Changes in DNA strand breaks in non-parenchymal cells following hepatocyte regeneration in CCl4-induced rat liver injury.

DNA strand breaks (nicks) in non-parenchymal cells (NPCs) in CCl4-induced acute or chronic liver injury in rats were detected using an in situ nick translation method; their dynamic changes were analysed in relation to the proliferation pattern of hepatocytes and NPCs, as revealed by bromodeoxyuridine (BrdU)-uptake. In acute injury, hepatocyte proliferation started before centrilobular necrosis had occurred, whereas BrdU-labeled sinusoidal NPCs markedly increased only after centrilobular necrosis was apparent. DNA breakages in NPCs paralleled the proliferation pattern of these cells, suggesting that nicks are physiological, and reflect proliferation and activated gene expression. In chronic injury, liver cirrhosis developed after 9 weeks, but BrdU-labeling of hepatocytes was almost the same level as that in untreated liver. The number of BrdU-labeled NPCs showed only a slight increase, while those with DNA breakages were much more frequent in the cirrhotic stage, suggesting a significant role for NPCs in the fibrotic process. These results indicate that DNA strand breaks in NPCs act as a marker for activation states such as proliferation, differentiation and/or activated gene expression.     

High levels of Myc expression are required for the robust proliferation of hepatocytes,but not for the sustained weak proliferation

In contrast to the robust proliferation exhibited following acute liver injury, hepatocytes exhibit long-lasting proliferative activity in chronic liver injury. The mechanistic differences between these distinct modes of proliferation are unclear. Hepatocytes exhibited robust proliferation that peaked at 2 days following partial hepatectomy in mice, but this proliferation was completely inhibited by hepatocyte-specific expression of MadMyc, a Myc-suppressing chimeric protein. However, Myc suppression induced weak but continuous hepatocyte proliferation, thereby resulting in full restoration of liver mass despite an initial delay. Late-occurring proliferation was accompanied by prolonged suppression of proline dehydrogenase (PRODH) expression, and forced PRODH overexpression inhibited hepatocyte proliferation. In hepatocytes in chronic liver injury, Myc was not activated but PRODH expression was suppressed in regenerating hepatocytes. In liver tumors, PRODH expression was often suppressed, especially in the highly proliferative tumors with distinct Myc expression. Our results indicate that the robust proliferation of hepatocytes following acute liver injury requires high levels Myc expression and that there is a compensatory Myc-independent mode of hepatocyte proliferation with the regulation of proline metabolism, which might be relevant to liver regeneration in chronic injury.     

Breakage of Chromosomal DNA with Aromatic Reductones

Strand breakages of mammalian cellular chromosomal DNA with aromatic reductones were ascertained by use of a cultured cell strain of the rat fetal lung (RFL). The mode of the breakages was investigated by ultracentrifugal analyses. The reductones induced the breakages of the cellular DNA in two different fashions; one is single strand breaks and another double strand breaks. Although the single strand breaks were rapidly repaired, double strand breaks were only partially repaired. Both breaks were not cytocidal. Some physiological alterations were observed to follow the strand breaks.     

CCl4-induced acute liver injury in mice is inhibited by hepatocyte growth factor overexpression but stimulated by NK2 overexpression

Hepatocyte growth factor (HGF) inhibits acute liver injury. NK2 acts as an antagonist to HGF in vitro, but its in vivo function has reached no consensus conclusions. We have investigated in vivo effects of HGF and NK2 on CCl4-induced acute liver injury. Elevation of the serum alanine aminotransferase level and extension of centrilobular necrosis were inhibited in HGF transgenic mice but were promoted in NK2 transgenic mice. Hepatocyte proliferation after liver injury was not inhibited in NK2 transgenic mice. Thus, this study indicates that HGF inhibits liver injury, and NK2 antagonizes HGF on liver injury, however, NK2 may not antagonize HGF on hepatocyte proliferation.     

A strong genotoxic effect in mouse skin of a single painting of coal tar in hairless mice and in MutaMouse

The dorsal skin of C3H/Tif/hr hairless mice was painted with coal tar, pharmacological grade. Epidermal cells and hepatocytes were isolated after 4, 24, 48 and 96 h and DNA strand breaks were determined as tail moment by the alkaline comet assay. The tail moment of epidermal cells was significantly greater at the time points 4, 24, 48 and 96 h after exposure compared to the controls, with the most DNA strand breaks at 24 h. The DNA strand breaks in epidermal cells increased linearly with the dose of coal tar. In hepatocytes, no difference in DNA strand breaks was found between exposed animals and controls. DNA adducts were determined by the 32P-postlabeling assay. For epidermal cells, the mean DNA adduct level was 12-fold greater in coal tar painted mice after 24 h than in controls. Again, a linear dose/response relationship was seen 24 h after painting. For liver DNA, the mean DNA adduct level was 3-fold greater than for controls. The mutation frequency in epidermal and liver cells was examined in lambdalacZ transgenic mice (MutaMouse). Thirty-two days after painting, the mutation frequency in epidermal cells was 16-fold greater in coal tar treated mice compared to controls. No effect was detected in hepatocytes. We found that a single painting of coal tar resulted in strong genotoxic effects in the murine epidermis, evidenced by induction of DNA strand breaks and DNA adducts in hairless mice and lambdalacZ mutations in the MutaMouse. This demonstrates that it is possible to detect genotoxic effects of mixtures with high sensitivity in mouse skin by these end-points.     

Plasma lysophosphatidic acid level and serum autotaxin activity are increased in liver injury in rats in relation to its severity

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological actions. We have reported that LPA stimulates hepatic stellate cell proliferation and inhibits DNA synthesis in hepatocytes, suggesting that LPA might play some role in the liver. We have found that plasma LPA level and serum autotaxin (ATX) activity were increased in patients with chronic hepatitis C. However, the clinical significance of LPA and its synthetic enzyme, autotaxin (ATX), is still unclear. To determine whether the increase of plasma LPA level and serum ATX activity might be found generally in liver injury, we examined the possible modulation of them in the blood in rats with various liver injuries. Plasma LPA level and serum ATX activity were increased in carbon tetrachloride-induced liver fibrosis correlatively with fibrosis grade, in dimethylnitrosamine-induced acute liver injury correlatively with serum alanine aminotransferase level or in 70% hepatectomy as early as 3 h after the operation. Plasma LPA level was correlated with serum ATX activity in rats with chronic and acute liver injury. ATX mRNA in the liver was not altered in carbon tetrachloride-induced liver fibrosis. Plasma LPA level and serum ATX activity are increased in various liver injuries in relation to their severity. Whether increased ATX and LPA in the blood in liver injury is simply a result or also a cause of the injury should be further clarified.     

Effect of ischemia-reperfusion on the heterogeneous lobular distribution pattern of glycogen content and glucose-6-phosphatase activity in human liver allograft.

In order to examine glucose metabolism in liver grafts after cold ischemia and reperfusion, the heterogeneous lobular distribution pattern of glycogen content and glucose-6-phosphatase activity was studied using histochemical methods. The characteristic heterogeneous lobular distribution pattern of glycogen and glucose-6-phosphatase was maintained after preservation and reperfusion. However, it appeared that glycogen content decreased in both periportal and centrilobular hepatocytes after reperfusion. The glycogen decrease was higher in periportal hepatocytes. Glucose-6-phosphatase activity was maintained after reperfusion in most of the cases in periportal hepatocytes. In centrilobular hepatocytes, more cases showed a decrease in enzyme activity. It is suggested that ischemia-reperfusion mainly affects the glycogen content in both periportal and centrilobular hepatocytes and that centrilobular glucose-6-phosphatase activity is more sensitive to ischemia-reperfusion injury than periportal hepatocytes.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Phenotype of single hepatocytes expressing an activated version of β-catenin in liver of transgenic mice

The gene CTNNB1 encoding β-catenin is mutated in about 30% of hepatocellular carcinoma, generally often combined with other genetic alterations. In transgenic mice, it has been shown that activation of β-catenin in more than 70% of all hepatocytes causes immediate proliferation leading to hepatomegaly. In this study we established a novel mouse model where β-catenin is activated only in individual, dispersed hepatocytes. Hepatocyte-specific expression of activated point-mutated β-catenin (human β-catenin^S33Y) was established using the Cre/loxP system. Expression of several downstream targets of β-catenin signaling such as glutamine synthetase and several cytochrome P450 isoforms was confirmed by immunostaining. Only a minor portion of hepatocytes expressed the β-catenin^S33Y transgene, which were mainly positioned as dispersed individual cells within the normal liver parenchyma. The hepatocytes with activated β-catenin did not show increased proliferation and the mice did not develop hepatomegaly. In conclusion, activated β-catenin in single hepatocytes induces a gene expression pattern in hepatocytes which is similar to that of Ctnnb1-mutated mouse liver tumors, but is apparently not sufficient to induce increased cell proliferation. Therefore, onset of proliferation seems to require concomitant activation of β-catenin in clusters of hepatocytes, suggesting a role of cell–cell communication in this process.     

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.     

Depletion of activated hepatic stellate cell correlates with severe liver damage and abnormal liver regeneration in acetaminophen-induced liver injury

Hepatic stellate cells (HSCs) are important part of the local 'stem cell niche' for hepatic progenitor cells (HPCs) and hepatocytes. However, it is unclear as to whether the products of activated HSCs are required to attenuate hepatocyte injury, enhance liver regeneration, or both. In this study, we performed 'loss of function' studies by depleting activated HSCs with gliotoxin. It was demonstrated that a significantly severe liver damage and declined survival rate were correlated with depletion of activated HSCs. Furthermore, diminishing HSC activation resulted in a 3-fold increase in hepatocyte apoptosis and a 66% decrease in the number of proliferating hepatocytes. This was accompanied by a dramatic decrease in the expression levels of five genes known to be up-regulated during hepatocyte replication. In particular, it was found that depletion of activated HSCs inhibited oval cell reaction that was confirmed by decreased numbers of Pank-positive cells around the portal tracts and lowered gene expression level of cytokeratin 19 (CK19) in gliotoxin-treated liver. These data provide clear evidence that the activated HSCs are involved in both hepatocyte death and proliferation of hepatocytes and HPCs in acetaminophen (APAP)-induced acute liver injury.     

Chromosomal fragmentation in dUTPase-deficient mutants of Escherichia coli and its recombinational repair

Recent findings suggest that DNA nicks stimulate homologous recombination by being converted into double-strand breaks, which are mended by RecA-catalysed recombinational repair and are lethal if not repaired. Hyper-rec mutants, in which DNA nicks become detectable, are synthetic-lethal with recA inactivation, substantiating the idea. Escherichia coli dut mutants are the only known hyper-recs in which presumed nicks in DNA do not cause inviability with recA, suggesting that nicks stimulate homologous recombination directly. Here, we show that dut recA mutants are synthetic-lethal; specifically, dut mutants depend on the RecBC-RuvABC recombinational repair pathway that mends double-strand DNA breaks. Although induced for SOS, dut mutants are not rescued by full SOS induction if RecA is not available, suggesting that recombinational rather than regulatory functions of RecA are needed for their viability. We also detected chromosomal fragmentation in dut rec mutants, indicating double-strand DNA breaks. Both the synthetic lethality and chromosomal fragmentation of dut rec mutants are suppressed by preventing uracil excision via inactivation of uracil DNA-glycosylase or by preventing dUTP production via inactivation of dCTP deaminase. We suggest that nicks become substrates for recombinational repair after being converted into double-strand DNA breaks.     

DNA damage response and cellular senescence in tissues of aging mice

The impact of cellular senescence onto aging of organisms is not fully clear, not at least because of the scarcity of reliable data on the mere frequency of senescent cells in aging tissues. Activation of a DNA damage response including formation of DNA damage foci containing activated H2A.X (γ-H2A.X) at either uncapped telomeres or persistent DNA strand breaks is the major trigger of cell senescence. Therefore, γ-H2A.X immunohistochemistry (IHC) was established by us as a reliable quantitative indicator of senescence in fibroblasts in vitro and in hepatocytes in vivo and the age dependency of DNA damage foci accumulation in ten organs of C57Bl6 mice was analysed over an age range from 12 to 42 months. There were significant increases with age in the frequency of foci-containing cells in lung, spleen, dermis, liver and gut epithelium. In liver, foci-positive cells were preferentially found in the centrilobular area, which is exposed to higher levels of oxidative stress. Foci formation in the intestine was restricted to the crypts. It was not associated with either apoptosis or hyperproliferation. That telomeres shortened with age in both crypt and villus enterocytes, but telomeres in the crypt epithelium were longer than those in villi at all ages were confirmed by us. Still, there was no more than random co-localization between γ-H2A.X foci and telomeres even in crypts from very old mice, indicating that senescence in the crypt enterocytes is telomere independent. The results suggest that stress-dependent cell senescence could play a causal role for aging of mice.     

Protein that induces cell differentiation causes nicks in double-stranded DNA

The growth and differentiation of myeloid hematopoietic cells are regulated by different macrophage and granulocyte inducing proteins, those that induce growth and others that induce differentiation. The proteins that induce differentiation but not those that induce growth bind to double-stranded DNA. We now report that purified myeloid cell differentiation-inducing protein causes single strand breaks (nicks) in double-stranded DNA. This DNA nicking may initiate the changes in gene expression that are required for differentiation.     

Chromosome demise in the wake of ligase-deficient replication

Bacterial DNA ligases, NAD⁺‐dependent enzymes, are distinct from eukaryotic ATP‐dependent ligases, representing promising targets for broad‐spectrum antimicrobials. Yet, the chromosomal consequences of ligase‐deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase‐deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double‐strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non‐allelic double‐strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double‐strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase‐deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double‐strand breaks and then into irreparable double‐strand gaps may be behind lethality of any DNA damaging treatment.     

Influence of DNA conformation on radiation-induced single-strand breaks

It is usually assumed that sparsely ionizing radiation produces randomly distributed DNA breakages. This seems to be supported by the finding that in some DNA fragments single-strand scissions occur uniformly at all nucleotide sites, regardless of sequence. We performed experiments on two DNA fragments of about 300 by having different conformation to test whether radiation-induced single-strand breakage is dependent on DNA conformation. Breakage analysis was carried out by denaturing polyacrylamide gel electrophoresis, which allows determination of the broken site at single nucleotide resolution. We found uniform cutting patterns in B-form regions. On the contrary, X- or-irradiation of curved fragments of kinetoplast DNA showed that the distribution of single-strand breaks was not uniform along the fragment, as the cleavage pattern was modulated in phase with the runs of A-T pairs. This modulation likely reflected the reduced accessibility of the sites which on hydroxyl-radical attack give rise to strand breaks. The cleavage pattern was phased with the runs of A-T pairs. Moreover, the overall yield of strand breaks was considerably lower in curved DNA fragments than in those with extended straight regions. The conformation effect found here indicates that the cleavage pattern reflects the fine structural features of DNA.     

The effect of Z-FA.FMK on D-galactosamine/TNF-alpha-induced liver injury in mice

Cathepsin B is a cysteine proteinase, considered to have an important role in apoptosis, which is activated by D-galactosamine and tumor necrosis factor-alpha (D-GalN/TNF-alpha). Benzyloxycarbonyl-L-phenylalanine fluoromethyl ketone (Z-FA.FMK) is a cathepsin B inhibitor used in research on apoptotic pathways. The aim of this study was to investigate the role of Z-FA.FMK on apoptotic cell death, cell proliferation and liver damage induced by a D-GalN/TNF-alpha combination in mice. In the study, 1 h after administration of 8 mg/kg Z-FA.FMK by intravenous injection, D-GalN (700 mg/kg) and TNF-alpha (15 microg/kg) were administered by a single intraperitoneal injection. In the group given D-GalN/TNF-alpha, the following results were found: Degenerative changes in the liver tissue, significant increase in the number of both TUNEL and activated caspase-3-positive hepatocytes, a decrease in the number of PCNA-positive hepatocytes, an increase in lipid peroxidation (LPO) levels and a decrease in glutathione (GSH) and DNA levels in the liver tissue. In contrast, in the group given D-GalN/TNF-alpha and Z-FA.FMK, a decrease in the damage of the liver tissue, a significant decrease in TUNEL and activated caspase-3-positive hepatocytes, a significant increase in the number of PCNA-positive hepatocytes, a decrease in the LPO levels, an increase in GSH and DNA levels in the liver tissue were found. As a result, microscopic and biochemical evaluations indicate that Z-FA.FMK plays a protective role against liver injury induced by D-GalN/TNF-alpha and it has an inverse effect on hepatocyte apoptosis and proliferation in BALB/c mice.     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

Early nuclear events in lymphocyte proliferation : The role of DNA strand break repair and ADP ribosylation

The previously reported extensive DNA strand breakage in resting murine splenic lymphocytes is not an artifact of the extraction or assay procedure. The benzamide inhibitors of poly(ADP ribose) synthetase (pADPRS), such as 5-methoxybenzamide (MBA), had been shown to block the strand break repair occurring within 2 h of activation of splenic lymphocytes by the mitogen concanavalin A (conA); the inhibitors also blocked early events in proliferation, such as blast formation, as well as entry into S phase. Inhibitors of pADPRS blocked lymphocyte proliferation by inhibiting the activity of this enzyme, rather than by non-specific effects. Aphidicolin, an inhibitor of alpha-polymerase, also prevented DNA strand break repair in conA-stimulated cells but, unlike MBA, did not prevent blast formation. DNA strand breaks accumulated in the presence of MBA at the same linear rate (300-400/h) in both resting and conA-treated cells. We and others had hypothesized that this accumulation was due to a continuous production of strand breaks in lymphocytes, leading to their accumulation in presence of repair inhibitors. However, incubation of the cells with aphidicolin at concentrations that inhibited repair did not result in any increase in strand breaks. The hypothesis of continuous cycling of breaks is incorrect; accumulation of breaks was due to some indirect effect of MBA, such as a possible disinhibition of an ADP-ribosylation-sensitive endonuclease described in other cell types. All of the early stages of lymphocyte proliferation, including blast transformation (but not DNA synthesis) require ADP ribosylation. Repair of DNA strand breaks is not a precondition for blast formation, though experiments involving the combined effects of MBA and aphidicolin showed that repair of the breaks is essential in order for the cells to replicate their DNA. Our data are consistent with a model suggesting that DNA strand breaks introduced into differentiated cells act as an additional safety-catch mechanism that restrains them from replicating their genetic material but not from undergoing the early stages of proliferation.