Liver cell proliferation induced by a single dose of lead nitrate.

Treatment of male Wistar rats with a single dose of lead nitrate caused a marked enlargement of the liver, which reached its maximum 3 days after the administration of the metal salt. This grossly anatomic effect was accompanied by biochemical changes such as an increase in total protein and DNA content, with a maximum at 3 and 4 days, respectively. A partial regression of liver weight and total DNA and protein content occurred 7 days after lead administration; a significant increase in DNA concentration was found after 1 week, while no variation in protein, when expressed as milligrams per gram liver, was observed in lead-treated rat liver. An increase in DNA synthesis, as monitored by the incorporation of labeled thymidine, was also observed. An enhancement in the specific radioactivity of DNA was evident at 24 hours and appeared maximal at 36 hours after the administration of lead nitrate. The ability of lead to stimulate liver cell proliferation was shown by a significant increase of cells entering mitosis, with a peak at 48 hours. This mitogenic stimulus occurred in parenchymal as well as in nonparenchymal cells, thus showing that this effect was not unique to a particular liver cell populations. No detectable cell necrosis, as monitored by histologic observation, was seen in the liver of lead-treated rats, thus indicating that the cellular proliferation induced by lead is not due to a regenerative response. Only a slight elevation in the levels of serum glutamate-pyruvate transaminase (GPT) was observed by biochemical analysis.     

Liver hyperplasia and regression after lead nitrate administration

The effect of a single intravenous injection of lead nitrate on liver, was investigated in male Wistar rats. Lead nitrate at 5 and 10 mumoles/100 g of body weight stimulated a 19-fold increase in the incorporation of 3H-thymidine into liver DNA and resulted in temporal changes in DNA synthesis, as determined by assays of specific activity. Thirty-six hours after lead nitrate administration, the incorporation of 3H-thymidine reached its maximum and returned to normal levels within 3 days. A significant increase in the number of cells entering mitosis at 36 hours indicated the capacity of lead to stimulate liver cell proliferation. Enlargement of the liver after lead treatment was also observed in both female Wistar rats as well in male Fischer rats. This stimulatory effect of lead on liver growth was reversible; during the involution of the liver, cell death morphologically similar to the one described as apoptosis was observed in histological sections of liver from animals sacrificed 4-7 days after lead treatment.     

Occurrence of cell death (apoptosis) during the involution of liver hyperplasia

A single intravenous injection of lead nitrate at a dose of 10 mumoles/100 gm of body weight caused liver enlargement associated with hepatic cell proliferation. In the present study the involution of liver hyperplasia which follows the withdrawal of lead was studied in male Wistar rats. Histologic examination of liver sections from rats killed during the regression of the liver did not show any sign of massive lytic cell necrosis; no variation in the levels of serum glutamate pyruvate transaminase could be observed during the same time period; however, light microscopic observation of sections from the involuting liver showed the presence of several apoptotic bodies; the occurrence of apoptotic bodies was also confirmed by ultrastructural examination. Their incidence was found to be markedly increased at 5 days after treatment, a time period when the liver is already regressing; very few apoptotic bodies were observed in control animals or in treated rats 2 days after lead injection, a time point when mitotic index reached its maximum, or at 15 days, when the liver had returned to control values. These findings suggest that removal of excess liver which follows the initial hyperplasia caused by lead is due to a controlled mode of cell death, namely, apoptosis.     

Ultrastructural and DNA damaging effects of lead nitrate in the liver

A ubiquitous environmental toxicant – lead is known to affect several organ systems. This study was designed to investigate the effects of lead nitrate exposure on liver structure and DNA fragmentation. Adult male Wistar rats were treated orally with lead nitrate at the dose levels of 0%, 0.5% and 1% for 60 days and sacrificed on the next day. The liver was processed for thick sections and evaluated after toludine blue staining and by electron microscopy after staining with uranyl acetate and lead citrate. The DNA damage was assessed by DNA fragmentation assay. The liver weight was not significantly affected in the experimental groups. Hepatocyte nuclei were not shrunk, instead lead was mitogenic to hepatocytes as indicated by an increase in the number of binucleated hepatocytes (P<0.05). The number of mitochondria per hepatocyte decreased in a dose-dependent manner (P<0.05). Qualitatively, the necrotic changes such as small to large-sized cytoplasmic vacuoles often displacing the organelles, decrease in hepatocyte microvilli, degeneration of mitochondria, and vacuolar encroachment of nuclei and dilatation of sinusoids were observed. The qualitative changes were induced in a dose-dependent manner. Kupffer cells or Ito cells did not present any notable structural changes. Although the electrophoretic flow of DNA fragments was observed in lead-treated groups, these changes were not significantly different from that in control as evaluated by optical density. In conclusion, lead induces necrotic changes with simultaneous mitogenic activity; however, it does not induce significant DNA damage in the liver.     

Dexamethasone inhibits induction of liver tumor necrosis factor-alpha mRNA and liver growth induced by lead nitrate and ethylene dibromide.

We have recently demonstrated that a single injection of the mitogen lead nitrate to rats induced a rapid increase of tumor necrosis factor-alpha (TNF-alpha) mRNA in the liver and suggested that this cytokine may be involved in triggering hepatocyte proliferation in this model of direct hyperplasia. In this study, we examined whether a similar induction of liver TNF-alpha mRNA could be observed preceding the onset of hepatocyte proliferation induced by ethylene dibromide, another hepatocyte mitogen. In addition, we used dexamethasone, a well known inhibitor of TNF-alpha production, to determine whether its administration could suppress hepatocyte proliferation induced by lead nitrate and ethylene dibromide. A single intragastric administration of ethylene dibromide (100 mg/kg) to male Wistar rats enhanced liver TNF-alpha mRNA after 4 and 7 hours, which then returned to control levels by 24 hours. TNF-alpha mRNA was detectable only in a nonparenchymal cell fraction of the liver. Pretreatment of rats with a single dose of dexamethasone (2 mg/kg) 60 minutes before lead nitrate (100 mumol/kg) or ethylene dibromide completely abolished the increased levels of liver TNF-alpha mRNA induced by these agents. Inhibition by dexamethasone of TNF-alpha mRNA was associated with an inhibition of liver cell proliferation induced by these mitogens, as measured by [3H]thymidine incorporation into hepatic DNA, mitotic index, and DNA content. These results further support the hypothesis that TNF-alpha may be involved in triggering hepatocyte proliferation induced by primary mitogens.     

Variations of serum lipoproteins during cell proliferation induced by lead nitrate

In the present study serum lipoproteins were investigated during cell proliferation induced by a potent mitogen, lead nitrate. A strong decrease in HDL2 and a concomitant increase in HDL3 were observed in lead-treated rats. The recovery of normal lipoprotein pattern took place together with the regression of hyperplastic process. Since a decrease in HDL also occurs under other conditions of cell growth, we hypothesize that a decrease in HDL, mainly in HDL2 subfraction, may represent a generalized phenomenon related to massive cell proliferation.     

Integration and proliferation of transplanted cells in hepatic parenchyma following D-galactosamine-induced acute injury in F344 rats

To determine whether liver repopulation with cell transplantation could be of therapeutic value in acute hepatic failure, it is necessary to establish the fate of transplanted hepatocytes. This study used dipeptidyl peptidase IV-deficient F344 rats as recipients to analyse the engraftment and proliferation of transplanted hepatocytes. Syngeneic hepatocytes were transplanted intrasplenically 24-30 h after induction of liver injury by D-galactosamine (GalN). Portosystemic shunting was analysed with 99m-Tc-labelled albumin microspheres. GalN-treated rats showed characteristic hepatic necrosis, inflammation, gamma-glutamyl transpeptidase activation, and regenerative activity, without increased portosystemic shunting (>99% 99m-Tc activity was in the liver in normal and GalN-treated rats). Transplanted cells entered hepatic sinusoids promptly and were observed in liver plates at 48 h. The number of transplanted cells increased in GalN-treated rats by approximately seven-fold (range two- to 12-fold), along with evidence for DNA synthesis between 3 and 14 days after cell transplantation and greater prevalence of larger transplanted cell clusters. These findings indicate that the liver can be safely repopulated in animals with acute liver failure, although the time required for regenesis of plasma membrane structures and proliferation in transplanted hepatocytes will need to be considered in developing therapeutic strategies.     

A study of endotoxin-associated hepatotoxicity on proliferating hepatocytes.

It is thought that regeneration of the liver provides a state of preparedness for the Shwartzman reaction and contributes to the development of endotoxin-associated massive hepatic necrosis following partial hepatectomy. Therefore we examined endotoxin hepatotoxicity in rats with hepatic regeneration after 35% hepatectomy and in rats with liver cell proliferation induced by lead nitrate. Biochemical and histopathological studies showed no enhanced endotoxin hepatotoxicity in either partially hepatectomized rats or in rats with lead nitrate-induced liver cell proliferation. These results indicate that the development of endotoxin-associated hepatic damage after partial hepatectomy may not relate to regeneration and proliferation of the liver.     

Induction of the placental form of glutathione S-transferase by lead nitrate administration in rat liver

The administration of a single dose of lead nitrate to male Wistar rats caused an increase of a polypeptide in the liver cytosol that cross reacted with the anti-rat antibody of the placental form of glutathione S-transferase (GST-P). GST-P appeared when doses of lead that induced liver cell proliferation were given (5 and 10 micromoles/100 g of body weight). Recently, it has been shown that rat hepatic nodules also exhibited an increased content of the placental form of GST-P. The induction of GST-P by lead together with other biochemical effects exerted in the liver by this metal, suggests that some chemicals may induce in rat liver a biochemical pattern similar, in some aspects, to that exhibited by carcinogen-induced hepatocyte nodules.     

Toxicosis associated with dual oral exposure of rats to lead and trichloroethylene

To determine if additive or synergistic toxic effects would occur, adult male rats were exposed orally to lead carbonate (2,000 mg/kg) for 9 days before trichloroethylene (TCE), 2,000 mg/kg, was given concurrently for an additional 7 days. Comparisons were made with groups of vehicle-treated rats and rats given only lead or only TCE. Potential neurotoxicity was evaluated by using the Functional Observational Battery (FOB) recommended for neurotoxicity screening. Rats were sacrificed on day 16, and brain, testes, spleen, kidney/adrenals, heart, and liver weighed and observed for pathological changes. Results of the FOB indicated that lead carbonate was more responsible than TCE for changes observed. Additive or synergistic neurotoxicities were not noted. Histological examination of the kidney from lead-treated rats revealed inclusions, an increased incidence of coagulated proteins, and tubular dilation that was generally more severe in the medullary segments. Gastric and testicular necrosis were found in rats given lead carbonate both with and without TCE (15/20 and 6/20 treated, respectively). The results suggest that, even when given concurrently, the toxicities of lead carbonate and TCE are expressed only as though one toxicant was given.     

Carbachol induces hepatocyte proliferation, but only in the presence of hepatic nonparenchymal cells

Vagal hyperactivity correlates with enhanced DNA synthesis and cell proliferation in the peripheral tissues of ventromedial hypothalamic (VMH)-lesioned rats. The infusion of an ACh receptor agonist, carbachol (Cch), induces rat duodenal and pancreatic cell proliferation to a degree comparable to the VMH lesions. Whereas the VMH lesions also induce the proliferation of hepatic cells, it is unclear whether Cch can also do this. Here we attempted to clarify the mechanism of hepatic cell proliferation induction by cholinergic stimulation. First, hepatic cell proliferation was examined in rats previously vagotomized and intraperitoneally administered with Cch via an osmotic minipump. Second, the sera from the Cch-infused rats were examined for a proliferative effect on isolated hepatic cells. And last, the effect of the presence of hepatic nonparenchymal cells (NPCs) on the proliferation of the cultured hepatocytes treated with Cch was investigated. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) showed that the 3-day Cch infusion significantly increased the number of PCNA-immunoreactive cells in the liver. Moreover, the sera from the Cch-infused rats increased the number of PCNA-immunoreactive hepatocytes in culture. However, Cch alone did not induce proliferation in monocultured hepatocytes. When compared with the monoculture of hepatocytes, the coculture of those with hepatic NPCs resulted in enhanced PCNA immunoreactivity after a 4-day treatment with 3 mM Cch. These findings suggest that ACh induces hepatocyte proliferation, which is mediated by unidentified humoral factor(s) possibly secreted from hepatic NPCs, and that it also participates in liver hypertrophy in the VMH-lesioned animals.     

Regulatory effects of senescence marker protein 30 on the proliferation of hepatocytes

Senescence marker protein 30 (SMP 30) is preferentially expressed in the liver. One of its remarkable functions is the protection of cells against various injuries by enhancement of membrane calcium-pump activity. We analyzed the role of SMP 30 in hepatocyte proliferation. SMP 30 expression was decreased initially, then increased along with hepatic regeneration, after carbon tetrachloride (CCl4) administration. SMP 30 expression was decreased in the necrotic phase and then gradually increased. Its increase was slightly delayed just after the mitotic phase. These results lead us to speculate that mitoses of hepatic cells induce enhanced SMP 30 expression. In contrast, administration of lead nitrate (LN) as a hepatic mitogen induced a more stable increase of SMP 30 expression. To estimate the effect of SMP 30 on cell proliferation, we evaluated hepatic mitosis in wild-type and SMP 30-deficient knockout (KO) mice after CCl4 administration. We found an increase in mitotic numbers in hepatocytes of KO mice. This result suggests that SMP 30 has a suppressive effect on cell proliferation. Suppressive activity of SMP 30 cDNA was shown in cultured hepatoblastic cells. Our results suggest that SMP 30 performs a regulatory function in liver regeneration.     

The Number and Dimensions of Small Airways in Emphysematous Lungs

Effects of a single dose of lead (0.04 mg lead g body weight) on the proliferation of proximal tubular epithelium in rat kidneys were investigated by autoradiography over a period of 72 hours, using ³H-thymidine as a label. The results demonstrate that cell proliferation was greatly stimulated within 2 days after lead was injected. The increase in DNA synthesis began about 20 hours after intraperitoneal injection of lead, reached a sharp peak at 30 hours, and declined rapidly thereafter. At the peak, the mean labeling activity was 40 times that observed in control rats. Cumulatively, an average of 14.5% of the proximal tubular epithelial cells were labeled 72 hours after lead was injected. When uninephrectomy was followed immediately by injection of lead, the stimulation of DNA synthesis in the remaining kidney was, on the average, greater than the sum of the separate effects of the two treatments. This indicates that the stimulatory effects of uninephrectomy and injection of lead on renal cell proliferation were additive.     

The effect of pancreatectomy and other agents on iron absorption and storage in the rat

Evidence from both animal and human studies had been presented by various authors to suggest that reduction of pancreatic function increased iron absorption. The present experimental studies in rats showed that there was increased radioiron ((59)Fe) absorption after pancreatectomy, but this appeared to be due to the operative procedure as there was a similar increased absorption in animals who had sham operations performed. There was no certain evidence that pancreatic enzymes or extract had a specific inhibitory effect on the absorption of iron. But with increased doses of pancreatic extract, above the normal doses, the amount of radioiron absorption was greatly enhanced.A significant increase in hepatic storage iron was found in the groups of rats who had been fed a diet supplemented with dl-ethionine. All these animals showed extensive damage and atrophy of the pancreas, with degeneration and considerable regenerative activity in the liver. The absolute increased hepatic storage iron was not due to the liberation and laying down of haemosiderin from the damaged cells or to decreased liver mass. Rats who had carbon tetrachloride-induced liver cirrhosis without pancreatic damage showed no increased hepatic storage iron.It was observed that in liver damage with considerable cellular proliferation there was increased liver iron content. As this was found only when there was associated pancreatic injury, it is suggested that decreased pancreatic function in the presence of liver cell injury with cellular proliferation leads to increased iron absorption and storage. The relationship of liver injury with cellular proliferation and pancreatic damage to increased hepatic storage iron merits further study.     

Thyroid and chemical hepatocarcinogenesis: further insights from the hepatocarcinogen ZAMI 1305

The beta-blocker DL-1-(2-nitro-3-methyl-phenoxy)-3-tert-butylaminopropan-2-ol (ZAMI 1305), oncogenic to the liver of the female but not of the male Wistar rat, was used to investigate some aspects of the relationship between liver and thyroid during chemical hepatocarcinogenesis. Thyroidectomy (TDX) strongly reduces the amount of hepatic DNA damage induced by a single administration of ZAMI 1305 in the female Wistar rat. One week of treatment with triiodothyronine (T3) completely restores the susceptibility of the liver of thyroidectomized animals to the genotoxic activity of the molecule. The amount of hepatic DNA damage in intact females varies with the age of the animal, being maximal in rats of 4-8 weeks of age, when T3 serum concentration are also maximal. An increase of relative thyroid weight, coupled with histological hyperplasia of the gland, is observed in female Wistar rats treated for 6 months with ZAMI 1305. Minimal changes of the thyroid are observed in ZAMI 1305-treated male rats. The increase of relative thyroid weight in female rats appears to be related to the severity of preneoplastic and neoplastic liver changes. These findings and several suggestions from the literature lead us to propose a model for the interaction between liver and thyroid during chemical hepatocarcinogenesis.     

Oxidative stress and oval cell accumulation in mice and humans with alcoholic and nonalcoholic fatty liver disease

In animals, the combination of oxidative liver damage and inhibited hepatocyte proliferation increases the numbers of hepatic progenitors (oval cells). We studied different murine models of fatty liver disease and patients with nonalcoholic fatty liver disease or alcoholic liver disease to determine whether oval cells increase in fatty livers and to clarify the mechanisms for this response. To varying degrees, all mouse models exhibit excessive hepatic mitochondrial production of H(2)O(2), a known inducer of cell-cycle inhibitors. In mice with the greatest H(2)O(2) production, mature hepatocyte proliferation is inhibited most, and the greatest number of oval cells accumulates. These cells differentiate into intermediate hepatocyte-like cells after a regenerative challenge. Hepatic oval cells are also increased significantly in patients with nonalcoholic fatty liver disease and alcoholic liver disease. In humans, fibrosis stage and oval cell numbers, as well as the number of intermediate hepatocyte-like cells, are strongly correlated. However, cirrhosis is not required for oval cell accumulation in either species. Rather, as in mice, progenitor cell activation in human fatty liver diseases is associated with inhibited replication of mature hepatocytes. The activation of progenitor cells during fatty liver disease may increase the risk for hepatocellular cancer, similar to that observed in the Solt-Farber model of hepatocarcinogenesis in rats.     

Hepatic arterial infusion of bevacizumab in combination with oxaliplatin reduces tumor growth in a rat model of colorectal liver metastases

Unresectable colorectal liver metastases are commonly treated with systemic chemotherapy (SCT). Clinical studies on the effect of additional systemic application of bevacizumab (BE), a monoclonal antibody directed against vascular endothelial growth factor, to SCT showed a slight increase of patient survival. Herein, we studied in a rat model of colorectal liver metastasis whether a locoregional application of oxaliplatin (OX) and BE via hepatic arterial infusion (HAI) is more effective to inhibit metastatic growth compared to systemic drug application. Ten days after implantation of CC531 colorectal cancer cells into the left liver lobe of WAG/Rij rats, animals underwent either HAI or systemic intravenous application of BE (5 mg/kg body weight), OX (85 mg/m² body surface) or a combination of both. Sham-treated animals received saline and served as controls. Tumor volume was measured at days 10 and 13 using three dimensional ultrasound. At day 13 tumor tissue was analyzed histologically and immunohistochemically. Systemic application of OX, BE or their combination did not affect tumor volume when compared to controls. In contrast, HAI of BE and particularly the combination of BE and OX significantly reduced tumor volume. In the tumor tissue this was associated with a decrease of vascularization and cell proliferation as well as an increase of cell apoptosis, as indicated by a decreased number of PECAM-1- and PCNA-positive cells and an increased number of cleaved caspase-3-positive cells. Locoregional administration of BE, particularly in combination with OX, enhances the inhibitory effect on hepatic metastatic growth compared to systemic application of the drugs.     

连接蛋白/缝隙连接细胞间通讯对大鼠肝卵圆细胞增殖调控作用

背景：缝隙连接蛋白介导的缝隙连接细胞间通讯(gap junction intercellular communication,GJIC)是细胞间最重要的信息交流形式。 目的：验证CX/GJIC对卵圆细胞的增殖调控作用。 方法：Wistar大鼠分为4组。对照组正常饮食;2-AAF/PH组按改良Solt-Farber法建立卵圆细胞增殖动物模型;苯巴比妥组予以苯巴比妥饮水7 d,第8天按2-AAF/PH组建模,苯巴比妥饮水持续至实验结束;三七总皂苷组按2-AAF/PH组建模时予以三七总皂苷25 mg/(kg•d)腹腔内注射,并持续实验结束。 结果与结论：造模后肝脏连接蛋白呈时空特异性表达,先下调后逐渐恢复,连接蛋白43表达于卵圆细胞,先升高后逐渐恢复;采用苯巴比妥改变大鼠2-AAF/PH模型肝脏的连接蛋白32、连接蛋白43时空表达模式后,可下调肝脏的GJIC,减少卵圆细胞与偶联细胞间GJIC,解除卵圆细胞生长抑制,促进卵圆细胞的增殖;三七总皂苷可以增加大鼠2-AAF/PH模型肝脏的连接蛋白32表达、滞后连接蛋白43的表达,增加卵圆细胞与偶联细胞间GJIC,使卵圆细胞增殖峰低、滞后、持续时间长;通过下调大鼠2-AAF/PH模型肝脏的GJIC,可使卵圆细胞增殖早、增殖水平高;上调GJIC使卵圆细胞增殖峰低、滞后、持续时间长,CX/GJIC可以调节体内卵圆细胞的增殖、分化过程。     

Dietary restriction: effects of short-term fasting on protein uptake and cell death/proliferation in the rat liver

Dietary restriction (DR) is known to prolong life in laboratory animals. Intermittent (alternate-day) fasting or short-term repeated fasting has also been reported to increase the life span of animals. In the present study, we investigated the changes or induction of abnormalities of protein metabolism in rats during fasting, and measured asialoglycoprotein uptake and cell death/proliferation in the liver of rats receiving fasting and refeeding. In the results, liver weight decreased significantly after 48 h of fasting and increased during the refeeding period, returning to the pre-fasting level by 12 h of refeeding. Cell death, determined by single stranded DNA (ssDNA) staining method, increased during the fasting period, and returned to the pre-fasting level during the refeeding period. Cell proliferation, determined using antibodies (Ab) against proliferating cell nuclear antigen, decreased during the fasting period, and increased during the refeeding period. Changes in cell death and cell proliferation were inversely related. However, there was no significant difference in asialoglycoprotein uptake by the whole liver between the ad libitum (AL)-fed rats and 48 h fasted rats. Thus, neither the changes in liver weight nor cell death/proliferation affected asialoglycoprotein uptake on a living body. These results suggest that episodes of 48 h fasting do not induce protein metabolism abnormalities in the liver.     

Sequential analysis of hepatic carcinogenesis. Regeneration of liver after carbon tetrachloride-induced liver necrosis when hepatocyte proliferation is inhibited by 2-acetylaminofluorene

The effect of inhibition of hepatocyte proliferation by dietary 2-acetylaminofluorene (2-AAF) on the restoration of liver in rats after a necrogenic dose of carbon tetrachloride has been studied. The liver weights remained low during the entire feeding period of the 2-AAF, and virtually no hepatocyte proliferation was seen, as determined autoradiographically after thymidine incorporation and by the absence of mitotic figures. Oval cell proliferation was extensive. Morphometric analysis showed (a) equal and maximum liver cell necrosis by 24 hours in both the experimental and control groups, (b) similar kinetics of removal of dead liver cells, and (c) similar values for the mean liver cell area. The distance between the portal triad and terminal hepatic vein in animals on the dietary 2-AAF was considerably reduced. Massive hepatocyte proliferation began after termination of the 2-AAF diet, and the liver returned to normal appearance within 14 days. The oval cells disappeared during this period of liver cell restoration. A new hypothesis for oval cell proliferation based on differential inhibition of hepatocyte proliferation resulting in unbalanced growth of ductular cells is presented.