Interindividual variation in the responses of cultured human lymphocytes to exposure from DNA damaging chemical agents: interindividual variation to carcinogen exposure.

Human population variability to standardized doses of N-acetoxy-2-acetylaminofluorene (NA-AAF) and 7, 12-dimethylbenz(a) anthracene (DMBA) was determined in cultured lymphocytes by measuring (a) differential stimulation of unscheduled DNA synthesis after 1 h induction of DNA damage by 10 micrometer NA-AAF, (b) the level of NA-AAF induced chromosome aberrations remaining after 8 h of DNA-repair synthesis, and (c) the level of [3H]DMBA bound to DNA after 18 h incubation of resting lymphocytes in 5 micrometer DMBA. All 3 parameters indicated individual variation to carcinogen exposure and were correlated to the population differences in age, sex, blood pressure and mortality rates. Males always had a greater potential to accumulate DNA-damage than did females regardless of the sampled population. DNA-damage potentials increased with increasing age, blood pressure or mortality rates. There was always proportionally greater DNA-damage potentials in the males than in females. The in vitro response of mature granulocytes to a 10 micrometer NA-AAF dose, as estimated by [3H] thymidine incorporation from unscheduled DNA synthesis, was much lower than lymphocyte response. Nevertheless, individual variations in granulocyte NA-AAF induced unscheduled DNA synthesis paralleled the inter-individual fluctuations observed in the lymphocyte responses to NA-AAF.     

Unscheduled DNA synthesis induced by N-acetoxy-2-acetylaminofluorene is not sensitive to regulation by ADP-ribosyl transferase

We have directly compared in resting human mononuclear leukocytes the DNA repair effects caused by ADP-ribosyl transferase (ADPRT) activity following DNA damage induction by gamma radiation, UV radiation, ethylene oxide (EO) and N-acetoxy-2-acetylaminofluorene (NA-AAF). The presence of inhibitors of ADPRT during the quantitation of unscheduled DNA synthesis (UDS) resulted in about a 2-fold increase of UDS when induced by gamma radiation, UV radiation or EO. The stimulation of UDS by EO, UV- or gamma-radiation in the presence of an ADPRT inhibitor was equally strong whether 1 mM or 10 mM hydroxyurea was used to suppress scheduled DNA synthesis. The level of NA-AAF induced UDS was not affected by inhibitors of ADPRT. In addition, direct estimation of ADPRT activity revealed that at doses giving maximal UDS, NA-AAF damage did not induce a measurable enzymatic activity whereas gamma-radiation, UV radiation and EO all showed a significant dose response increase. We have interpreted our data to mean that NA-AAF induced UDS estimates DNA repair relating mainly to DNA lesions that are recognized with difficulty, and hence, the rate of endonuclease-induced DNA strand break accumulation is not sufficient to allow a stimulation of ADPRT and affect the quantitation of UDS.     

Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

In vivo and in vitro ethylene oxide exposure of human lymphocytes assessed by chemical stimulation of unscheduled DNA synthesis

Factory workers exposed to ethylene oxide (EO), 0.5–1.0 ppm in factory air, together with matched controls from the same factory, were examined for evidence of toxic exposure by measurement of unscheduled DNA synthesis (UDS) induced by N-acetoxy-2-acetylaminofluorene (NA-AAF) and of chromosome aberrations in peripheral lymphocytes.The total chromatid gaps plus breaks were significantly elevated and NA-AAF-induced UDS was significantly reduced in the EO-exposed group as compared with the unexposed control group. The NA-AAF-induced UDS values negatively correlated to the duration (yr) of EO exposure (r = ?0.45, p < 0.02) and the number of chromosome breaks (r = ?0.61, p < 0.05), indicating an inhibition in vivo of DNA-repair capacity by EO. These data were verified in vitro by biochemical and autoradiographic studies of EO-induced UDS in human blood cells. Above 2 mM EO, UDS was inhibited in lymphocytes whether they were cultured for 24 or 122 h after alkylation with EO. Even at the subtoxic EO dose of 0.1 mM, lymphocytes were sensitized to additional exposures of NA-AAF, so that cytotoxicity was increased to 40% compared with 5% for the controls even though UDS was unaffected.It is concluded that EO was toxic to lymphocytes, even when they were sensitized at non-toxic EO doses to the cytotoxic action of other mutagens (e.g. NA-AAF), and the cells that did survive above 2 mM EO were inhibited in their DNA-repair capacity as judged by reduced UDS.     

Repair of DNA damage induced by oxygen radicals in human non-proliferating and proliferating lymphocytes.

Repair of DNA lesions induced by oxygen radicals, generated by xanthine/xanthine oxidase (X/XO), was studied in human peripheral blood lymphocytes and in PHA-stimulated proliferating lymphocytes from 4 healthy subjects. The lesions included DNA-strand breaks (SSB) and other lesions that are converted to SSB under alkaline conditions. The frequencies of SSB were estimated by fluorometric analysis of DNA unwinding. Maximum production of SSB occurred within 10 min of incubation with X/XO at 22 degrees C; with 0.5 mM or higher concentrations of xanthine; and with 0.1-0.5 units/ml of xanthine oxidase. Proliferating lymphocytes repaired X/XO-induced SSB about 4 times more rapidly than lymphocytes. Lymphocytes repaired X/XO-induced SSB more slowly than SSB caused by gamma-radiation. These findings are consistent with the evidence that a number of DNA-repair enzymes have greater activity in proliferating cells than in resting cells. These findings also support the view that there are differences between the DNA damage due to oxygen radicals and that due to ionizing radiation.     

Genotoxic effects of inhaled ethylene oxide, propylene oxide and butylene oxide on germ cells: sensitivity of genetic endpoints in relation to dose and repair status

We report here results on forward mutation induction (recessive lethal mutations, RL) in Drosophila spermatozoa and spermatids by the three 1,2-alkyl-epoxides ethylene oxide (EO), propylene oxide (PO) and butylene oxide (BO), at doses ranging from 47 to 24,000 ppm h for EO, 375 to 48,000 ppm h for PO, and 24,000 to 91,200 ppm h for BO. The results indicate for EO mutation induction at doses 500-fold below the LD₅₀. In crosses of mutagenized NER⁺ males with NER⁺ females, the 500-fold increase in EO dose from 47 ppm h to 24,000 ppm h resulted in no more than a 17-fold enhanced mutant frequency in spermatozoa. This flat dose–response relationship is primarily the result of efficient repair of EO-induced DNA adducts in the fertilized egg, as was evident from the up to 40-fold or 240-fold increased mutant frequencies above NER⁻ or NER⁺ background levels, respectively, in crosses with NER⁻ females. With decreasing dose, / ratios decreased from 9 to 14 at high doses down to ≈1 at the two lowest doses, indicating that a small fraction of premutagenic lesions induced by EO cannot be repaired by the NER system of Drosophila. Linear extrapolation from high to low EO exposure led to an underestimation of the mutation frequency actually observed at low doses. The pattern of EO-induced ring chromosome loss (CL) differed in two respects from that observed for forward mutations: (a) an increase in CL frequencies was observed only at the two highest EO exposure levels, and (b) inactivation of the NER pathway by the mus201 mutant had no measurable effect on the occurrence of CL. The absence of a potentiating effect of mus201 on EO-induced clastogenicity suggests the formation of clastogenic DNA lesions not causing point mutations, and which are not repaired by NER. Consistent with an inversed correlation of reactivities towards N7-guanine and chain length of 1,2-alkyl-epoxides, the relative mutagenic efficiencies of EO:PO:BO are 100:7.2:1.8 for the NER⁺ groups, and 100:20:0.7 in the absence of NER. Although in Drosophila germ cells EO is also more effective as a clastogen than PO, the difference (EO:PO=100:58) is much smaller than for recessive mutations. These results provide another argument that DNA lesions generating base substitutions as opposed to those causing clastogenic damage may not be the same for these agents.     

Parallel increases in sister-chromatid exchanges at base level and with UV treatment in human opiate users

The SCE base level frequency and SCE levels induced by far-UV (254 nm) treatment of cells in early G1 and early S phases of the cell cycle were significantly higher in leukocytes from heroin addicts as compared to controls. The increased SCE levels in addicts was greatest at base level and smallest after UV irradiation of cells in S phase. These results corroborate and extend our previous findings of increased chromosome damage and reduced DNA-repair synthesis in heroin users. Since opiates do not directly damage DNA, the elevated cytogenetic effects associated with opiate use probably arise from secondary promotional effects related to opiate-mediated alterations in leukocyte metabolism.     

Recovery of mitomycin C-treated mouse 10T1/2 cells during confluent holding

The relationship between cytotoxicity, sister-chromatid exchanges (SCE) and the repair of DNA crosslinks was studied in mouse 10T1/2 cells during confluent holding following either acute or protracted MMC treatment. No cytotoxic effects were observed with increasing doses of MMC until SCE frequencies 1.8 times background levels were induced. Protracted MMC treatments were less cytotoxic than acute MMC exposure at doses which yielded similar frequencies of SCE. The kinetics of recovery during confluent holding in acute MMC-treated cells were similar for cytotoxicity and the repair of DNA interstrand crosslinks. These results suggest that a type of non-lethal DNA damage which causes SCE may persist for long periods of time in MMC-treated cells. This non-lethal damage may accumulate during protracted MMC exposure while damage leading to cell killing is repaired.     

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.     

Quantification of unscheduled DNA synthesis in mononuclear leukocytes of the horse

This study compares the relationship between N-acetoxy-2-acetylaminofluorene (NA-AAF) and u.v. induced unscheduled DNA synthesis (UDS) and their respective relationships to age and blood pressure in horse mononuclear leukocytes with earlier, similar investigations on human leukocytes. U.v. induced UDS was found to proceed more rapidly than NA-AAF induced UDS. A pronounced lag period associated with the rapid demand for 3H-dThd into DNA after u.v. damage was observed. NA-AAF induced UDS correlated significantly with NA-AAF binding, age and the blood pressure of male horses. UDS values, induced by either method, were about half the level calculated for human leukocytes.     

5-氮胞苷对贵州小型猪淋巴细胞DNA损伤及修复的影响

目的　研究贵州小型猪淋巴细胞对化学物或药物引起的DNA损伤及修复影响的反应。方法　用单细胞凝胶电泳技术检测比较 5 氮胞苷对PHA刺激和未刺激淋巴细胞的DNA损伤及其修复过程。结果　 5 氮胞苷引起未刺激淋巴细胞明显的DNA泳动 (彗星尾 ) ,经修复孵育 2h后 ,DNA泳动与孵育前比较无显著差异 ,而 5 氮胞苷引起的刺激细胞DNA泳动经 2h修复孵育后与孵育前比较显著减少。结论　 5 氮胞苷引起贵州小型猪未刺激淋巴细胞DNA损伤经 2h孵育未能修复 ,而刺激细胞的DNA损伤明显修复。     

The repair of bleomycin-induced DNA damage and its relationship to chromosome aberration repair.

Previous studies using the technique of premature chromosome condensation indicated that nearly one-half of the bleomycin-induced chromatid breaks and gaps in CHO cells could be repaired within 1 h (repair starting at 30 min) after treatment. Cycloheximide and streptovitacin A (but not hydroxyurea or hycanthone) inhibited chromosome repair. The purpose of this study was to measure the kinetics of DNA repair after bleomycin treatment using the alkaline elution technique and to determine whether various inhibitors could block this repair. After bleomycin treatment, the major proportion of the repair of DNA damage occurred within 15 min, with significant repair evident by 2 min. This fast repair component was inhibited by 0.2% EDTA. A slower repair component was observed to occur up to 60 min after bleomycin treatment. None of the inhibitors tested were found to have a significant effect on the repair of bleomycin damage at the DNA level. Since chromosome breaks were observed not to begin repair until after 30 min while over 50% of the DNA was repaired by 15 min, these results suggest that the DNA lesions that are repaired quickly are not important in the formation of chromosome aberrations. Further, since cycloheximide and streptovitacin A blocked chromosome repair but had little measurable effect on DNA repair, these results suggest that the DNA lesions responsible for chromosome damage represent only a small proportion of the total DNA lesions produced by bleomycin.     

Hypersensitivity of Bloom's syndrome fibroblasts to N-ethyl-N-nitrosourea

Fibroblast cells from two Japanese patients with Bloom's syndrome (BS) and normal donors were studied for the inactivation of colony-forming ability and the induction of sister-chromatid exchanges (SCEs) after N-ethyl-N-nitrosourea (ENU) treatment. The reduction of ENU-induced SCEs as a function of post-treatment incubation time was also compared between BS and normal fibroblasts. BS cells were approximately 4 times more sensitive than normal cells to the lethal effect of ENU and remarkably hypersensitive to the SCE induction by ENU. The post-treatment incubation of ENU-treated normal cells in the fresh medium resulted in a time-dependent decrease of the SCE level until 6 h after which time the SCE level remained the plateau of about 50% of the initial level. In contrast, the ENU-induced SCEs in BS cells decreased much more slowly with post-treatment incubation time and its half life was 24 h. These results collectively support the view that BS cells may be defective in the rapid repair of certain type(s) of DNA damages induced by ENU.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

The fate of U.V.-induced lesions affecting SCEs,chromosome abberrations and survival of CHO cells arrested by deprivation of arginine

CHO cells undergo proliferative arrest when incubated in medium deficient in the amino acid arginine (ADM). Cells arrested in this way can be released and resume mitotic activity after a brief lag period. The incidence of U.V.-induced sister chromatid exchanges (SCEs) induced in cells arrested in ADM was reduced when the cells were incubated in ADM after irradiation and prior to release. Periods of incubation in ADM of 24 and 48 h prior to release reduced the resulting SCE levels (relative to the SCE levels present in cells irradiated immediately prior to release) by an average of 35 and 45% respectively. A similar time-dependent decrease in the incidence of chromosome aberrations induced in CHO cells arrested in ADM was not observed. Despite the decrease in SCEs over time in ADM, the survival of ADM-arrested cells was not enhanced by a period of incubation in ADM after irradiation of 48 h. These observations are consistent with the hypothesis that the U.V.-induced lesions responsible for the induction of SCE are repaired in time in ADM-arrested CHO cells. Repair of those lesions resulting in chromosome aberrations was not detected in ADM-arrested CHO cells. This absence of repair of certain lesions was apparently reflected in the absence of any enhancement of cell survival.     

Aging and sister chromatid exchange : II. The effect of the in vitro passage level of human fetal lung fibroblasts on baseline and mutagen-induced sister chromatid exchange frequencies

The frequencies of baseline and mutagen-induced sister chromatid exchanges (SCE) were examined in human fetal lung fibroblasts (IMR-90, WI38) as a function of in vitro serial passage (in vitro aging). Although baseline SCE levels remained relatively constant throughout the in vitro lifespan of these cell cultures, a significant decline was observed at middle and late passage in the levels of SCE induced by mitomycin-C, ethyl methane-sulfonate and N-acetoxy-2-acetylaminofluorene. These findings indicate that cellular aging results in an altered response to certain types of induced DNA damage.     

Cytogenetic damage in workers exposed to ethylene oxide

Sister-chromatid exchanges (SECs) and chromosomal aberrations (CAs) were detected in the peripheral lymphocytes of 41 sanitary workers exposed to ethylene oxide (EO) in the sterilizing units of 8 hospitals in the Venice Region. The first group (19 workers) was exposed to 10.7 +/- 4.9 ppm EO, expressed as the time-weighted average concentration for an 8-h working day (TWA/8 h conc.), and the second group (22 workers) to 0.35 +/- 0.12 ppm. Each exposed worker was paired with a control of similar age and smoking habits. A highly significant (P less than 0.001) increase in the mean frequency of SCEs was found in the higher exposure group, 14 (74%) exposed subjects having significantly increased levels of SCEs compared to their matched controls. In the lower exposure group, the increase in mean frequency of SCEs was lower, though still significant (P less than 0.05): 7 (33%) exposed subjects had higher and 1 (5%) had a lower SCE level than the matched controls. From the first group, 10 subjects, 7 of whom had increased SCE levels, were reanalysed 12-18 months after their exposure had been lowered or interrupted: in only 2 of them the SCE level was significantly decreased. A statistically significant correlation between SCE frequency and level of EO exposure (TWA/8 h conc.), as well as a multiple correlation between SCE level and EO exposure, smoking and age were found. However, no interaction could be detected between EO exposure and smoking in the induction of SCEs. In controls, SCE frequency was correlated with smoking and age. In the higher exposure group, the number of both chromatid- and chromosome-type aberrations, independent of gaps, was significantly increased, whereas in the lower exposure group only the frequency of chromosome-type aberrations, excluding gaps, was statistically higher than in controls. The level of CAs remained to a great extent unchanged in the 10 subjects re-examined at a later stage after lowering or halting exposure. Taking the group as a whole, the frequency of cells with total CAs was found to be weakly (P = 0.05) correlated with EO exposure, and was not correlated with smoking, age or SCE frequency.     

In vivo repair of rat liver DNA damaged by dimethylnitrosamine or diethylnitrosamine.

Effects of hepatocarcinogens dimethylnitrosamine (DMN) and diethylnitrosamine (DEN) on the sedimentation pattern of rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. Both DMN (10 mg/kg body weight) and den (13.4 or 134 mg/kg) induced appreciably decreased DNA sedimentation rates at 24 h after injection. DMN at 10 mg/kg was as effective in decreasing the DNA sedimentation rate at 24 h after injection as was the higher dose of DEN (134 mg/kg). Sedimentation patterns at 1, 6 and 14 days after injection indicated that damage induced by DEN (134 mg/kg) was repaired at a substantially lower rate than DMN (10 mg/kg) induced damage. When effects of equimolar doses of DMN (10 mg/kg) and DEN (13.4 mg/kg) were compared at 1, 6 and 14 days after injection, it was observed that the more pronounced damage of rat liver DNA induced by DMN was repaired at a faster rate than was the DEN-induced damage. At the molecular level this difference in repair between damage induced by the two nitrosamines is probably related to different DNA alkylation patterns. The relatively persistent nitrosamine-induced DNA lesions (observed especially after DEN administration) are thought to represent phosphotriesters which give rise to single strand DNA breaks at strongly alkaline conditions of lysis on top of the gradient. The results are discussed in relation to the possible significance of alkylation and repair of DNA in the formation of (pre)cancerous lesions in rat liver.     

Genotoxic effects of paracetamol in V79 Chinese hamster cells

Paracetamol was studied for possible genotoxic effects in V79 Chinese hamster cells. Paracetamol (0.5 mM for 30 min) reduced the rate of DNA synthesis in exponentially growing V79 cells to about 50% of control. A further decrease in the DNA synthesis was seen during the first 30 min after termination of paracetamol exposure. Paracetamol (3 and 10 mM for 2 h) caused a small increase in DNA single-strand breaks, as measured by the alkaline elution technique. After 16 h elution, the amount of DNA retained on the filters was 79 and 70% of controls in cells treated with 3 and 10 mM paracetamol respectively. No indication of DNA damage was seen in measuring the effect of paracetamol (0.25-10 mM for 2 h) on unscheduled DNA synthesis in growth-arrested cultures of V79 cells. At the highest concentrations (3 and 10 mM paracetamol), decreased unscheduled DNA synthesis was observed. Also UV-induced DNA-repair synthesis was inhibited by 3 and 10 mM paracetamol. DNA-repair synthesis was, however, inhibited at a much higher concentration than that inhibiting replicative DNA synthesis. The number of sister-chromatid exchanges (SCE) increased in a dose-dependent manner on 2 h exposure to paracetamol from 1 mM to 10 mM. At the highest dose tested (10 mM), the number of SCE increased to 3 times the control value. Co-culturing the V79 cells with freshly isolated mouse hepatocytes had no further effect on the paracetamol induced sister-chromatid exchanges. The present study indicates that paracetamol may cause DNA damage in V79 cells without any external metabolic activation system added.     

In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells

The difference in efficiency of methylnitrosourea (MNU) and ethylnitrosourea (ENU) to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation during G1. The repair during G1 was determined by treating the cells in early or late G1. Treatment was in the first cycle (G1 before incorporation of 5-bromodeoxyuridine (BrdU)) or in G1 of the second cycle (after a single round of BrdU incorporation). It was observed that 50% of the lesions induced by MNU that elicit SCE are repaired during G1. BrdU incorporation into DNA increases the sensitivity of the cell to SCE induction by MNU nearly 40%; however under this circumstance a slightly lower SCE frequency was observed in the cells exposed to MNU at early G1, indicating that during G1 only few lesions are repaired. The ENU-induced DNA-lesions involved in SCE production are nearly 100% persistent along G1; besides, a slight but significantly higher SCE frequency was observed in cells exposed at early G1, suggesting the formation of SCE-inducing lesions during G1. BrdU incorporation to DNA sensitizes the cell to SCE induction by ENU, increasing the SCE frequency to nearly to a 40%, although these additional lesions involved in SCE induction seem to be susceptible to repair during G1.