A conjugate of doxorubicin with lactosaminated albumin enhances the drug concentrations in all the forms of rat hepatocellular carcinomas independently of their differentiation grade.

BACKGROUND/AIMS: Doxorubicin (DOXO) was coupled to lactosaminated human serum albumin (L-HSA) in order to enhance the drug concentration in the well differentiated hepatocellular carcinomas (HCCs), which can accumulate L-HSA through the asialoglycoprotein receptor. In the present experiments we compared the DOXO concentrations produced by this conjugate (L-HSA-DOXO) and by the uncoupled drug in the well, moderately, and poorly differentiated rat HCCs. METHODS: The same dose (1 microg/g) of free or L-HSA coupled-DOXO was injected in rats with HCCs induced by diethylnitrosamine. At different times, the animals were killed and the neoplastic nodules of liver were isolated. Their differentiation grade was determined histologically and their DOXO content was measured. RESULTS: Unexpectedly, we found that also in the poorly differentiated forms of HCCs, which display no or only a poor capacity of accumulating L-HSA, the conjugate raised DOXO levels that were approximately twofold higher than those produced by the free drug. CONCLUSIONS: The conjugate L-HSA-DOXO could improve the potential of DOXO in the treatment of all HCCs, including the poorly differentiated tumors that are the common forms in the advanced disease for which an effective chemotherapy is particularly needed.     

Enhanced uptake of lactosaminated human albumin by rat hepatocarcinomas: implications for an improved chemotherapy of primary liver tumors.

BACKGROUND/AIMS: The hepatocyte receptor for asialoglycoproteins (ASGP-R) internalizes macromolecules exposing galactosyl residues (MEGRs) which can be used as liver-addressed drug carriers. This receptor was also found on the cells of the large majority of well differentiated hepatocarcinomas (HCCs). The aim of the present experiments was to ascertain whether ASGP-R of HCCs is functionally active and these tumors can internalize higher quantities of MEGRs than extra-hepatic tissues. METHODS: We injected radioactive lactosaminated human albumin (L-HSA) in rats with HCCs produced by nitroso-diethylamine and measured the radioactivity of tumors, surrounding liver, heart, intestine and kidney. L-HSA is a MEGR successfully used in humans as a hepatotropic drug carrier. RESULTS: The levels of radioactivity of HCCs were two to three times lower than those of surrounding liver, but several times higher than those of extra-hepatic tissues. L-HSA accumulation in the tumors mainly occurred via the ASGP-R, as indicated by the 20 times lower penetration of non-lactosaminated HSA. L-HSA uptake by the well-differentiated tumors were four times higher compared with that by the poorly differentiated forms. CONCLUSIONS: The present results suggest that in the chemotherapy of HCCs expressing the ASGP-R the extra-hepatic toxicity of anticancer agents can be reduced by conjugation to L-HSA.     

Inhibitors of hepatic DNA synthesis in fulminant hepatic failure

In certain etiological groups of patients with fulminant hepatic failure, poor survival may be due to lack of liver regeneration.In vitro experiments have shown that fulminant hepatic failure serum is cytotoxic to rabbit hepatocytes and inhibits DNA synthesis on short-term incubation with isolated regenerating rat hepatocytes. When fulminant hepatic failure serum is injected into partially hepatectomized rats at the time of maximal DNA synthesis, [³H]thymidine incorporation into hepatic DNA is reduced significantly. The effect is greater with sera obtained from patients with fulminant hepatic failure due to non-A, non-B hepatitis or an adverse drug reaction and is associated with a<10,000-dalton fraction. No stimulation of DNA synthesis is observed with injection of the >10,000-dalton serum fraction into normal rats. In preliminary experiments, no increase in epidermal growth factor production has been found in liver failure. Overall, the substances present in fulminant hepatic failure serum appear to be inhibitory rather than stimulatory for liver cell regeneration.Presented at the Proceedings of the International Meeting on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989.     

Floxuridine coupling with lactosaminated human albumin to increase drug efficacy on liver micrometastases

BACKGROUND: Conjugates of nucleoside analogues with galactosyl terminating peptides selectively enter hepatocytes through the asialoglycoprotein receptor. After intracellular release from the carrier, the drugs partly exit from hepatic cells into hepatic blood. AIMS: To establish whether administration of a conjugate of floxuridine with lactosaminated human albumin selectively enhances drug concentrations in hepatic blood. Floxuridine is a fluoropyrimidine active on human colorectal cancer, a tumour which metastasises first to the liver. METHODS: In rats injected with free or conjugated floxuridine, plasma levels of the drug were determined in hepatic veins and in inferior vena cava, in order to measure drug concentrations in hepatic blood and in the systemic circulation, respectively. RESULTS: Ratios between floxuridine levels in hepatic veins and those in systemic circulation were found to be seven times higher in rats injected with the conjugate (p=0.000). CONCLUSIONS: The present results suggest that coupling to lactosaminated albumin might improve the effect of floxuridine in adjuvant chemotherapy of colorectal cancer by exposing the cells of liver micrometastases (nourished by hepatic sinusoids) to enhanced drug concentrations.     

Stimulation of DNA synthesis in hepatocytes by Kupffer cells after partial hepatectomy

The role of Kupffer cells during reparative regeneration of rat liver was investigated with an in vitro experimental model. Conditioned media from primary cultures of Kupffer cells isolated from intact and regenerating liver were added to primary cultures of hepatocytes, and [3H]thymidine incorporation into DNA was studied. Kupffer cell-conditioned media from intact liver and regenerating remnant liver significantly stimulated DNA synthesis in hepatocytes as compared with control media (p less than 0.05). Moreover, the stimulating activity of Kupffer cells prepared from regenerating liver at 6 and 12 hr after partial hepatectomy was significantly higher than that of Kupffer cells from untreated rats (p less than 0.05). The activity was found in serum-free conditioned media. This stimulating activity exponentially increased as the increase of the number of the cultured cells, indicating that the stimulating activity was released directly by cultured Kupffer cells. These results suggest that Kupffer cells stimulate DNA synthesis in hepatocytes by producing and releasing certain factor(s) at an early stage of liver regeneration after partial hepatectomy.     

Liver Regeneration in Donors Evaluated by Tc-99m-GSA Scintigraphy after Living Donor Liver Transplantation

The impact of hepatic steatosis on regeneration of the remnant liver after living donor liver transplantation is unclear. We evaluated the impact of steatosis on regeneration and function of the remnant liver by using technetium-99m-diethylenetriaminepentaacetic acid-galactosyl human serum albumin scintigraphy. Twelve living donors were classified into groups with or without mild hepatic steatosis according to the liver-to-spleen attenuation ratio on computed tomography: 6 donors had a ratio ≥ 1.20 (control group) and 6 donors had a ratio < 1.20 (fatty liver group). Scintigraphy was performed to determine the hepatic uptake ratio of the tracer (corrected for disappearance from the blood) and the maximum removal rate of the tracer by hepatocytes as parameters of the hepatic functional reserve. The fatty liver group had a significantly lower corrected hepatic uptake ratio and removal rate compared with the control group at 6 and 12 months after partial hepatectomy. These parameters were decreased at 1 month after surgery in both groups. However, both parameters returned more rapidly to prehepatectomy levels in the control group. The regenerated liver volume estimated by scintigraphy did not differ significantly between the two groups at any time. Liver scintigraphy may be useful for evaluating the regeneration of functioning hepatocytes. Because donors with mild hepatic steatosis showed impaired liver regeneration at 1 year after partial hepatectomy, management of these donors requires more care.     

Involvement of retinoic acid-induced transglutaminase activity in zonal differences of hepatocyte proliferation after partial hepatectomy

BACKGROUND: The authors have recently demonstrated that there is inverse correlation between transglutaminase (TGase) activity and DNA synthesis in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) at 1 day after partial hepatectomy in rats. In order to ensure the involvement of TGase in the differential growth capacities between periportal and perivenous regions of regenerating liver, the aim of this study was to investigate the effect of retinoic acid, an inducer of TGase expression, on zonal differences of hepatocyte proliferation between PPH and PVH isolated from regenerating rat liver. METHODS: Regenerating liver was prepared by 70% partial hepatectomy. PPH and PVH subpopulations were isolated by the digitonin/collagenase perfusion technique. Cell cycle was evaluated for incorporation of BrdU into hepatocytes and detected by flow cytometric analysis. TGase activity was determined by incorporation of 14C-putrescine into dimethylcasein. RESULTS: When retinoic acid was injected immediately after partial hepatectomy, TGase activity greatly increased in both PPH and PVH at 1 day after partial hepatectomy, and activity was higher in PPH than in PVH. DNA synthesis in both subpopulations did not increase 1 day after partial hepatectomy, with peaks of DNA synthesis shifting to 2 days, and synthesis was higher in PVH than in PPH. CONCLUSION: These results suggest that TGase might be involved in differential growth capacities between periportal and perivenous regions of regenerating rat liver after partial hepatectomy.     

Detection of proliferating hepatocytes in patients with acute hepatic failure by mitotic figures and a monoclonal antibody against DNA polymerase alpha

ABSTRACT— Information on the ultrastructure and phenotypes of proliferative hepatocytes is scarce, so we set out to detect proliferating hepatocytes immunohistochemically by use of a monoclonal antibody against DNA polymerase alpha (DNA-PA). The findings from this method were compared with conventional features, such as mitotic figures, and hepatic regeneration after injury was considered in the light of these findings. The subjects of the basic study were 23 patients with acute hepatic failure. There were 6.8 ± 5.5 (mean ± SD) mitotic hepatocytes per 1000 hepatocytic nuclei, and 209 ± 158 hepatocytes stained for DNA-PA per 1000 hepatocytic nuclei. By light and electron microscopy (n=4), hepatocytes stained for DNA-PA showed various morphological features, including development of organelles, but some resembled hepatocytes in mitosis. Accordingly, this histochemical method may be useful in studies of hepatic regeneration. In acute confluent necrosis, when hepatocytic proliferation is urgently needed for survival, small hepatocytes next to necrotic areas (probably immature cells, to judge from the development of their organelles) were predominant in hepatic regeneration. These findings suggest that hepatocytes in different stages of development can easily enter the mitotic cell cycle repeatedly when rapid regeneration is needed.     

Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation.

BACKGROUND: Expression of neurotrophins (NTs) and their receptors is increased during hepatic regeneration, but their role is not well understood. METHODS: NTs and their receptors were investigated by RT-PCR and immunostaining in regenerating livers after two-thirds hepatectomy (PH) and in hepatocytes and hepatic stellate cells (HSCs) isolated from regenerating livers in mice. Induction of apoptosis after treatment with NGF and the expression of alpha-smooth muscle actin (SMA), interleukin 6 (IL-6) and hepatocyte growth factor (HGF) were also investigated in regenerating HSCs. RESULTS: Nerve growth factor (NGF) and p75 NT receptor (p75NTR) mRNA were elevated after PH, while other NTs and NT receptors showed no remarkable change. NGF was detected in regenerating hepatocytes, but not in normal hepatocytes. Regenerating HSCs expressed increased p75NTR and SMA in vivo and showed an activated phenotype and the high expression of HGF and IL-6 in vitro. Enhanced cell death was seen in HSCs, both from normal and regenerating liver, after treatment with NGF. CONCLUSIONS: Although activated HSCs may produce the factors that regulate liver regeneration, the de novo NGF production by regenerating hepatocytes may induce the death of activated HSCs via p75NTR, leading to termination of hepatic regeneration.     

The extent of synchronous initiation and termination of DNA synthesis in regenerating mouse liver is dependent on connexin32 expressing gap junctions

BACKGROUND/AIMS: It has previously been shown in rat liver that the gap junctional proteins connexin32 and connexin26 are downregulated when murine hepatocytes are in the S-phase of the cell cycle. Therefore, it has been hypothesized that loss of functional gap junctions could affect proliferation of hepatocytes. This study aimed to check this hypothesis. METHODS: We searched for differences in liver regeneration after two-thirds partial hepatectomy between connexin32-deficient and wild-type mice. RESULTS: The ratio of liver to body weight in regenerating liver was not affected by loss of the connexin32 gene. The peak of DNA synthesis occurred at the same time, i.e. 36 to 96 h after partial hepatectomy, in connexin32-deficient and wild-type liver. During this time, however, only about half as many nuclei of hepatocytes in connexin32-deficient liver incorporated bromodeoxyuridine, compared to wild-type liver. Furthermore, 1-2 weeks after full recovery of liver mass, we detected a higher level of bromodeoxyuridine incorporation into hepatocytes of connexin32-deficient than in wild-type liver. CONCLUSIONS: Loss of connexin32 protein and/or diminished expression of connexin26 did not promote G0/1-S transition of hepatocytes in two-thirds hepatectomized mouse livers. Instead, the extent of synchronous initiation and termination of DNA synthesis in regenerating liver was altered in connexin32-deficient mice.     

Mild hypothermia does not affect liver regeneration after partial hepatectomy in mice

Background: The use of mild hypothermia has been suggested to be therapeutically useful in treating acute liver failure. It is not known if hypothermia influences liver regeneration. Aim: To assess the effect of hypothermia on liver regeneration in mice. Methods: After partial (70%) hepatectomy (PHx), C57BL6/J mice were randomly assigned to either a hypothermic group or a normothermic group. Controlled mild hypothermia was maintained for up to 3 h after surgery. In addition, assessment of liver mass restitution was examined by studying the induction of key cell cycle proteins (cyclin A, D1 and E) and hepatocyte proliferation [assessment of proliferating cell nuclear antigen (PCNA) protein expression] by Western blotting and DNA synthesis by measuring 5‐bromo‐2‐deoxyuridine (BrdU) incorporation by immunohistochemical techniques 45 h after PHx. Results: Partial hepatectomy induced a vigorous proliferative response in the remnant livers of both groups of mice (normothermic and hypothermic groups), as evidenced by the induction of key cyclins, PCNA and incorporation of BrdU after PHx. The liver/body weight ratio and both cyclin and PCNA protein expression as well as BrdU incorporation did not differ between the regenerating livers of hypothermic and normothermic groups. Conclusion: Mild hypothermia does not influence liver regeneration in mice.     

Effect of platelet-activating factor (PAF) receptor antagonist (BN52021) on acetaminophen-induced acute liver injury and regeneration in rats.

BACKGROUND: Platelet-activating factor (PAF) is an endogenous lipid mediator that plays a key role in catalyzing various pro-inflammatory processes associated with acute liver injury. In the present study, the possible influence of PAF-R antagonist (BN52021) on the protection of liver injury after 4-hydroxyacetanilide, N-acetyl-p-aminophenol, paracetamol (APAP) intoxication was investigated. METHODS: Thereby, one group of rats was treated with a toxic dose of APAP (3.5 g/kg body weight (b.w.). The animals were killed at 56, 66, 72, 84 and 96 h after treatment. RESULTS: APAP was found to cause an acute hepatic injury, evident by alterations of biochemical (serum enzymes: aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase) and liver histopathological (degree of necrosis and apoptosis) indices, which was followed by liver regeneration, evident by three independent indices ([3H] thymidine incorporation into hepatic DNA, liver thymidine kinase activity and hepatocyte mitotic index). The protective effects of BN52021 were qualified during post-treatment time by: (1) significant reduction of hepatic injury as showed by all biochemical and histological parameters, (2) high decrease of regenerating activity showed by three regenerative markers and (3) remarkable increase of PAF-acetylhydrolase (PAF-AH) activity. CONCLUSION: These results suggest that PAF may play an important role in APAP-induced liver injury and regeneration, and PAF-R antagonist (BN52021) attenuates liver damage.     

Research review: DNA polymerases as molecular markers of the regenerating capacity of hepatocytes

Hepatocyte regeneration has been widely investigated, with the mitotic index and the incorporation of [³H]thymidine being used as regeneration markers. We focused on the induction of DNA replication enzymes, particularly DNA polymerases (pol) α, δ, and ε. Using rat models, we have shown that the activity of pol α in crude liver extract well represents the regenerating capacity of hepatocytes. Using pol α as an indicator, we analyzed liver regeneration in rat models under various conditions: obstructive jaundice, external or internal biliary drainage, and the obstruction of portal vein branches. It has been revealed that the ligation of the common bile duct alone induces a certain amount of hepatocyte proliferation. It was striking that external biliary drainage suppressed regeneration capacity in cholestatic rat liver after partial hepatectomy. The strong regeneration in nonligated lobes induced by portal branch ligation was similar to the liver regeneration seen after partial hepatectomy with respect to the induction of DNA polymerases. Taken together, the aspects of DNA replication, particularly the induction of DNA polymerases, may contribute to shedding new light on the regeneration of human liver.     

Doxorubicin coupled to lactosaminated albumin inhibits the growth of hepatocellular carcinomas induced in rats by diethylnitrosamine

BACKGROUND/AIMS: The hepatocyte receptor for asialoglycoproteins internalizes galactosyl terminating macromolecules which can be used as hepatotropic drug carriers. Since this receptor is also expressed on the cells of well differentiated human hepatocellular carcinomas (HCCs), we studied whether conjugation of doxorubicin (DOXO) with lactosaminated human albumin (L-HSA) increases the drug efficacy on HCCs induced in rats by diethylnitrosamine (DENA). METHODS: DENA was given in the drinking water for 8 weeks. One week after the last day of DENA administration, animals were randomly assigned to three groups. Each group was administered with either saline, free or coupled DOXO (1 microg/g). Rats received 4 weekly intravenous injections. One week after the last administration, rats were killed and HCC development was evaluated by counting the tumor nodules on the surface of hepatic lobes. RESULTS: In rats treated with L-HSA coupled DOXO the number of neoplastic nodules was significantly lower (P < 0.05) than that counted in animals injected with saline or with free DOXO. Coupled DOXO did not decrease body rat weight, which was markedly reduced by the free drug. CONCLUSIONS: Conjugation with L-HSA increased the antineoplastic efficacy and decreased the systemic toxicity of DOXO administered to rats with HCCs produced by DENA.     

Impairment of liver regeneration correlates with activated hepatic NKT cells in HBV transgenic mice

A fraction of HBV carriers have a risk to develop liver cancer. Because liver possesses a strong regeneration capability, surgical resection of cancerous liver or transplantation with healthy liver is an alternate choice for HBV-caused hepatocarcinoma therapy. How HBV infection affects the regeneration of hepatectomized or transplanted liver remains elusive. We report that partial hepatectomy (PHx)-induced liver regeneration was reduced in HBV transgenic (HBV-tg) mice, a model of human HBV infection. PHx markedly triggered natural killer T (NKT) cell accumulation in the hepatectomized livers of HBV-tg mice, simultaneously with enhanced interferon gamma (IFN-gamma) production and CD69 expression on hepatic NKT cells at the early stage of liver regeneration. The impairment of liver regeneration in HBV-tg mice was largely ameliorated by NKT cell depletion, but not by natural killer (NK) cell depletion. Blockage of CD1d-NKT cell interaction considerably alleviated NKT cell activation and their inhibitory effect on regenerating hepatocytes. Neutralization of IFN-gamma enhanced bromodeoxyuridine incorporation in HBV-tg mice after PHx, and IFN-gamma mainly induced hepatocyte cell cycle arrest. Adoptive transfer of NKT cells from regenerating HBV-tg liver, but not from normal mice, could inhibit liver regeneration in recipient mice. CONCLUSION: Activated NKT cells negatively regulate liver regeneration of HBV-tg mice in the PHx model.     

Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a common malignant tumour worldwide, and its differential diagnosis from benign lesions of the liver is often difficult yet of great clinical importance. In the present study, we analysed whether glypican-3 is useful in differentiating between benign and malignant liver diseases and whether it influences the growth behaviour of HCC. METHODS: Northern blot analysis and in situ hybridisation. RESULTS: Northern blot analysis indicated that expression of glypican-3 mRNA was either low or absent in normal liver, in focal nodular hyperplasia (FNH), and in liver cirrhosis. In contrast, expression of glypican-3 mRNA was markedly increased in 20 of 30 and moderately increased in five of 30 HCC samples. The average increase in glypican-3 mRNA expression in HCC was significant compared with expression in normal liver (21.7-fold increase, p<0.01). In comparison with FNH or liver cirrhosis, glypican-3 mRNA expression in HCC was increased 7.2- (p<0.05) and 10.8-fold (p<0.01), respectively. In addition, pushing HCCs exhibited significantly higher glypican-3 mRNA expression than invading tumours (p<0.05). In situ hybridisation analysis demonstrated weak expression of glypican-3 mRNA in normal hepatocytes and bile ductular cells, and weak to occasionally moderate signals in hepatocytes forming nodules of liver cirrhosis and in regenerated hepatic nodules of FNH. In contrast, glypican-3 in situ hybridisation signals were intense in hepatic cancer cells with even higher levels in pushing HCCs than in invading HCCs. CONCLUSIONS: These findings suggest that glypican-3, in many cases, has the potential to differentiate between benign and malignant liver diseases.     

Protection of regenerating liver after partial hepatectomy from carbon tetrachloride hepatotoxicity in rats: Role of hepatic stimulator substance

BACKGROUND: In the present study, we examined the effect of hepatic stimulator substance (HSS) on modulating hepatotoxicity induced by carbon tetrachloride (CCl4) in the regenerating rat liver. METHODS: Hepatotoxicity was induced in vivo by administering CCl4 to rats that had undergone a 68% partial hepatectomy (PH). In vitro studies were also performed in hepatocytes isolated from PH rats. RESULTS: Hepatic stimulator substance was extracted from regenerating rat liver 96 h after PH and its activity, as determined according to the method of LaBrecque, reached its maximum 96 h after PH. At this time, the mortality induced by CCl4 was significantly decreased in PH rats compared with sham-operated rats (18 vs 59%, P < 0.01). Likewise, changes in serum alanine aminotransferase (ALT) or bilirubin induced by CCl4 were less in rats after 96 h PH. The resistance of regenerating hepatocytes to CCl4 was retained in in vitro samples. Thus, leakage of intracellular ALT or aspartate aminotransferase induced by CCl4 in hepatocytes from 96 h hepatectomised rats was less than in control hepatocytes. HSS demonstrated a protective effect on hepatocytes against CCl4 both in vivo and in vitro. In additional studies, regenerating liver showed increased mitochondrial respiratory activity and enhanced plasma membrane fluidity. The HSS was also shown to increase hepatic mitochondrial respiratory activity and enhance plasma membrane fluidity. Further, the protective effect induced by HSS was correlated with the restoration of mitochondrial respiratory activity and plasma membrane fluidity induced by CCl4. CONCLUSIONS: Regenerating rat liver exhibits resistance to CCl4-induced hepatotoxicity, and the protection afforded by the regenerating state can be attributed, at least in part, to HSS-induced increases in mitochondrial respiratory activity and plasma membrane fluidity.     

Impaired liver regeneration following partial hepatectomy using the Pringle maneuver: Protective effect of mesna

Background and Aim: We investigated the role of the prophylactic administration of the antioxidant 2‐mercaptoethane sulfonate (mesna) on the hepatocyte‐regenerating capacity following partial hepatectomy (PH) with concurrent Pringle maneuver. Methods: Wistar rats were subjected to PH (70% hepatectomy), 30 min Pringle maneuver, PH plus Pringle with or without mesna pretreatment (400 mg/kg, per os, 3 h before Pringle), or sham operation. At 24 h, 48 h, 72 h, and 1 week after operation, relative liver weight, hepatocyte mitotic activity (mitotic index), the histopathological score and serum aspartate aminotransferase, and alanine aminotransferase concentrations were assessed. At 1 h after operation, oxidative stress markers (glutathione to glutathione disulfide ratio, malondialdehyde concentration, and superoxide dismutase activity) and nuclear factor‐κB (NF‐κB) activity were assessed. Results: Hepatectomy stimulated the regenerating process and induced mild oxidative stress and the activation of NF‐κB in hepatocytes, while causing tissue injury in the remnant liver. When PH was performed under Pringle maneuver, hepatocyte mitotic activity was substantially suppressed, although Pringle alone initiated a delayed regenerating response. Furthermore, Pringle maneuver deteriorated oxidative stress markers, markedly increased NF‐κB activity, and aggravated tissue injury, as compared to hepatectomy alone. Mesna pretreatment prevented the Pringle‐induced antimitotic effect and the induction of oxidative stress, inhibited the activation of NF‐κB, while attenuating liver injury after PH under Pringle. Conclusion: The excessive activation of NF‐κB is related to the suppression of hepatocyte‐regenerating activity following PH with concurrent liver ischemia. Mesna pretreatment protects the liver against the Pringle‐induced antimitotic effect after PH via the prevention of oxidative stress and the inhibition of NF‐κB activation.     

In situ cellular analysis of alpha-fetoprotein gene expression in regenerating rat liver after partial hepatectomy

Cellular analysis of hepatic alpha-fetoprotein gene expression in normal adult rat and during regeneration induced by partial hepatectomy was performed at the cellular level by in situ hybridization using 35S-labeled complementary DNA probes and immunoperoxidase techniques. In normal adult rat liver sections, a few alpha-fetoprotein mRNA-cDNA hybrids are detected over all hepatocytes. No protein is detected with routine immunoperoxidase methods. However, after in vivo colchicine blockade of alpha-fetoprotein secretion, 10 to 20% alpha-fetoprotein-positive hepatocytes are observed. In regenerating livers, at 2,6 and 24 hr (before and at the time of the peak of DNA synthesis in the periportal zones), a rise of the nuclear signal level is observed selectively in periportal hepatocytes, without modification of the cytoplasmic signal. At 48 hr (when most hepatocytes have completed at least one replicative cycle), almost all hepatocytes throughout the liver lobule display a rise of the nuclear (2- to 3-fold) and cytoplasmic (1.5- to 2-fold) signal level compared to nonoperated rats. These data show that all hepatocytes in the adult liver express a small number of alpha-fetoprotein mRNA sequences; they appear to be translated in protein whose secretion can be blocked by colchicine. The moderate increase in alpha-fetoprotein gene expression induced by liver regeneration takes place in all hepatocytes, in apparently two distinct steps: a very early nuclear accumulation of alpha-fetoprotein mRNA sequences and a late cytoplasmic accumulation of alpha-fetoprotein mRNA molecules.