Inhibitory Effects of Trientine, a Copper-Chelating Agent, on Induction of DNA Strand Breaks in Kidney Cells of Long-Evans Cinnamon (LEC) Rats.

The effects of treatment with trientine, a specific copper-chelating agent, on the accumulation of copper and induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson's disease. Copper accumulated in the kidneys of LEC rats in an age-dependent manner from 12 to 18 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, renal copper contents did not increase and were maintained at the same levels as those in 4-week-old LEC rats. Estimation of the amounts of DNA single-strand breaks (SSBs) by comet assay showed that SSBs of DNA were induced in a substantial population of LEC rat renal cortex cells around 12 weeks of age and that the amounts of SSBs increased in an age-dependent manner from 12 to 18 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased, suggesting that induction of SSBs of DNA was inhibited and/or SSBs were repaired during the period of treatment with trientine. The results show that SSBs of DNA in LEC rat kidney cells are induced prior to occurrence of clinical signs of hepatic injury and that treatment of LEC rats with trientine decreases the number of DNA strand breaks.     

Accumulation of Copper Induces DNA Strand Breaks in Brain Cells of Long-Evans Cinnamon (LEC) Rats, An Animal Model for Human Wilson Disease.

Copper accumulation and induction of DNA strand breaks were investigated in the brain of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease that is a heritable disease of copper accumulation and copper toxicity in the liver, kidney and brain. Copper contents in the brain of LEC rats increased from 20 weeks of age and were approximately 3.5 to 6 folds higher than those in the brain of WKAH rats at 24 weeks of age. Hepatic copper contents in LEC rats increased from 4 to 12 weeks of age in an age-dependent manner, and then decreased from 16 to 20 weeks of age. Thus, we consider that copper accumulated in the liver was released from severely damaged hepatocytes and deposited in the brain, although copper contents in the brain were 1/20-fold lower than those in the liver. We also evaluated the amounts of DNA single-strand breaks (SSBs) in the brain by comet analysis. The proportions of nuclei in the cerebrum and cerebellum without DNA damage decreased, and nuclei with severe DNA damage appeared in LEC rats at 24 weeks of age. The comet scores of cerebrum and cerebellum cells significantly increased in LEC rats and were significantly higher than those in WKAH rats at 24 weeks of age. The results show that SSBs in LEC rat brain cells are induced at a lower concentration of copper than are SSBs in hepatic cells.     

Hepatic iron accumulation is not directly associated with induction of DNA strand breaks in the liver cells of Long-Evans Cinnamon (LEC) rats.

Effects of accumulation of copper and iron on induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant hepatitis. Copper and iron accumulated in the liver of LEC rats in an age-dependent manner from 4 to 15 weeks. Low-iron diet prevented the accumulation of iron in the liver, but did not prevent accumulation of copper. The amounts of DNA strand breaks that were estimated by comet assay in the liver cells of rats fed standard diet increased with age from 4 to 15 weeks. No significant differences were observed in the proportions of LEC rat liver cells without tail and the average lengths of tail momentum in the comet images between LEC rats that had been fed standard MF diet and low-iron diet. These results support the idea that accumulation of iron is not directly associated with the induction of DNA damage in the liver cells of LEC rats.     

Monte Carlo simulation of strand-break induction on plasmid DNA in aqueous solution by monoenergetic electrons

The radiation-induced process of strand breaks on pBR322 plasmid DNA in aqueous solution for different energy electrons was studied by Monte Carlo simulation. Assumptions of induction mechanisms of single- and double-strand breaks (SSBs and DSBs) used in the simulation are that SSB is induced by OH or H reaction with DNA and that DSB is induced by two SSBs on the opposite strands within 10 bp. Dose-response relationships of SSBs and DSBs were demonstrated for monoenergetic electrons of 100 eV, 10 keV, 1 keV and 1 MeV, and the yields of SSB and DSB were calculated. The dose-response relationships of SSBs and DSBs can be fitted by linear and linear-quadratic functions, respectively. The ratio of quadratic to linear components of DSB induction changes due to the electron energy. A high contribution of the linear component is observed for 1 keV electrons in the dose range below 160 Gy. The yields of SSBs and DSBs for all examined electron energies lie well within the experimental data when the probability of strand-break induction by OH and H is assumed to be around 0.1-0.2. The yield of SSBs has a minimum at 1 keV, while the yield of DSBs has a maximum at 1 keV in the examined energies. The strand breaks are formed most densely for 1 keV electrons.     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

High sensitivity to 5-azacytidine in LEC rats, a strain with a metabolic predisposition to hepatitis and hepatoma: possible involvement of DNA methylation in the expression of cytochrome P-450 and gamma-glutamyl transpeptidase.

The 5-methylcytosine (5-mCyt) content in hepatic DNA of LEC rats was measured in order to know the mechanism by which changes in the cytochrome P-450 content and gamma-glutamyl transpeptidase activity occur. At the age of 10 or 16 weeks, there was no difference in the extent of DNA methylation as compared with that of control strain (LEA) rats. However, in the hepatoma tissues that developed later in LEC animals, the percentage of 5-mCyt in the liver of LEC rats was markedly reduced. A single i.p. dose of 5-azacytidine brought about a significant reduction of 5-mCyt content with a concomitant decrease of cytochrome P-450 and an increase in gamma-glutamyl transpeptidase activity in LEC rats, whereas no such changes occurred in the control LEA rats. These results suggest that LEC rats are highly sensitive to 5-azacytidine and that a reduction in hepatic DNA methylation may play some role in the predisposition of the rats to hepatitis or hepatoma.     

Azaserine: further evidence for DNA damage in Escherichia coli

Azaserine causes DNA damage in stationary-phase cells. In our investigation of this damage, we used strains of Escherichia coli differing in repair capabilities to study azaserine-induced DNA damage, detected as DNA strand breaks by sucrose gradient sedimentation techniques. Reduced sedimentation in alkaline and neutral sucrose gradients indicated the presence of both alkali-labile sites and in situ strand breaks. Azaserine induced DNA single-strand breaks (SSBs) abundantly in all but the recA strain, in which SSBs were greatly reduced. Treatment of purified DNA with azaserine from bacteriophages T4 and PM2 produced no detectable SSBs. Several other studies also failed to detect DNA damage induced directly by azaserine. Increased levels of beta-galactosidase were induced in an E. coli strain possessing a rec::lac fusion, providing further evidence for azaserine induction of the recA gene product. In addition, azaserine induced adaptation against killing but not against mutagenesis in wild-type E. coli strain.     

Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G₁ phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci.     

No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro

Using the in vitro human diploid fibroblast model, we tested theories of aging which hypothesize that either accumulation of DNA damage or decreased DNA repair capacity is causally related to cellular senescence. Between population doubling level (PDL) 32 and 71, fetal lung-derived normal diploid human fibroblasts (IMR 90) were assayed for both DNA single-strand breaks (SSBs, spontaneous and induced by 6 Gy) and DNA double-strand breaks (DSBs, spontaneous and induced by 100 Gy). After gamma-irradiation cells were kept on ice unless undergoing repair incubation at 37 degrees C for 7.5-120 min or 18-24 h. To assay DNA strand breaks we used the filter elution technique in conjunction with a fluorometric determination of DNA which is not biased in favor of proliferating aging cells as are radioactive labelling methods. We found no change with in vitro age in the accumulation of spontaneous SSBs or DSBs, nor in the kinetics or completeness of DNA strand rejoining after gamma-irradiation. Cells at varying PDLs rejoined approx. 90% of SSBs and DSBs after 60 min repair incubation and 100% after 18-24 h repair incubation. We conclude that aging and senescence as measured by proliferative lifespan in IMR 90 cells are neither accompanied nor caused by accumulation of DNA strand breaks or by diminished capacity to rejoin gamma-radiation-induced SSBs or DSBs in DNA.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. VI. The correlation between UV-induced DNA lesions and chromosomal aberrations, and their modulations with inhibitors of DNA repair synthesis

The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.     

Spontaneous cholangiofibrosis in Long-Evans Cinnamon rats: a rodent model for Wilson's disease

The Long-Evans Cinnamon (LEC) rat is a rodent model of Wilson's disease characterized by ceruloplasmin deficiency, hepatic copper accumulation, and hepatocellular injury. So far, the LEC rat appears to be the only strain in which cholangiofibrosis develops spontaneously. The aim of the study reported here was to characterize the time course of development and investigate the structural and ultrastructural features of cholangiofibrosis and their possible relationship to hepatic copper and iron content. The livers of 54 rats (22 males), ages 5 to 113 weeks, were examined by light microscopy and graded for statistical analysis, with respect to extent of replacement of liver tissue by cholangiofibrosis. The study was complemented by electron microscopy, and by measurements of copper and iron contents by atomic absorption spectroscopy. Cholangiofibrosis was present in LEC rats by 20 weeks of age. The hyperplastic biliary epithelial cells varied markedly in size and shape, ranging from flat to cuboidal or elongated. Epithelial cells did not exhibit characteristics of intestinal cells. Some basement membranes had splits, duplications, or multiplications. Cytoplasmic organelles within hyperplastic biliary cells appeared unremarkable in contrast to the characteristic mitochondrial abnormalities present in neighboring hepatocytes. There was a positive correlation between histologic grades of cholangiofibrosis and ages of the animals (r = 0.68, P < 0.001), but no significant correlation between histologic grade and hepatic copper or iron content. We conclude that cholangiofibrosis is the predominant pathologic response to chronic liver injury induced by excess copper in LEC rats. The pathogenic role of copper in the development of cholangiofibrosis requires clearer definition.     

Radiation-induced DNA strand breaks and their repair in the developing rat brain.

Rats, 5, 10 or 25 days old, were 60 Co gamma irradiated. The induction of DNA strand breaks was studied after killing the rats within 1 min after irradiation, and the repair of the induced breaks after various intervals up to 180 min. Cell suspensions were prepared from the brain and samples were transferred into alkaline solutions. The fraction of DNA remaining double-stranded after 30 min alkali treatment was estimated after separation of single- and double-stranded DNA on hydroxylapatite. The amount of DNA strand breaks induced per Gray (1--8 Gray) was found to be in accordance with earlier in vivo studies of the mouse small intestine and mouse spleen. The DNA strand breaks in the rat brain induced by 4 Gray 60Co gamma irradiation were repaired 30 min after irradiation in all age groups studied.     

Quantitative assessment of the contribution of clustered damage to DNA double-strand breaks induced by 60Co gamma rays and fission neutrons

The induction of DNA strand breaks by fission neutrons was studied in aqueous plasmid (pBR322) DNA under aerobic conditions for a wide range of hydroxyl radical (*OH) scavenger concentrations and was compared to the induction of strand breaks by 6OCo gamma rays. Strand breaks were measured using agarose gel electrophoresis coupled with sensitive 32P-based phosphor imaging. Yields are reported for DNA single-strand breaks (SSBs) and double-strand breaks formed linearly with dose (alphaDSBs). The fraction of alphaDSBs that were dependent on the multiply damaged site (MDS) or clustered damage mechanism was also calculated using a model. G values for SSBs and alphaDSBs declined with increasing *OH scavenging capacity. However, with increasing *OH scavenging capacities, the decrease in yields of strand breaks for fission neutrons was not as pronounced as for gamma rays. The percentage of alphaDSBs for gamma rays was dependent on *OH scavenging capacity, appearing negligible at low scavenging capacities but increasing at higher scavenging capacities. In contrast, fission neutrons induced high percentages of alphaDSBs that were approximately independent of *OH scavenging capacity. The levels of alphaDSBs formed by the MDS mechanism after exposure to fission neutrons are consistent with the expected distinctive features of high-LET energy deposition events and track structure. The results also confirm observations made by others that even for low-LET radiation, the MDS mechanism contributes significantly to DNA damage at cell-like scavenging conditions.     

The semi-quantitative comparison of oxidative stress mediated DNA single and double strand breaks using terminal deoxynucleotidyl transferase mediated end labeling combined with a slot blot technique

The accumulation of DNA damages by environmental stresses is represented by the steady state level of single strand breaks (SSBs) and double strand breaks (DSBs). Terminal deoxynucleotidyl transferase (TdT) mediated end labeling is suitable in detecting DSBs, but is unsuitable for SSBs due to its catalyzing characteristics. However, the sensitivity of TdT to detect SSBs may be significantly improved by first denaturing the double strands and expose all the DNA nicks as potential substrates for TdT. By coupling DNA denaturation to slot blot southern hybridization, the authors demonstrate the sensitive detection of SSBs as well as DSBs in 20 ng DNA samples derived from a retinal pigment epithelial cell line treated with tert-butyl hydroperoxide. The signal intensity of denatured and TdT-treated DNA in slot blot hybridization correlated to the amount of SSBs calculated in an S1 nuclease digestion assay. The signal ratio between denatured and non-denatured DNA likely approximates the SSBs/DSBs ratio in genomic DNA. The combination of DNA denaturing, TdT treatment and slot blot hybridization could be a useful method to assess oxidative stress-induced DNA strand damages.     

Hepatic Oxidative Stress,Genotoxicity and Vascular Dysfunction in Lean or Obese Zucker Rats

Metabolic syndrome is associated with increased risk of cardiovascular disease, which could be related to oxidative stress. Here, we investigated the associations between hepatic oxidative stress and vascular function in pressurized mesenteric arteries from lean and obese Zucker rats at 14, 24 and 37 weeks of age. Obese Zucker rats had more hepatic fat accumulation than their lean counterparts. Nevertheless, the obese rats had unaltered age-related level of hepatic oxidatively damaged DNA in terms of formamidopyrimidine DNA glycosylase (FPG) or human oxoguanine DNA glycosylase (hOGG1) sensitive sites as measured by the comet assay. There were decreasing levels of oxidatively damaged DNA with age in the liver of lean rats, which occurred concurrently with increased expression of Ogg1. The 37 week old lean rats also had higher expression level of Hmox1 and elevated levels of DNA strand breaks in the liver. Still, both strain of rats had increased protein level of HMOX-1 in the liver at 37 weeks. The external and lumen diameters of mesenteric arteries increased with age in obese Zucker rats with no change in media cross-sectional area, indicating outward re-modelling without hypertrophy of the vascular wall. There was increased maximal response to acetylcholine-mediated endothelium-dependent vasodilatation in both strains of rats. Collectively, the results indicate that obese Zucker rats only displayed a modest mesenteric vascular dysfunction, with no increase in hepatic oxidative stress-generated DNA damage despite substantial hepatic steatosis.     

Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.     

In vivo evidence for accelerated generation of hydroxyl radicals in liver of Long-Evans Cinnamon (LEC) rats with acute hepatitis

The Long-Evans Cinnamon (LEC) rats accumulate excess copper (Cu) in the liver in a manner similar to patients with Wilson's disease (WD) and spontaneously develop acute hepatitis with severe jaundice. Although hydroxyl radicals (*OH) have been proposed to be a cause of hepatitis by the accumulation of Cu, it is not clear whether or not *OH can be produced in the liver of hepatitic LEC rats in vivo and also can be involved in the onset of hepatitis. In the present study, *OH production in plasma and liver of hepatitic LEC rats was quantified by trapping *OH with salicylic acid (SA) as 2, 3-dihydroxybenzoic acid (2, 3-DHBA). The ratios of 2, 3-DHBA/SA were significantly higher in plasma and liver of hepatitic LEC rats than those of Wistar rats and LEC rats showing no signs of hepatitis. Furthermore, the ratios of 2, 3-DHBA/SA in plasma and liver of hepatitic LEC rats were almost the same as those of Wistar rats treated orally with CuSO(4) (0.5 mmol/kg) 2 h before acetylsalicylic acid (ASA) injection. We also evaluated the protective effects of D-mannitol (a *OH scavenger) treatment against acute hepatitis in LEC rats. D-mannitol (500 mg/kg) was administered intraperitoneally to 10-week-old LEC rats for 3 weeks. D-mannitol treatment suppressed the increases in serum aspartate aminotransferase activity and total bilirubin concentration. In addition, D-mannitol treatment significantly reduced hepatic mitochondrial lipid peroxidation, which is thought to be important in the pathogenesis of Cu-induced hepatotoxicity. These observations suggest that accelerated generation of *OH catalyzed by free Cu in the liver may, at least in part, play a role in the pathogenesis of acute hepatitis in LEC rats.     

Spontaneous porphyria of the Long-evans cinnamon rat: an animal model of Wilson's disease

To confirm and extend our previous microspectrophotometric observations of 30-week-old male Long-Evans Cinnamon (LEC) rats, an animal model of human Wilson's disease, we analyzed the porphyrin patterns of the organs, urine, and plasma of LEC rats. Abnormal accumulation of porphyrins, especially highly carboxylated porphyrins (uro- and heptaporphyrin), in the kidneys and liver was seen in male and female LEC rats aged 30 weeks and also in 10-week-old rats, before the onset of spontaneous hepatic dysfunction. Accumulation of copper and iron in the kidneys was not observed in the 10-week-old rats. Massive accumulation of porphyrins was observed only in the kidneys of the 30-week-old male LEC rat, indicating that this symptom is related to sex and age. Renal accumulation of porphyrins was reflected in the rate of urinary porphyrin excretion. Hepatic accumulation of porphyrins appeared to be independent of sex and age. These results indicate that neither renal nor hepatic porphyrin accumulation is the result of renal deposition of metals or of spontaneous hepatic dysfunction and that porphyrinuria in the LEC rat is closely related to the renal accumulation of porphyrins. In contrast to these organs, a reduction in the porphyrin levels was observed in the brain of the LEC rat. This was independent of sex and age. The present work stresses the existence of an abnormal heme metabolism in the LEC rat, and thus, the necessity to study the heme metabolism in human Wilson's disease is strongly suggested.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

Effects of soy protein isolate on LEC rats,a model of Wilson disease: mechanisms underlying enhancement of liver cell damage

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.