Induction and repair of DNA strand breaks in cultured human fibroblasts exposed to various phenols and dihydrodiols of benzo[a]pyrene

Cultured human fibroblasts from healthy donors were incubated for 30 min with nine different benzo[a]pyrene (BP) derivatives in the presence or absence of liver microsomes from 3-methylcholanthrene treated rats. The induction and repair of DNA strand breaks were analysed by alkaline unwinding and separation of double and single stranded DNA (SS-DNA) by hydroxylapatite chromatography immediately after the incubation or at various times after the treatment. In the absence of microsomes DNA stand breaks were detected in fibroblasts exposed to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP) and the three BP dihydrodiols (BP-4,5-, BP-7,8- or BP-9,10-dihydrodiol). After removal of the BP derivatives from the medium the DNA strand breaks disappeared within 24 h. alpha-Naphthoflavone (alpha-NF) caused a decrease in the induction of strand breaks by 1-, 3- and 9-OH-BP but did not affect the induction of strand breaks in cells exposed to BP-7,8-dihydrodiol. In the presence of microsomes DNA strand breaks were found after exposure to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP), as well as BP-7,8- and 9,10-dihydrodiol. In contrast BP-4,5-dihydrodiol did not induce strand breaks under these conditions. The induction of strand breaks by BP-7,8-dihydrodiol was enhanced in the presence of cytosine-1-beta-D-arabinofuranoside (AraC). In all cases the DNA strand breaks had disappeared 24 h after removal of the BP derivatives and microsomes except after treatment with BP-7,8-dihydrodiol.     

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.     

Mechanism of protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide- and menadione-induced DNA single strand breaks in Caco-2 cells

Protection by the flavonoids, quercetin and rutin, against tert-butylhydroperoxide (tert-BOOH)- and menadione-induced DNA single strand breaks was investigated in Caco-2 cells. Both tert-BOOH and menadione induced DNA single strand breaks in a concentration-dependent manner. Pre-incubation of Caco-2 cells with either quercetin or rutin for 24 h significantly decreased the formation of DNA single strand breaks evoked by tert-BOOH (P <.05). Iron chelators, 1,10-phenanthroline (o-Phen) and deferoxamine mesylate (DFO), also protected against tert-BOOH-induced DNA damage, whereas butylated hydroxytoluene (BHT) had no effect. Quercetin, and not rutin, decreased the extent of menadione-induced DNA single strand breaks. DFO and BHT, and not o-Phen, protected against menadione-induced DNA strand break formation (P <.05). From the results of this study, iron ions were involved in tert-BOOH-induced DNA single strand break formation in Caco-2 cells, whereas DNA damage evoked by menadione was far more complex. We demonstrated that the flavonoids, quercetin and rutin, protected against tert-BOOH-induced DNA strand breaks by way of their metal ion chelating mechanism. However, quercetin, and not rutin, protected against menadione-induced DNA single strand breaks by acting as both a metal chelator and radical scavenger.     

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.     

Increased Sensitivity of DNA Damage Response-Deficient Cells to Stimulated Microgravity-Induced DNA Lesions

Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG). In this study we used mouse embryonic stem (MES) and mouse embryonic fibroblast (MEF) cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs) in Rad9 ^-/- MES and Mdc1 ^-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9 ^-/- MES. As the exposure to SMG was prolonged, Rad9 ^-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9 ^-/- MES were due to SMG-induced reactive oxygen species (ROS). Interestingly, Mdc1 ^-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1 ^-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR) defects.     

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.     

Breakage of Chromosomal DNA with Aromatic Reductones

Strand breakages of mammalian cellular chromosomal DNA with aromatic reductones were ascertained by use of a cultured cell strain of the rat fetal lung (RFL). The mode of the breakages was investigated by ultracentrifugal analyses. The reductones induced the breakages of the cellular DNA in two different fashions; one is single strand breaks and another double strand breaks. Although the single strand breaks were rapidly repaired, double strand breaks were only partially repaired. Both breaks were not cytocidal. Some physiological alterations were observed to follow the strand breaks.     

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.     

Quantitation of radiation-, chemical-, or enzyme-induced single strand breaks in nonradioactive DNA by alkaline gel electrophoresis: application to pyrimidine dimers

We have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controlled scanner. The median lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determine pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.     

Accumulation of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants of mouse FM3A cells

Thymidylate synthase-negative mutants of cultured mouse cells were immediately committed to cell death upon thymidine deprivation, especially when the cells were synchronized in the S phase. Thymidylate deprivation induced single strand breaks in chromosome-size DNA strands, as measured by alkaline sucrose gradient sedimentation, giving rise to two peaks, one with large and the other with small fragments, the latter about the size of T4 DNA. An increase in the small DNA fragments paralleled that of thymineless death. Thymidine deprivation also produced double strand DNA fragments as determined by a method of neutral filter elution, and their extent paralleled that of cell death. Double-stranded DNA eluted through the filter sedimented as a single peak both in a neutral and in an alkaline sucrose gradient that coincided with that of the above small DNA fragments. Therefore, the strand breaks seemed to occur in some defined portions of the genome and in a specific manner compared to breaks induced by x-rays, which occurred rather randomly. Cycloheximide blocked both thymineless death and the production of the small DNA fragments. The strand breaks induced by thymidine starvation were not repaired but instead advanced on subsequent incubation of the cells in growth medium containing thymidine.     

Deoxyribonucleoside triphosphate imbalance. 5-Fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

The mechanism of cytotoxic action of 5-fluorodeoxyuridine (FdUrd) in mouse FM3A cells was investigated. We observed the FdUrd-induced imbalance of intracellular deoxyribonucleoside triphosphate (dNTP) pools and subsequent double strand breaks in mature DNA, accompanied by cell death. The imbalance of dNTP pools was maximal at 8 h after 1 microM FdUrd treatment; a depletion of dTTP and dGTP pools and an increase in the dATP pool were observed. The addition of FdUrd in culture medium induced strand breaks in DNA, giving rise to a 90 S peak by alkaline sucrose gradient sedimentation. The loss of cell viability and colony-forming ability occurred at about 10 h. DNA double strand breaks as measured by the neutral elution method were also observed in FdUrd-treated cells about 10 h after the addition. These results lead us to propose that DNA double strand breaks play an important role in the mechanism of FdUrd-mediated cell death. A comparison of the ratio of single and double strand breaks induced by FdUrd to that observed following radiation suggested that FdUrd produced double strand breaks exclusively. Cycloheximide inhibited both the production of DNA double strand breaks and the FdUrd-induced cell death. An activity that can induce DNA double strand breaks was detected in the lysate of FdUrd-treated FM3A cells but not in the untreated cells. This suggests that FdUrd induces the cellular DNA double strand breaking activity. The FdUrd-induced DNA strand breaks and cell death appear to occur in the S phase. Our results indicate that imbalance of the dNTP pools is a trigger for double strand DNA break and cell death.     

High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor

Previous studies have demonstrated that phenolic compounds, including genistein (4',5,7-trihydroxyisoflavone) and resveratrol (3,4',5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in phi X-174 plasmid DNA. H(2)O(2)/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H(2)O(2)/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H(2)O(2) suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H(2)O(2)/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H(2)O(2) were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro.     

Caged single and double strand breaks

Ionizing radiation and radiomimetic drugs such as bleomycin, calichieamycin, neocarzinostatin chromophore, and other synthetic agents can produce both single and double strand breaks in DNA. The ability to study the structure-activity relationships of single and double-strand break repair, lethality, and mutagenesis in vivo is complicated by the numerous types and sites of DNA cleavage products that can be induced by such agents. The ability to "cage" such breaks in DNA might help to further such studies and additionally afford a mechanism for activating and deactivating nucleic acid based drugs and probes. The major type of single strand break induced by ionizing radiation is a 3'- and 5'-phosphate terminated single nucleotide gap. Previously, a caged strand break of this type had been developed that was designed to produce the 5'-phosphate directly upon irradiation with 366 nm light, and the 3'-phosphate by a subsequent beta-elimination reaction [Ordoukhanian, P., and Taylor, J.-S. (1995) J. Am. Chem. Soc. 117, 9570]. Unfortunately, the release of the 3'-phosphate group was quite slow at pH 7. To circumvent this problem, a second caged strand break has been developed that produces the 3'-phosphate directly upon irradiation, and the 5'-phosphate by a subsequent beta-elimination reaction. When this caged strand break was used in tandem with the previous caged strand break, 5'- and 3'-phosphate terminated gaps could be directly produced by irradiation with 366 nm light. These caged single strand breaks were also incorporated in tandem into hairpin substrates to demonstrate that they could be used to cage double strand breaks. These caged single strand breaks should be generally useful for generating site-specific DNA single and double strand breaks and gaps, using wavelengths and doses of light that are nondetrimental to biological systems. Because the position of the single strand break can be varied, it should now be possible to examine the effect of the sequence context and cleavage pattern of single and double strand breaks on the lethality and mutagenicity of this important class of DNA damage.     

Regulation of the activity and expression of ERK8 by DNA damage

We have investigated the agonists that activate transfected extracellular signal-regulated kinase 8 (ERK8) in cells, and have found that the most potent activators are hydrogen peroxide, DNA alkylating and cross-linking agents and the poly (ADP-ribose) polymerase inhibitor KU-0058948. The feature shared by all these agents is that they lead to the accumulation of single strand breaks in DNA, suggesting a role for ERK8 in the response to, or repair of, DNA single strand breaks. The DNA alkylating agent MMS also induced the disappearance of endogenous ERK8 by a proteasome-dependent mechanism.     

Determination of G-values for single and double strand break induction in plasmid DNA using agarose gel electrophoresis and a curve-fitting procedure

Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine the yield of single strand breaks (ssb) and double strand breaks (dsb) as a consequence of X-irradiation. One ssb transforms DNA of the CC form to the nicked circular form (NC), whereas one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments. DNA (30-800 micrograms/ml) was irradiated in air-saturated sodium phosphate buffer. The different forms of DNA were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered by using a curve-fitting procedure. From the quantitative changes of each conformation D37 values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots. The following yields were determined: Gssb 3.4 X 10E-8 molJ-1, and Gdsb 3.3 X 10E-10 molJ-1. Gdsb refers only to those dsb produced directly. Yields are related to strand breaks without further treatment by heat or alkali.     

X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution.

This work presents a neutral filter elution method for detecting DNA double strand breaks in mouse L1210 cells after X-ray. The assay will detect the number of double strand breaks induced by as little as 1000 rad of X-ray. The rate of DNA elution through the filters under neutral conditions increases with X-ray dose. Certain conditions for deproteinization, pH, and filter type are shown to increase the assay's sensitivity. Hydrogen peroxide and Bleomycin also induce apparent DNA double strand breaks, although the ratios of double to single strand breaks vary from those produced by X-ray. The introduction of double strand cuts by HpA I restriction endonuclease in DNA lysed on filters results in a rapid rate of elution under neutral conditions, implying that the method can detect double strand breaks if they exist in the DNA. The eluted DNA bands with a double stranded DNA marker in cesium chloride. This evidence suggests that the assay detects DNA double strand breaks. L1210 cells are shown to rejoin most of the DNA double strand breaks induced by 5-10 krad of X-ray with a half-time of about 40 minutes.     

Cigarette smoke-induced DNA damage in cultured human lung cells: role of hydroxyl radicals and endonuclease activation.

Cigarette smoke can cause DNA single strand breaks in cultured human lung cells (T. Nakayama et al., Nature, 314 (1985) 462-464) but the mechanisms behind this DNA damage have not been clearly elucidated. In the present study we have investigated the possibility that one of the major constituents in cigarette smoke, hydroquinone, may be important for mediating smoke-induced DNA damage in the human epithelial lung cell line, A 549, and the mechanisms behind this damage. Cells were exposed to cigarette smoke, hydrogen peroxide, or hydroquinone, in the absence and presence of different inhibitors, and the resulting DNA damage was assessed either as DNA single strand break formation or formation of the oxidative DNA adduct, 8-hydroxydeoxyguanosine. It was found that (i) exposure to cigarette smoke, hydrogen peroxide or hydroquinone causes a rapid decrease in the intracellular thiol level and a considerable DNA single strand break formation, (ii) the formation of DNA single strand breaks in cells exposed to cigarette smoke is inhibited by catalase, dimethylthiourea, and o-phenantroline, suggesting that hydroxyl radicals generated from iron-catalyzed hydrogen peroxide dissociation are involved in the DNA damage, (iii) hydroquinone causes considerable DNA strand break formation that is blocked by aurintricarboxylic acid, an inhibitor of endonuclease activation, and by BAPTA, an intracellular calcium chelator, (iv) addition of hydroquinone to a smoke condensate greatly enhances its ability to cause DNA single strand breaks, and (v) smoke, but not hydroquinone, causes formation of 8-hydroxydeoxyguanosine, a DNA damage product induced by the action of hydroxyl radicals on the DNA base, deoxyguanosine. These findings suggest that the ability of cigarette smoke to cause DNA single strand breaks in cultured lung cells is due to mechanisms involving hydroxyl radical attack on DNA and endonuclease activation. They also suggest that hydroquinone is an important contributor to the DNA damaging effect of cigarette smoke on human lung cells.     

DNA strand breaks in mammalian tissues induced by methylarsenics

DNA damage induced by administration of dimethylarsinic acid (DMAA) to rats and mice was investigated. At 12 h after administration of DMAA, DNA single-strand breaks were induced markedly in lung. The majority of dimethylarsine, one of the main metabolites, in the expired air was excreted within 6–18 h after administration of DMAA to rats. In vitro experiments using nuclei isolated from lung of mice indicated that DNA strand breaks were caused by dimethylarsine. Furthermore, the strand breaks after exposure to dimethylarsine were reduced in the presence of catalase and/or superoxide dismutase. These results strongly suggest that the strand breaks are induced not by dimethylarsine itself but by active oxygen, e.g., O ₂ ^? and ·OH, produced both by dimethylarsine and molecular oxygen. When DNA was exposed to dimethylarsine, thiobarbituric acid (TBA)-reactive intermediates andcis-thymine glycol were produced. Dimethylarsine appears to induce DNA damage by the mechanism similar to the damage produced by ionizing radiation.     

53BP1-mediated DNA double strand break repair: insert bad pun here

53BP1 is an established player in the cellular response to DNA damage and is a canonical component of ionizing-radiation induced foci--that cadre of proteins which assemble at DNA double strand breaks following radiation exposure and which are readily visualized by immunofluorescence microscopy. While its roles in p53 regulation and cell cycle checkpoint activation have been studied for some time, the impact of 53BP1 on DNA double strand break rejoining has only come to light in the past few years. Convincing evidence now exists for 53BP1 significantly affecting the outcome of DNA double strand break repair in several contexts, many of which hint to an important role in modulating chromatin structure surrounding the break site. Here, we highlight the known and emerging roles of 53BP1 in DNA double strand break repair, including the repair of lesions induced within heterochromatin, following telomere uncapping, in long-range V(D)J recombination, during immunoglobulin class switch recombination and its much debated role in regulating resection during homologous recombination.     

Differences in the repair of DNA strand breaks induced by 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene in cultured human fibroblasts

Two benzo(a)pyrene metabolites were found to induce DNA strand breaks in cultured human fibroblasts. DNA strand breaks induced by the non- or weakly carcinogenic 9-hydroxy-benzo(a)pyrene were repaired within two hours, while those induced by the strongly carcinogenic trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene were repaired at a much slower rate.