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PubMed1991

Phytohemagglutinin-stimulated lymphocytes from patients with systemic lupus erythematosus demonstrate DNA damage.

DNA samples from control and lupus lymphocytes were studied for DNA integrity and single-strand breaks by agarose gel electrophoresis following digestion with the enzyme S1 nuclease. S1 nuclease digests single-strand gaps in double-stranded DNA. Gel patterns of phytohemagglutinin (PHA)-stimulated control and lupus lymphocyte DNAs were identical in the absence of S1 nuclease incubation. DNA isolated from PHA-stimulated control lymphocytes was relatively resistant to S1 nuclease digestion in 14 of 16 samples. However, 15 of 16 DNA samples from PHA-stimulated lupus lymphocytes demonstrated dramatically greater S1 nuclease digestion than paired control DNAs from lymphocytes analyzed at the same time under the same conditions. Increased S1 sensitivity suggests that more single-strand DNA breaks were found in PHA-stimulated lupus lymphocytes and/or the lupus DNA was more damaged than control DNA. We suggest that structural changes found in DNA from stimulated T lymphocytes of lupus patients are consistent with an endogenous antigen-mediated disorder.

DNA↗

PubMed1992

Isolation and characterization of the Schizosaccharomyces pombe rad3 gene, involved in the DNA damage and DNA synthesis checkpoints.

We have cloned the Schizosaccharomyces pombe rad3 gene which is involved in G2 arrest following DNA damage, and in the dependence of mitosis on the completion of DNA replication. The gene was cloned by complementation of the sensitivity to UV light and gamma rays of the rad3-136 mutant with an Sz. pombe genomic library. Sublocalization of the complementing activity and sequencing of the clone identified an intronless 3210-bp open reading frame capable of encoding a 1070-amino acid protein with an M(r) of 121974. The rad3 gene is a new gene with no homologs in existing sequence databases. The gene is poorly expressed, with a codon bias index of -0.01. A disruption mutant affecting the coding region was only slightly more sensitive to UV light than the original rad3-136 mutant. The rad3 gene was mapped to NotI fragment C on chromosome II.

Base Sequence↗

PubMed2005

Base-excision repair of oxidative DNA damage by DNA glycosylases.

Oxidative damage to DNA caused by free radicals and other oxidants generate base and sugar damage, strand breaks, clustered sites, tandem lesions and DNA-protein cross-links. Oxidative DNA damage is mainly repaired by base-excision repair in living cells with the involvement of DNA glycosylases in the first step and other enzymes in subsequent steps. DNA glycosylases remove modified bases from DNA, generating an apurinic/apyrimidinic (AP) site. Some of these enzymes that remove oxidatively modified DNA bases also possess AP-lyase activity to cleave DNA at AP sites. DNA glycosylases possess varying substrate specificities, and some of them exhibit cross-activity for removal of both pyrimidine- and purine-derived lesions. Most studies on substrate specificities and excision kinetics of DNA glycosylases were performed using oligonucleotides with a single modified base incorporated at a specific position. Other studies used high-molecular weight DNA containing multiple pyrimidine- and purine-derived lesions. In this case, substrate specificities and excision kinetics were found to be different from those observed with oligonucleotides. This paper reviews substrate specificities and excision kinetics of DNA glycosylases for removal of pyrimidine- and purine-derived lesions in high-molecular weight DNA.

DNA↗

PubMed1984

Genotoxicity of apomorphine and various catecholamines in the Salmonella mutagenicity test (Ames test) and in tests for primary DNA damage using DNA repair-deficient B. subtilis strains (rec assay).

Apomorphine, N-nor-N-propyl-apomorphine, dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were evaluated for genotoxicity using the Ames test and DNA repair-deficient and DNA repair-proficient Bacillus subtilis strains (rec assay, H17/M45; HLL3g/HJ-15). In the absence of an S9 liver homogenate, apomorphine induced frame-shift mutations in Salmonella typhimurium, mainly in strain TA1537; no indication of DNA-damaging effects in B. subtilis was observed. N-Nor-N-propyl-apomorphine was tested using strain TA1537 only and found to be mutagenic. Dopamine, L-DOPA, 6-hydroxydopamine and adrenaline were non-mutagenic when tested without S9, whereas they were all more toxic for DNA repair-deficient than for DNA repair-proficient B. subtilis strains, indicating a DNA-damaging potential. In a second set of experiments the mode of action of apomorphine and the relevance of the positive Ames test data were investigated. Glutathione in physiological concentrations reduced the mutagenic effect of apomorphine in a dose-dependent way, both in the presence and the absence of S9. S9 also reduced the mutagenicity of apomorphine. By comparing the effects of a complete S9 mix with those of a preparation without glucose-6-phosphate and NADP, it became clear that S9 also had an activating effect, overshadowed under standard conditions by its deactivating activity. Apomorphine was not mutagenic under anaerobic conditions. Superoxide dismutase and catalase reduced the mutagenic effect of apomorphine. All test conditions which reduced the mutagenic effect also inhibited the dark discoloration of the tester plates, indicating a retardation of apomorphine oxidation. It can, therefore, be concluded that oxidation of apomorphine leads to mutagenic products which induce frame-shift mutations in Salmonella typhimurium. This oxidation was prevented both by glutathione in concentrations well below physiological levels and/or by catalase and superoxide dismutase. Under these conditions, apomorphine was non-mutagenic in therapeutic concentrations as well as at higher dose levels. The possibility of genotoxic side effects occurring in patients treated with apomorphine as an emetic drug is therefore considered to be very unlikely.

Animals↗

PubMed2002

Elevated levels of oxidative DNA damage and DNA repair enzymes in human atherosclerotic plaques.

BACKGROUND: The formation of reactive oxygen species is a critical event in atherosclerosis because it promotes cell proliferation, hypertrophy, growth arrest, and/or apoptosis and oxidation of LDL. In the present study, we investigated whether reactive oxygen species-induced oxidative damage to DNA occurs in human atherosclerotic plaques and whether this is accompanied by the upregulation of DNA repair mechanisms. METHODS AND RESULTS: We observed increased immunoreactivity against the oxidative DNA damage marker 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in plaques of the carotid artery compared with the adjacent inner media and nonatherosclerotic mammary arteries. Strong 8-oxo-dG immunoreactivity was found in all cell types of the plaque including macrophages, smooth muscle cells, and endothelial cells. As shown by competitive ELISA, carotid plaques contained 160+/-29 8-oxo-dG residues/10(5) dG versus 3+/-1 8-oxo-dG residues/10(5) dG in mammary arteries. Single-cell gel electrophoresis showed elevated levels of DNA strand breaks in the plaque. The overall number of apoptotic nuclei was low (1% to 2%) and did not correlate with the amount of 8-oxo-dG immunoreactive cells (>90%). This suggests that initial damage to DNA occurs at a sublethal level. Several DNA repair systems that are involved in base excision repair (redox factor/AP endonuclease [Ref 1] and poly(ADP-ribose) polymerase 1 [PARP-1]) or nonspecific repair pathways (p53, DNA-dependent protein kinase) were upregulated, as shown by Western blotting and immunohistochemistry. Overexpression of DNA repair enzymes was associated with elevated levels of proliferating cell nuclear antigen. CONCLUSIONS: Our findings provide evidence that oxidative DNA damage and repair increase significantly in human atherosclerotic plaques.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2006

Bioactivation of carboxylic acid compounds by UDP-Glucuronosyltransferases to DNA-damaging intermediates: role of glycoxidation and oxidative stress in genotoxicity.

Nonenzymatic modification of proteins by acyl glucuronides is well documented; however, little is known about their potential to damage DNA. We have previously reported that clofibric acid undergoes glucuronidation-dependent bioactivation to DNA-damaging species in cultured mouse hepatocytes. The aim of this study was to investigate the mechanisms underlying such DNA damage, and to screen chemically diverse carboxylic acid drugs for their DNA-damaging potential in glucuronidation proficient murine hepatocytes. Cells were incubated with each aglycone for 18 h, followed by assessment of compound cytotoxicity using the MTT assay and evaluation of DNA damage using the Comet assay. Relative cytotoxic potencies were ketoprofen > diclofenac, benoxaprofen, nafenopin >> gemfibrozil, probenecid > bezafibrate > clofibric acid. At a noncytotoxic (0.1 mM) concentration, only benoxaprofen, nafenopin, clofibric acid, and probenecid significantly increased Comet moments (P < 0.05 Kruskal-Wallis). Clofibric acid and probenecid exhibited the greatest DNA-damaging potency, producing significant DNA damage at 0.01 mM concentrations. The two drugs produced maximal increases in Comet moment of 4.51 x and 2.57 x control, respectively. The glucuronidation inhibitor borneol (1 mM) abolished the induction of DNA damage by 0.5 mM concentrations of clofibric acid and probenecid. In an in vitro cell-free system, clofibric acid glucuronide was 10 x more potent than glucuronic acid in causing DNA strand-nicking, although both compounds showed similar rates of autoxidation to generate hydroxyl radicals. In cultured hepatocytes, the glycation inhibitor, aminoguanidine, and the iron chelator, desferrioxamine mesylate, inhibited DNA damage by clofibric acid, whereas the free radical scavengers Trolox and butylated hydroxytoluene, and the superoxide dismutase mimetic bis-3,5-diisopropylsalicylate had no effect. In conclusion, clinically relevant concentrations of two structurally unrelated carboxylic acids, probenecid and clofibric acid, induced DNA damage in isolated hepatocytes via glucuronidation- dependent pathways. These findings suggest acyl glucuronides are able to access and damage nuclear DNA via iron-catalyzed glycation/glycoxidative processes.

Animals↗

PubMed1998

Supercoiling affects the accessibility of glutathione to DNA-bound molecules: positive supercoiling inhibits calicheamicin-induced DNA damage.

DNA superhelical tension, an important feature of genomic organization, is known to affect the interactions of intercalating molecules with DNA. However, the effect of torsional tension on nonintercalative DNA-binding chemicals has received less attention. We demonstrate here that the enediyne calicheamicin gamma1I, a strand-breaking agent specific to the minor groove, causes approximately 50% more damage in negatively supercoiled plasmid DNA than in DNA with positive superhelicity. Furthermore, we show that the decrease in damage in positively supercoiled DNA is controlled at the level of thiol activation of the drug. Our results suggest that supercoiling may affect both the activity of nonintercalating genotoxins in vivo and the accessibility of glutathione and other small physiologic molecules to DNA-bound chemicals or reactions occurring in the grooves of DNA.

Aminoglycosides↗

PubMed2007

Evidence for the receipt of DNA damage stimuli by PML nuclear domains.

Promyelocytic leukaemia nuclear domains (PML-NDs) comprise a shell of PML protein and many labile cargo proteins. The nature of their cargo, their juxtaposition to foci of damaged DNA following ionizing radiation (IR), and the altered DNA damage responses in PML null cells all implicate PML-NDs in the DNA damage response. In this work, the propensity of PML-NDs to increase in number and decrease in size following IR has been studied. Serial quantitative studies of endogenous PML-NDs prove that the PML-ND response to IR is not the result of the asymmetry in cell cycle distribution that can follow IR, but reflects more directly the process of DNA damage. The response is swift, sensitive (evident after 1 Gy), and potentially reversible in untransformed fibroblasts. In these cells and in HCT116 colon cancer cells, failure to restore PML-ND number within 24 h correlates with later loss of growth potential--in fibroblasts, through prolonged cell cycle arrest and in HCT116 cells, through apoptosis. Failure to express an intact ATM/CHK2 DNA damage signalling pathway in either cell type leads to a delay in the PML-ND response to IR. Conversely, cell cycle progression following IR in cells that detect damaged DNA accelerates PML-ND reorganization. Collectively, these data show that the increase in PML-ND number seen after irradiation is, in part, triggered by the receipt of the DNA damage stimulus. The senescent cell state is also associated with chronic DNA damage and Hayflick-limited fibroblasts were found to express nuclei with elevated numbers of PML-NDs before IR that remained unresponsive to IR. Though the underlying reasons for damage-induced PML alteration remain obscure, it is noteworthy that significant numbers of PML-NDs juxtapose with ionizing radiation-induced foci after IR. The co-regulation of these structures may necessitate the stereotyped increases in PML-ND number following damage.

Apoptosis↗

PubMed2004

The DNA damage response: sensing and signaling.

The protein kinases ATM and ATR are central components of the checkpoint mechanisms that signal the presence of damaged DNA and stalled replication forks. Recent studies have provided important new insights into how these kinases work together with their regulatory subunits, DNA repair proteins and adaptor proteins to sense abnormal DNA structures and implement the appropriate DNA damage response. These advances have provided a more detailed understanding of the interface between damaged DNA and the checkpoint sensor proteins.

Animals↗

PubMed2002

Human Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its overexpression sensitizes brain tumor cells to apoptosis following alkylation damage of DNA.

The human Dkk-1 (hDkk-1) gene, a transcriptional target of the p53 tumor suppressor, encodes a powerful inhibitor of the Wnt signaling pathway and regulates the spatial patterning/morphogenesis of the mammalian central nervous system. We investigated the p53-related functions of the hDkk-1 gene by studying its response to DNA damage and its modulation of apoptosis in human glioma cells. Various chemotherapeutic and other agents that induce DNA adducts and compromise its integrity (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, H(2)O(2) and UV rays) enhanced the expression of hDkk-1 significantly. The damage-induced increase in hDkk-1 mRNA levels occurred in many human tumor cell lines, irrespective of their p53 gene status. The human glioblastoma cell line, U87MG, which had undetectable hDkk-1 expression, was engineered to express moderate levels of the hDkk protein by stable transfection. The engineered cells did not show any morphological changes, but underwent marked apoptosis after ceramide treatment. Further, the DNA cross-linking drugs BCNU and cisplatin, but not the microtubule poison vincristine, induced significant cell death in U87MG/hDkk cells, and this was accompanied by altered Bcl-2/Bax expression and a reduction in the amount of telomere DNA as visualized by fluorescence in situ hybridization. These results show that hDkk-1 is a pro-apoptotic gene and suggest that it may play important roles in linking the oncogenic Wnt and p53 tumor suppressor pathways.

Alkylation↗

PubMed2002

Interaction between UV-damaged DNA binding activity proteins and the c-Abl tyrosine kinase.

The c-Abl tyrosine kinase is activated by some forms of DNA damage, including ionizing radiation, but not UV radiation. The functions of this activation in the damage response pathways remain obscure. To identify potential targets of c-Abl kinase, we utilized the yeast two-hybrid system to screen a murine cDNA library. One c-Abl binding protein of particular interest was the large subunit (DDB1) of the heterodimeric complex with UV-damaged DNA binding activity (UV-DDB). This complex binds with high specificity to DNA damaged by UV, is absent in a subset of xeroderma pigmentosum group E cells, and is required for global genomic repair of UV-induced damage. The association of c-Abl with DDB1 required the kinase domain of c-Abl and preserved the interaction between DDB1 and the small subunit (DDB2) of the UV-DDB complex. Significantly, overexpression of c-Abl increased tyrosine phosphorylation of DDB2 and suppressed UV-DDB activity. Conversely, a dominant negative, kinase-deficient allele of c-Abl decreased tyrosine phosphorylation of DDB2 and dramatically stimulated UV-DDB activity. These results suggest that one role of c-Abl may be to negatively regulate UV-DDB activity by phosphorylation of DDB2.

Cell Line↗

PubMed1996

Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications.

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.

Ascorbic Acid↗

PubMed2005

Farnesyltransferase inhibitors induce DNA damage via reactive oxygen species in human cancer cells.

Farnesyltransferase inhibitors (FTIs) possess antitumor activity. Based on recent findings, we hypothesized that FTIs induce reactive oxygen species (ROS) that damage DNA, leading to DNA damage responses. To test this hypothesis, we investigated the effects of FTIs on the generation of ROS, DNA double-strand breaks (DSB), DNA damage responses, and RhoB, and the effects of quenching ROS on these FTI effects. We evaluated four FTIs in human cancer cell lines of different tissue origins. We found that FTIs induced ROS and DSBs. Suppressing expression of the beta-subunit of farnesyltransferase with siRNA did not induce ROS, but slightly attenuated the ROS induced by FTIs. N-acetyl-L-cysteine (NAC), but not caspase inhibitors, blocked FTI-induced DSBs, suggesting that the DSBs were caused by ROS and did not result from apoptosis. The DSBs led to DNA damage responses. H2AX became phosphorylated and formed nuclear foci. The DNA-damage-sensing molecules involved were probably ataxia-telangiectasia mutated protein (ATM) and DNA-dependent protein kinase (DNA-PK) but not ATM- and Rad3-related protein (ATR). Key components of the homologous recombination and nonhomologous end joining repair pathways (DNA-PK, BRCA1, and NBS1) underwent phosphorylation and formed nuclear foci. RhoB, a mediator of the antineoplastic effect of FTIs and a protein inducible by DNA damage, was increased by FTIs. This increase was blocked by NAC. We concluded that FTIs induced oxidative DNA damage by inducing ROS and initiated DNA damage responses, including RhoB induction, and there was a complex relationship among FTIs, farnesyltransferase, ROS, and RhoB. Our data also imply that inhibitors of DNA repair may accentuate the clinical efficacy of FTIs.

Alkyl and Aryl Transferases↗

PubMed2003

MDC1 is a mediator of the mammalian DNA damage checkpoint.

To counteract the continuous exposure of cells to agents that damage DNA, cells have evolved complex regulatory networks called checkpoints to sense DNA damage and coordinate DNA replication, cell-cycle arrest and DNA repair. It has recently been shown that the histone H2A variant H2AX specifically controls the recruitment of DNA repair proteins to the sites of DNA damage. Here we identify a novel BRCA1 carboxy-terminal (BRCT) and forkhead-associated (FHA) domain-containing protein, MDC1 (mediator of DNA damage checkpoint protein 1), which works with H2AX to promote recruitment of repair proteins to the sites of DNA breaks and which, in addition, controls damage-induced cell-cycle arrest checkpoints. MDC1 forms foci that co-localize extensively with gamma-H2AX foci within minutes after exposure to ionizing radiation. H2AX is required for MDC1 foci formation, and MDC1 forms complexes with phosphorylated H2AX. Furthermore, this interaction is phosphorylation dependent as peptides containing the phosphorylated site on H2AX bind MDC1 in a phosphorylation-dependent manner. We have shown by using small interfering RNA (siRNA) that cells lacking MDC1 are sensitive to ionizing radiation, and that MDC1 controls the formation of damage-induced 53BP1, BRCA1 and MRN foci, in part by promoting efficient H2AX phosphorylation. In addition, cells lacking MDC1 also fail to activate the intra-S phase and G2/M phase cell-cycle checkpoints properly after exposure to ionizing radiation, which was associated with an inability to regulate Chk1 properly. These results highlight a crucial role for MDC1 in mediating transduction of the DNA damage signal.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Spindle checkpoint function is required for mitotic catastrophe induced by DNA-damaging agents.

Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents that damage DNA. This process is facilitated by defects in the G1 and G2 checkpoints of the cell cycle that are apparent in most cancer cells and which allow the cells to enter mitosis with DNA damage. We have now characterized the dynamics of mitotic catastrophe induced by DNA-damaging agents in p53-deficient cancer cells. Cells that entered mitosis with DNA damage transiently arrested at metaphase for more than 10 h without segregation of chromosomes and subsequently died directly from metaphase. In those metaphase arrested precatastrophic cells, anaphase-promoting complex appeared to be inactivated and BubR1 was persistently localized at kinetochores, suggesting that spindle checkpoint is activated after the DNA damage. Furthermore, suppression of spindle checkpoint function by BubR1 or Mad2 RNA interference in the DNA damaged cells led to escape from catastrophic death and to subsequent abnormal mitosis. Dysfunction of the spindle checkpoint in p53-deficient cancer cells is thus likely a critical factor in resistance to DNA-damaging therapeutic agents.

Base Sequence↗

PubMed2004

Relationships between cagA, vacA, and iceA genotypes of Helicobacter pylori and DNA damage in the gastric mucosa.

Helicobacter pylori (H. pylori) is believed to predispose carriers to gastric cancer by inducing chronic inflammation. The inflammatory processes may result in the generation of reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the relationships between DNA damage in the gastric mucosa and cagA, vacA, and iceA genotypes of H. pylori. The study was conducted with biopsies from the gastric antrum and corpus of 98 H. pylori-infected and 26 uninfected control patients. H. pylori genotypes were determined by PCR and DNA damage was measured in gastric mucosal cells by the Comet assay (single cell gel electrophoresis). All patients were nonsmokers, not abusing alcohol, and not using prescription or recreational drugs. Levels of DNA damage were significantly higher (P < 0.0001) in the H. pylori-infected patients than in uninfected patients. In comparison with the level of DNA damage in the uninfected controls, the extent of DNA damage in both the antrum (OR = 8.45; 95% CI = 2.33-37.72) and the corpus (OR = 6.55; 95% CI = 2.52-17.72) was related to infection by cagA+/vacAs1m1 and iceA1 strains. The results indicate that the genotype of H. pylori is related to the amount of DNA damage in the gastric mucosa. These genotypes could serve as biomarkers for the risk of extensive DNA damage and possibly gastric cancer.

Adult↗

PubMed1996

Comparative assessment of DNA adduct formation, Salmonella mutagenicity, and chromosome aberration assays as short-term tests for DNA damage.

DNA adduct formation assay (DAFA) was carried out to compare dose responses with the Ames test and chromosomal aberration test using aflatoxin B1 (AFB1) and benzo[a]pyrene (BaP). In the bacterial mutation test, AFB1 and BaP (0-1 microgram/plate) were all positive in TA97a and TA100 with dose-related revertants. However, the slopes of the dose-response curves were gradual (slope 0.55-3.73, r = .84-.98). In the chromosome aberration test, a significant increase in the percentage of chromosomal aberrations was obtained from male ICR mouse spleen cells treated with AFB1 and BaP, but a dose-related increase was insensitive (slope 0.09-0.23, r = .75-.78). The incidence of chromosomally aberrant spleen cells treated with BaP was significantly increased compared with AFB1. DAFA was performed in vitro with [3H]-AFB1 and [3H]BaP. These two carcinogens were able to induce genotoxicity and showed good dose-related increases in terms of DNA adduct formation (slope 0.78-1.28, r = 1.00). Coefficients of variation (CV) for the slope of each dose-response curve were much lower in DAFA in vitro (CV 15.09- 18.34%) than those in any other test (CV 19.69-99.33%, Ames test; 18.89-44.58%, chromosome aberration test). Furthermore, DAFA in vivo was performed to investigate organotropic DNA adduct formation and persistence in Sprague-Dawley rats ip or orally treated with AFB1 and BaP. DNA adducts were monitored for 48-96 h by enzyme-linked immunosorbent assay (ELISA) using corresponding monoclonal antibodies, 6A10 and 8E11. DAFA in vivo demonstrated that the liver and kidney might be the probable target organs for AFB1 with the highest formation and persistence of DNA adducts and the lung and liver for BaP regardless of the route of administration. The results suggest that DAFA in vitro could be useful for detecting genotoxic compounds, and DAFA in vivo should also be considered as a good alternative method for the screening of organ-specific chemical carcinogens.

Aflatoxin B1↗

PubMed2001

Activation of DNA damage checkpoints in CHO cells requires a certain level of DNA damage.

DNA damage activates checkpoint controls in eukaryotic cells. It is not clear, however, whether a certain level of DNA damage is required for the activation of DNA damage checkpoints. We show here that low levels of DNA damage in Chinese hamster ovary (CHO) cells induced by short exposure to hydroxyurea (HU) did not trigger checkpoints, whereas higher levels of DNA damage caused by longer exposure to HU resulted in a cell cycle arrest. Our results argue that a threshold of DNA damage is necessary for activation of DNA damage checkpoints.

Animals↗