rat--a hypothesis

Biliverdin has been observed to occur in the blood plasma of a 90% hepatectomized rat. It is also shown that the bile pigment induces a rise in mitotic index in the hepatic parenchymal cells of an intact rat at about 30 h and an elevated rate of hepatic DNA synthesis at 26 h after a single intraperitoneal injection. Hemoglobin, bilirubin, hemin, and protoporphyrin exhibited some of the inducing activity at lower degrees. It is hypothesized that biliverdin initiates the liver-cell-multiplication.     

大鼠反复肝切除后再生肝组织的实验形态学研究

９０只雄性ＳＤ大鼠随机分为三组,每组各３０只,以第一次切肝量５０％（Ａ组）,７０％（Ｂ组）,８０％（Ｃ组）为分组标准,均作反复四次肝切除。动态观察其再生肝组织的酶组织化学,组织学及超微结构变化。结果显示：Ａ、Ｂ组三次术后累计存活率（８５％）明显高于Ｃ组（５７％）,Ｐ＜０．０１。Ａ、Ｂ、Ｃ各组三次术后累计切肝率各为１１４．２８％,１２０．７２％,１２５．８１％。Ａ、Ｂ组ＳＤＨ、ＡＴＰａｓｅ、ＬＤＨ活性改变相近,优于Ｃ组。各组ＣＨＥ活性均处于低水平。ＡＣＰ活性随切量,切次增加而增强。肝再生的方式既有小叶数量增多,面积增大,又有细胞肥大,细胞数量增加。电镜显示了增生活跃的肝细胞的核发裂像。所示线粒体结构受损与ＳＤＨ活性下降相对应。研究证明;大鼠反复肝切除是可行的,第一次切肝量,切次多少是影响大鼠耐受反复肝切除的重要因素。     

Effect of the parenteral administration of amino acid solutions in different phases after partial hepatectomy on the initiation and development of liver regeneration in rats

Amino acid solutions enriched with branched-chain amino acids or pure branched-chain amino acid solutions were administered parenterally to female laboratory rats (pre-operative weight 230 +/- 30 g) which had been subjected to 65-70% partial hepatectomy (PH), and specific liver DNA activity, the hepatocyte mitotic index and other indicators of the initiation of liver regeneration were studied. Both solutions were infused in an hourly dose of 3.3 ml/kg body weight, during the following postoperative intervals: 1-6, 7-12, 1-12, 1-18 and 1-24 hours. The control rats continued to be fed on the standard laboratory diet after the operation. The results show that the infusion of an amino acid solution enriched with branched-chain amino acids had an inhibitory effect on the onset of DNA synthesis in the liver 18 hours after partial hepatectomy whatever the administration interval. The situation in the case of pure branched-chain amino acid solutions was the same. Twenty-four hours after PH, neither type of solution, irrespective of the infusion interval, was followed by an increase in DNA synthesis compared with the controls fed on the standard laboratory diet. Neither the hepatocyte mitotic index, nor the total liver DNA concentration, showed any changes indicative of stimulation of the initiation of liver regeneration. An infusion stress effect, evaluated from the decrease in the weight of the thymus, was found chiefly in the case of infusions lasting 12 h or longer.     

Nicotinamide nucleotide synthesis in regenerating rat liver

1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors.     

Relation between Water Stress and DNA Synthesis during Germination of Zea mays L.

Mitotic index, percent nuclei in DNA synthesis and the relativeDNA content per nucleus were determined from cells of the Zeamays radicle at various times after the beginning of germination.Nuclear DNA synthesis was initiated after 45 h and mitosis wasfirst observed after 74 h from sowing. Most of the dormant nucleiwere in the pre-synthetic or G₁ phase of the mitotic cycle.By 72 h most cells were in S and 77 h after the beginning ofgermination, the cells of the primary root were observed inall phases of the mitotic cycle. Dehydration of karyopses after45–74 h of imbibition progressively reduced the percentof germination to zero upon dehydration and subsequent replantingdemonstrating that drought sensitivity was related to the onsetof nuclear DNA synthesis and genome duplication.     

Poly(ADP-ribose) polymerase activity in regenerating liver of hypothyroid rats

The role of PARP, a nuclear enzyme involved in DNA synthesis, repair and cell transformation, was studies during liver regeneration in hypothyroid animals. Hypothyroidism was induced by in vivo administration of propylthiouracil. In regenerating euthyroid animals PARP activity is stimulated showing an early and significant increase at 1.5 h with a maximum at 6 h after partial hepatectomy. Such an increase returns to control values within 18 h preceding the onset of DNA synthesis. A markedly different behavior, with respect to euthyroids, has been evidenced in hypothyroid rats. At first, liver PARP level was about 2-fold higher in non regenerating hypothyroid rats with respect to control euthyroids. During regeneration, PTU-treated animals show a net decrease in PARP activity, with a minimum at 6-9 h after partial hepatectomy. The activity returns to control levels within 24 days. The minimum in PARP activity anticipates, also in this case, the onset of DNA synthesis, which exhibits a maximum at 15-18 h. During liver regeneration PARP activity shows modifications related to the beginning of de novo DNA synthesis. Furthermore, these variations in turn undergo the effects of hypothyroidism.     

Mitotic activity and its 24-hour rhythm in the ovarian follicles during the estrous cycle of a wild rat, Bandicota bengalensis

Mitotic activity in ovarian follicles was studied in relation to the size of the follicles during a 24-hour period (10.00, 16.00, 22.00 and 04.00 h) throughout the estrous cycle of the wild bandicoot rat (Bandicota bengalensis) to ascertain the cell proliferation rate and its 24-hour rhythm in the follicular tissue. In the bandicoot ovary, mitotic activity in the granulosa and thecal cells was highest in the follicles ranging from 201 to 400 micron in diameter. During the estrous cycle, mitotic activity of the granulosa cells was highest at estrus in follicles less than 601 micron, and at diestrus in follicles greater than 600 micron; while the mitotic trough was at proestrus in all the follicles. Thecal mitosis was significantly lower than mitosis of the granulosa cells. In most of the follicles, mitotic activity in the thecal cells was highest at diestrus and lowest at metestrus. In both the granulosa and thecal cells, the timing of mitotic peaks and troughs varied according to the size of the follicles and the stages of the estrous cycle. In the granulosa cells mitotic peaks were maximal in the daytime (10.00 h, 16.00 h) and in some cases at night (04.00 h); and mitotic troughs were primarily during the night (22.00 h, 04.00 h) and in some cases in the day (10.00 h). In the thecal cells, however, mitotic activity in most of the follicles was distinctly higher in the daytime (16.00 h) than at night (22.00 h, i.e., evening). Thus, a prominent 24-hour mitotic rhythm was noticed in the ovarian follicles of the bandicoot rat.