Influence of age and gamma irradiation on the proliferative activity in regenerating rat liver.

The effect of aging (2-14 months) and total body irradiation (5.7 Gy of gamma radiation) on liver regeneration was investigated in rats 30 h after partial hepatectomy. Exposure of rats to irradiation 30 min before partial hepatectomy caused latent injury in the remaining liver cells. During the course of liver regeneration this became manifested as a delay in increasing the nucleic acid concentration and content and liver weight and, furthermore, as inhibition of the increase in the mitotic index and cellularity and pronounced increase in the frequency of chromosome aberrations in the postmetaphase. The pattern of age-related changes during liver regeneration was the same as that after irradiation, so that the differences between irradiated and nonirradiated animals became smaller with age.     

The induction of latent liver damage by cyclophosphamide

The effect of the cyclophosphamide on the changes of the mitotic index (MI), metaphase-prophase ratio (M/P) and chromosomal aberrations in postmetaphase in regenerating rat liver was studied. Cyclophosphamide was injected in the single doses of 100 and 200 mg/kg i.p. 2 hours before partial hepatectomy (PHE). The results have shown that cyclophosphamide caused latent liver damage which was manifested after PHE in reduced MI and increased frequency of chromosomal aberrations. The extent of the changes induced by cyclophosphamide corresponds to that due to the effect of 3 or 6 Gy of X-rays, respectively.     

Effects of hepato-protective agents thioctacid and flavobion on histones in intact and regenerating liver of irradiated rats]

The changes in concentration, total content of histones and relative proportion of individual histone fractions in intact and regenerating liver were followed in rats after administration of hepatoprotective agents flavobion and thioctacid and after whole-body gamma irradiation with a dose 5.7 Gy. We found that thioctacid alone caused an increase in histone concentration in intact liver whereas flavobion alone did not produce significant quantitative changes. Irradiation alone decreased markedly the concentration and total content of histones in intact as well as regenerating liver of unprotected rats. Administration of thioctacid or flavobion protected from these quantitative histone changes or alleviated them considerably. In relative proportion of individual histone fractions, the most profound changes were found in H1 histone after flavobion application.     

Transmission of genome damage from irradiated male rats to their progeny

The effect of gamma-radiation (3Gy) on slowly proliferating liver tissue of male rats and their progeny was investigated with respect to induction and duration of latent damage. The irradiation caused latent cytogenetic damage in the liver in irradiated males of the F(0) generation, which manifested itself in different ways during proliferation of hepatocytes induced by partial hepatectomy: a reduced proliferating activity, a higher frequency of chromosomal aberrations and a higher proportion of cells with apoptotic DNA fragments were observed, compared with non-irradiated rats. In the progeny of irradiated males (F(1) and F(2) generation), the latent genome damage manifested itself during regeneration of the liver after partial hepatectomy by similar, but less pronounced changes compared with those seen in irradiated males of the parental generation. This finding gave evidence of the transfer of part of the radiation-induced genome damage from parents to their offspring. Irradiation of F(1) and F(2) progeny of irradiated males (their total radiation load being 3 + 3 and 3 + 0 + 3 Gy, respectively) caused less change as irradiation of progeny of non-irradiated control males (their total radiation load being 0 + 3 and 0 + 0 + 3 Gy, respectively).     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo.

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activity in rat kidney. Administration of diaminopropane 60 min before partial hepatectomy only marginally inhibited ornithine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant. An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed S-adenosyl-L-methionine decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy. Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals. Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life. Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [14C]methionine 4 h after partial hepatectomy or after administration of porcine growth hormone. Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornithine decarboxylase activity and spermidine synthesis.     

Nonlatent and latent hepatic glutathione-insulin transhydrogenase activity during perinatal development and liver regeneration in rats and in rat placenta

We have studied glutathione-insulin transhydrogenase (GIT) activity in differentiating rat liver during parturition and neonatal growth and during compensatory liver growth. Parturition is characterized by a rapid but transient increase in total (i.e., nonlatent plus latent) hepatic GIT activity resulting from changes in the quantity (Vm) of the enzyme while neonatal growth is characterized by an increase in the nonlatent (active) form which persists until just prior to weaning. During liver regeneration following partial hepatectomy, GIT activity/mg protein is lowest after 12 h of regeneration and then progressively increases exceeding the control levels after 72 h of regeneration. Placenta from near-term rats contain a significant concentration of GIT which is immunologically similar to hepatic GIT.     

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.     

Specific inhibition of the synthesis of putrescine and spermidine by 1,3-diaminopropane in rat liver in vivo

Chronic administration of 1,3-diaminopropane, a compound inhibiting mammalian ornithine decarboxylase (EC 4.1.1.17) in vivo, effectively prevented the large increases in the concentration of putrescine that normally occur during rat liver regeneration. Furthermore, repeated injections of diaminopropane depressed by more than 85% ornithine decarboxylase activtivity in rat kidney.Adminsitration of diaminopropane 60 min before partial hepatectomy only marginally inhibited orthine decarboxylase activity at 4 h after the operation. However, when the compound was given at the time of the operation (4 h before death), or any time thereafter, it virtually abolished the enhancement in ornithine decarboxylase activity in regenerating rat liver remnant.An injection of diaminopropane given 30 to 60 min after operation, but not earlier or later, depressed $\text{S-}\text{adenosyl}\text{-}\text{l}\text{-}\text{methionine}$ decarboxylase activity (EC 4.1.1.50) 4 h after partial hepatectomy.Diaminopropane likewise inhibited ornithine decarboxylase activity during later periods of liver regeneration. In contrast to early regeneration, a total inhibition of the enzyme activity was only achieved when the injection was given not earlier than 2 to 3 h before the death of the animals.Diaminopropane also exerted an acute inhibitory effect on adenosylmethionine decarboxylase activity in 28-h regenerating liver whereas it invariably enhanced the activity of tyrosine aminotransferase (EC 2.6.1.5), used as a standard enzyme of short half-life.Treatment of the rats with diaminopropane entirely abolished the stimulation of spermidien synthesis in vivo from [¹⁴C] methionine 4 h after hepatectomy or after administration of porcine growth hormone.Both partial hepatectomy and the treatment with growth hormone produced a clear stimulation of hepatic RNA synthesis, the extent of which was not altered by injections of diaminopropane in doses sufficient to prevent any enhancement of ornitine decarboxylase activity and spemedicine synthesis.     

alpha-Fetoprotein and albumin mRNA levels in liver regeneration and carcinogenesis

Using a titration procedure, we measured the proportion of alpha-fetoprotein (AFP) and albumin mRNA in normal, regenerating, and preneoplastic rat livers. AFP mRNA constitutes approximately 0.006% of the polysomal polyadenylated RNA of normal livers and this proportion increases only slightly before the onset of DNA synthesis in liver regeneration induced by partial hepatectomy or CCl4 injury. In either model of liver regeneration, the proportion of AFP mRNA in polysomal RNA is highest approximately 24 h after the peak of DNA synthesis. The increase in the proportion of AFP mRNA in polysomal RNA is relatively small during liver regeneration (2-4-fold) but is larger (30-50-fold) in preneoplastic livers of rats fed a choline-deficient diet containing 0.1% ethionine. In contrast to those changes in AFP mRNA, albumin mRNA levels remain unchanged during liver regeneration and double in preneoplastic livers. Our results indicate that the concept of "retrodifferentiation" as it applies to liver regeneration and certain types of hepatic neoplasia needs reevaluation.     

Hepatic injury after whole-liver irradiation in the rat

Radiation-induced hepatic injury in rats, which is characterized by marked ascites accompanied by liver necrosis, fibrosis, and vein lesions, is described in this study. These adverse sequelae are produced within 30 days after irradiation if there is surgical removal of two-thirds of the liver immediately after whole-liver irradiation. The LD50/30 day and median survival time after liver irradiation and two-thirds partial hepatectomy is 24 Gy and 17 days, respectively. Death is preceded by reduction in liver function as measured by [131I]-labeled rose bengal clearance. Prior to death, liver sepsis and endotoxemia were detected in most irradiated, partially hepatectomized animals. Pretreatment of the animals with endotoxin and/or antibiotic decontamination of the GI tract, which increase the host resistance to infection and endotoxemia, resulted in increased survival time, but no irradiated, partially hepatectomized animal survived beyond 63 days. The combination of these treatments resulted in additive effects leading to 38% survival at 100 days. These treatments did not, however, prevent the eventual development of radiation-induced liver pathology. This suggests that sepsis and endotoxemia resulting from the bacteria in the intestine are the immediate cause of death after 30-Gy liver irradiation and partial hepatectomy. It is concluded that the hepatectomized rat model is an economical and scientifically manageable experimental system to study a form of radiation hepatitis that occurs in compromised human livers.     

Prolongation of occult damage to the liver after cessation of chronic gamma irradiation

A study was made of the effect of continuous gamma-irradiation on changes in the mitotic index (MI), the ratio of the number of metaphases to that of prophases (M/P), and the frequency of chromosome aberrations at the post-metaphase in the regeneration rat liver. The data obtained indicate that the injury to the intact liver of exposed rats persists throughout the entire period of observation which is displayed by a relative stability of M/P ratio and the frequency of chromosome aberrations. On the other hand, the MI changes indicate the trend toward the repair of the injury.     

Consideration on the mechanism of liver regeneration on experimental results]

Liver has enough functional capacity and regeneration ability. Liver parenchymal cells are usually stable, and it is thought being at stage of Go in cell cycle. But liver cells are easily into progressive stage and the liver recovers its functions and volume after partial hepatectomy or liver injury. Usually the studies of liver regeneration are done by studying control of growth of isolated hepatocytes in primary culture in vitro, and by pathological considerations of experimental injured models of liver in vivo. In this paper, we considered known several hepatotrophic factors including our experimental results, and regeneration mechanisms by noticing appearance of albumin positive hepatocytes in injured Nagase analbuminemic rat (NAR).     

Increased synthesis of low-molecular-weight nuclear ribonucleic acids of rat liver after gamma irradiation and hepatectomy.

Incorporation of [3H]orotic acid into low-molecular-weight nRNA of rat liver, fractionated on polyacrylamide gels, increased 6-12h after partial hepatectomy and 6h after gamma-irridation at 2000 R. The incorporation of orotic acid was particularly increased into the 4.5S, 5S and approx. 10S nRNA fractions. If the irradiation was given after 6h of regeneration and RNA was isolated from the nucleus 12h after hepatectomy then the incorporation of orotic acid into these low-molecular-weight nRNA components was greater than after hepatectomy or irradiation alone.     

Effects of flavobion on nucleic acids in tissues of rats irradiated with gamma rays]

Flavobion /SPOFA/ is a hepatoprotective preparation containing an effective constituent--the flavonoids, namely, silybin, silydianin and silychristin, collectively referred to as silymarin. Silymarin can influence certain metabolic processes including RNA synthesis and stabilize cell membranes. Suspension of this preparation was given p. o. by tube in a dose of 70 mg/kg, one hour before whole-body irradiation with a 5.7 Gy dose of gamma radiation (60Co). Animals were subjected to partial hepatectomy/by 30 min. after irradiation/and examined on hour 30 after operation. The changes in concentration and total content of RNA and DNA in the liver (both intact and regenerating), spleen and bone marrow were followed. We found that Flavobion administration influenced the concentration and total content of RNA and DNA in the spleen and bone marrow. However, in a target organ, liver, changes in nucleic acids were less pronounced.     

CXC Chemokines Function as a Rheostat for Hepatocyte Proliferation and Liver Regeneration

Background

Our previous in vitro studies have demonstrated dose-dependent effects of CXCR2 ligands on hepatocyte cell death and proliferation. In the current study, we sought to determine if CXCR2 ligand concentration is responsible for the divergent effects of these mediators on liver regeneration after ischemia/reperfusion injury and partial hepatectomy.

Methods

Murine models of partial ischemia/reperfusion injury and hepatectomy were used to study the effect of CXCR2 ligands on liver regeneration.

Results

We found that hepatic expression of the CXCR2 ligands, macrophage inflammatory protein-2 (MIP-2) and keratinocyte-derived chemokine (KC), was significantly increased after both I/R injury and partial hepatectomy. However, expression of these ligands after I/R injury was 30-100-fold greater than after hepatectomy. Interestingly, the same pattern of expression was found in ischemic versus non-ischemic liver lobes following I/R injury with expression significantly greater in the ischemic liver lobes. In both systems, lower ligand expression was associated with increased hepatocyte proliferation and liver regeneration in a CXCR2-dependent fashion. To confirm that these effects were related to ligand concentration, we administered exogenous MIP-2 and KC to mice undergoing partial hepatectomy. Mice received a “high” dose that replicated serum levels found after I/R injury and a “low” dose that was similar to that found after hepatectomy. Mice receiving the “high” dose had reduced levels of hepatocyte proliferation and regeneration whereas the “low” dose promoted hepatocyte proliferation and regeneration.

Conclusions

Together, these data demonstrate that concentrations of CXC chemokines regulate the hepatic proliferative response and subsequent liver regeneration.