Repair of X-ray induced DNA strand damage by isolated rat splenic lymphocytes.

Although a number of chemicals can alter DNA repair function, little is known about the effect of chronic, low dose exposure to environmental agents on DNA repair capacity. Lymphocytes provide a potential target population to study the effects of chronic exposures to low doses of toxic chemicals since they are an easily obtainable cell population. Prior to investigating the repair capacity of chemically exposed lymphocytes, the repair by chemically naive lymphocytes has been characterized. In the present study, the DNA repair capacity of isolated rat lymphocytes was characterized. The capacity of these cells to repair single-strand DNA breaks (SSB) was determined after in vitro treatments with X-rays. The effect of in vitro exposure to 3-aminobenzamide (3-AB) on DNA repair capacity was also assessed. The levels of induced SSB and their repair were determined using the alkaline elution technique. Splenic lymphocytes were isolated and placed in culture medium 18 h prior to assessment of repair capacity, but were not stimulated with mitogens. A dose-dependent increase in SSB was observed following exposure of lymphocytes to 300 or 600 rad. The rate of SSB repair was analyzed after a dose of 400 rad. Approximately 80% of the DNA strand break repair was completed within 60 min. The half-time for repair of these lesions by lymphocytes was determined to be 21.3 min. Exposure to 3-AB resulted in a decrease in the rate of repair of the X-ray-induced strand breakage. Although no SSB were detected at the end of a 1-h 3-AB treatment of non-irradiated cells, significant accumulation of SSB was observed after a 2-h treatment. The characterization of DNA repair in rat lymphocytes following in vitro exposure to X-rays will allow us to investigate the effects of chronic, in vivo toxicant exposure on the capacity of isolated lymphocytes to repair DNA damage produced by X-rays.     

A microgel electrophoresis technique for the direct quantitation of DNA damage and repair in individual fibroblasts cultured on microscope slides

We demonstrate by single-cell microgel electrophoresis that the 2 main techniques, trypsinization and scraping, used to collect normal diploid mammalian cells cultured in monolayer induce DNA damage. To minimize this potential interference with studies on DNA damage and repair, we have standardized the single-cell gel electrophoretic (SCG) technique for the in situ quantitation of DNA single-strand breaks and alkali-labile sites in cultured human-fibroblasts. To demonstrate the utility of this technique, human neonatal foreskin-derived fibroblasts were allowed to attach to frosted microscope slides and then either irradiated with X-rays (25-200 rad) or treated for 1 h with hydrogen peroxide (2.2-140.8 mumoles). Treatment with either agent induced a dose-dependent increase in DNA migration. At equal levels of DNA damage, cell-to-cell variability in DNA migration was more heterogeneous for hydrogen peroxide-treated cells than for X-irradiated cells. A time course study to evaluate the kinetics of DNA repair for X-ray (200 rad)-induced damage indicated that the damage was completely repaired within 2 h. Applications of this technique for in vitro toxicology are discussed.     

Genotoxicity of inhalation anesthetics halothane and isoflurane in human lymphocytes studied in vitro using the comet assay

The alkaline single cell gel electrophoresis (comet) assay was applied to study genotoxic properties of two inhalation anesthetics-halothane and isoflurane-in human peripheral blood lymphocytes (PBL). The cells were exposed in vitro to either halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) or isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether) at concentrations 0.1-10 mM in DMSO. The anesthetics-induced DNA strand breaks as well as alkali-labile sites were measured as total comet length (i.e., increase of a DNA migration). Both analysed drugs were capable of increasing DNA migration in a dose-dependent manner. In experiments conducted at two different electrophoretic conditions (0. 56 and 0.78 V/cm), halothane was able to increase DNA migration to a higher extent than isoflurane. The comet assay detects DNA strand breaks induced directly by genotoxic agents as well as DNA degradation due to cell death. For this reason a contribution of toxicity in the observed effects was examined. We tested whether the exposed PBL were able to repair halothane- and isoflurane-induced DNA damage. The treated cells were incubated in a drug-free medium at 37 degrees C for 120 min to allow processing of the induced DNA damage. PBL exposed to isoflurane at 1 mM were able to complete repair within 60 min whereas for halothane a similar result was obtained at a concentration lower by one order of magnitude: the cells exposed to halothane at 1 mM removed the damage within 120 min only partly. We conclude that the increase of DNA migration induced in PBL by isoflurane at 1 mM and by halothane at 0.1 mM was not a result of cell death-associated DNA degradation but was caused by genotoxic action of the drugs. The DNA damage detected after the exposure to halothane at 1 mM was in part a result of DNA fragmentation due to cell death.     

The evaluation of protective effect of lycopene against genotoxic influence of X-irradiation in human blood lymphocytes

Many studies suggest that exogenous antioxidants may protect cells against DNA damage caused with ionizing radiation. One of the most powerful antioxidants is lycopene (LYC), a carotenoid derived from tomatoes. The aim of this study was to investigate, using the comet assay, whether LYC can act as protectors/modifiers and prevent DNA damage induced in human blood lymphocytes, as well as to mitigate the effects of radiation exposure. In this project, LYC, dissolved in DMSO at a concentration of 10, 20 or 40 μM/ml of cell suspension, was added to the isolated lymphocytes from human blood at appropriate intervals before or after the X-irradiation at doses of 0.5, 1 and 2 Gy. Cell viability in all groups was maintained at above 70%. The results showed the decrease of DNA damage in cells treated with various concentrations of LYC directly and 1 h before exposure to X-rays compared to the control group exposed to irradiation alone. Contrary results were observed in cells exposed to LYC immediately after exposure to ionizing radiation. The studies confirmed the protective effect of LYC against DNA damage induced by ionizing radiation, but after irradiation the carotenoid did not stimulate of DNA repair and cannot act as modifier. However, supplementation with LYC, especially at lower doses, may be useful in protection from radiation-induced oxidative damage.     

Exposure of mammalian cells to 60-Hz magnetic or electric fields: analysis of DNA repair of induced, single-strand breaks

DNA damage was induced in isolated human peripheral lymphocytes by exposure at 5 Gy to 60Co radiation. Cells were permitted to repair the DNA damage while exposed to 60-Hz fields or while sham-exposed. Exposed cells were subjected to magnetic (B) or electric (E) fields, alone or in combination, throughout their allotted repair time. Repair was stopped at specific times, and the cells were immediately lysed and then analyzed for the presence of DNA single-strand breaks (SSB) by the alkaline-elution technique. Fifty to 75 percent of the induced SSB were repaired 20 min after exposure, and most of the remaining damage was repaired after 180 min. Cells were exposed to a 60-Hz ac B field of 1 mT; an E field of 1 or 20 V/m; or combined E and B fields of 0.2 V/m and 0.05 mT, 6 V/m and 0.6 mT, or 20 V/m and 1 mT. None of the exposures was observed to affect significantly the repair of DNA SSB.     

Human lymphocytes exposed to low doses of ionizing radiations become refractory to high doses of radiation as well as to chemical mutagens that induce double-strand breaks in DNA

Human lymphocytes exposed to low doses of ionizing radiation from incorporated tritiated thymidine or from X-rays become less susceptible to the induction of chromatid breaks by high doses of X-rays. This response can be induced by 0.01 Gy (1 rad) of X-rays, and has been attributed to the induction of a repair mechanism that causes the restitution of X-ray-induced chromosome breaks. Because the major lesions responsible for the induction of chromosome breakage are double-strand breaks in DNA, attempts have been made to see if the repair mechanism can affect various types of clastogenic lesions induced in DNA by chemical mutagens and carcinogens. When cells exposed to 0.01 Gy of X-rays or to low doses of tritiated thymidine were subsequently challenged with high doses of tritiated thymidine or bleomycin, which can induce double-strand breaks in DNA, or mitomycin C, which can induce cross-links in DNA, approximately half as many chromatid breaks were induced as expected. When, on the other hand, the cells were challenged with the alkylating agent methyl methanesulfonate (MMS), which can produce single-strand breaks in DNA, approximately twice as much damage was found as was induced by MMS alone. The results indicate that prior exposure to 0.01 Gy of X-rays reduces the number of chromosome breaks induced by double-strand breaks, and perhaps even by cross-links, in DNA, but has the opposite effect on breaks induced by the alkylating agent MMS. The results also show that the induced repair mechanism is different from that observed in the adaptive response that follows exposure to low doses of alkylating agents.     

Influence of environmental exposure to PAHs on the susceptibility of lymphocytes to DNA-damage induction and on their repair capacity

The influence of occupational exposure to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) on DNA damage detected in lymphocytes of exposed people (city policemen) was studied. The cellular susceptibility to the induction of the DNA damage and the repair capacity of exposed donors are presented in comparison with matched controls. Monitoring was performed and blood samples (164 donors) were collected in Prague, Czech Republic, during the winter and summer seasons. The single-cell gel electrophoresis (SCGE) assay with an internal standard was applied to evaluate the DNA damage. A challenging dose of 2Gy of X-rays was used to study cellular capacities. In the results of studies of the DNA damage induced in vivo or as an immediate response to the challenging treatment no significant difference was found between exposed and unexposed subgroups. The percentage of non-repaired X-ray-induced DNA damage (residual damage, RD) overall in both seasons was significantly higher in lymphocytes of policemen exposed to c-PAHs than in matched controls (RD(T-DNA), %DNA in the comet tail: winter 36.4+/-22.1 versus 22.7+/-10.8, p < 0.001; summer 47.7+/-22.9 versus 34.7+/-15.2, p < 0.001). The results suggest that occupational exposure to environmental c-PAHs significantly reduces the cellular capacity to repair the DNA damage induced by a challenging treatment. A significant decrease of repair efficiency in donors occupationally exposed to environmental c-PAHs was also observed when subgroups were stratified according to smoking history. In conclusion, our results suggest that environmental exposure to c-PAHs affects the cellular repair processes and can lead to harmful effects hazardous to human health.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25+/-8.45 microm; non-smokers, 21.6+/-2.06 microm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5+/-20.34 microm; non-smokers, 79.2+/-11.59 microm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13+/-10.73 microm; non-smokers, (27.2+/-4.13 microm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.     

DNA repair after gamma irradiation in lymphocytes exposed to low-frequency pulsed electromagnetic fields

The effect of exposure to extremely low-frequency pulsed electromagnetic fields (EMFs) on DNA repair capability and on cell survival in human lymphocytes damaged in vitro with gamma rays was studied by two different micromethods. In the first assay, which measures DNA repair synthesis (unscheduled DNA synthesis, UDS), lymphocyte cultures were stimulated with phytohemagglutinin (PHA) for 66 h and then treated with hydroxyurea (which blocks DNA replication), irradiated with 100 Gy of 60Co, pulsed with [3H]thymidine ([3H]TdR), and then exposed to pulsed EMFs for 6 h (the period in which cells repaired DNA damage). In the second assay, which measures cell survival after radiation or chemical damage, lymphocytes were first irradiated with graded doses of gamma rays or treated with diverse antiproliferative agents, and then stimulated with PHA, cultured for 72 h, and pulsed with [3H]TdR for the last 6 h of culture. In this case, immediately after the damage induced by either the radiation or chemicals, cultures were exposed to pulsed EMFs for 72 h, during which cell proliferation took place. Exposure to pulsed EMFs did not affect either UDS or cell survival, suggesting that this type of nonionizing radiation--to which humans may be exposed in the environment, and which is used for both diagnostic and therapeutic purposes--does not affect DNA repair mechanisms.     

Influence of environmental exposure to PAHs on the susceptibility of lymphocytes to DNA-damage induction and on their repair capacity

The influence of occupational exposure to environmental carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) on DNA damage detected in lymphocytes of exposed people (city policemen) was studied. The cellular susceptibility to the induction of the DNA damage and the repair capacity of exposed donors are presented in comparison with matched controls. Monitoring was performed and blood samples (164 donors) were collected in Prague, Czech Republic, during the winter and summer seasons. The single-cell gel electrophoresis (SCGE) assay with an internal standard was applied to evaluate the DNA damage. A challenging dose of 2 Gy of X-rays was used to study cellular capacities. In the results of studies of the DNA damage induced in vivo or as an immediate response to the challenging treatment no significant difference was found between exposed and unexposed subgroups. The percentage of non-repaired X-ray-induced DNA damage (residual damage, RD) overall in both seasons was significantly higher in lymphocytes of policemen exposed to c-PAHs than in matched controls (RD_T-DNA, %DNA in the comet tail: winter 36.4 ± 22.1 versus 22.7 ± 10.8, p < 0.001; summer 47.7 ± 22.9 versus 34.7 ± 15.2, p < 0.001). The results suggest that occupational exposure to environmental c-PAHs significantly reduces the cellular capacity to repair the DNA damage induced by a challenging treatment. A significant decrease of repair efficiency in donors occupationally exposed to environmental c-PAHs was also observed when subgroups were stratified according to smoking history. In conclusion, our results suggest that environmental exposure to c-PAHs affects the cellular repair processes and can lead to harmful effects hazardous to human health.     

Repair of products of oxidative DNA base damage in human cells.

Oxidative DNA damage is the most frequent type of damage encountered by aerobic cells and may play an important role in biological processes such as mutagenesis, carcinogenesis and aging in humans. Oxidative damage generates a myriad of modifications in DNA. We investigated the cellular repair of DNA base damage products in DNA of cultured human lymphoblast cells, which were exposed to oxidative stress by H2O2. This DNA-damaging agent is known to cause base modifications in genomic DNA of mammalian cells [Dizdaroglu, M., Nackerdien, Z., Chao, B.-C., Gajewski, E. and Rao, G. (1991) Arch. Biochem. Biophys. 285, 388-390]. Following treatment with H2O2, the culture medium was freed from H2O2 and cells were incubated for time periods ranging from 10 min to 6 h. DNA was isolated from control cells, hydrogen peroxide-treated cells and cells incubated after H2O2 exposure. DNA samples were analyzed by gas chromatography/isotope-dilution mass spectrometry. Eleven modified bases were identified and quantified. The results showed a significant formation of these DNA base products upon H2O2-treatment of cells. Subsequent incubation of cells caused a time-dependent excision of these products from cellular DNA. The cell viability did not change significantly by various treatments. There were distinct differences between the kinetics of excision of individual products. The observed excisions were attributed to DNA repair in cells. The rate of repair of purine lesions was slower than that of pyrimidine lesions. Most of the identified products are known to possess various premutagenic properties. The results of this work may contribute to the understanding of the cellular repair of oxidative DNA damage in human and other mammalian cells.     

Kinetics of division of human and rabbit lymphocytes stimulated by phytohemagglutinin, studied by means of the bromodeoxyuridine (BUDR) technic]

The BUDR-Giemsa technique has been used to distinguish the first from later divisions for man and rabbit lymphocytes stimulated by treatment with phytohemagglutinin. At 48 hours, 99% of human lymphocytes exposed to 200 rads of X-rays are in first division whereas 45% of the rabbit lymphocytes are already in second division and 28% in third division.     

Inhalation of ozone induces DNA strand breaks and inflammation in mice

Ozone (O3) is a well-known oxidant pollutant present in photochemical smog. Although ozone is suspected to be a respiratory carcinogen it is not regulated as a carcinogen in most countries.The genotoxic and inflammatory effects of ozone were investigated in female mice exposed to ozone for 90 min. The tail moment in bronchoalveolar lavage (BAL) cells from BALB/c mice was determined by the comet assay as a measure of DNA strand breaks. Within the first 200 min after exposure, the BAL cells from the mice exposed to 1 or 2 ppm ozone had 1.6- and 2.6-fold greater tail moments than unexposed mice. After 200 min there was no effect. It could be ruled out that the effect during the first 200 min was due to major infiltration of lymphocytes or neutrophils. Unexpectedly, ozone had no effect on the content of 8-oxo-deoxyguanosine (8-oxo-dG) in nuclear DNA or on oxidised amino acids in the lung tissue. The mRNA level of the repair enzyme ERCC1 was not increased in the lung tissue. Inflammation was measured by the cytokine mRNA level in lung homogenates. An up to 150-fold induction of interleukin-6 (IL-6) mRNA was detected in the animals exposed to 2 ppm ozone compared to the air-exposed control mice. Also at 1 ppm ozone, the IL-6 mRNA was induced. The large induction of IL-6 mRNA in the lung took place after DNA strand breaks were induced in BAL. This does not support the notion that inflammatory reactions are the cause of DNA damage. To determine whether these exposures were mutagenic, Muta Mice were exposed to 2 ppm ozone, 90 min per day for 5 days. No treatment-related mutations could be detected in the cII transgene.These results indicate that a short episode of ozone exposure at five times the threshold limit value (TLV) in US induces lung inflammatory mediators and DNA damage in the cells in the lumen of the lung. This was not reflected by an induction of mutations in the lung of Muta Mice.     

Imatinib (STI571) induces DNA damage in BCR/ABL-expressing leukemic cells but not in normal lymphocytes

Imatinib (STI571) is a 2-phenylaminopyrimidine derivative used mostly in the treatment of chronic myeloid leukaemia. It targets the BCR/ABL oncogenic tyrosine kinase, inhibiting its activity. Using the alkaline comet assay we showed that STI571 at concentrations ranging from 0.2 to 2 microM induced DNA damage in human leukemic K562 and BV173 cells expressing the BCR/ABL oncogene, whereas it had no effect in normal human lymphocytes and leukemic CCRF-CEM cells without the expression of BCR/ABL. Imatinib did not induce DNA strand breaks in the direct interaction with DNA as examined by the circular plasmid relaxation assay. Because the extent of DNA damage observed in the neutral and pH 12.1 versions of the comet assay was much lesser than in the alkaline version, we concluded that the drug induced DNA alkali-labile sites rather than strand breaks. K562 cells were unable to repair H(2)O(2)-induced DNA damage during a 120-min incubation, if they had been preincubated with STI571, whereas normal lymphocytes did so within 60 min. Pre-treatment of K562 cells with Vitamins A, C and E reduced the extent of DNA damage evoked by STI571. Similar results brought experiments with the nitrone spin traps POBN and PBN, suggesting that free radicals may be involved in the formation of DNA lesions induced by STI571 in K562 cells. These cells exposed to imatinib and treated with endonuclease III, formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, the enzymes recognizing oxidized and alkylated bases, displayed greater extent of DNA damage than those not treated with these enzymes. Therefore, the mechanism of the anti-leukemic action of STI571 may involve not only the inhibition of BCR/ABL, but also DNA damage in the cells expressing this fusion protein. DNA damage induced by STI571 may follow from oxidative and alkylative base modifications.     

Role of senataxin in DNA damage and telomeric stability

Ataxia with oculomotor apraxia type 2 (AOA2) is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia and oculomotor apraxia. The gene mutated in AOA2, SETX, encodes senataxin (SETX), a putative DNA/RNA helicase. The presence of the helicase domain led us to investigate whether SETX might play a role in DNA damage repair and telomere stability. We analyzed the response of AOA2 lymphocytes and lymphoblasts after treatment with camptothecin (CPT), mitomycin C (MMC), H?O? and X-rays by cytogenetic and Q-FISH (quantitative-FISH) assays. The rate of chromosomal aberrations was normal in AOA2 cells after treatment with CPT, MMC, H?O? and X-rays. Conversely, Q-FISH analysis showed constitutively reduced telomere length in AOA2 lymphocytes, compared to age-matched controls. Furthermore, CPT- or X-ray-induced telomere shortening was more marked in AOA2 than in control cells. The partial co-localization of SETX with telomeric DNA, demonstrated by combined immunofluorescence-Q-FISH and chromatin immunoprecipitation, suggests a possible involvement of SETX in telomere stability.     

Alterations in histone acetylation following exposure to <Superscript>60</Superscript>Co γ-rays and their relationship with chromosome damage in human lymphoblastoid cells

Chromosome damage is related to DNA damage and erroneous repair. It can cause cell dysfunction and ultimately induce carcinogenesis. Histone acetylation is crucial for regulating chromatin structure and DNA damage repair. Ionizing radiation (IR) can alter histone acetylation. However, variations in histone acetylation in response to IR exposure and the relationship between histone acetylation and IR-induced chromosome damage remains unclear. Hence, this study investigated the variation in the total acetylation levels of H3 and H4 in human lymphocytes exposed to 0–2 Gy ⁶⁰Co γ-rays. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to modify the histone acetylation state of irradiated cells. Then, the total acetylation level, enzyme activity, dicentric plus centric rings (dic?+?r) frequencies, and micronucleus (MN) frequencies of the treated cells were analyzed. Results indicated that the acetylation levels of H3 and H4 significantly decreased at 1 and 24 h, respectively, after radiation exposure. The acetylation levels of H3 and H4 in irradiated groups treated with SAHA were significantly higher than those in irradiated groups that were not treated with SAHA. SAHA treatment inhibited HDAC activity in cells exposed to 0–1 Gy ⁶⁰Co γ-rays. SAHA treatment significantly decreased dic?+?r/cell and MN/cell in cells exposed to 0.5 or 1.0 Gy ⁶⁰Co γ-rays relative to that in cells that did not receive SAHA treatment. In conclusion, histone acetylation is significantly affected by IR and is involved in chromosome damage induced by ⁶⁰Co γ-radiation.     

Occupational exposure to mercury vapour on genotoxicity and DNA repair

We have conducted a population study to investigate whether current occupational exposure to mercury can cause genotoxicity and can affect DNA repair efficiency. Blood samples from 25 exposed workers and 50 matched controls were investigated for the expression of genotoxicity. The data indicate that mercury exposure did not cause any significant differences between the workers and controls in the baseline levels of DNA strand breaks (as measured by the alkaline version of the single cell gel electrophoresis [SCGE] assay) or sister chromatid exchanges (SCE). However, the exposure produced elevated average DNA tails length in the SCGE assay and frequency of chromosome aberrations. In the studies, isolated lymphocytes were exposed to 6J/m2 UV-C light or 2 Gy dose of X-rays in a challenge assay and repair of the induced DNA damage was evaluated using the SCGE assay. Results from the UV-light challenge assay showed no difference between the workers and controls in the expression of DNA strand breaks after exposure followed by incubation in the absence or presence of the cellular mitogen (phytohemagglutinin, PHA). No difference in DNA strand breaks between the workers and controls was seen immediately after the X-ray challenge, either. However, significant differences were observed in cells that were incubated for 2h with and without phytohemagglutinin. Data from the X-rays challenge assay were further used to calculate indices that indicate DNA repair efficiency. Results show that the repair efficiencies for the workers (69.7% and 83.9% in un-stimulated and stimulated lymphocytes, respectively) were significantly lower than that of matched controls (85.7% and 90.4%, respectively). In addition, the repair efficiency showed a consistent and significant decrease with the duration of occupational exposure to mercury (from 75.7% for <10 years employment, to 65.1% for 11-20 years and to 64.1% for 21-35 years) associated with increase of cytogenetic damage. Our study suggests that the occupational exposure to mercury did not cause a direct genotoxicity but caused significant deficiency in DNA repair. Our observations are consistent with previous studies using the standard chromosome aberration assay to show that exposure to hazardous environmental agents can cause deficiency in DNA repair. Therefore, these affected individuals may have exposure-related increase of health risk from continued exposure and in combination with exposure to other genotoxic agents.     

Adaptive response of human lymphocytes for the repair of radon-induced chromosomal damage

In human lymphocytes low doses of X-rays can decrease the number of chromatid deletions induced by subsequent high doses of sparsely ionizing X-rays. Because of the concern with the carcinogenic effects of low doses of -particles from radon in homes, experiments were carried out to see if low doses of X-rays could also decrease the yield of chromosomal aberrations induced by subsequent exposure to radon. Human peripheral blood lymphocytes were irradiated with low doses of X-rays (2 cGy) at 48 h of culture, exposed to radon at 72 h of culture, and analyzed for the presence of chromatid aberrations at subsequent intervals. The frequency of chromatid aberrations induced by radon alone increased with time after exposure, indicating exaggerated differences in the stage sensitivity of cell cycle stages to high-LET radiation. Furthermore, the numbers of aberrations per cell did not follow a Poisson distribution but were over dispersed, as might be expected since high-LET radiations have a high relative biological effectiveness compared with low-LET radiations. Nevertheless, lymphocytes exposed to 2 cGy of X-rays before radon exposure contained approximately one-half the number of chromatid deletions compared with lymphocytes treated with radon alone and analzed at the same time. Thus, the putative chromosomal repair mechanism induced by low doses of sparsely ionizing radiation is also effective in reducing chromosomal aberrations induced by radon, which hitherto had been thought to be relatively independent of repair processes.     

DNA damage and repair with age in individual human lymphocytes

Previous biochemical studies on DNA repair competence and aging have been limited to techniques, such as alkaline elution or nucleoid sedimentation, involving mass cell populations. These techniques provide no information about the distribution of DNA damage and repair among individual cells and are unlikely to detect age-dependent changes affecting a minor fraction of the cell population. We have recently described a microgel electrophoretic assay (Singh et al., 1988) that measures, at the level of the individual cell, single-strand DNA breaks and alkali-sensitive sites. Here, we employ this method to analyze DNA damage and repair in lymphocytes isolated from the peripheral blood of 31 subjects (23 males and 8 females aged 25-91 years) and exposed in vitro to 200 rads of X-irradiation. While basal (pre-irradiation) levels of damage were independent of the age of the donor, an age-dependent increase in DNA damage was observed immediately following irradiation. For all subjects, the mean level of DNA damage was restored to pre-irradiation control levels within 2 h of incubation at 37 degrees C. However, a distribution analysis of DNA damage among cells within each sample indicated the presence of a few highly damaged cells (4-16%) in the 2-h sample, the occurrence of which was significantly more common among aged individuals. These data indicate an age-related decline in DNA repair competence among a small subpopulation of lymphocytes.     

DNA damage and integrity of UV-induced DNA repair in lymphocytes of smokers analysed by the comet assay

DNA damage was assessed in smoker lymphocytes by subjecting them to the single cell gel electrophoresis (SCGE) assay. In addition to the appearance of comet tails, smoker cells exhibited enlarged nuclei when analysed by the comet assay. On comparing basal DNA damage among smokers and a non-smoking control group, smoker lymphocytes showed higher basal DNA damage (smokers, 36.25±8.45 μm; non-smokers, 21.6±2.06 μm). A significant difference in DNA migration lengths was observed between the two groups at 10 min after UV exposure (smokers, 65.5±20.34 μm; non-smokers, 79.2±11.59 μm), but no significant differences were seen at 30 min after UV exposure (smokers, 21.13±10.73 μm; non-smokers, (27.2±4.13 μm). The study thus implies that cigarette smoking perhaps interferes with the incision steps of the nucleotide excision repair (NER) process. There appeared be no correlation between the frequency of smoking and DNA damage or the capacity of the cells to repair UV-induced DNA damage that suggests inherited host factors may be responsible for the inter-individual differences in DNA repair capacities. The study also suggests monitoring NER following UV insult using the SCGE assay is a sensitive and simple method to assess DNA damage and integrity of DNA repair in human cells exposed to chemical mutagens.