Inhibitory effects of trientine, a copper-chelating agent, on induction of DNA strand breaks in hepatic cells of Long-Evans Cinnamon rats

Effects of treatment with trientine, a specific copper-chelating agent, on 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 livers of LEC rats in an age-dependent manner from 4 to 13 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, hepatic copper contents did not increase and were maintained at the same levels as those in 10-week-old LEC rats. When the amounts of DNA single-strand breaks (SSBs) were estimated by a comet assay, SSBs of DNA were induced in a substantial population of LEC rat hepatic cells around 8 weeks of age and the amounts of SSBs increased in an age-dependent manner from 8 to 15 weeks of age. When LEC rats were treated with trientine from 10 weeks of age, the observed number of cells with DNA damage decreased dramatically, 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 treatment of LEC rats with trientine decreases the number of DNA strand breaks observed, although copper contents remain high in the liver.     

Camptothecin cytotoxicity in mammalian cells is associated with the induction of persistent double strand breaks in replicating DNA.

Camptothecin is a specific topoisomerase I poison and is highly cytotoxic to eukaryotic cells. In the present study, we show, using a pulse field gel electrophoresis assay, that camptothecin induces DNA double strand breaks (DSBs) specifically in newly replicated DNA. Camptothecin induces these replication associated DNA DSBs in a dose-dependent manner. At levels of the drug which are toxic to the cell, these breaks are long-lived, and still measurable 24 hr after treatment. Both camptothecin induced DSBs and cytotoxicity are prevented by co-exposure with aphidicolin--a result which indicates that ongoing DNA synthesis is required for the production of DNA DSBs and cell killing. It has been proposed that camptothecin toxicity involves an interaction between the replication machinery and a drug-mediated topoisomerase I-DNA cleavable complex. The present work indicates, for the first time in mammalian cellular DNA, that one possible outcome of this interaction is a replication-associated DSB, a lesion which is likely to be highly cytotoxic.     

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.     

The antioxidant effect of DL-alpha-lipoic acid on copper-induced acute hepatitis in Long-Evans Cinnamon (LEC) rats

The Long-Evans Cinnamon (LEC) rats, due to a genetic defect, accumulate excess copper (Cu) in the liver in a manner similar to patients with Wilson's disease and spontaneously develop acute hepatitis with severe jaundice. In this study we examined the protective effect of DL-alpha-Lipoic acid (LA) against acute hepatitis in LEC rats. LA was administered to LEC rats by gavage in doses of 10, 30 and 100 mg/kg five times per week, starting at 8-weeks-old and continuing till 12-weeks-old. Although LA had little effect against the increases in serum transaminase activities, it suppressed the loss of body weight and prevented severe jaundice in a dose-dependent manner. Antioxidant system analyses in liver showed that LA treatment significantly suppressed the inactivations of catalase and glutathione peroxidase, and the induction of heme oxygenase-1, an enzyme which is inducible under oxidative stress. Furthermore, LA showed dose-dependent suppressive effect against increase in nonheme iron contents of both cytosolic and crude mitochondrial fractions in a dose-dependent manner. Although at the highest dose, LA slightly suppressed the accumulation of Cu in crude mitochondrial fraction, it had no effect on the accumulation of Cu in cytosolic fraction. While LA completely suppressed the increase in lipid peroxidation (LPO) in the microsomal fraction at the highest dose, the suppressive effect against LPO in crude mitochondrial fractions was slight. From these results, it is concluded that LA has antioxidant effects at the molecular level against the development of Cu-induced hepatitis in LEC rats. Moreover, mitochondrial oxidative damage might be involved in the development of acute hepatitis in LEC rats.     

Persistence and accumulation of (potential) single strand breaks in liver DNA of rats treated with diethylnitrosamine or dimethylnitrosamine: correlation with hepatocarcinogenicity.

Effects of diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) on the sedimentation pattern of [3H]thymidine-labelled Sprague-Dawley female rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. In experiments at 1--56 days after a single injection it was observed that (potential) single strand breaks induced by DEN were repaired at a low rate. At 56 days the sedimentation pattern was still grossly abnormal. Half-life values of 27 and 46 days were observed after 134 mg/kg DEN (approx. 45% of the LD50) and 13.4 mg/kg DEN, respectively. Identical experiments after DMN (10 mg/kg, corresponding to about 35% of the LD50) showed return to (almost) completely control sedimentation patterns within 56 days after injection (t 1/2 = 8 days). Experiments at 6 or 56 days after the last of a series of 5 or 10 weekly injections of DEN (13.4 mg/kg) showed that a major part of DEN-induced damage (measured as single strand breaks) is of a persistent and accumulating character. No accumulation of DMN-induced rat liver lesions was observed. It is concluded that DNA fragmentation and lack of DNA repair is not a consequence of hepatotoxicity. Since at equimolar doses DEN gives appreciably less DNA alkylation (including O6-alkylguanine) but is much more effective both as an inducer of preneoplastic liver lesions and as a hepatocarcinogen when compared with DMN, we believe that the formation of persistent (and accumulating) DNA damage after DEN administration might be relevant in the process of liver tumour formation.     

Copper-metallothionein induction in the liver of LEC rats.

Recently, copper (Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.     

Deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins in hyper-radiosensitive Long-Evans Cinnamon (LEC) rat cells after X irradiation

The DNA-dependent protein kinase (DNA-PK) complex has been implicated in the repair of DNA double-strand breaks (DSBs). DNA-PK is a heterotrimeric protein complex comprised of two components: a large catalytic subunit, Prkdc, with serine/threonine kinase activity and a DNA-targeting component, G22p1 and Xrcc5. In previous report, we showed that approximately 80% of the G22p1 and Xrcc5 proteins were observed in the cytoplasm of rat fibroblasts, and that nuclear translocation of the proteins from the cytoplasm is important for the repair of DNA DSBs. In the present study, we showed that nuclear accumulation of the G22p1 and Xrcc5 proteins was not observed in fibroblasts from a mutant strain of Long-Evans Cinnamon (LEC) rat that has an enhanced radiosensitivity and a reduced level of repair of DSBs after X irradiation. Nuclear translocation of the proteins was observed in both LEC rat cells and control rat cells with normal radiosensitivity at 5 min after X irradiation. Although high levels of G22p1 and Xrcc5 proteins were observed in the nuclei of control rat cells until 60 min postirradiation, the amounts of the proteins decreased rapidly in the nuclei of LEC rat cells in the first 10 min after X irradiation. These findings suggest that there are some defects in maintaining the levels of G22p1 and Xrcc5 proteins in the nuclei of LEC rat cells. An analysis of fibroblasts from backcross rats showed that the deficiency in nuclear accumulation of G22p1 and Xrcc5 proteins is genetically linked to enhanced radiosensitivity. Since the nucleotide sequences of the G22p1 and Xrcc5 genes of the LEC rats coincided with those of the control rats, the deficiency in nuclear accumulation may not be caused by mutations of the G22p1 and Xrcc5 proteins.     

Proteomic analysis of the hepatic tissue of Long-Evans Cinnamon (LEC) rats according to the natural course of Wilson disease

Copper-induced toxicity is important in the pathogenic process of Wilson's disease (WD). Using Long-Evans Cinnamon (LEC) rats, an animal model of WD, the study was undertaken to identify proteins involved in the process of WD and to investigate their functional roles in copper-induced hepatotoxicity. In early stages, expression levels of mitochondrial matrix proteins including agmatinase, isovaleryl coenzyme A dehydrogenase, and cytochrome b5 were downregulated. As mitochondrial injuries progressed, along with subsequent apoptotic processes, expressions of malate dehydrogenase 1, annexin A5, transferrin, S-adenosylhomocysteine hydrolase, and sulfite oxidase 1 were differentially regulated. Notably, the expression of malate dehydrogenase 1 was downregulated while the annexin A5 was overexpressed in an age-dependent manner, indicating that these proteins may be involved in the WD process. In addition, pronounced under-expression of S-adenosylhomocysteine hydrolase in elderly LEC rats, also involved in monoamine neurotransmitter metabolism, indicates that this protein might be related to the development of neurological manifestations in WD. The results of our study help to understand the pathogenic process of WD in hepatic tissues, identifying the important proteins associated with the disease process of WD, and to investigate the molecular pathogenic process underlying the development of neurological manifestations in WD.     

Unusual accumulation of copper related to induction of metallothionein in the liver of LEC rats.

Copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn) levels in organs of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis, were determined by instrumental neutron activation analysis method. Unusual accumulations of Cu in the liver of LEC rats were found, depending on the age of the animals, the metal concentration being more than approximately 20-40 times those of normal LEA rats (Long-Evans rats with an agouti coat color). Fe and Zn were also accumulated, in addition to Cu, significantly in the LEC rats. The unusual Cu accumulations in the liver of LEC rats were associated with the induction of metallothionein, estimated by radioimmunoassay method, in the liver of LEC rats, rather than that of superoxide dismutase, estimated by electron spin resonance -spin trapping method. These findings suggest that the unusual Cu accumulation in LEC rats is involved in the development of jaundice, hepatic injury and hepatocellular carcinoma.     

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.     

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.     

DNA strand breaks signal the induction of DNA double-strand break repair in Saccharomyces cerevisiae

Genotoxic stress induces a checkpoint signaling cascade to generate a stress response. Saccharomyces cerevisiae shows an altered radiation response under different type of stress. Although the induction of repair has been implicated in enhanced survival after exposure to the challenging stress, the nature of the signal remains poorly understood. This study demonstrates that low doses of gamma radiation and bleomycin induce RAD52-dependent recombination repair pathway in the wild-type strain D-261. Prior exposure of cells to DNA-damaging agents (gamma radiation or bleomycin) equips them better for the subsequent damage caused by challenging doses. However, exposure to UV light, which does not cause strand breaks, was ineffective. This was confirmed by PFGE studies. This indicates that the strand breaks probably serve as the signal for induction of the recombination repair pathway while pyrimidine dimers do not. The nature of the induced repair was investigated by mutation scoring in special strain D-7, which showed that the induced repair is essentially error free.     

Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease.

Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.     

The accumulation of MMS-induced single strand breaks in G1 phase is recombinogenic in DNA polymerase beta defective mammalian cells

DNA polymerase (Pol) β null mouse embryonic fibroblasts provide a useful cell system to investigate the effects of alterations in base excision repair (BER) on genome stability. These cells are characterized by hypersensitivity to the cytotoxic effects of methyl methanesulfonate (MMS) and by decreased repair of the MMS-induced DNA single strand breaks (SSB). Here, we show that, in the absence of Pol β, SSB accumulate in G₁ phase cells, accompanied by the formation of proliferating cell nuclear antigen foci in the nuclei. When replicating Pol β null cells are treated with MMS, a rapid phosphorylation of histone H2AX is detected in the nuclei of S phase cells, indicating that double strand breaks (DSB) are formed in response to unrepaired SSB. This is followed by relocalization within the nuclei of Rad51 protein, which is essential for homologous recombination (HR). These findings are compatible with a model where, in mammalian cells, unrepaired SSB produced during BER are substrates for the HR pathway via DSB formation. This is an example of a coordinated effort of two different repair pathways, BER and HR, to protect mammalian cells from alkylation-induced cytotoxicity.     

Repair of radiation-induced DNA strand breaks does not occur preferentially in transcriptionally active DNA.

Certain DNA base lesions induced by ionizing radiation or oxidative stress are repaired faster from the transcribed strand of active genes compared to the genome overall. In this study, it was investigated whether radiation-induced DNA strand breaks are preferentially repaired in active genes compared to the genome as a whole in CHO cells. The alkaline unwinding technique coupled to slot-blot hybridization with specific DNA probes was used to study the induction and repair of DNA strand breaks in defined DNA sequences. Results using this technique showed a linear dose response for the formation of radiation-induced DNA strand breaks in the dihydrofolate reductase (DHFR) gene. Furthermore, the half-life of radiation-induced strand breaks was less than 5 min in the DHFR gene, in the ribosomal genes, and in the genome as a whole. These results suggest that the repair of DNA strand breaks is fast and uniform in the genome of mammalian cells.     

DNA maintenance following bleomycin-induced strand breaks does not require poly(ADP-ribosyl)ation activation in Drosophila S2 cells

BackgroundPoly-ADP ribosylation (PARylation) is a post translational modification, catalyzed by Poly(ADP-ribose)polymerase (PARP) family. In Drosophila, PARP-I (human PARP-1 ortholog) is considered to be the only enzymatically active isoform. PARylation is involved in various cellular processes such as DNA repair in case of base excision and strand-breaks.ObservationsStrand-breaks (SSB and DSB) are detrimental to cell viability and, in Drosophila, that has a unique PARP family organization, little is known on PARP involvement in the control of strand-breaks repair process. In our study, strands-breaks (SSB and DSB) are chemically induced in S2 Drosophila cells using bleomycin. These breaks are efficiently repaired in S2 cells. During the bleomycin treatment, changes in PARylation levels are only detectable in a few cells, and an increase in PARP-I and PARP-II mRNAs is only observed during the recovery period. These results differ strongly from those obtained with Human cells, where PARylation is strongly activating when DNA breaks are generated. Finally, in PARP knock-down cells, DNA stability is altered but no change in strand-breaks repair can be observed.ConclusionsPARP responses in DNA strands-breaks context are functional in Drosophila model as demonstrated by PARP-I and PARP-II mRNA increases. However, no modification of the global PARylation profile is observed during strand-breaks generation, only changes at cellular levels are detectable. Taking together, these results demonstrate that PARylation process in Drosophila is tightly regulated in the context of strands-breaks repair and that PARP is essential during the maintenance of DNA integrity but dispensable in the DNA repair process.     

Inhibition of poly(ADP-ribose) polymerase causes increased DNA strand breaks without decreasing strand rejoining in alkylated HeLa cells

Treatment of alkylated HeLa cells with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, increased the number of DNA strand breaks but did not affect the rate of strand rejoining. This suggests that an increase in DNA incision, not a decrease in ligation, results from the inhibition ofpoly(ADP-ribose) polymerase in cells recovering from DNA damaged by alkylating agents. Poly(ADP-ribose) DNA strand break DNA repair     

Assessment of radiation-induced DNA strand breaks and their repair in unlabeled cells.

Radiation induced damage, i.e., the induction of DNA strand breaks, was studied on the level of single, unlabeled cells. DNA strand breaks were determined by direct partial alkaline unwinding in intact cell nuclei followed by staining with acridine orange, a development of a proposal first described by B. Rydberg (Int J Radiat Biol 46:521-527, 1984). The ratio of green fluorescence (double-stranded DNA) to red fluorescence (single-stranded DNA) in single cells was taken as a measure of DNA strand breaks. CHO-K1 and M3-1 cells irradiated with X-rays show a dose dependent induction of DNA strand breaks. Incubation at 37 degrees C after irradiation leads to repair of breaks. A repair halflife of about 10-11 min can be determined. Cell cycle specific differences in the induction of DNA strand breaks or repair behavior are not detectable at the resolution achieved so far. This new method offers two major advantages: the resolution of DNA damage and repair on the level of single cells and no need for labeling, thereby allowing for DNA damage and repair to be assessed in biopsy material from tumor patients.     

Suppression of tumor suppressor Tsc2 and DNA repair glycosylase Nth1 during spontaneous liver tumorigenesis in Long-Evans Cinnamon rats

Chronic inflammation and oxidative stress are arguably associated with an increased risk of cancer. Certain diseases that are characterized by oxyradical overload, such as Wilson’s disease (WD), have also been associated with a higher risk of liver cancer. The Long-Evans Cinnamon (LEC) rat, an animal model for WD, is genetically predisposed to the spontaneous development of liver cancer and has been shown to be very useful for studying the mechanisms of inflammation-mediated spontaneous carcinogenesis. Endonuclease III (Nth1) plays a significant role in the removal of oxidative DNA damage. Nth1 and a tumor suppressor gene Tuberous sclerosis 2 (Tsc2) are bi-directionally regulated in humans, mice, and rats by a common minimal promoter containing two Ets-binding sites (EBSs). In this study, we examined the expression of Nth1 and Tsc2 genes during disease progression in the LEC rat liver. During the period of acute hepatitis (16–17 weeks), we observed decreased Nth1 and Tsc2 mRNA levels and a continued decrease of the Tsc2 gene in 24 weeks in LEC rats, while the effect was minimal in Long-Evans Agouti (LEA) rats. This reduction in the mRNA levels was due to the reduced binding of EBSs in the Nth1/Tsc2 promoter. Increase in protein oxidation (carbonyl content) during the same time period (16–24 weeks) may have an effect on the promoter binding of regulatory proteins and consequent decrease in Nth1 and Tsc2 gene expressions during tumorigenesis.     

The repair of DNA single strand breaks in human cells with genetical disorders

The aim of this work was to make a preliminary examination of some hereditary diseases, related to precocious aging in order to determine whether the defects involved resulted from an impairment of the DNA repair capacity. To test for an impairment in DNA single strand capacity, in cells from patients with different diseases, we have followed the rate of the DNA after X-irradiation by sedimentation on alkaline sucrose gradients. It was found that all cells observed from lesch nyhan, cystic fibrosis, trisomy 15 et 21, retinoblastoma, were able to repair the single strand breaks in their DNA to the same extent and at the same initial rate.