Stress protein expression in rat liver during tumour promotion: induction of heat-shock protein 27 in hepatocytes exposed to 2-acetylaminofluorene

Exposure of cells to a variety of stresses such as heat, radiation and xenobiotics leads to increased expression of heat-shock proteins (HSPs). HSPs protect cells against irreversible protein damage and are involved in adaptive responses to stress stimuli. Some HSPs are overexpressed in neoplasias, possibly contributing to the increased drug tolerance often observed in such lesions. We have studied HSP expression in two experimental rat hepatocarcinogenesis models. Our aim was to clarify whether they are involved in stress adaptation in hepatocytes during carcinogen exposure, and whether HSPs may contribute to xenobiotic resistance in preneoplastic lesions. The complete carcinogen 2-acetylaminofluorene (AAF) was used in a continuous feeding protocol, and in the resistant hepatocyte model where the growth of diethylnitrosamine initiated lesions is efficiently promoted. Of the HSPs tested, only heat-shock protein 27 (hsp27) was induced during continuous AAF exposure. After 4 weeks of feeding AAF, increased hsp27 expression was noted in hepatocytes in perivenous areas of the liver lobule, possibly mediating an adaptive response to stress caused by reactive AAF metabolites. Enzyme altered preneoplastic foci were not found to overexpress HSPs. Thus, HSP induction does not seem to be a general mechanism underlying the increased stress tolerance observed in such lesions.     

Dichloroacetic acid induction of peroxisome proliferation in cultured hepatocytes

Trichloroethylene is a widespread industrial solvent and one of the most common environmental contaminants. Trichloroethylene causes hepatocarcinoma in the B6C3F1 mouse in a dose-dependent manner. Trichloroethylene's hepatocarcinogenicity is thought to be mediated through its metabolites trichloroacetate and dichloroacetate. Although the mechanism of action is not well understood, hepatic tumors are thought to arise as a result of excessive peroxisome-dependent active oxygen production or secondary to enhanced cell replication. The peroxisome proliferative activity of trichloroacetate has been replicated in cultured rodent hepatocytes, while that of dichloroacetate has not been demonstrated. The present experiments were designed to characterize the peroxisome proliferative response to dichloroacetate in hepatocyte cultures from male B6C3F1 mice and male Long Evans rats. The cultured hepatocytes were treated after attachment with 0.1, 0.5, 1.0, 2.0, or 4.0 mM dichloroacetate for 72 hours. Peroxisome proliferation was assessed by measuring palmitoyl-CoA oxidation and by immunoquantitation of peroxisomal bifunctional enzyme. Palmitoyl CoA oxidation increased in a concentration-dependent manner, with maximal induction of 5.5- and 5-fold in mouse and rat hepatocytes, respectively, after treatment with 2.0 mM dichloroacetate. Peroxisomal bifunctional enzyme protein levels also increased in a concentration-dependent manner in both rat and mouse hepatocytes in response to dichloroacetate exposure. These results indicate that the peroxisomal response observed in vivo in response to dichloroacetate administration can be reproduced in primary cultures of rat and mouse hepatocytes treated with dichloroacetate. Further studies using this model system will help elucidate mechanisms of dichloroacetate-induced hepatocarcinogenesis.     

Effects of iodinated contrast agents in MR imaging

At least two classes of mRNA for the GH receptor (GHR) and GH binding protein (GH BP) with different 5' untranslated first exons exist in the rat. One such class, the GHR1 is predominantly expressed in the liver of female rats. The hepatic expression of the GHR1 mRNA in normal and hypophsectomized rats of both sexes was studied by employing an RNase protection/solution hybridization assay. Normal females expressed 10-fold more GHR1 mRNA than males, hypophysectomy of female rats decreased the GHR1 level to that observed in male rats. Continuous GH treatment of hypophysectomized male and female rats for 6 days increased the expression of GHR1 mRNA to levels found in normal females, whereas intermittent GH treatment without effect. Bovine GH(bGH) induced the GHR1 expression in a time- and dose-dependent manner in primary cultures of adult rat hepatocytes as determined by solution hybridization. Maximal induction was achieved after 72 h of treatment with 50 ng bGH/ml medium. Female enriched expression of receptor and binding protein mRNAs raises the possibility that they participate in determining the ability of the liver to respond differently to the male and female GH secretory patterns. Our in vitro model utilizing cultures of primary adult rat hepatocytes could be used to address this issue as well as explore a hormonal interplay in regulation of GHR1 expression.     

gamma-Glutamyl transpeptidase-dependent lipid peroxidation in isolated hepatocytes and HepG2 hepatoma cells

Gamma-glutamyltranspeptidase (GGT), a plasma membrane-bound enzyme, provides the only activity capable to effect the hydrolysis of extracellular glutathione (GSH), thus favoring the cellular utilization of its constituent amino acids. Recent studies have shown however that in the presence of chelated iron prooxidant species can be originated during GGT-mediated metabolism of GSH, and that a process of lipid peroxidation can be started eventually in suitable lipid substrates. The present study was undertaken to verify if a GGT-dependent lipid peroxidation process can be induced in the lipids of biological membranes, including living cells, and if this effect can be sustained by the GGT highly expressed at the surface of HepG2 human hepatoma cells. In rat liver microsomes (chosen as model membrane lipid substrate) exposed to GSH and ADP-chelated iron, the addition of GGT caused a marked stimulation of lipid peroxidation, which was further enhanced by the addition of the GGT co-substrate glycyl-glycine. The same was observed in primary cultures of isolated rat hepatocytes, where the lipid peroxidation process did not induce acute toxic effects. GGT-stimulation of lipid peroxidation was dependent both on the concentration of GSH and of ADP-chelated iron. In GGT-rich HepG2 human hepatoma cells, the exposure to GSH, glycyl-glycine, and ADP-chelated iron resulted in a nontoxic lipid peroxidation process, which could be prevented by means of GGT inhibitors such as acivicin and the serine-boric acid complex. In addition, by co-incubation of HepG2 cells with rat liver microsomes, it was observed that the GGT owned by HepG2 cells can act extracellularly, as a stimulant on the GSH- and iron-dependent lipid peroxidation of microsomes. The data reported indicate that the lipid peroxidation of liver microsomes and of living cells can be stimulated by the GGT-mediated metabolism of GSH. Due to the well established interactions of lipid peroxidation products with cell proliferation, the phenomenon may bear particular significance in the carcinogenic process, where a relationship between the expression of GGT and tumor progression has been envisaged.     

The role of hepatocyte heterogeneity in the initiation of hepatocarcinogenesis

N-Nitrosodimethylamine (NDMA) is a carcinogen in rat liver while N-nitrosomethylbenzylamine (NMBzA) produces no liver tumors but is a potent esophageal carcinogen in the rat. Both nitrosamines, however, are metabolically activated in the liver and methylate hepatic DNA. The reasons for their different carcinogenic properties in rat liver are unclear. Here we show that as expected, NDMA produces large numbers of putative initiated hepatocytes that overexpress the placental form of glutathione S-transferase (GST-P+ hepatocytes). Hepatocyte division induced by the hepatotoxic effect of NDMA occurs principally in the periportal region of the liver lobule, while O6-methylguanine formation is principally in the DNA of perivenous cells. These two effects lead to the production of GST-P+ hepatocytes in roughly equal numbers throughout the liver lobule. NMBzA also induces the formation of a small, but significant number of GST-P+ hepatocytes. The NMBzA-induced GST-P+ hepatocytes are localized within the perivenous zone of the liver lobule. Since, unlike NDMA, NMBzA produces no hepatocellular necrosis and hence does not induce regenerative cell division, these results suggest that NMBzA initiates only those hepatocytes adjacent to the hepatic vein that are spontaneously dividing at the time their DNA becomes methylated by the nitrosamine. We used partial hepatectomy to stimulate cell division in specific regions of the liver lobule. When the peak of DNA methylation produced by NMBzA in the perivenous cells coincided with a peak of cell division in that region, an increased number of GST-P+ hepatocytes was induced. Our results suggest that the potency of initiating agents in the liver depends both on their ability to form mutagenic lesions in DNA and to induce division in the specific hepatocytes that contain the modified DNA.     

Monosialoganglioside GM1 inhibits neurotoxicity after hypothermic circulatory arrest

BACKGROUND/AIM: A herbal medicine, Sho-saiko-to (TJ-9), has recently been orally administered to patients with chronic liver disease in Japan and has been reported to inhibit the development of hepatocellular carcinoma. The aim of this study was to investigate whether TJ-9 has an inhibitory effect on the development of preneoplastic lesions and liver fibrosis in rats. METHODS: The effects of the TJ-9 were examined using the choline-deficient L-amino acid-defined (CDAA) diet-induced liver fibrosis model in 16-week-old male Wistar rats. RESULTS: TJ-9 (1% w/w) prevented fibrosis, as indicated by reduced hydroxyproline content in the liver and inhibition of the increase in a serum marker of fibrosis (hyaluronic acid), without reducing the increase in serum alanine aminotransferase and aspartate aminotransferase. TJ-9 also reduced the expression of type III procollagen alpha 1 mRNA in the liver, as well as the proliferation of myofibroblast-like cells (activated stellate cells, activated Ito cells). Furthermore, TJ-9 reduced the number of preneoplastic lesions, detected as enzyme-altered (glutathione S-transferase placental form-positive) lesions, in the liver. CONCLUSIONS: These results indicate that the herbal medicine Sho-saiko-to (TJ-9) prevents fibrosis as well as preneoplastic lesions, not by inhibiting hepatocyte cell death but by inhibiting the activation of stellate cells, which are considered to be the main collagen-producing cells, leading to a reduction in the development of preneoplastic lesions.     

The influence of medium composition on the maintenance of cytochrome P-450, glutathione content and urea synthesis: a comparison of rat and sheep primary hepatocyte cultures

Rat and sheep primary hepatocytes have been cultured in four different medium formulations: Williams' E, Chee's, Medium 199 and Modified Earle's. The total cytochrome P450 content, intracellular concentration of reduced glutathione, rate of urea synthesis and total protein content of cultures of cells from both species in each medium have been determined. Modified Earle's and Chee's medium proved to be the most favourable formulations for the culture of rat hepatocytes. After 48 h, cells cultured in Modified Earle's had significantly more cytochrome P450 and a significantly greater rate of urea synthesis than cells in any other medium. After 6 days in culture the difference in cytochrome P450 levels between rat hepatocytes in Chee's medium and those in Modified Earle's medium was abrogated. The difference in the rate of urea synthesis between rat hepatocytes cultured in each of these two media was shown to be more dependent on the medium in which the cells were maintained during the period of urea synthesis measurement than on the medium in which the cells had been previously cultured. Sheep hepatocytes cultured in Chee's medium ruptured and died within 24 h. Apart from this, sheep cells were less sensitive to changes in medium formulation than were rat hepatocytes. The initial plating efficiency was lower in sheep cells. Total cytochrome P450 content was the most discriminatory of the four parameters for evaluating the status of rat hepatocyte cultures. However, urea synthesis may be the most useful parameter for assessment of hepatocyte function in hybrid liver devices such as bioartificial liver support systems where access to the cells during operation of the device is restricted.     

Cytochrome P-450 induction by phenobarbital and 3-methylcholanthrene in primary cultures of hepatocytes

The characteristic hepatocellular changes resulting from phenobarbital administration in vivo, namely an increase in the levels of cytochrome P-450 and proliferation of membranes of the smooth endoplasmic reticulum, have been demonstrated in primary cultures of nonreplicating hepatocytes on floating collagen membranes. Addition of methylcholanthrene to the medium resulted in an increase in cytochrome P-448 within 48 hours, whereas the phenobarbital induction of P-450 required 5 days. These results demonstrate that responses induced in adult liver cells in vivo by phenobarbital can be reporoduced in cultured hepatocytes, contrary to previous reports.     

Multidrug resistance gene expression in rodents and rodent hepatocytes treated with mitoxantrone

Overexpression of P-glycoprotein in tumor cells can represent a severe drawback for cancer chemotherapy. P-glycoprotein acts as an efflux transporter for a variety of chemotherapeutic agents. It is encoded by multidrug resistance (mdr) genes of the subfamily 1 in humans (MDR1) and rodents (mdr1a and 1b). Because mdr1 gene expression is inducible in cultured rat hepatocytes and in rat liver with chemical carcinogens such as 2-acetylaminofluorene or aflatoxin B1, which form DNA-binding electrophiles during their metabolism, we investigated whether the DNA-damaging chemotherapeutic drug mitoxantrone may induce multidrug resistance in rodents and in hepatocytes in primary culture. In H4IIE rat hepatoma cells stably transfected with a luciferase construct containing the rat mdr1b promoter, mitoxantrone caused a concentration-dependent increase in promoter activity. Mdr1 gene expression in cultured rat hepatocytes was enhanced at mitoxantrone concentrations greater than or equal to 0.1 microM and in mouse hepatocytes at 5 microM. In hepatocytes from both species, a correlation was found between mdr1 induction and the inhibition of protein synthesis. In vivo, mitoxantrone was a very powerful inducer of mdr1 gene expression in rat liver and small intestine. In rat kidney, induction of mRNA was lower, and a marginal effect was seen in lung. In contrast with rats, no significant induction of mdr1 gene expression was obtained in mouse liver. Probably as a consequence of inhibition of protein synthesis, mitoxantrone did not lead to a pronounced elevation of P-glycoprotein levels in rat liver and kidney.     

Localization of oxidative damage by a glutathione-gamma-glutamyl transpeptidase system in preneoplastic lesions in sections of livers from carcinogen-treated rats

Previous studies from our laboratories have shown that catabolism of glutathione (GSH) by gamma-glutamyl transpeptidase (GGT) in the presence of transition metals leads to oxidative damage (OD). This damage is exemplified in vitro by GGT-dependent GSH mutagenesis which involves reactive oxygen species and by GGT-dependent accumulation of lipid peroxidation (LPO) products in systems containing polyunsaturated fatty acid and GSH. In order to test whether catabolism of GSH by membranal GGT in enzyme-altered preneoplastic hepatic lesions can induce oxidative damage in situ, and to test whether the OD is localized in these lesions, 21 day old Fischer rats were treated with 12 mg/kg diethylnitrosamine (DEN) followed by 0.1% or 0.25% phenobarbital (PB) in the diet. Cryostat sections were examined histochemically for GGT-rich hepatic lesions. Adjacent sections were incubated with GSH and iron and examined for areas staining for lipid peroxidation. Distinct LPO-positive areas were shown to correspond well with the GGT-positive hepatic lesions. Promotion with 0.25% PB led to increasing proportions of LPO-positive lesions with time among GGT-positive lesions. The visualization of LPO in GGT-rich hepatic lesions depended on the presence of GSH and iron, and was not observed following chelation of iron by diethyl triaminopentaacetic acid (DTPA), in the presence of acivicin, an inhibitor of GGT, or in the presence of the radical scavenger butylated hydroxytoluene (BHT). The factors affecting GSH-GGT-dependent LPO in the GGT-rich foci were identical to those affecting GSH-GGT-driven LPO in vitro, and were similar to those affecting oxidative GSH-mutagenesis catalyzed by GGT. The results indicate that metabolism of GSH by GGT in preneoplastic liver foci can initiate an oxidative process leading to a radical-rich environment and to oxidative damage. Such damage may contribute to the processes by which cells within such foci progress to malignancy.     

Inhibition by phenyl N-tert-butyl nitrone of early phase carcinogenesis in the livers of rats fed a choline-deficient, L-amino acid-defined diet

Male Wistar rats were fed a choline-deficient, L-amino acid-defined (CDAA) diet alone or in combination with a nitrone-based free radical trapping agent, phenyl N-tert-butyl nitrone (PBN) in the drinking water at the concentrations of 0.013, 0.065, and 0.130% for 12 weeks. PBN inhibited the changes that are normally induced in the livers of rats by the CDAA diet feeding, i.e., development of putative preneoplastic lesions, proliferation of connective tissue, reduction of glutathione S-transferase activity, formation of 8-hydroxyguanine in DNA, and an increase in inducible cyclo-oxygenase (COX2) activity. PBN, however, did not prevent the increases in the COX2 mRNA or protein levels brought on by the CDAA diet These results indicate that the loss of glutathione S-transferase activity and COX2 induction may play significant roles in rat liver carcinogenesis by the CDAA diet and that PBN prevents neoplasia not only by its radical scavenging activity but also by inhibiting COX2 activity at the catalytic level.     

Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-alpha-induced apoptosis by inducing heat shock protein 70 expression

Nitric oxide (NO) and tumor necrosis factor-alpha (TNFalpha) play important roles in the pathogenesis of liver disease during acute inflammation. The present study was designed to elucidate the effect of NO pre-exposure on TNFalpha-induced hepatotoxicity. Pretreatment of primary cultures of rat hepatocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of heat shock protein 70 (HSP70) mRNA and protein, which was associated with thermotolerance and cytoprotection from TNFalpha+actinomycin D-induced hepatotoxicity and apoptosis. SNAP transiently changed the intracellular redox state by inducing glutathione (GSH) oxidation associated with the formation of S-nitrosoglutathione (GSNO). HSP70 mRNA was also induced by the GSH-oxidizing agent diamide and the GSH-conjugating agent N-ethylmaleimide, suggesting that NO induces HSP70 expression through GSH oxidation. The protective effect of SNAP pretreatment on TNFalpha-induced apoptosis correlated with the level of HSP70 expression. SNAP pretreatment inhibited reactive oxygen intermediate generation and lipid peroxidation effects that were reversed by blocking HSP70 expression using an antisense oligonucleotide to HSP70. Finally, endogenous NO formation, induced in hepatocytes stimulated with interferon-gamma and interleukin-1beta, led to the formation of GSNO and GSSG, induced HSP70, and attenuated TNFalpha-mediated cytotoxicity. These findings demonstrated that NO can induce resistance to TNFalpha-induced hepatotoxicity, possibly through the stimulation of HSP70 expression.     

Ha-ras mutations in N-nitrosomorpholine-induced lesions and inhibition of hepatocarcinogenesis by antisense sequences in rat liver

To evaluate the application of Ha-ras mRNA antisense oligonucleotide therapy for liver tumors, we examined the frequency and types of mutation in codon 61 of the Ha-ras oncogene in preneoplastic lesions and hepatocellular carcinomas induced by N-nitrosomorpholine (NNM) in rats. Thirty-seven percent of preneoplastic lesions and 50% of hepatocellular carcinomas contained mutations, mostly CAA-CTA and CAA-AAA transversions. We also investigated the effects on NNM-induced lesions of an antisense oligonucleotide directed against a point mutation (CAA-CTA) in codon 61 of Ha-ras mRNA. In this experiment, Sprague-Dawley rats were given free access to water containing NNM for 8 weeks and received twice-weekly i.p. injections of a mutated Ha-ras antisense oligonucleotide with a 5' phosphorothioate linkage or a sense oligonucleotide in oligonucleotide-liposome complexes. At week 16, rats that had received the mutated Ha-ras antisense oligonucleotides had significantly fewer and smaller preneoplastic lesions positive for glutathione-S-transferase, placental type, and had smaller hepatocellular carcinomas than rats that had received the sense oligonucleotide. Mean cellular fluorescence in the liver was found to increase with higher doses of mutated, fluorescein-isothiocyanate-labeled antisense or sense oligonucleotides. Moreover, mutated Ha-ras antisense oligonucleotide decreased the expression of mutated Ha-ras mRNA (CAA-CTA). Our findings indicate that mutated Ha-ras antisense oligonucleotide significantly inhibits hepatocarcinogenesis in rats and could be an effective therapy against liver tumors.     

Enhanced oxygen delivery reverses anaerobic metabolic states in prolonged sandwich rat hepatocyte culture

It must be assumed that current petri dish primary hepatocyte culture models do not supply sufficient amounts of oxygen and thus cause anaerobic metabolism of the cells. This is contrary to the physiologic state of the cells. In vivo the liver is a highly vascularized organ with a rather high blood flow rate of a mixture of arterial and venous blood. The aim of the present study was to show the oxygen dependence of primary rat hepatocytes in long-term culture and to define appropriate conditions that could allow hepatocytes to maintain tissue specific functions in an aerobic environment. To this purpose matrix overlaid hepatocytes were either cultured on gas-permeable (fluorinated hydrocarbon films) or gas-impermeable (polystyrene) supports at 10% and 20% ambient oxygen concentration (v/v), respectively. Tissue-specific functions were assessed by studying albumin and urea secretion as well as xenobiotic metabolism. The mRNA expression and catalytic activities of the cytoprotective antioxidant enzymes mitochondrial manganese superoxide dismutase (MnSOD), cytosolic copper and zinc superoxide dismutase, peroxisomal catalase, and cytosolic glutathione peroxidase were investigated to assess intracellular responses to the defined variations in oxygen supply. Hepatocytes could successfully be maintained at aerobic conditions in long-term culture on gas-permeable PTFE films. At 50% (10%, v/v) of currently used oxygen levels lactate accumulation was prevented, a plateau-like albumin secretion reestablished, urea secretion improved, and xenobiotic metabolism proceeded at physiological rates. mRNA expression of cytoprotective enzymes responded to the pericellular availability of oxygen and was most pronounced in the case of MnSOD. However, the biggest stress factor for the hepatocytes still appeared to be the isolation procedure, as mRNA expression and catalytic activities were most elevated shortly thereafter. In conclusion, this study clearly shows the oxygen dependence of primary rat hepatocytes in long-term culture and indicates means to establish appropriate conditions for the aerobic culture of primary rat sandwich hepatocytes with full maintenance of function. The long-term culture of hepatocytes on oxygenating supports at in vivo-like oxygen tensions therefore appears to be more physiologic and beneficial for the cells.     

Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase

Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.     

Evaluation of auramine genotoxicity in primary rat and human hepatocytes and in the intact rat

Auramine, a dye previously found to be a liver carcinogen in both mice and rats, was evaluated for its DNA-damaging and clastogenic activities in primary cultures of rats and human hepatocytes and for the induction of DNA single-strand breaks in the liver and urinary bladder mucosa of intact rats. A similar dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic concentrations ranging from 10 to 32 microM. In contrast, neither rat nor human hepatocytes displayed an increased frequency of micronuclei after a 48-h exposure to the same auramine concentrations. In rats given a single oral dose of 125, 250 or 500 mg kg-1 auramine, the Comet assay revealed a significant increase in the frequency of DNA lesions in the liver and in the urinary bladder mucosa, the effect being slightly more marked in the liver. Taken as a whole and compared with previous findings, these results suggest that auramine is biotransformed into reactive species in target organs of both rats and humans, and that this dye might play by itself the main role in the increased incidence of bladder cancer which has been judged as causally related to its manufacture.     

Importance of and approaches to quantification of hepatocyte apoptosis

Abnormal regulation of the life cycle of cells is a key feature of neoplasia. The net increase and growth of initiated cells, preneoplastic lesions, and tumors is highly dependent on rates of both cell proliferation and cell death. Studies of mechanisms involved in regulation of cell death and the development of methods to detect dying and dead cells thus appear to be as important as measurements of cell proliferation in understanding the growth of both normal, preneoplastic and neoplastic lesions. This article describes apoptosis in the mouse liver and its potential role in liver carcinogenesis. Quantitation of hepatocyte apoptosis is a emerging and evolving research area that will require evaluations as thoroughly as those performed with cell proliferation in order to understand all the variables that might influence its occurrence, measurement, and interpretations. Utilizing available data, various methodologies for identifying hepatocyte apoptosis are presented and compared. Aspects important for the quantitation of apoptosis in liver are emphasized. Accurate quantitation of apoptosis, in conjunction with proliferation measurements, is critical for investigations of the mechanisms of chemically induced carcinogenesis and the development of assays for growth alterations and can be applied to biologically based cancer models.     

Bacterial lipopolysaccharide induces uncoupling protein-2 expression in hepatocytes by a tumor necrosis factor-alpha-dependent mechanism

The liver is a target for bacterial lipopolysaccharide (LPS) and participates in the metabolic response to endotoxemia. Recently published evidence indicates that LPS increases the expression of mitochondrial uncoupling protein-2 (UCP-2) mRNAs in several tissues, including the liver. Because hepatocytes in the healthy liver do not express UCP-2, LPS was thought to induce UCP-2 in liver macrophages, which express UCP-2 constitutively. However, the present studies of cultured peritoneal macrophages indicate that LPS reduces steady state levels of UCP-2 mRNAs in these cells. In contrast, UCP-2 mRNAs are induced in hepatocytes isolated from LPS treated rats and transfection of these hepatocytes with UCP-2 promoter-reporter constructs demonstrates substantial increases in UCP-2 promoter activity. LPS induction of hepatocyte UCP-2 expression is virtually abolished by prior treatment of rats with neutralizing antibodies to tumor necrosis factor alpha (TNF). Futhermore, TNFalpha treatment induces UCP-2 mRNA accumulation in primary cultures of hepatocytes from healthy rats. Thus, hepatocytes are likely to be important contributors to endotoxin-related increases in liver UCP-2 via a mechanism that involves the LPS-inducible cytokine, TNFalpha.     

Resuming of thermoregulation in rats during deep hypothermia with EDTA without rewarming the body

Carcinogen-resistant inbred DRH rats developed from the Donryu strain showed a remarkably low incidence of liver tumors when they were fed diets containing hepatocarcinogens such as 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). In this work, we examined various characteristics of male DRH and Donryu rats during 3'-Me-DAB administration for 8 weeks. 32P-Postlabeling analysis showed that essentially similar levels of DNA-adducts were generated by the metabolites of 3'-Me-DAB in the livers of these two strains of rats at several time points. However, both GADD45 (growth arrest and DNA damage-inducible) and O6-methylguanine methyltransferase (putatively DNA damage-inducible) mRNA levels were increased significantly in Donryu rat livers, but were increased to a lesser extent in DRH rats. [3H]Thymidine incorporation into hepatic DNA began to increase around 10 to 20 days after the start of 3'-Me-DAB administration in Donryu rats probably due to DNA repair, while no significant change occurred in DRH rats under the same conditions. Furthermore, inductions of heme oxygenase (due to degradation of heme-proteins) and hepatocyte growth factor (HGF; cell death and regeneration of hepatocytes) mRNAs were greater in Donryu rat livers than those of DRH, suggesting that the former were more sensitive to cytotoxic effects of 3'-Me-DAB than the latter. Another remarkable difference observed between these two strains was the significant induction of cytochrome P-450 2E1 mRNA in Donryu rat livers; this may contribute to the generation of reactive oxygen intermediates. Finally, increases of glutathione S-transferase (P-form) and gamma-glutamyltranspeptidase mRNAs as marker enzymes of preneoplastic changes of hepatocytes were clearly seen only in Donryu rat livers at 6 to 8 weeks after the start of 3'-Me-DAB administration. These results indicate that the different susceptibility to hepatocarcinogenesis between these two strains of rats may arise from events other than the DNA adduct formation.