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.     

Immunohistochemical detection of activated caspases in apoptotic hepatocytes in rat liver

In our study we tested the utility of antibodies that specifically recognize the cleaved large (active) subunits of caspase-3 and caspase-9 for immunohistochemical detection of apoptotic hepatocytes in rat liver sections using archival material from cyproterone acetate (CPA)-treated and control rats. CPA blocks apoptosis of hepatocytes and discontinuation of CPA treatment results in a syncronized wave of hepatocyte apoptosis. By comparing liver sections from CPA-treated and control rats with high and low rates of apoptosis we observed a close correlation between the occurrence of cleaved caspase-positive apoptotic figures and H&E-stained apoptotic bodies when evaluated in parallel sections. Caspase-stained figures were either immuno-positive apoptotic bodies or pre-apoptotic hepatocytes showing cytoplasmic and/or nuclear caspase-staining with otherwise normal cellular appearance. In extension of these observations we developed a double-immunohistochemical staining procedure which enables the detection of caspase-3-positive apoptotic hepatocytes within glutathione-S-transferase-P (GST-P)-positive preneoplastic liver foci. By use of this technique, inhibition of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin as detected by counting of H&E-stained apoptotic bodies was found to correlate with a strong reduction of cleaved caspase-positive hepatocytes in GST-P-positive preneoplastic foci. In summary, this study demonstrates that cleaved caspase-positive apoptotic hepatocytes could be reliably identified and quantified both in normal and neoplastically transformed liver tissue.     

Molecular mechanisms of apoptosis in the liver of rats after portal branch ligation with and without retrorsine

The mechanisms accounting for the atrophy of the portal blood-deprived liver lobes after portal branch ligation (PBL) are still unclear. The first aim of this study was to confirm the role of apoptosis in this process and to determine which apoptotic pathways are involved. The second aim of the study was to evaluate the effect of blocking compensatory hyperplasia of the nonligated lobes with retrorsine on the mechanisms of apoptosis in the ligated lobes. Mitochondrial Bax, Bcl-2 and Bcl-X(L), cytosolic cytochrome c, caspase-3, -8 and -9 activities and TNF-alpha levels were assessed in the liver of rats before and at various time points, ranging from 30 min to 7 days, after PBL. Caspase activities were also measured in rats pretreated with retrorsine. Both the mitochondrial and the death receptor-mediated pathways are activated in the ligated liver lobes after portal branch ligation. Caspase activation is inhibited by retrorsine pretreatment, resulting in fewer apoptotic bodies. Apoptosis accounts for the atrophy of the ligated lobes after PBL. It is inhibited by retrorsine, suggesting an attempt to reduce the loss of liver mass when hyperplasia of the nonligated lobes is impaired     

Regression mechanism of cyanamide-induced inclusion bodies in the rat: a useful experimental pattern to study the beta-glycogen metabolization of hepatocytes.

Cyanamide, a drug used in alcohol aversion therapy, induces a distinctive liver cell lesion, both in human beings and rats. The lesion consists of cytoplasmic inclusion bodies which give a ground-glass appearance to the hepatocytes. In human beings the inclusion bodies do not persist but disappear some time after withdrawal of the drug. In order to confirm their disappearance and determine how they regress rats were treated with cyanamide (32 mg/kg) for 6 months before partial lobectomy. At this time, inclusion bodies were observed. After a period without further treatment (5-19 weeks) the animals were killed and a marked decrease in the number of inclusion bodies was observed, paralleling the period of time without treatment. Inclusion bodies regress as a result of glycogen removal by enzymatic activity of the smooth endoplasmic reticulum which then undergoes hyperplasia, plus a process of autophagocytosis and necrosis of inclusion-body-bearing hepatocytes which are then phagocytosed by macrophages.     

Induction of two different modes of cell death, apoptosis and necrosis, in rat liver after a single dose of thioacetamide.

A sequential study of the appearance of liver cell death after thioacetamide (TH) administration was performed in male Wistar rats. Within 3 hours of a single dose of TH, occurrence of cell death by apoptosis was evident around the centrilobular area. Light as well as electron microscopic examination demonstrated the presence of eosinophilic globules, often containing nuclear remnants (apoptotic bodies); they frequently were found within the cytoplasm of intact hepatocytes. The number of apoptotic bodies (ABs) was further enhanced at 6 hours, resulting in a 70-fold increase over the control values. Although necrosis or inflammation could not be observed at this time, as monitored by microscopic analysis as well as by determination of serum glutamate pyruvate transaminase levels, centrilobular necrosis accompanied by massive inflammatory reaction was evident at 12 hours and even more pronounced at 24 to 36 hours. Evidence of liver regeneration was found to occur at 48 hours, and the liver regained its normal architecture between 72 and 96 hours. Studies performed to analyze the activity of 'tissue' transglutaminase (tTG), a presumptive marker of apoptosis, showed that, 1 hour after treatment, TH caused a drastic dose-dependent inhibition of the enzyme activity. This early inhibition was followed by a rapid recovery in tTG activity that paralleled the induction of apoptosis in the liver. Treatment with cycloheximide (CH) 2 hours after TH partially inhibited the incidence of ABs at 6 hours (approximately 30% inhibition). The present study indicates that two different modes of cell death, apoptosis and necrosis, may be induced in a sequential fashion by a single dose of TH.     

Lead and liver cell proliferation. Effect of repeated administrations

The effect of repeated treatments with lead on hepatic cell proliferation was investigated in male Wistar rats. The animals were given intravenous injections of lead nitrate once every 10 days for 30 and 80 days. At the end of the experimental regimen, enlargement of the liver, accompanied by an increase in hepatic DNA content, was observed. A significant enhancement in the incorporation of labeled thymidine into hepatic DNA was found in lead-treated rats at the time intervals mentioned above, when compared with controls. An increase in the number of liver cells involved in mitosis was also observed in lead-treated animals. Analysis of serum glutamic-pyruvic transaminase and histologic observations did not show any sign of cell death at the time points examined. These results indicate that liver cells exposed to repeated treatments with lead undergo proliferation. However, a progressive reduction in the capacity of hepatic cells to divide was found in rats given repeated administrations of the metal, when compared with the extent of cell proliferation induced by a single dose of lead nitrate.     

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.     

Appearance of ductular hepatocytes in rat liver after bile duct ligation and subsequent zone 3 necrosis by carbon tetrachloride.

Intrahepatic biliary cell plasticity was investigated in a rat model that combined prior bile ductular cell hyperplasia after bile duct ligation with subsequent CCl4-induced hepatonecrosis. Morphometric analysis of histologic liver sections from rats at 4 to 6 weeks after bile duct ligation and 3 to 5 days after CCl4 demonstrated the total section area to be occupied by near-equal amounts of hyperplastic bile ductular tissue area and hepatonecrotic area. Of particular significance was the unique presence, albeit infrequent, of newly appearing hepatic cell cholangioles composed of both biliary epithelial cells and one or more 'ductular hepatocytes' exclusively within the hyperplastic bile ductular tissue area of liver sections from the bile-duct-ligated/CCl4-treated rats, but not observed in control liver sections. This finding is compatible with the possibility of a 'transdifferentiation' of some hyperplastic biliary epithelial cells into 'ductular hepatocytes' in response to an extreme hepatic injury.     

Comparative ultrastructural hepatic alterations induced by free and liposome-encapsulated mefenamic acid

Mefenamic acid (MFA) is used as an anti-inflammatory, antinociceptive, and antipyretic agent for treatment of a wide range of pathological disorders. While the uncertainty of its safety and the poor oral bioavailability constitute the major limiting factors of its medical use, considerable efforts including liposomal encapsulation are needed to achieve maximum therapeutic advantages. The current work was conducted to investigate the ultrastructural alterations in the liver induced by free MFA and its liposomal preparation. Female Sprague-Dawley rats were treated with daily oral doses of either free MFA or MFA entrapped in Tween 80 inoculated liposomes at the concentration of 80 mg/kg for 28 days. Ultrathin sections were prepared from biopsies taken from the liver of each member of all animals under study and subjected to examination by transmission electron microscopy. The liver of rats that were exposed to liposomal MFA showed more ultrastructural alterations than the rats treated with the free drug. While both groups of rats demonstrated sinusoidal dilatation, Kupffer cell hyperplasia, mitochondrial damage, and nuclear alterations, rats treated with liposome-encapsulated MFA induced an increase in the multiple lysosomes formation, hepatocytic steatosis, and apoptotic activity than free MFA-treated rats. The ultrastructural findings of the present study indicate that the use of liposomal MFA induces more hepatic damage than the use of free MFA.     

Zonal distribution of cell proliferation in the liver of untreated B6C3F1 and C57BL mice

To determine the lobular distribution of hepatocellular proliferation, S-phase response was measured in untreated adult male B6C3F1 and C57BL mice at ages 11, 14, and 23 weeks. The percentage of cells in S-phase (labeling index, LI) was evaluated using immunohistochemical detection of 5-bromo-2'-deoxyuridine (BrdU). The BrdU was delivered either by a single ip injection 2 hr prior to sacrifice or via an osmotic minipump implanted subcutaneously for 3 or 7 days. Mitotic and apoptotic indices were determined on H&E stained sections. Animals of both strains and all ages revealed highest LI in the intermediate compartment of the liver acinus (zone 2) irrespective of the length of BrdU application. Cell proliferation in this zone was at all time points significantly higher than elsewhere in the liver. The mitotic index confirmed the data obtained by the S-phase response study. Apoptosis was rarely observed. With regard to rodent data in chemical carcinogenesis and the way they should be evaluated, this study proved that the acinar distribution of proliferating cells in liver of mice is different from that in rats, since, according to the literature, rats reveal highest cell proliferative activity in the periportal zone.     

不同途径移植羊膜上皮细胞在肝脏的定居**○

背景：细胞移植对肝脏疾病具有一定的疗效,与肝脏移植相比有其自身的优点,但有诸多问题尚待解决。 目的：探讨不同途径移植的人羊膜上皮细胞在肝脏内定居情况。 方法：从剖腹产后的人胎盘羊膜中分离人羊膜上皮细胞,PKH26荧光标记后计数1×107细胞通过大鼠腹腔、门静脉、尾静脉及肝脏内直接注入方式移植入大鼠体内。 结果与结论：门静脉、尾静脉及肝脏直接注入途径移植的细胞均在肝脏内定居,但移植细胞数过量时造成局部肝组织的缺血坏死等不良反应。腹腔途径移植入体内的细胞未转移肝脏内。通过门静脉移植入体内的人羊膜上皮细胞在大鼠肝脏内维持存活至少16 d。移植细胞过量时,导致肝脏血管堵塞及坏死。证明人羊膜上皮细胞至少在大鼠肝脏中存活2周,提示低免疫原性的羊膜来源细胞可成为肝脏病治疗的候选细胞之一。     

Apoptosis of the intestinal principal cells of Xenopus larvae exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In our previous work using paraffin-embedded sections, we determined that Xenopus larvae exposed to 200 ppb 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 5 days from shortly after fertilization to the early larval stage showed a shortening of the digestive tract and a loss of mucosal epithelium cells due to exfoliation into the gut cavity. In the current study, we ultrastructurally examined the mucosal epithelium of the gut of TCDD-exposed Xenopus larvae 12 days after fertilization. Exfoliated cell structures at the villus tip and in the lumen were equipped with a microvillus portion and occasionally had terminal web-like structures seen by ultramicroscopy. As these exfoliated structures had nuclear fragments with condensed chromatin, they were considered to be apoptotic bodies derived from the principal cells of the epithelium. In addition, many membrane-bound cell fragments-identified as apoptotic bodies derived from the principal cells based on their ultrastructural features-were observed at the basal side of the mucosal epithelium. These apoptotic bodies were phagocytized and digested chiefly by the neighboring intact principal cells. No such cells and/or cell fragments showing apoptotic features were observed in the controls. Our observations indicate that marked apoptosis occurs in the intestinal principal cells of TCDD-exposed larvae, which may result in the shortening of the gut.     

Cell death by apoptosis during involution of the lactating breast in mice and rats

The role of cell death in involution of lactating breast was investigated in mice and rats by light and electron microscopy. Apoptosis, recognized by sharply demarcated compaction of chromatin against the nuclear envelope and by shrinkage and budding of the whole cell to form membrane-bounded apoptotic bodies, was responsible for major loss of cells in both species. In the mouse, rapid involution during the first 2 days was associated with shedding of large numbers of apoptotic bodies derived from alveolar epithelial cells into alveolar lumens. This was followed by more gradual regression, during which the bodies were mostly phagocytosed by macrophages within the epithelium. In the rat, glandular involution was a more gradual and uniform process, with shedding of apoptotic epithelial cells into alveolar lumens being much less conspicuous. Apoptosis of myoepithelial cells was observed in mice, the resulting apoptotic bodies being phagocytosed by intraepithelial macrophages, but was not detected in rats. Apoptosis of capillary endothelial cells caused rapid regression of the capillary beds in both mice and rats. Intraepithelial macrophages increased in number during involution, developed cytoplasmic lipofuscin pigment, and either remained within the epithelium or migrated to the interstitium and regional nodes. Cell loss by apoptosis has been demonstrated during involution and atrophy of a variety of other glands. It characteristically results in shrinkage of a tissue without disruption of its basic architecture.     

Cure of colon cancer metastasis in rats with the new lipid A OM 174. Apoptosis of tumor cells and immunization of rats

The antitumoral effect of the new lipid A OM 174 was investigated in a model of colon cancer in rats. Peritoneal carcinomatosis were induced in BDIX rats by intraperitoneal injection of syngeneic PROb cancer cells. The treatment started 2 weeks later, when rats had macroscopic peritoneal nodules. An antitumoral effect was first obtained with OM 174 intraperitoneally injected, then an intravenous treatment was developed. When injected 15 times intravenously, at the dose of 1 mg/kg, 2 days apart, OM 174 induced the complete regression of tumors and hemorrhagic ascitis in 90% of the tumor-bearing rats, whereas all the untreated rats died of their tumors. To our knowledge, this treatment is the most effective ever applied to macroscopic tumors. Furthermore, the treatment induced the immunization of rats since the reinjection of PROb tumor cells in OM 174-cured rats did not cause the formation of new tumors while injection of another syngenic colon tumor cells did. Only in treated rats tumors were infiltrated with lymphocytes, macrophages and fibroblasts. The treatment did not increase necrosis but generated apoptotic areas. OM 174 was not directly toxic for tumor cells, and thus the observed effect involved the host-mediated antitumor reaction. Therefore we hypothesize that OM 174 therapy induces tumor cell apoptosis, stimulates the phagocytosis of apoptotic bodies and then activates immune system by antigen presentation.     

Facilitation by partial hepatectomy of tumor growth within the rat liver following intraportal injection of syngeneic tumor cells

The effects of both mechanical trauma and regeneration on the growth of intraportally injected tumor in the rat liver were investigated using two-thirds partial hepatectomy (PH). Tumor grew at the excision scar when PH was performed less than 2 days before tumor injection (34/34 animals). However, when the PH was performed 4–7 days before injection, tumor developed within the regenerating lobe, but not at the scar (50/51). Injecting the same cell dose into rats with intact livers caused few tumors to develop in 12/30 animals. Intraportally injected⁵¹Cr-labelled tumor cells distributed uniformly in the liver irrespective of the time after PH. Patterns of tumor take seen at different times after PH were not due to selective trapping of the injected cells. Liver extracts showed that epidermal growth factor-like activity was unaltered by PH, while heparin-binding growth factor activity peaked at 2 days post-PH, before the incidence of tumor growth in the parenchyma increased. We observed two peaks of DNA synthesis at days 1 and 4 post-PH by pulse labeling with [¹²⁵I]deoxyuridine and bromodeoxyuridine. Bromodeoxyuridine immunohistochemistry showed the first peak to be confined to hepatocytes. The second peak involved non-hepatocytes and coincided with the beginning of enhanced tumor take in the regenerating lobe.     

Granule neuron DNA damage following deafferentation in adult rats cerebellar cortex: a lesion model

Neuronal programmed cell death is regulated by a neurotrophic supply from targets and afferent inputs. The relative contribution of each component varies according to neuronal type and age. We have previously reported that primary cultures of cerebellar granule cells undergo apoptosis when deprived of depolarising KCl concentrations, suggesting a significant role of afferent inputs in the control of cerebellar granule cells survival. This issue was investigated by setting up various in vivo lesional paradigms in order to obtain partial or total deafferentation of the cerebellar granule layer in adult rats. At different times after surgery, cerebellar sections were subjected to TUNEL staining in order to detect possible DNA damage. One week after unilateral pedunculotomy, few scattered groups of apoptotic granule neurons were observed in the homolateral hemisphere. On the contrary, total deafferentation obtained by a new experimental paradigm based on an “L-cut” lesion induced massive and widespread apoptotic death in the granule layer of the deafferentated area. The time window of DNA fragmentation in granule layer was one to seven days after the “L-cut”. Selective Purkinje cell deafferentation obtained by 3-acetylpyridine injection did not result in TUNEL staining in the cerebellar cortex.The current finding that mossy fiber axotomy induces granule cell apoptotic death points out for the first time the crucial role of afferent inputs in mature granule cell survival. Moreover, the in vivo lesional model described here may prove to be an useful tool for investigating cellular and molecular mechanisms of neuronal death triggered by deafferentation.     

Sustainability of forestomach hyperplasia in rats treated with ethyl acrylate for 13 weeks and regression after cessation of dosing.

In a chronic study conducted by the National Toxicology Program (NTP), gavage administration of 100 or 200 mg ethyl acrylate (EA)/kg/day, 5 days/week, to F344 rats and B6C3F1 mice resulted in a significant dose-dependent increase in the incidence of squamous cell papillomas and carcinomas of the forestomach of both sexes of rats and mice. No increase in the incidence of tumors was observed at any other site in these rats. Chemically-induced cell proliferation is currently thought to play a role in the development and progression of chemically-induced neoplasia. Therefore, a stop-study was initiated where 100 or 200 mg EA/kg (in corn oil) was administered daily, 5 days/week, for 13 weeks. Rats sacrificed at the end of the treatment regimen had severe epithelial hyperplasia of the forestomach. No lesions were observed in the glandular stomach or liver of EA-treated rats. Forestomach hyperplasia induced by EA included upward and downward cell proliferation. However, forestomachs of rats treated for 13 weeks and sacrificed 8 weeks after the last EA dose exhibited a significant decline in the incidence and severity of forestomach mucosal hyperplasia. Histopathologic evaluation of forestomachs of EA-treated rats (13 weeks) which were allowed a 19-month-recovery (with no exposure to EA) showed further decline in the incidence and severity of mucosal cell hyperplasia. These results indicate that gavage administration of EA to rats results in extensive and sustained forestomach mucosal hyperplasia. The sustainability of forestomach hyperplasia is apparently dependent on the continued exposure of rats to ethyl acrylate, and regressed after cessation of dosing. Furthermore, although enough post-treatment time was allowed for tumors to develop after cessation of EA administration, forestomachs exhibited a nearly complete recovery with no increased incidence of papillomas or carcinomas. It, therefore, remains to be determined what duration of exposure or other factors are critical for reversibility or progression of EA-induced forestomach mucosal hyperplasia to neoplasia.     

Response of liver to lipopolysaccharide treatment in male and female rats

Gender is considered to be an important factor in endotoxin-induced tissue damage. Our aim was to examine the role of sex on the prooxidant–antioxidant status, necrotic and apoptotic events in the liver of lipopolysaccharide (LPS)-treated rats. We determined levels of lipid peroxides, non-enzymatic and enzymatic antioxidants, and expressions of apoptosis-related proteins, antiapoptotic B cell lymphoma-2 (Bcl-2) and proapoptotic Bax, caspase-3 activity and apoptotic cell numbers in the liver. Hepatic histopathology and serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were also investigated. Male and female Wistar rats (180–200 g) were injected with LPS (10 mg/kg, i.p.) and examinations were performed 6 h after the injection. Significant increases in hepatic thiobarbituric acid reactive substances and diene conjugate levels were observed in male and female rats following LPS treatment. However, there were no changes in hepatic glutathione, vitamin E and vitamin C levels together with superoxide dismutase, glutathione peroxidase and glutathione transferase activities. LPS treatment caused significant increases in serum ALT and AST activities and lymphocyte infiltration and necrotic changes in the livers. Bcl-2 and Bax expressions, caspase-3 activity and apoptotic cell numbers were also found to be increased in both groups. In conclusion, no sex-dependent difference was observed in the changed hepatic prooxidant–antioxidant status of rats following LPS treatment. Besides, the process leading to apoptosis and necrosis in the liver showed a similar pattern in both gender of rats.     

Cell deletion by apoptosis during regression of renal hyperplasia.

Regression of renal hyperplasia after withdrawal of the mitogenic stimulus induced by a single injection of lead nitrate was studied in male Wistar rats. Lead nitrate administration (10 mumol/100 g body weight) resulted in a ninefold increase in the incorporation of labeled thymidine into renal DNA and in an enhancement in the mitotic index; these changes were accompanied by an increase in the organ weight and DNA content that reached a maximum at 2 days. Regression of the renal hyperplasia was observed as early as 3 days after treatment and was completed within 2 weeks. Although lytic necrosis was not responsible for cell loss, the elimination of the excess renal cells took the form of apoptosis. This distinctive mode of cell death, which has been implicated in the involution of hyperplasia in other tissues and organs, was characterized by the occurrence of intracellular and extracellular membrane-bounded eosinophilic globules that often contained nuclear fragments. It affected mainly cells of the proximal tubules, and it was not detected once the kidney had regressed to its original mass. These results support the hypothesis that apoptosis is involved in the regulation of organ size.