Recombination induced by triple-helix-targeted DNA damage in mammalian cells.

Gene therapy has been hindered by the low frequency of homologous recombination in mammalian cells. To stimulate recombination, we investigated the use of triple-helix-forming oligonucleotides (TFOs) to target DNA damage to a selected site within cells. By treating cells with TFOs linked to psoralen, recombination was induced within a simian virus 40 vector carrying two mutant copies of the supF tRNA reporter gene. Gene conversion events, as well as mutations at the target site, were also observed. The variety of products suggests that multiple cellular pathways can act on the targeted damage, and data showing that the triple helix can influence these pathways are presented. The ability to specifically induce recombination or gene conversion within mammalian cells by using TFOs may provide a new research tool and may eventually lead to novel applications in gene therapy.     

The kinetics of DNA damage by bleomycin in mammalian cells.

The kinetics of DNA damage by bleomycin (BLM) was assessed by measuring the amount of DNA breakage induced by BLM at different doses, treatment lengths, and treatment temperatures. DNA degradation was measured with the alkaline unwinding method. Comparison of the curves of DNA cleavage by BLM leads to the conclusion that low doses (1-5 micrograms/ml) and short treatments (5-15 min) produce marked damage in the DNA. High increases in BLM concentration produce relatively small increases in DNA damage above the levels obtained with low doses. Extension of treatment times does not increase the DNA degradation above the rate observed with 15-min treatments. The repair of DNA damage starts at about 15 min after the initiation of treatment. The mending of DNA breaks is very fast and extensive when BLM is no longer present. Repair not only implies the closing of DNA nicks, but very likely the degradation of the BLM molecules intercalated in the DNA interrupting the reactions responsible for the generation of free radicals. Persistence of BLM in the cell environment facilitates the replacement of degraded BLM molecules by new ones. This produces the persistent production of free radicals and the establishment of a balance between the processes of DNA damage and repair.     

Oxidative damage to DNA in mammalian chromatin.

Efforts have been made to characterize and measure DNA modifications produced in mammalian chromatin in vitro and in vivo by a variety of free radical-producing systems. Methodologies incorporating the technique of gas chromatography/mass spectrometry have been used for this purpose. A number of products from all four DNA bases and several DNA-protein cross-links in isolated chromatin have been identified and quantitated. Product formation has been shown to depend on the free radical-producing system and the presence or absence of oxygen. A similar pattern of DNA modifications has also been observed in chromatin of cultured mammalian cells treated with ionizing radiation or H2O2 and in chromatin of organs of animals treated with carcinogenic metal salts.     

Paraquat-induced DNA damage in mammalian cells

We have examined the possibility that paraquat (PQ) may exert its toxicity by inducing DNA damage. Mouse lymphoblasts in culture exhibited inhibition of colony forming ability and DNA single strand breaks following a 2 hour exposure to PQ. These phenomenon are dose dependent and increase when a rat liver S9 fraction is included in the incubation mixture. The presence of superoxide dismutase and catalase did not prevent the effects of PQ. Our data indicate that DNA should be considered as a possibile macromolecular target for the lethal effects of paraquat.     

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Long-term DNA damage and mammalian cell survival

On the basis of our own data and those reported in the literature we have made an attempt to follow the fate of the DNA lesions which remain unrepaired during a long period of time, and their possible role in the fate of irradiated cells. The presence of long-lived ("residual") damages is determined by the changes in survival of exposed cells treated, at different times after irradiation, with a mixture of arabinoside cytosine and hydroxyurea. It is shown that "residual" damages can probably exist in the exposed generation and be retained in that following the irradiated one, i.e. after the first mitosis. The nearest descendants of exposed cells (the 3d-5th generations) exhibit a 50% decrease in the rate of DNA synthesis and fall of their proliferative activity, as well as a decrease in the rate of reproduction of their remote descendants. The comparison of the results obtained with those reported by other authors enable us to assume that "residual" DNA lesions play an important role in the fate of exposed cells, that is, in reproductive death, radiation mutagenesis, and malignant transformations.     

Critical DNA damage and mammalian cell reproduction

Synchronous Chinese hamster cells accumulated ¹²⁵I-induced DNA damage in the G₂ + M2 period at 4 °C. The position of the ¹²⁵I within the nuclear DNA was varied by incorporating [¹²⁵I]iododeoxyuridine at various times during the previous DNA replication period. When the initial cell inactivation efficiency was compared for damage accumulated in various regions of nuclear DNA, it was found that the efficiency of inactivation was least in early replicating DNA, and that it gradually increased, reaching a maximum during the fourth and fifth hours of the six-hour DNA replication period. Because the DNA that replicated during this maximum corresponds to that DNA which later forms centromeric and near-centromeric regions of the chromosomes, damage in centromeric region DNA may be critical in causing mammalian cell inactivation.     

Repair of methylation damage in DNA and RNA by mammalian AlkB homologues

Human and Escherichia coli derivatives of AlkB enzymes remove methyl groups from 1-methyladenine and 3-methylcytosine in nucleic acids via an oxidative mechanism that releases the methyl group as formaldehyde. In this report, we demonstrate that the mouse homologues of the alpha-ketoglutarate Fe(II) oxygen-dependent enzymes mAbh2 and Abh3 have activities comparable to those of their human counterparts. The mAbh2 and mAbh3 release modified bases from both DNA and RNA. Comparison of the activities of the homogenous ABH2 and ABH3 enzymes demonstrate that these activities are shared by both sets of enzymes. An assay for the detection of alpha-ketoglutarate Fe(II) dioxygenase activity using an oligodeoxyribonucleotide with a unique modification shows activity for all four enzymes studied and a loss of activity for eight mutant proteins. Steady-state kinetics for removal of methyl groups from DNA substrates indicates that the reactions of the proteins are close to the diffusion limit. Moreover, mAbh2 or mAbh3 activity increases survival in a strain defective in alkB. The mRNAs of AHB2 and ABH3 are expressed most in testis for ABH2 and ABH3, whereas expression of the homologous mouse genes is different. The mAbh3 is strongly expressed in testis, whereas highest expression of mAbh2 is in heart. Other purified human AlkB homologue proteins ABH4, ABH6, and ABH7 do not manifest activity. The demonstration of mAbh2 and mAbh3 activities and their distributions provide data on these mammalian homologues of AlkB that can be used in animal studies.     

Bacterial mutagenicity and mammalian cell DNA damage by several substituted anilines

Several substituted alkyl- and haloanilines were tested for their ability to mutate Salmonella typhimurium and to damage the DNA of mammalian (V79) cells. These results were correlated with their reported carcinogenicity. Of 9 suspected carcinogens, 4 were bacterial mutagens and 4 (out of 7 tested) damaged DNA of V79 cells. The following compounds were weakly mutagenic (<150 revertants/μmole): 4-fluoroaniline, 2,3-, 2,4-, 2,5- and 3,4-dimethylaniline, and 2-methyl-4-fluoroaniline. The following compounds were strong mutagens: 2,4,5-trimethylaniline, 2-methyl-4-chloro-, 2-methyl-4-bromo-, 4-methyl-2-chloro-, 4-methyl-2-bromo- and 2-ethyl-4-chloroaniline. The compounds which damaged DNA in V79 cells were: 2-methyl-4-chloroaniline, 2-methyl-4-bromoaniline, 2,4,5- and 2,4,6-trimethylaniline.     

DNA breakage induced by 1,2,4-benzenetriol: relative contributions of oxygen-derived active species and transition metal ions

We report here the relative roles of metals and selected reactive oxygen species in DNA damage by the genotoxic benzene metabolite 1,2,4-benzenetriol, and the interactions of antioxidants in affording protection. 1,2,4-Benzenetriol induces scission in supercoiled phage DNA in neutral aqueous solution with an effective dose (ED(50)) of 6.7 microM for 50% cleavage of 2.05 microg/ml supercoiled PM2 DNA. In decreasing order of effectiveness: catalase (20 U/ml), formate (25 mM), superoxide dismutase (20 U/ml), and mannitol (50 mM) protected, from 85 to 28%. Evidently, H(2)O(2) is the dominant active species, with O(2)(*)(-) and *OH playing subordinate roles. Desferrioxamine or EDTA inhibited DNA breakage by 81-85%, despite accelerating 1,2,4-benzenetriol autoxidation. Consistent with this suggestion of a crucial role for metals, addition of cupric, cuprous, ferric, or ferrous ions enhanced DNA breakage, with copper being more active than iron. Combinations of scavengers protected more effectively than any single scavenger alone, with implications for antioxidants acting in concert in living cells. Synergistic combinations were superoxide dismutase with *OH scavengers, superoxide dismutase with desferrioxamine, and catalase with desferrioxamine. Antagonistic (preemptive) combinations were catalase with superoxide dismutase, desferrioxamine with *OH scavengers, and catalase with *OH scavengers. The most striking aspect of synergism was the extent to which metal chelation (desferrioxamine) acted synergistically with either catalase or superoxide dismutase to provide virtually complete protection. Concluding, 1,2,4-benzenetriol-induced DNA damage occurs mainly by site-specific, Fenton-type mechanisms, involving synergism between several reactive intermediates. Multiple antioxidant actions are needed for effective protection.     

Phase resetting of the mammalian circadian clock by DNA damage

To anticipate the momentum of the day, most organisms have developed an internal clock that drives circadian rhythms in metabolism, physiology, and behavior [1]. Recent studies indicate that cell-cycle progression and DNA-damage-response pathways are under circadian control [2-4]. Because circadian output processes can feed back into the clock, we investigated whether DNA damage affects the mammalian circadian clock. By using Rat-1 fibroblasts expressing an mPer2 promoter-driven luciferase reporter, we show that ionizing radiation exclusively phase advances circadian rhythms in a dose- and time-dependent manner. Notably, this in vitro finding translates to the living animal, because ionizing radiation also phase advanced behavioral rhythms in mice. The underlying mechanism involves ATM-mediated damage signaling as radiation-induced phase shifting was suppressed in fibroblasts from cancer-predisposed ataxia telangiectasia and Nijmegen breakage syndrome patients. Ionizing radiation-induced phase shifting depends on neither upregulation or downregulation of clock gene expression nor on de novo protein synthesis and, thus, differs mechanistically from dexamethasone- and forskolin-provoked clock resetting [5]. Interestingly, ultraviolet light and tert-butyl hydroperoxide also elicited a phase-advancing effect. Taken together, our data provide evidence that the mammalian circadian clock, like that of the lower eukaryote Neurospora[6], responds to DNA damage and suggest that clock resetting is a universal property of DNA damage.     

Arsenic induces oxidative DNA damage in mammalian cells

Although arsenic is a well-established human carcinogen, the underlying carcinogenic mechanism(s) is not known. Using the human-hamster hybrid (A_L) cell mutagenic assay that is sensitive in detecting mutagens that induce predominately multilocus deletions, we showed previously that arsenite is indeed a potent gene and chromosomal mutagen and that oxyradicals may be involved in the mutagenic process. In the present study, the effects of free radical scavenging enzymes on the cytotoxic and mutagenic potential of arsenic were examined using the A_L cells. Concurrent treatment of cells with either superoxide dismutase or catalase reduced both the cytotoxicity and mutagenicity of arsenite by an average of 2–3 fold, respectively. Using immunoperoxidase staining with a monoclonal antibody specific for 8-hydroxy-2-deoxyguanosine (8-OHdG), we demonstrated that arsenic induced oxidative DNA damage in A_L cells. This induction was significantly reduced in the presence of the antioxidant enzymes. Furthermore, reducing the intracellular levels of non-protein sulfhydryls (mainly glutathione) using buthionine S-R-Sulfoximine increased the total mutant yield by more than 3-fold as well as the proportion of mutants with multilocus deletions. Taken together, our data provide clear evidence that reactive oxygen species play an important causal role in the genotoxicity of arsenic in mammalian cells.     

Biophysical mechanism of radiation damage to mammalian cells by X- and gamma-rays

Published information on the reproductive death in mammalian cells irradiated by a wide range of X- and gamma-ray energies has been re-analysed to extract intrinsic efficiencies of damage for the secondary electrons in transient equilibrium. On a log-log plot, a linear dependence on the track average l.e.t. and the average specific primary ionization is found, indicating that either serves as a good quality parameter. The soft X-ray data are consistent with this conclusion. Upon comparison with data for fast heavy ion irradiations, the average specific primary ionization is shown to be applicable independently of radiation type whereas track average l.e.t. is not. Furthermore it is revealed that electrons are most damaging near the end of their range but their efficiency is only about 10-20 per cent of that of fast ions at the same quality, possibly due to the influence of multiple scatter on the electron penetration depth. It is deduced that, for the dose rates involved, the damage by electrons is predominantly by intra-track action and not inter-track action. The results are consistent with the suggestion that optimum damage occurs when the mean free path between ionizations is equivalent to the strand separation in the double-stranded DNA.     

Cell killing and DNA damage induced in cultured mammalian cells by some tetrahydrobenzopsoralenquinones

The mechanism of action of two tetrahydrobenzopsoralenquinones: 4-methyl-tetrahydrobenzopsoralenquinone (compound 3) and 4-hydroxymethyltetrahydrobenzopsoralenquinone (compound 4) was studied in mammalian cells. These agents differ structurally from earlier benzo and tetrahydrobenzopsoralen derivatives 4-hydroxymethylbenzopsoralen (compound 1) and 4-hydroxymethyltetrahydrobenzopsoralen (compound 2) by the replacement of the benzopyranone with a quinonepyranone. In this study, we evaluated the antiproliferative activity of such derivatives in normal human lymphocytes and CHO cells cultivated in vitro. Compound 4 showed a noticeable antiproliferative activity. Studying the induction of chromosomal aberrations and of SCEs, we demonstrated that compound 4 has a clastogenic effect on mammalian cells. By means of DNA filter elution and protein precipitation techniques we evaluated the DNA damage produced by the tested compounds. Some experiments performed in presence of a DNA synthesis inhibitor showed that ongoing DNA synthesis is involved in cell killing by derivative 4. All data obtained suggest that compound 4 can interfere with the activity of topoisomerase II. Catalytic studies carried out with purified topoisomerase II and bacteriophage DNA confirmed this hypothesis.     

Relationships between the dynamics of iatrogenic DNA damage and genomic design in mammalian spermatozoa from eleven species

The dynamic onset of DNA fragmentation in mammalian sperm populations varies widely in different species when the spermatozoa are incubated in vitro at body temperature for several hours, and recent studies have shown that the dynamic rate of DNA fragmentation within a species has considerable predictive value in terms of fertility. The reasons for such variation are unclear, but here we show that differences in protamine sequence and identity could be partially responsible. Sets of 10 normal semen samples from 11 species (ram, goat, boar, white-tailed deer, rabbit, human, domestic and Spanish fighting bull, horse, donkey, rhinoceros, and koala) were cryopreserved, thawed, diluted in an appropriate extender for each species, and then incubated for 4 hr at 37 °C. Semen samples from human infertility patients were also included for comparison with the donors. DNA fragmentation analysis was undertaken immediately after thawing (t(0)) and after 4 hr (t(4)) using the Halomax/Halosperm procedure, and the differences in DNA fragmentation between t(0) and t(4) were examined in the context of the respective protamine genomes. The expression of protamine 2 in a species significantly enhanced the likelihood of sperm DNA fragmentation; greater numbers of cysteine residues in protamine 1 tended to confer increased sperm DNA stability, and there were logical evolutionary relationships between species in terms of their sperm DNA stability. Human spermatozoa from infertility patients exhibited considerably higher DNA instability than the normal semen donors, a difference that could be indirectly attributed to unbalanced protamine 1-to-protamine 2 ratios.     

Arsenic induces oxidative DNA damage in mammalian cells

Although arsenic is a well-established human carcinogen, the underlying carcinogenic mechanism(s) is not known. Using the human-hamster hybrid (A(L)) cell mutagenic assay that is sensitive in detecting mutagens that induce predominately multilocus deletions, we showed previously that arsenite is indeed a potent gene and chromosomal mutagen and that oxyradicals may be involved in the mutagenic process. In the present study, the effects of free radical scavenging enzymes on the cytotoxic and mutagenic potential of arsenic were examined using the AL cells. Concurrent treatment of cells with either superoxide dismutase or catalase reduced both the cytotoxicity and mutagenicity of arsenite by an average of 2-3 fold, respectively. Using immunoperoxidase staining with a monoclonal antibody specific for 8-hydroxy-2'-deoxyguanosine (8-OHdG), we demonstrated that arsenic induced oxidative DNA damage in A(L) cells. This induction was significantly reduced in the presence of the antioxidant enzymes. Furthermore, reducing the intracellular levels of non-protein sulfhydryls (mainly glutathione) using buthionine S-R-Sulfoximine increased the total mutant yield by more than 3-fold as well as the proportion of mutants with multilocus deletions. Taken together, our data provide clear evidence that reactive oxygen species play an important causal role in the genotoxicity of arsenic in mammalian cells.