Evidence to support the existence of efficient DNA double-strand break rejoining in a radiosensitive mutant of V79-4 following irradiation with 250 kVp X-rays or neutrons

The repair of ionising-radiation-induced DNA double-strand break type damage was measured by Kohn neutral elution in an X-ray-sensitive mutant of V79-4, irsl. This was done in order to investigate further the likelihood that irsl carries a defect which leads to error-prone repair of DNA damage, and not simply a reduced ability to rejoin DNA double-strand breaks.The mutant displayed an equal increase in sensitivity to the lethal of neutrons, as compared to X-rays. Both irsl and V79-4 showed an increased sensitivity to the killing effects of neutrons of around 2 at 10% survival. irsl also showed an exponential survival after either X-rays or neutrons.The induction of DNA double-strand breaks was measured in both cell lines over a dose range of 10–40 Gy using Kohn neutral filter elution. Induction of breaks by X-rays in irsl seemed to increase slightly with dose, relative to induction in V79-4, so that at 40 Gy 1.5 times more DNA double-strand breaks were measured in irsl cells than in V79-4. Neutron irradiation resulted in a more similar level of induction in either strain after 10–40 Gy. This difference in induction of damage may be due to a different cell-cycle composition in either cell line.The rejoining of X-ray induced double-strand breaks showed a very similar pattern (on a percentage rejoined basis) in both cell lines, although from the induction data at 40 Gy, the dose at which rejoining was measured, fewer breaks were rejoined in V79-4 but also fewer breaks remained unsealed. Neutron-induced breaks, however, were rejoined more efficiently in irsl again on a percentage basis, but also in absolute terms since similar induction was seen after 40 Gy. This data, together with the differences seen in the rejoining of X-ray compared to neutron induced breaks, may indirectly support the proposal that irsl is a misrepair mutant.     

Theory of relative biological effectiveness (RBE) of tritium beta rays: bacteria killing effects of tritiated water

A new model is applied to bacteria exposed to tritiated water. In this model, the relation between the radiation quality, induction of single- and double-strand breaks in DNA and their repair in vivo can be reasonably described in terms of the microdosimetric distribution and the modification factors for single- and double-strand breaks in DNA. First, a mathematical formulation of RBE of tritium beta rays relative to an arbitrary reference radiation for killing effect on bacteria is derived on this model. Typical theoretical results on the survival curve parameters for bacteria exposed to tritiated water and RBE of tritium beta rays relative to 60Co gamma rays are presented. It is found that RBE of tritium beta rays depends on the ability of the cell to repair DNA damage. The present model is applied to the survival of Escherichia coli Bs-1 exposed to tritiated water and 7 MeV electrons. Although the average LET of tritium beta rays is remarkably higher than that of 7 MeV electrons, the experimental result that RBE of tritium beta rays relative to 7 MeV electrons is close to unity, is reasonably explained by the present model.     

A description of the damaged DNA produced during tissue injury

Warm ischemia produces DNA damage which is characterized by both single- and double-strand breaks with 5'-PO4 and 3'-OH ends. In contrast, cold ischemia produces mostly single-strand breaks for the first 60 hr and then, abruptly, double-strand damage is produced. Cold ischemia produces both 5'-OH and 5'-PO4 termini, but 5'-OH ends do not appear until after 24 hr of storage. Cold ischemia, also produces 3'-PO4 ends but we have not found any 3'-OH termini. Mechanisms for DNA degradation during warm and cold ischemia are presented to account for these results. It is suggested that damage to the genetic code may be prevented if degradation of the DNA can be confined to the production of the easily repairable 5'-PO4/3'-OH opposed ends.     

Adaptive response to DNA and chromosomal damage induced by X-rays in human blood lymphocytes

Nucleoid sedimentation, single-cell gel electrophoresis (comet assay) and premature chromosome condensation (PCC) technique were utilized to estimate the involvement of DNA strand breaks and chromosomal damage in radio-adaptive response of stimulated human lymphocytes. Conditioning of cells with 0.02 Gy X-rays rendered them more resistant to single- and double-strand DNA breaks produced by 1 Gy challenging treatment as revealed by the sedimentation behaviour of the nucleoids and the comet assay. Nucleoid sedimentation also demonstrated that adaptive reaction towards X-ray-induced DNA damage is favoured in the presence of oxygen. A concomitant decrease in the amount of interphase chromosomal breaks visualized by PCC under the same experimental conditions was observed. Data indicate that adaptation of human lymphocytes to X-rays is tightly linked to the reduced susceptibility towards generation of DNA and chromosomal breaks. It is proposed that the very persistence of DNA strand discontinuities might serve as a triggering signal for the adaptation of human lymphocytes against ionizing radiation exposure.     

Rejoining of DNA strand breaks induced by propylene oxide and epichlorohydrin in human diploid fibroblasts

The repair kinetics of DNA single- and double-strand breaks (SSBs, DSBs) induced with two carcinogenic epoxides, propylene oxide (PO) and epichlorohydrin (ECH), was studied in human diploid fibroblasts. The methods used were: alkaline DNA unwinding (ADU), the comet assay, and pulsed field gel electrophoresis (PFGE). About 70% of SSBs, measured by ADU, were rejoined after the treatment with 5 mMh and 10 mMh of PO within 20 hr, and the half-life was estimated to be ∼15 hr. On the other hand, effective rejoining of SSBs after ECH treatment was observed only at a dose of 1 mMh (a half-life of ∼15 hr), whereas after 2 mMh treatment, only 26% of SSBs could be rejoined within 20 hr. Furthermore, the use of the comet assay demonstrated that DNA strand breaks were effectively rejoined after PO and ECH treatment at doses of 5–10 mMh and 0.5–1 mMh, respectively. About 76% and 83% of DSBs induced by 5 and 10 mMh of PO, respectively, were rejoined within 4 hr after the treatment (a half-life of ∼2.5 hr), with little further repair thereafter. At lower dose of ECH (1 mMh) a half-life for DSBs rejoining was estimated to be ∼2 hr; however, only 29% of DSBs were rejoined within 2 hr at the higher dose of 2 mMh. After 18 hr, the rejoining following treatment with a lower dose was negligible. At a higher dose, a rapid accumulation of DSBs was observed, probably as the result of cell death and DNA degradation. The results demonstrate the capability of human diploid fibroblasts to repair DNA SSBs and DSBs at low-to-moderate doses of the epoxides. A weak capacity to rejoin DNA strand breaks induced by higher doses of ECH may be a consequence of its higher DNA alkylation activity and approximately 10 times higher toxicity compared to PO. Environ. Mol. Mutagen. 32:223–228, 1998 © 1998 Wiley-Liss, Inc.     

Electromagnetic fields and DNA damage

A major concern of the adverse effects of exposure to non-ionizing electromagnetic field (EMF) is cancer induction. Since the majority of cancers are initiated by damage to a cell's genome, studies have been carried out to investigate the effects of electromagnetic fields on DNA and chromosomal structure. Additionally, DNA damage can lead to changes in cellular functions and cell death. Single cell gel electrophoresis, also known as the ‘comet assay’, has been widely used in EMF research to determine DNA damage, reflected as single-strand breaks, double-strand breaks, and crosslinks. Studies have also been carried out to investigate chromosomal conformational changes and micronucleus formation in cells after exposure to EMF. This review describes the comet assay and its utility to qualitatively and quantitatively assess DNA damage, reviews studies that have investigated DNA strand breaks and other changes in DNA structure, and then discusses important lessons learned from our work in this area.     

Kinetics of gamma-H2AX focus formation upon treatment of cells with UV light and alkylating agents

Histone H2AX is rapidly phosphorylated in response to DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). Here we show that DNA damage induced by alkylating agents [methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)] and ultraviolet light (UV-C) leads to a dose and time dependent accumulation of phosphorylated H2AX (gamma-H2AX). Time course experiments revealed that the number of gamma-H2AX foci reached peak levels 8 hr after MMS or MNNG treatment and declined to almost control values within 24 hr after exposure. Upon UV-C treatment, a biphasic response was observed with a maximum 12 hr after treatment. In 43-3B cells deficient in nucleotide excision repair (NER) the number of gamma-H2AX foci increased steadily. gamma-H2AX foci were preferentially formed in BrdU labeled cells. In proliferation compromised cells, the gamma-H2AX level was significantly reduced, indicating that most of the gamma-H2AX foci induced by UV-C and alkylating agent treatments were replication dependent. The data are in line with the view that DNA damage induced by UV-C light and simple alkylating agents, leads to the formation of DSBs during DNA replication giving rise to H2AX phosphorylation. In replicating NER defective cells, DSBs accumulate due to nonrepaired primary DNA lesions that produce a high level of DSBs during replication. The data support that gamma-H2AX foci are a useful marker of DSBs that are induced by S-phase dependent genotoxins during replication.     

Comparison of radon-daughter-induced effects in repair-proficient and repair-deficient cho cell lines

The radiobiological effects of the radon daughter ²¹²Bi were investigated in the Chinese hamster ovary cell line AA8 and its radiosensitive derivative EM9. EM9 cells rejoin radiation-induced DNA strand breaks more slowly than do AA8 cells. Three endpoints were examined: cell killing, G2-induced chromosome aberration frequency, and mutation induction at the hypoxanthine (guanine) phosphoribosyltransferase (HGPRT) locus. Cells were exposed to the alpha-emitter ²¹²Bi chelated to diethylenetriaminepentaacetic acid (²¹²Bi-DTPA). As expected, ²¹²Bi-DTPA was more effective than X-rays in producing cytotoxicity, chromosome aberrations, and gene mutations. The relative biological effectiveness (RBE) for all three endpoints ranged from about 2 for chromosome aberrations to 4.4 for mutation induction. EM9 was more sensitive than AA8 cells to the cytotoxic and clastogenic effects of both X-rays and ²¹²Bi-DTPA, suggesting that the repair deficiency in EM9 cells affects response to low-and high-linear energy transfer (LET) radiation for these endpoints. There was no significant difference between these two cell lines in their mutagenic response to X-rays and AA8 was slightly more sensitive to the mutagenic effects of alpha radiation. These results suggest that alterations in DNA repair ability may affect response of cells to both low- and high-LET radiation-induced cytotoxicity and clastogenicity, but they appear to have little effect on gene mutation induction.     

DNA-damage processing in a radiation-sensitive mouse cell line

The induction and repair of radiation-induced DNA damage was assessed in 3 mouse cell lines, including the parental L cell line, a radiation-sensitive, SL3-147 mutant cell line and the H5 revertant to radiation resistance. The yield of neither radiation-induced DNA single- nor double-strand breaks could explain the variable sensitivity of the 3 cell lines. Closure of DNA single-strand breaks proceeded at a similar rate in both the L and SL3-147 cell lines. Closure of DNA double-strand breaks however was significantly slower and less complete in the SL3-147 cell line than in either of the radiation-resistant cell lines. The results are consistent with the increased radiation sensitivity of the SL3-147 cell line resulting from a defect in their ability to repair radiation-induced DNA double-strand breaks.     

X-irradiation induces cell death in fetal fibroblasts

The impact of ionizing radiation on developing organisms has been widely studied for risk assessment purposes. Even though efforts have been made to decrease received doses to as low as reasonably achievable, the possibility of accidental exposure has to be considered as well. Mammalian gestation is usually divided into three periods. Radiation exposure during the 'pre-implantation period' may essentially result in embryonic mortality while exposure during the 'organogenesis period' may characteristically lead to malformations. In humans, the 'fetal period' is one of particular sensitivity to radiation induction of mental retardation, especially if the exposure occurs between weeks 8-15 of gestation. It is also admitted that prenatal irradiation may increase the risk of leukemia and childhood cancer, with an equal risk over the whole pregnancy. The aim of this study was to investigate the effects of moderate to high doses of X-irradiation on mouse skin fetal fibroblasts, one of the cell types subjected to the highest dose of radiation. Exposure of the cells to X-rays led to a rapid and significant increase in γ-H2AX foci, indicative of high levels of DNA double strand breaks. High doses (>2 Gy) also led to a pronounced G2-arrest and a decrease in the number of cells in the S phase, which was followed after 24 h by a decrease in cell survival and an increase in the level of apoptosis and necrosis. This study shows that mouse fetal skin fibroblasts are sensitive to high doses of X-irradiation. Furthermore, we report a better repair for higher doses than lower, which seems to indicate that little DNA damage is not necessarily repaired immediately. However, more sensitive approaches are necessary to identify the risk associated with low doses of radiation.     

Defective repair of DNA single- and double-strand breaks in the bleomycin- and X-ray-sensitive Chinese hamster ovary cell mutant,BLM-2

Using filter elution techniques, we have measured the level of induced single- and double-strand DNA breaks and the rate of strand break rejoining following exposure of two Chinese hamster ovary (CHO) cell mutants to bleomycin or neocarzinostatin. These mutants, designated BLM-1 and BLM-2, were isolated on the basis of hypersensitivity to bleomycin and are cross-sensitive to a range of other free radical-generating agents, but exhibit enhanced resistance to neocarzinostatin. A 1-h exposure to equimolar doses of bleomycin induces a similar level of DNA strand breaks in parental CHO-K1 and mutant BLM-1 cells, but a consistently higher level is accumulated by BLM-2 cells. The rate of rejoining of bleomycin-induced single- and double-strand DNA breaks is slower in BLM-2 cells than in CHO-K1 cells show normal strand break repair kinetics.The level of single- and double-strand breaks induced by neocarzinostatin is lower in both BLM-1 and BLM-2 cells than in CHO-K1 cells. The rate of repair of neocarzinostatin-induced strand breaks is normal in BLM-1 cells but retarded somewhat in BLM-2 cells.Thus, there is a correlation between the level of drug-induced DNA damage in BLM-2 cells and the bleomycin-sensitive, neocarzinostatin resistant phenotype of this mutant. Strand breaks induced by both of these agents are also repaired with reduced efficiency by BLM-2 cells. The neocarzinostatin resistance of BLM-1 cells appears to be a consequence of a reduced accumulation of DNA damage. However, the bleomycin-sensitive phenotype of BLM-1 cells does not apparently correlate with any alterations in DNA strand breaks induction or repair, as analysed by filter elution techniques, suggesting an alternative mechanism of cell killing.     

Factors affecting the spontaneous cell-mediated cytotoxicity of intestinal mononuclear cells.

This study was performed to determine what factors related to the enzymatic isolation technique and assay conditions may influence the measurement of spontaneous cell-mediated cytotoxicity (SCMC) of mononuclear cells (MNC) isolated from human intestinal mucosa. In 18 studies, the SCMC of freshly isolated cells was 1.8 +/- 0.4% but increased to 12 +/- 3% following 24 hr culture without a change in the proportion of cells with the NK phenotype (Leu-7+). The SCMC tended to plateau with more prolonged culture. Culturing peripheral blood (PB) MNC for 24 hr did not alter SCMC nor the proportion of Leu-7+ cells. However, the suppression of the SCMC of PBMNC preincubated with the supernatant of the collagenase digestion of intestinal mucosa was completely reversed by 24 hr culture. Intestinal MNC were found to suppress the SCMC of autologous PBMNC in mixing experiments. However, 24 hr culture did not affect the degree of suppression and the proportion of T cells of the suppressor-cytotoxic phenotype (Leu-2a+) was also unchanged. It is concluded that the SCMC of intestinal MNC may be accurately assessed following 24 hr culture of the cells to allow recovery from the suppressive influences of the isolation process and that this does not introduce other artefactual problems. However, suppression of cytotoxicity within the assay may result in an underestimation of the SCMC of intestinal MNC when compared to that of PBMNC.     

Joining of correct and incorrect DNA double-strand break ends in normal human and ataxia telangiectasia fibroblasts

Chromosomal aberrations are believed to result from the incorrect joining of DNA double-strand breaks (DSBs). In an attempt to investigate induction and rejoining quality of DSBs following ionizing radiation exposure in specific genomic locations of mammalian DNA, an experimental approach based on Southern hybridization of single-copy probes to NotI restriction fragments was developed. Induction of DSBs is measured from the decrease of the band intensity representing the unbroken restriction fragment. An increase in intensity of the hybridization band following repair incubation determines reconstitution of the original restriction fragment and thus rejoining of correct DNA ends. We investigated the dose dependence of DSB misrejoining using X-ray doses of 5, 10, 20, 40, and 80 Gy and provide evidence that the number of misrejoined DSBs exceeds, for the same doses used, the number of cytogenetically visible aberrations by an order of magnitude, reflecting the higher resolution of our assay. Induction of DSBs and joining of correct and incorrect break ends were further investigated in cells from a patient with the cancer-prone disease ataxia telangiectasia (AT) and in heterozygous AT cells. We found, compared to normal cells, identical induction rates and identical kinetics for joining correct ends following an 80-Gy X-ray exposure. After 5 and 10 Gy, however, AT homozygotes showed a 50% elevation in the proportion of breaks that are not correctly rejoined. These data indicate a defect in the accuracy of DSB rejoining in AT cells that may account for radiation sensitivity and the occurrence of the high level of chromosomal aberrations observed in AT cells. Genes Chromosomes Cancer 27:59-68, 2000.     

Does exhaustive exercise result in oxidative stress and associated DNA damage in the chub (Leuciscus cephalus)?

DNA strand breaks [as determined by the conventional and formamidopyrimidine glycosylase (FPG)-modified Comet assay] and antioxidant defense status [as indicated by superoxide dismutase (SOD) activity and reduced glutathione (GSH) concentration] were evaluated in healthy adult chub (Leuciscus cephalus) after exhaustive exercise [swimming to their critical swimming speed (U(crit)), twice in succession with a 40 min rest period between] vs. confined (unexercised) control fish. The conventional Comet assay revealed significantly higher DNA strand breaks in all the tissues (blood, liver, and gill), with the highest increase over background evident in the epithelial gill cells of swum fish compared to the controls. Moreover, when the FPG-modified Comet assay was conducted to reveal specific oxidative lesions, the gill cells of exercised fish sustained the highest level of oxidative DNA damage in comparison to the control. Data on tissue antioxidant defense mechanism were less conclusive, with no significant differences in the tissue levels of SOD or GSH. This suggests that either the degree of oxidative stress was not great enough to evoke a response in terms of defense mechanisms or the timescale of antioxidant defense response was somewhat different from the time between the application of stress and subsequent tissue sampling. From the swimming data, U(crit) was significantly lower on the second assessment compared to the first (repeat ratio: 0.76), suggesting that the fish were exercised to a level which was not sustainable. Overall, these findings support the theory that acute extreme exercise could result in oxidative stress and associated DNA damage in fish. These observations suggest that fish living in fast flowing and polluted rivers are at increased risk of DNA damage.     

Heavy-ion-induced mutations in the gpt delta transgenic mouse: comparison of mutation spectra induced by heavy-ion,X-ray,and gamma-ray radiation

Heavy-ion radiation accounts for the major component of absorbed cosmic radiation and is thus regarded as a significant risk during long-term manned space missions. To evaluate the genetic damage induced by heavy particle radiation, gpt delta transgenic mice were exposed to carbon particle irradiation and the induced mutations were compared with those induced by reference radiations, i.e., X-rays and gamma-rays. In the transgenic mouse model, deletions and point mutations were individually identified as Spi(-) and gpt mutations, respectively. Two days after 10 Gy of whole-body irradiation, the mutant frequencies (MFs) of Spi(-) and gpt were determined. Carbon particle irradiation significantly increased Spi(-) MF in the liver, spleen, and kidney but not in the testis, suggesting an organ-specific induction of mutations by heavy-ion irradiation. In the liver, the potency of inducing Spi(-) mutation was highest for carbon particles (3.3-fold increase) followed by X-rays (2.1-fold increase) and gamma-rays (1.3-fold increase), while the potency of inducing gpt mutations was highest for gamma-rays (3.3-fold increase) followed by X-rays (2.1-fold increase) and carbon particles (1.6-fold increase). DNA sequence analysis revealed that carbon particles induced deletions that were mainly more than 1,000 base pairs in size, whereas gamma-rays induced deletions of less than 100 base pairs and base substitutions. X-rays induced various-sized deletions and base substitutions. These results suggest that heavy-ion beam irradiation is effective at inducing deletions via DNA double-strand breaks but less effective than X-ray and gamma-ray irradiation at producing oxidative DNA damage by free radicals.     

Multiple abnormalities in the ultraviolet light response of cultured fibroblasts derived from patients with the basal cell nevus syndrome

Basal Cell Nevus Syndrome (BCNS) is a rare autosomal-dominant inherited disorder associated with a marked hypersusceptibility to spontaneous and radiation-induced skin cancer. We examined the changes in cell survival, unscheduled DNA synthesis (UDS) and the frequency of sister chromatid exchanges (SCE) induced by ultraviolet light (UVL) in confluent normal and BCNS fibroblasts. BCNS cells appeared slightly hypersensitive to the cytotoxic effects of UVL. The rate of UDS induced by UVL exposure in normal cell strains increased linearly following doses up to 30 J/m2, whereas in BCNS cells UDS became saturated at doses of 10 J/m2 showing no further increase with doses up to 30 J/m2. UDS activity persisted for longer periods after UVL exposure in BCNS as compared with normal cells. The dose-response relationship for UVL-induced SCE was similar in normal and BCNS fibroblasts. However, the frequencies of UVL-induced SCE declined to near background levels in normal cells following 12-24 hr of confluent holding prior to subculture whereas they remained elevated in BCNS cells with holding times up to 24 hr after UVL exposure. Overall, these results suggest that BCNS fibroblasts may have a diminished capacity for the repair of some type of DNA damage as compared with normal fibroblasts.     

Kinetics of induction of DNA damage and lacZ gene mutations in stomach mucosa of mice treated with beta-propiolactone and N-methyl-N'-nitro-N-nitrosoguanidine, using single-cell gel electrophoresis and MutaMouse models.

beta-Propiolactone (BPL) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are two direct alkylating agents that induce multiple genetic lesions and tumors in the rodent stomach. We measured the kinetics of the induction of DNA damage by using the single-cell gel electrophoresis assay (SCGE) and the induction of gene mutations by using the MutaMouse model in the glandular stomach mucosa of mice exposed to a single oral administration of BPL or MNNG. The aims were to determine the optimal sampling time and to investigate the cause-effect relationship between DNA damage and gene mutations. The induction of comets, evaluated in individual cells with the tail moment, was analyzed 1, 2, 4, 24, and 72 hr after a single oral administration of 25 mg/kg BPL or 20 mg/kg MNNG. The effects of both compounds were most intense at the earlier sampling times (1-2 hr), tailing off 4 hr after treatment and becoming undetectable at 72 hr. The lacZ mutant frequency (MF) was measured 3, 7, 14, 28, and 50 days after a single oral administration of 150 mg/kg BPL or 100 mg/kg MNNG, and 3 and 14 days after a single administration of 25 mg/kg BPL or 20 mg/kg MNNG. The MF was strongly enhanced at the highest doses and all sampling times, the most marked effects being observed 14 days (11.1-fold) and 28 days (19.0-fold) after BPL and MNNG administration, respectively. At the lowest doses, only a small increase in MF ( approximately 2.5- to 3.5-fold) was found at both sampling times. Primary DNA damage detected with SCGE shortly after treatment (1-2 hr) was rapidly (3 days) transformed into stable gene mutations that remained detectable for 50 days. These results illustrate the ability and complementarity of the SCGE and MutaMouse models to assess the genotoxicity of direct alkylating agents in the mouse gastric mucosa in vivo.     

Assessment of DNA strand breaks induced by bleomycin in barley by the comet assay

Comet assay was applied to study induction and repair of DNA damage produced by bleomycin in barley genome. Experimental conditions were adapted to achieve efficient detection of both DNA single- and double-strand breaks. Substantial increase of the parameter "% of DNA in tail" was observed coupled with almost linear dependence from bleomycin concentration, more pronounced for the induction of DNA double-strand breaks. Data obtained at different recovery periods displayed rapid restoration of breakage, revealing that efficient mechanisms for repair of strand discontinuities induced by bleomycin are functional in barley DNA loop domains.     

Molecular cytogenetic evaluation of the mechanism of micronuclei formation induced by camptothecin,topotecan, and irinotecan

We used the conventional bone marrow micronucleus test complemented with the fluorescent in situ hybridization with the minor satellite DNA probe to investigate the mechanisms of induction of micronuclei in mice treated with camptothecin and its clinical antineoplastic analogues topotecan and irinotecan. All experiments were performed with male Swiss albino mice. Single doses of 1 mg/kg camptothecin or 0.6 mg/kg topotecan were injected intraperitoneally and bone marrow was sampled at 30 hr (camptothecin) or 24 hr (topotecan) after treatment. A dose of 60 mg/kg irinotecan was injected intravenously, once every fourth day for 13 days and bone marrow was sampled 24 hr after the last treatment. In animals treated with camptothecin, a total of 1.07% micronuclei were found and 70% of them were centromere‐negative, indicating their formation by DNA strand breaks and reflecting the predominant clastogenic activity of camptothecin. Exposure to topotecan and irinotecan yielded 1.71 and 0.83% micronuclei, respectively. About 52.7 and 48.8% of the induced micronuclei, respectively, were centromere‐positive, indicating their formation by whole chromosomes and reflecting the aneugenic activity of both compounds. Correspondingly, about 47.3 and 51.2% of the induced micronuclei, respectively were centromere‐negative, demonstrating that topotecan and irinotecan not only induce chromosome loss but also DNA strand breaks. Both the clastogenic and aneugenic potential of these drugs can lead to the development of secondary tumors and abnormal reproductive outcomes. Therefore, the clinical use of these agents must be weighed against the risks of secondary malignancies in cured patients and persistent genetic damage of their potential offspring. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Mutation induction in haploid yeast after split-dose radiation exposure. II. Combination of UV-irradiation and X-rays

Split-dose protocols can be used to investigate the kinetics of recovery from radiation damage and to elucidate the mechanisms of cell inactivation and mutation induction. In this study, a haploid strain of the yeast, Saccharomyces cerevisiae, wild-type with regard to radiation sensitivity, was irradiated with 254-nm ultraviolet (UV) light and then exposed to X-rays after incubation for 0-6 hr. The cells were incubated either on nutrient medium or salt agar between the treatments. Loss of reproductive ability and mutation to canavanine resistance were measured. When the X-ray exposure immediately followed UV-irradiation, the X-ray survival curves had the same slope irrespective of the pretreatment, while the X-ray mutation induction curves were changed from linear to linear quadratic with increasing UV fluence. Incubations up to about 3 hr on nutrient medium between the treatments led to synergism with respect to cell inactivation and antagonism with respect to mutation, but after 4-6 hr the two treatments acted independently. Incubation on salt agar did not cause any change in the survival curves, but there was a strong suppression of X-ray-induced mutation with increasing UV fluence. On the basis of these results, we suggest that mutation after combined UV and X-ray exposure is affected not only by the induction and suppression of DNA repair processes, but also by radiation-induced modifications of cell-cycle progression and changes in the expression of the mutant phenotype.