Comet assay with nuclear extract incubation

Alkaline comet assay is a simple sensitive method for detecting DNA strand breaks. However, at the time of cell lysis, only a fraction of the entire DNA damage appears as DNA strand breaks, while some DNA strand breaks may have been rejoined and some DNA lesions may still remain unexcised. We showed that nuclear extract (NE) prepared from human cells could excise the DNA adducts induced by UVC, X-ray, and methyl methanesulfonate (MMS). Thus, the comet assay with NE incubation allows a closer estimation of total DNA damage. Among the human urothelial carcinoma cell lines we tested, the NE of NTUB1 cells showed higher activity in excising the DNA adducts induced by UVC, but with a lower activity in excising the DNA adducts induced by MMS than the NE of BFTC905 cells. Moreover, under the same dose of X-ray irradiation, a larger difference in total DNA damage between two cell lines was revealed in comet assay incubated with NE than without NE. Therefore, the comet assay with NE incubation may be useful in the research of cancer risk, drug resistance, and DNA repair proteins.     

Changes of 8-OH-dG levels in DNA and its base excision repair activity in rat lungs after inhalation exposure to hexavalent chromium

The co-genotoxic effects of cadmium are well recognized and it is assumed that most of these effects are due to the inhibition of DNA repair. We used the comet assay to analyze the effect of low, non-toxic concentrations of CdCl2 on DNA damage and repair-induced in Chinese hamster ovary (CHO) cells by UV-radiation, by methyl methanesulfonate (MMS) and by N-methyl-N-nitrosourea (MNU). The UV-induced DNA lesions revealed by the comet assay are single-strand breaks which are the intermediates formed during nucleotide excision repair (NER). In cells exposed to UV-irradiation alone the formation of DNA strand breaks was rapid, followed by a fast rejoining phase during the first 60 min after irradiation. In UV-irradiated cells pre-exposed to CdCl2, the formation of DNA strand breaks was significantly slower, indicating that cadmium inhibited DNA damage recognition and/or excision. Methyl methanesulfonate and N-methyl-N-nitrosourea directly alkylate nitrogen and oxygen atoms of DNA bases. The lesions revealed by the comet assay are mainly breaks at apurinic/apyrimidinic (AP) sites and breaks formed as intermediates during base excision repair (BER). In MMS treated cells the initial level of DNA strand breaks did not change during the first hour of recovery; thereafter repair was detected. In cells pre-exposed to CdCl2 the MMS-induced DNA strand breaks accumulated during the first 2h of recovery, indicating that AP sites and/or DNA strand breaks were formed but that further steps of BER were blocked. In MNU treated cells the maximal level of DNA strand breaks was detected immediately after the treatment and the breaks were repaired rapidly. In CdCl2 pre-treated cells the formation of MNU-induced DNA single-strand breaks was not affected, while the repair was slower, indicating inhibition of polymerization and/or the ligation step of BER. Cadmium thus affects the repair of UV-, MMS- and MNU-induced DNA damage, providing further evidence, that inhibition of DNA repair is an important mechanism of cadmium induced mutagenicity and carcinogenicity.     

Kinetics of excision repair of UV-induced DNA damage, measured using the comet assay

The kinetics of UV- (254 nm) irradiation-induced DNA single-strand breaks (SSBs), generated during the excision repair of UV-induced DNA damage, in leukemic lymphocytes and in normal blood mononuclear cells (MNCs) were studied using the alkaline comet assay. The cells were isolated by density gradient centrifugation from peripheral blood of patients with chronic lymphocytic leukemia (CLL) and from healthy study subjects. The cytotoxicity of UV irradiation was determined in vitro in peripheral blood mononuclear lymphocytes from 36 CLL patients and from eight healthy donors using the incorporation of radioactive leucine in 4-day cultures. A remarkable difference in excision repair capability was observed between normal and leukemic lymphocytes. In contrast to normal lymphocytes, there was always a subpopulation of CLL cells that did not complete the repair of UV-induced DNA damage during the 24-h repair period. Furthermore, differences were also recorded between UV-sensitive and UV-resistant CLL cases. The differences in DNA migration between the maximum increase (59-77 microm) and that at 24 h after irradiation (21-66 microm) was statistically significant in two of three patients exhibiting UV-resistance. Correspondingly, only in one of three patients exhibiting UV-sensitivity was the difference in DNA migration statistically significant (maximum increase: 44-107 microm, vs. 24 h after: 42-100 microm). Our results confirm an abnormal pattern of the CLL cell response to UV irradiation. Furthermore, we identified defective processing of UV-induced DNA damage in CLL versus normal lymphocytes, particularly in UV-sensitive cases.     

Rejoining kinetics of DNA single- and double-strand breaks in normal and DNA ligase-deficient cells after exposure to ultraviolet C and gamma radiation: an evaluation of ligating activities involved in different DNA repair processes

The repair kinetics for rejoining of DNA single- and double-strand breaks after exposure to UVC or gamma radiation was measured in cells with deficiencies in DNA ligase activities and in their normal counterparts. Human 46BR cells were deficient in DNA ligase I. Hamster EM9 and EM-C11 cells were deficient in DNA ligase III activity as a consequence of mutations in the XRCC1 gene. Hamster XR-1 cells had mutation in the XRCC4 gene, whose product stimulates DNA ligase IV activity. DNA single- and double-strand breaks were assessed by the comet assay in alkaline conditions and by the technique of graded-field gel electrophoresis in neutral conditions, respectively. 46BR cells, which are known to re-ligate at a reduced rate the DNA single-strand breaks incurred during processing of damage induced by UVC but not gamma radiation, were shown to have a normal repair of radiation-induced DNA double-strand breaks. EM9 cells exhibited a reduced rate of rejoining of DNA single-strand breaks after exposure to ionizing radiation, as reported previously, as well as UVC radiation. EM-C11 cells were deficient in the repair of radiation-induced-DNA single-strand breaks but, in contrast to EM9 cells, demonstrated the same kinetics as the parental cell line in the resealing of DNA breaks resulting from exposure to UVC radiation. Both EM9 and EM-C11 cells displayed a significant defect in rejoining of radiation-induced-DNA double-strand breaks. XR-1 cells were confirmed to be highly deficient in the repair of radiation-induced DNA double-strand breaks but appeared to rejoin DNA single-strand breaks after UVC and gamma irradiation at rates close to normal. Taken together these results indicate that: (1) DNA ligase I is involved only in nucleotide excision repair; (2) DNA ligase IV plays an important role only in repair of DNA double-strand breaks; and (3) DNA ligase III is implicated in base excision repair and in repair of DNA double-strand breaks, but probably not in nucleotide excision repair.     

Vanadate induces DNA strand breaks in cultured human fibroblasts at doses relevant to occupational exposure

To study possible genotoxic effects of occupational exposure to vanadium pentoxide, we determined DNA strand breaks (with alkaline comet assay), 8-hydroxy-2'deoxyguanosine (8-OHdG) and the frequency of sister chromatid exchange (SCE) in whole blood leukocytes or lymphocytes of 49 male workers employed in a vanadium factory in comparison to 12 non-exposed controls. In addition, vanadate has been tested in vitro to induce DNA strand breaks in whole blood cells, isolated lymphocytes and cultured human fibroblasts of healthy donors at concentrations comparable to the observed levels of vanadium in vivo. To investigate the impact of vanadate on the repair of damaged DNA, co-exposure to UV or bleomycin was used in fibroblasts, and DNA migration in the alkaline and neutral comet assay was determined. Although, exposed workers showed a significant vanadium uptake (serum: median 5.38microg/l, range 2.18-46.35microg/l) no increase in cytogenetic effects or oxidative DNA damage in leukocytes could be demonstrated. This was consistent with the observation that in vitro exposure of whole blood leukocytes and lymphocytes to vanadate caused no significant changes in DNA strand breaks below concentrations of 1microM (50microg/l). In contrast, vanadate clearly induced DNA fragmentation in cultured fibroblasts at relevant concentrations. Combined exposure of fibroblasts to vanadate/UV or vanadate/bleomycin resulted in non-repairable DNA double strand breaks (DSBs) as seen in the neutral comet assay. We conclude that exposure of human fibroblasts to vanadate effectively causes DNA strand breaks, and co-exposure of cells to other genotoxic agents may result in persistent DNA damage.     

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.     

DNA damage in zebrafish larvae induced by exposure to low-dose rate gamma-radiation: detection by the alkaline comet assay

This study has determined the sensitivity of the alkaline comet assay for the detection of strand breaks in the DNA of cells taken from a whole organism rather than a single cell type as in previously reported studies. The assay has been performed on cells from whole zebrafish larvae irradiated for 1 or 24 h at dose rates of 0.4, 1.2 or 7.2 mGy/h. Zebrafish larvae exposed to only 1.2 mGy/h of gamma-radiation for 1h showed a statistically significant increase in DNA damage compared to controls. This represents a high sensitivity of this animal model for DNA damage and of the comet assay protocol used for detecting such damage. Increasing the exposure time from 1 to 24 h caused significant increases in DNA damage in zebrafish larvae, although the modest size of these increases in damage for the relatively large increases (24 times) in total absorbed dose indicates that dose rate may be the major factor in determining the level of DNA damage observed under the conditions of these experiments.     

Possible repair action of Vitamin C on DNA damage induced by methyl methanesulfonate, cyclophosphamide, FeSO4 and CuSO4 in mouse blood cells in vivo

Interaction between Vitamin C (VitC) and transition metals can induce the formation of reactive oxygen species (ROS). VitC may also act as an ROS scavenger and as a metal chelant. To examine these possibilities, we tested in vivo the effect of two doses of VitC (1 and 30 mg/kg of mouse body weight) on the genotoxicity of known mutagens and transition metals. We used the alkaline version of the comet assay to assess DNA damage in peripheral white blood cells of mice. Animals were orally given either water (control), cyclophosphamide (CP), methyl methanesulfonate (MMS), cupric sulfate or ferrous sulfate. A single treatment with each VitC dose was administered after treatment with the mutagens or the metal sulfates. Both doses of VitC enhanced DNA damage caused by the metal sulfates. DNA damage caused by MMS was significantly reduced by the lower dose, but not by the higher dose of VitC. For CP, neither post-treatment dose of VitC affected the DNA damage level. These results indicate a modulatory role of Vitamin C in the genotoxicity/repair effect of these compounds. Single treatment with either dose of VitC showed genotoxic effects after 24 h but not after 48 h, indicating repair. Double treatment with VitC (at 0 and 24 h) induced a cumulative genotoxic response at 48 h, more intense for the higher dose. The results suggest that VitC can be either genotoxic or a repair stimulant, since the alkaline version of the comet assay does not differentiate "effective" strand breaks from those generated as an intermediate step in excision repair (incomplete excision repair sites). Further data is needed to shed light upon the beneficial/noxious effects of VitC.     

The role of short-lived DNA lesions in the production of chromosome-exchange aberrations

Human lymphocytes were treated with combined UVC radiation and X-rays or they were X-irradiated and incubated for 60–90 min in the presence of DNA-repair inhibitor ara-C. The X-ray induced chromosome exchange aberration yield was enhanced both by UVC and ara-C by approximately a factor of two in the linear (low dose) portion of the dose-response curve. The enhancement was small in the dose squared (high dose) portion where previous dose-fractionation experiments have shown that X-ray-induced lesions leading to aberrations exist for several hours. The yield of aberrations in lymphocytes incubated after irradiation in the presence of ara-C reaches a saturation level almost immediately after irradiation (5–15 min). These cytogenetic observations together with a previous finding (Holmberg and Strausmanis, 1983) give direct and indirect evidence that the enhanced aberration yield is due to short-lived DNA breaks formed immediately after X-irradiation.

Measurements on the repair kinetics of the DNA breaks induced by X-irradiation show that ara-C strongly impairs the repair of short-lived X-ray-induced DNA breaks. It was also observed that the DNA breaks generated after UVC irradiation occur almost immediately after irradiation and the level of these transient DNA breaks reaches saturation even for short incubation times. Thus, the repair of these breaks can compete with the repair of short-lived X-ray-induced DNA-breaks in combined irradiation with UVC and X-rays.

The experimental results can be explained on the assumption that X-ray-induced aberrations originate from exchange complexes formed in interactions between both short-lived DNA breaks. The short-lived DNA breaks give rise to exchange complexes mainly within single ionization tracks where the DNA breaks are close together. The time between irradiation and exchange complex formation is of the order of 5–15 min within such a track, and short-lived breaks might be repaired before complexes have been formed. If the DNA repair of these breaks is delayed by UVC or ara-C treatment this results in a higher probability of exchange-complex formation. In contrast, interactions between breaks in different tracks originate from long-lived DNA breaks and the probability for complex formation from these breaks is not markedly affected by UVC or ara-C.     

The comet assay: a method to measure DNA damage in individual cells

We present a procedure for the comet assay, a gel electrophoresis-based method that can be used to measure DNA damage in individual eukaryotic cells. It is versatile, relatively simple to perform and sensitive. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-links, base damage and apoptotic nuclei. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell. DNA damage and its repair in single-cell suspensions prepared from yeast, protozoa, plants, invertebrates and mammals can also be studied using this assay. Originally developed to measure variation in DNA damage and repair capacity within a population of mammalian cells, applications of the comet assay now range from human and sentinel animal biomonitoring (e.g., DNA damage in earthworms crawling through toxic waste sites) to measurement of DNA damage in specific genomic sequences. This protocol can be completed in fewer than 24 h.     

PARP-1 inhibition induces a late increase in the level of reactive oxygen species in cells after ionizing radiation

Poly(ADP-ribose) polymerase 1 (PARP1), an enzyme activated by DNA strand breaks, synthesizes polymers of poly(ADP-ribose) (PAR) that modify chromatin and other proteins and play a role in DNA repair. Inhibition of PARP1 activity is considered a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radio-therapy. Here we examined the influence of inhibition of PARP1 on formation of reactive oxygen species (ROS) and on DNA repair in cells exposed to ionizing radiation (IR). K562 (human myelogenous leukaemia) cells were grown and exposed to 4 or 12Gy of ionizing radiation in presence or absence of the PARP inhibitor NU1025 (100μM). Intracellular ROS were assayed using the probe 2,7-dichlorofluorescein with detection by flow cytometry and the rejoining of DNA strand breaks were followed by alkaline single cell gel electrophoresis (comet) assays. In untreated cells a significant increase in PAR formation occurred during the first 5min after IR, followed by a gradual decrease up to 30min. Addition of a PARP inhibitor arrested the production of PAR almost completely and decreased the rate of rejoining of DNA strand breaks significantly; however, 3h after irradiation we observed no difference in the amount of DNA strand breaks between PARP inhibitor-treated and untreated cells. Twelve to 48h after irradiation, an increase of ROS concentration was observed in irradiated cells and ROS levels in PARP inhibitor-treated cells were significantly higher than in cells without inhibitor. Irradiated cells grown in the presence or absence of PARP inhibitor did not differ in the frequencies of apoptotic and necrotic cells or in the activity of caspases at 24, 48 and 72h after irradiation. Poly(ADP-ribosylation) and inhibition of PARP1 appeared to modulate DNA strand break rejoining and influence the concentration of ROS in irradiated cells.     

Primary DNA damage in human blood lymphocytes exposed in vitro to 2450 MHz radiofrequency radiation

Human peripheral blood samples collected from three healthy human volunteers were exposed in vitro to pulsed-wave 2450 MHz radiofrequency (RF) radiation for 2 h. The RF radiation was generated with a net forward power of 21 W and transmitted from a standard gain rectangular antenna horn in a vertically downward direction. The average power density at the position of the cells in the flask was 5 mW/cm(2). The mean specific absorption rate, calculated by finite difference time domain analysis, was 2.135 (+/-0.005 SE) W/kg. Aliquots of whole blood that were sham-exposed or exposed in vitro to 50 cGy of ionizing radiation from a (137)Cs gamma-ray source were used as controls. The lymphocytes were examined to determine the extent of primary DNA damage (single-strand breaks and alkali-labile lesions) using the alkaline comet assay with three different slide-processing schedules. The assay was performed on the cells immediately after the exposures and at 4 h after incubation of the exposed blood at 37 +/- 1 degrees C to allow time for rejoining of any strand breaks present immediately after exposure, i.e. to assess the capacity of the lymphocytes to repair this type of DNA damage. At either time, the data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to the comet tail length, fluorescence intensity of the migrated DNA in the tail, and tail moment. The conclusions were similar for each of the three different comet assay slide-processing schedules examined. In contrast, the response of lymphocytes exposed to ionizing radiation was significantly different from RF-radiation- and sham-exposed cells. Thus, under the experimental conditions tested, there is no evidence for induction of DNA single-strand breaks and alkali-labile lesions in human blood lymphocytes exposed in vitro to pulsed-wave 2450 MHz radiofrequency radiation, either immediately or at 4 h after exposure.     

Nitric oxide inhibits DNA-adduct excision in nucleotide excision repair

Sustained induction of nitric oxide (NO) in chronic inflammation may be mutagenic, through DNA damage induction and/or DNA repair inhibition. Although there is good evidence that NO can cause DNA damage, how NO is involved in DNA repair remains elusive. By using DNA synthesis inhibitors to accumulate DNA strand breaks in comet assay, we show that NO and peroxynitrite inhibit DNA-adduct excision in human fibroblasts damaged by UVC, 4-nitroquinoline 1-oxide, benzo[a]pyrene dihydrodiol epoxide, cisplatin, or mitomycin C, but not with methyl methane sulfonate. Treating cells with arsenite increased NO production and also inhibited the DNA-adduct excision induced by UVC, 4-nitroquinoline 1-oxide, benzo[a]pyrene dihydrodiol epoxide, cisplatin, and mitomycin C, but not by methyl methane sulfonate, H(2)O(2), sodium nitrosoprusside, or 3-morpholinosydnonimine. Arsenite inhibition of DNA-adduct excision was decreased by NO synthase inhibitors and NO scavengers. The nuclear extract prepared from fibroblasts pretreated with sodium nitrosoprusside, dipropylenetriamine NONOate, 3-morpholinosydnonimine, or arsenite also showed decreased activity in excising the DNA adducts induced by UVC and cisplatin but not by methyl methane sulfonate or H(2)O(2) plus Fe. These results are consistent with the notion that NO, peroxynitrite, and arsenite inhibit the DNA-adduct excision in nucleotide excision repair but not that in base excision repair.     

Evaluation of UV damage at DNA level in <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis> protoplasts using single cell gel electrophoresis

The aim of the present study was to observe the induction and repair of single strand breaks (Ssbs) and double strand breaks (Dsbs) in mesophyll protoplasts of Nicotiana plumbaginifolia, irradiated with UV-C and cultured under light or dark conditions. DNA damage and repair was determined by the neutral and alkaline comet assay to reveal Dsbs and Ssbs respectively. Subculturing protoplasts for 4 h at low temperature was essential to reduce the amount of Dsbs to the detection limit of the assay procedure. Light-cultured protoplasts showed a significant increase of Ssbs and Dsbs compared to dark cultured protoplasts, in which the number of Ssbs and Dsbs remained very constant throughout the experiments. UV treatment significantly enhanced the levels of Ssbs and Dsbs in light and dark cultured protoplasts. On average, equal levels of DNA damage were observed under light or dark conditions. Formulations introduced to evaluate the contribution of UV-C or light treatment in repair kinetics of DNA damage, showed that the number of Ssbs, but not of Dsbs, evolved differently for light and dark cultured protoplasts. DNA repair was more rapidly observed under light conditions and occurred in different repair phases. Observations are discussed in relation to the involvement of chromatin remodelling, photosynthetic active radiation and DNA repair mechanisms.     

Niacin deficiency delays DNA excision repair and increases spontaneous and nitrosourea-induced chromosomal instability in rat bone marrow

We have shown that niacin deficiency impairs poly(ADP-ribose) formation and enhances sister chromatid exchanges and micronuclei formation in rat bone marrow. We designed the current study to investigate the effects of niacin deficiency on the kinetics of DNA repair following ethylation, and the accumulation of double strand breaks, micronuclei (MN) and chromosomal aberrations (CA). Weanling male Long-Evans rats were fed niacin deficient (ND), or pair fed (PF) control diets for 3 weeks. We examined repair kinetics by comet assay in the 36 h following a single dose of ethylnitrosourea (ENU) (30 mg/kg bw). There was no effect of ND on mean tail moment (MTM) before ENU treatment, or on the development of strand breaks between 0 and 8 h after ENU. Repair kinetics between 12 and 30 h were significantly delayed by ND, with a doubling of area under the MTM curve during this period. O₆-ethylation of guanine peaked by 1.5 h, was largely repaired by 15 h, and was also delayed in bone marrow cells from ND rats. ND significantly enhanced double strand break accumulation at 24 h after ENU. ND alone increased chromosome and chromatid breaks (four- and two-fold). ND alone caused a large increase in MN, and this was amplified by ENU treatment. While repair kinetics suggest that ND may be acting by creating catalytically inactive PARP molecules with a dominant-negative effect on repair processes, the effect of ND alone on O₆-ethylation, MN and CA, in the absence of altered comet results, suggests additional mechanisms are also leading to chromosomal instability. These data support the idea that the bone marrow cells of niacin deficient cancer patients may be more sensitive to the side effects of genotoxic chemotherapy, resulting in acute bone marrow suppression and chronic development of secondary leukemias.     

Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.     

Comparative in vitro and in vivo assessment of genotoxic effects of etoposide and chlorothalonil by the comet assay.

The alkaline single cell gel electrophoresis (comet) assay was used to assess in vitro and in vivo genotoxicity of etoposide, a topoisomerase II inhibitor known to induce DNA strand breaks, and chlorothalonil, a fungicide widely used in agriculture. For in vivo studies, rats were sacrificed at various times after treatment and the induction of DNA strand breaks was assessed in whole blood, bone marrow, thymus, liver, kidney cortex and in the distal part of the intestine. One hour after injection, etoposide induced DNA damage in all organs studied except kidney, especially in bone marrow, thymus (presence of HDC) and whole blood. As observed during in vitro comet assay on Chinese hamster ovary (CHO) cells, dose- and time-dependent DNA effects occurred in vivo with a complete disappearance of damage 24 h after administration. Even though apoptotic cells were detected in vitro 48 h after cell exposure to etoposide, such a result was not found in vivo. After chlorothalonil treatment, no DNA strand breaks were observed in rat organs whereas a clear dose-related DNA damage was observed in vitro. The discrepancy between in vivo and in vitro models could be explained by metabolic and mechanistic reasons. Our results show that the in vivo comet assay is able to detect the target organs of etoposide and suggest that chlorothalonil is devoid of appreciable in vivo genotoxic activity under the protocol used.     

Relationship of DNA repair to chromosome aberrations, sister-chromatid exchanges and survival during liquid-holding recovery in X-irradiated mammalian cells

The repair of X-ray induced DNA single strand breaks and DNA—protein cross-links was investigated in stationary phase, contact-inhibited mouse cells by the alkaline-elution technique. Approx. 90% of X-ray induced single strand breaks were rejoined during the first hour of repair, whereas most of the remaining breaks were rejoined more slowly during the next 5 h. At early repair times, the number of residual non-rejoined sungle strand breaks was approx. proportional to the X-ray dose. DNA—protein cross-links were removed at a slower rate (T1/2 approx. 10–12 h). Cells were held in stationary growth for various periods of time after irradiation before subculture at low density to score for colony survival (potentially lethal damage repair), chromosome aberrations in the first mitosis, and sister-chromatid exchanges in the second mitosis. Both cell killing and the frequency of chromosome aberrations decreased during the first several hours of recovery, reaching a minimum level by 6 h; this decrease correlated temporally with the repair of the slowly rejoining DNA-strand breaks. Relatively few sister-chromatid exchanges were observed when the cells were subcultured immediately after X-ray. The exchange frequency rose to maximum levels after a 4-h recovery interval, and returned to control levels after 12 h of recovery. The possible relationship of DNA repair to these changes in survival, chromosome aberrations, and sister-chromatid exchanges during liquid-holding recovery is discussed.     

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.