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)-up-take. 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.     

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.     

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.     

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.     

Proliferation and differentiation of ductular progenitor cells and littoral cells during the regeneration of the rat liver to CCl4/2-AAF injury

Restoration of centrolobular injury induced by carbon tetrachloride (CCl4), when hepatocyte proliferation is inhibited by treatment with N-2-acetylaminofluorene (AAF), is accomplished by proliferation of ductular progenitor cells, that arise intraportally and extend into the liver lobule. This pattern contrasts to the restitutive proliferation of hepatocytes when AAF is not administered, and the proliferation of non-ductular periportal oval cells follows periportal necrosis induced by allyl alcohol. The expanding ducts stain for alphafetoprotein (AFP), OV-6, pan-cytokeratin (CKPan), and laminin. The neoductular proliferation is accompanied by fibronectin-positive Kupffer cells and desmin-positive stellate (Ito) cells, which may play critical roles not only in controlling proliferation and differentiation of ductular progenitor cells, but also in reestablishing hepatic cord structure. When AAF is discontinued 7 days after injury, clusters of small hepatocytes appear next to the neoductules. Some of these small hepatocytes, as well as some larger hepatocytes adjacent to the ducts, stain for AFP and for carbamoylphosphate synthetase I (CPS-I), suggesting that the ductular progenitor cells may differentiate into hepatocytes when AAF is withdrawn. The restitutive process is facilitated by clearing of the central necrotic zone by infiltrating macrophages and co-migration of mature hepatocytes, with Kupffer cells and stellate cells, into the necrotic zone.     

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.     

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.     

Liver inflammation and cytokine production, but not acute phase protein synthesis, accompany the adult liver progenitor (oval) cell response to chronic liver injury

Oval cells are facultative liver progenitor cells, which are invoked during chronic liver injury in order to replenish damaged hepatocytes and bile duct cells. Previous studies have observed inflammation and cytokine production in the liver during chronic injury. Further, it has been proposed that inflammatory growth factors may mediate the proliferation of oval cells during disease progression. We have undertaken a detailed examination of inflammation and cytokine production during a time course of liver injury and repair, invoked by feeding mice a choline-deficient, ethionine-supplemented (CDE) diet. We show that immediately following initial liver injury, B220-expressing leucocytes transiently infiltrate the liver. This inflammatory response occurred immediately before oval cell numbers began to expand in the liver, suggesting that the two events may be linked. Two waves of liver cytokine production were observed during the CDE time course. The first occurred shortly following commencement of the diet, suggesting that it may represent a hepatic acute phase response. However, examination of acute phase marker expression in CDE-fed mice did not support this hypothesis. The second wave of cytokine expression correlated with the expansion of oval cell numbers in the liver, suggesting that these factors may mediate oval cell proliferation. No inflammatory signalling was detected following withdrawal of the injury stimulus. In summary, our results document a close correlation between inflammation, cytokine production and the expansion of oval cells in the liver during experimental chronic injury.     

60 Hz magnetic field exposure induces DNA crosslinks in rat brain cells

In previous research, we found an increase in DNA strand breaks in brain cells of rats acutely exposed to a 60 Hz magnetic field (for 2 h at an intensity of 0.5 mT). DNA strand breaks were measured with a microgel electrophoresis assay using the length of DNA migration as an index. In the present experiment, we found that most of the magnetic field-induced increase in DNA migration was observed only after proteinase-K treatment, suggesting that the field caused DNA-protein crosslinks. In addition, when brain cells from control rats were exposed to X-rays, an increase in DNA migration was observed, the extent of which was independent of proteinase-K treatment. However, the X-ray-induced increase in DNA migration was retarded in cells from animals exposed to magnetic fields even after proteinase-K treatment, suggesting that DNA-DNA crosslinks were also induced by the magnetic field. The effects of magnetic fields were also compared with those of a known DNA crosslink-inducing agent mitomycin C. The pattern of effects is similar between the two agents. These data suggest that both DNA-protein and DNA-DNA crosslinks are formed in brain cells of rats after acute exposure to a 60 Hz magnetic field.     

Partial purification and characterisation of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy.

We have investigated the influences that nonparenchymal cells from regenerating rat liver exert on hepatocyte proliferation. When primary adult rat hepatocytes isolated from resting liver were co-cultured with nonparenchymal cells (NPCs) from resting liver of a different syngeneic animal, the proliferative response of hepatocytes to epidermal growth factor (EGF) was unaffected by the presence of NPCs. In the presence of NPCs taken from livers that had undergone partial hepatectomy 24 hours before (regen-NPCs), the response of hepatocytes from resting liver to EGF, TGF-alpha, and hepatocyte growth factor (HGF) was markedly inhibited. Inhibitory activity was not dependent on cell-to-cell contact, and conditioned-medium from regen-NPCs, but not normal NPCs, inhibited EGF-induced hepatocyte DNA synthesis by approximately 50%. After concentration by gel chromatography and lyophilisation, inhibition was 98%. The inhibitory activity migrated on SDS-PAGE gel electrophoresis with an apparent molecular weight of 14 to 17 kDa and was trypsin-sensitive but relatively heat-stable. The effects of blocking antibodies established that it was not TGF-beta 1, IL1-beta, or IL6. Investigations of regen-NPCs taken at different time points demonstrated that inhibitory activity was released into conditioned medium of cells harvested at 24 and 48 hours after partial hepatectomy, but not 10 or 72 hours. This powerful inhibitor of hepatocyte response to proliferogens is released by cultures of NPCs with a time course suggesting that it may be involved in terminating the surge of hepatocyte replication induced by partial hepatectomy.     

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.     

Enzymatic detection of x-ray-induced strand breaks in nuclear DNA on sections of mouse tissue.

An enzyme-histochemical method was used to detect X-ray-induced strand breaks in the DNA of mammalian tissue cells. Paraffin sections of ethanol-fixed mouse brain and liver were incubated with three kinds of exogenous DNA polymerizing enzymes, and the amount of in situ incorporation of 3H-deoxyribonucleotides into nuclear DNA was examined by autoradiography. No increase in labelling intensity was observed over nuclei of neurons and astrocytes in cerebral cortex, or over hepatocytes in liver immediately after X-irradiation when compared with unirradiated specimens. In liver Kupffer cells, heavily-labelled nuclei appeared from 30 min to 6 hours after, but were not observed immediately after X-irradiation. This method cannot, therefore, be applied to detect the strand breaks directly induced by X-rays, but it is useful in detecting secondary DNA degradation occurring as a result of nuclear degradation.     

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.     

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.     

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.     

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.     

The zinc fingers of human poly(ADP-ribose) polymerase are differentially required for the recognition of DNA breaks and nicks and the consequent enzyme activation. Other structures recognize intact DNA

The recognition of double-stranded DNA breaks and single-stranded nicks by human poly(ADP-ribose) polymerase and the consequent enzymic activation were examined using derivatives of the enzyme expressed in Escherichia coli. The N-terminal 162 residues encompass two zinc fingers. Deletion or mutation of the first finger results in a loss of activation by DNA with either single-stranded or double-stranded damage. Destruction of the second finger reduces activation by double-stranded DNA breaks only slightly, but eliminates activation by single-stranded DNA nicks. These data suggest that activation by single-stranded DNA nicks requires two zinc fingers, but activation by double-stranded DNA breaks requires only the finger closer to the N terminus. Variant proteins that lack both zinc fingers are enzymically inactive but still exhibit weak DNA binding, which is independent of DNA damage. Thus, other regions are also capable of binding intact DNA, but the recognition of a strand nick or break which occasions the synthesis of poly(ADP-ribose) specifically requires the zinc fingers.     

Stem and progenitor cells in liver regeneration and repair

Mammalian liver has a unique capacity to regenerate following resection or injury, and recovery of liver mass is mainly through proliferation of remaining adult hepatocytes. However, in pathologic conditions, especially during acute liver failure (ALF) and advanced stages of chronic liver disease (CLD), regeneration eventually fails and orthothopic liver transplantation (OLT) represents the only curative approach. The clinical scenario of a world-wide increasing incidence of end-stage CLD and an associated lack of organ availability has led several laboratories to explore the feasibility and efficiency of experimental alternatives to OLT involving cellular therapy. This review presents experimental and clinical studies performed in the last 10-15 years where adult and embryonic hepatocytes, hepatic stem/progenitor cells and extrahepatic stem cells have been used as transplantable cell sources.     

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.