Baseline Sister Chromatid Exchanges Research Articles

A new cytogenetic approach for the evaluation of mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase.

The aim of the present study was to develop and standardize a cytogenetic approach for evaluation of the mutagenic potential of chemicals that induce cell cycle arrest in the G2 phase. Even though cytogenetic end-points such as sister chromatid exchange (SCE) have been extensively used to indirectly assess the DNA-damaging potential of various chemicals, they are based on metaphase chromosome analysis. Cells delayed in G2 phase after chemical exposure are not included in conventional SCE analysis. The yield of SCEs obtained, therefore, can be biased, since predominantly undamaged cells proceed to metaphase without delay. To overcome this shortcoming of conventional SCE analysis, the use of a new cytogenetic approach for genotoxic studies is presented that enables the analysis of SCEs directly in G2 phase using drug-induced premature chromosome condensation in cultured peripheral blood lymphocytes. By means of this method, firstly, the possibility that SCE analysis in metaphase chromosomes underestimates the mutagenic potential of various chemicals was tested. Secondly, whether the genotoxic potential of suspected carcinogens could be evaluated using SCE analysis in G2 phase, even at exposures that arrest cells in G2 phase, was examined. Thirdly, whether an important part of the background variation in SCE frequency among individuals is due to the delay of affected cells in G2 phase, rather than to a true biological variation in the cytogenetic end-point used, was tested. The results showed that a higher SCE frequency was scored in G2 phase than in metaphase. Subsequently, the mutagenic potential of chemicals that temporarily arrest cells in G2 phase could now be evaluated more accurately. In addition, it may be of interest to further examine the involvement of cell cycle kinetics in the baseline SCE variation among individuals since a lesser SCE variability was observed when the analysis was carried out in G2 phase rather than at metaphase.

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium.

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

RecA stimulates sister chromatid exchange and the fidelity of double-strand break repair, but not gene targeting, in plants transformed by Agrobacterium

Expression of the bacterial RecA protein in plants stimulates homologous recombination in tobacco. Here we show that RecA plays a direct role in DNA strand exchange in vivo. The number of sister chromatid exchanges (SCEs) was increased 2.4-fold over wild type in transgenic tobacco plants expressing a nuclear-targeted RecA (nt-RecA) protein and could not be increased further by DNA damage, which caused a doubling of the baseline SCE frequency in wild-type plants. Although gene targeting requires homologous recombination, the number of targeted gene replacements was not increased markedly by the presence of nt-RecA by using Agrobacterium-mediated transformation. However, the number of double-strand breaks that were repaired at both sides by homologous recombination was increased 3.3-fold. Stimulation of SCE and fidelity of double-strand break repair by nt-RecA, but not by gene targeting, suggests that the stimulatory activity of RecA is linked to active DNA synthesis. Therefore, nascent replication-associated single strands may be a prerequisite for RecA action in plant cells.

Meeting report

Meeting report

Sister chromatid exchange in human lymphocytes exposed to isoflurane and nitrous oxide in vitro

The question of whether or not inhalation anaesthetics are genotoxic remains controversial. Therefore, we have studied the in vitro genotoxic potential of isoflurane and nitrous oxide in human lymphocytes. Blood samples were obtained from eight healthy male, non-smoking volunteers, which were incubated and exposed to increasing concentrations of isoflurane (0.0, 0.3, 0.6 and 1.2 mmol litre-1) or 50% nitrous oxide in oxygen. Baseline sister chromatid exchange (SCE) rate per cell was mean 7.65 (SD 1.5) which increased to 9.15 (1.0), 9.55 (1.4) and 9.95 (1.8) SCE/cell during exposure to isoflurane 0.3, 0.6 and 1.2 mmol litre-1, respectively. During 50% nitrous oxide exposure, SCE rate was 9.26 (1.4). The difference between the control and exposed cells was statistically significant (P < or = 0.05). We conclude that exposure to nitrous oxide and subanaesthetic concentrations of isoflurane can produce genetic damage in peripheral lymphocytes in vitro.

Monocytes affect cell‐cycle progression but not baseline frequency of sister chromatid exchanges of pig lymphocytes

The effect of pig monocytes (MNs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression of pig lymphocytes was studied in plasma leukocyte (PLCs) and whole blood leukocyte cultures (WBCs). No variation in SCE frequency was observed between control WBC and PLC, nor did the addition of pig MNs to PLCs modify the baseline frequency of SCEs. Cell proliferation was slower in PLCs than in WBCs. Variations in cell-cycle progression of pig lymphocytes from PLC were observed both in the absence and presence of adherent cells in the culture. In MN-free cultures, lymphocytes proliferated faster than in parallel PLC cultures. However, when MNs were seeded into the cultures, cell-cycle progression gradually slowed as a function of the concentration of adherent cells present in the cultures. This finding shows that pig MNs modulate the in vitro cell-cycle progression of pig lymphocytes in a dose-dependent manner and that the low baseline SCE frequency of pig lymphocytes is independent of the presence or absence of MNs in the culture. Environ. Mol. Mutagen. 33:86–90, 1999 © 1999 Wiley-Liss, Inc.

Monocytes affect cell‐cycle progression but not baseline frequency of sister chromatid exchanges of pig lymphocytes

The effect of pig monocytes (MNs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression of pig lymphocytes was studied in plasma leukocyte (PLCs) and whole blood leukocyte cultures (WBCs). No variation in SCE frequency was observed between control WBC and PLC, nor did the addition of pig MNs to PLCs modify the baseline frequency of SCEs. Cell proliferation was slower in PLCs than in WBCs. Variations in cell-cycle progression of pig lymphocytes from PLC were observed both in the absence and presence of adherent cells in the culture. In MN-free cultures, lymphocytes proliferated foster than in parallel PLC cultures. However, when MNs were seeded into the cultures, cell-cycle progression gradually slowed as a function of the concentration of adherent cells present in the cultures. This finding shows that pig MNs modulate the in vitro cell-cycle progression of pig lymphocytes in a dose-dependent manner and that the low baseline SCE frequency of pig lymphocytes is independent of the presence or absence of MNs in the culture.

Chromosome Breaks and Sister Chromatid Exchange as Predictors of Second Cancers in Hodgkin's Disease

Hodgkin's disease (HD) survivors face an increased risk of developing second cancers. We evaluated baseline cytogenetic biomarkers, sister chromatid exchange (SCE) and chromosome breaks [spontaneous (SCB) and bleomycin-induced (BIB)], as predictors of second cancer risk in a cohort of 105 adult HD patients. During follow-up, seven second cancers occurred. SCBs and BIBs showed no association with risk of second primaries. Multivariate Cox regression revealed that high levels of SCEs (relative risk (RR) = 11.3, p = 0.02) and age (RR = 1.08, p = 0.02) predicted second cancer risk. Histology, stage, and treatment were not associated with elevated risk. In conclusion, baseline SCE frequencies may be a useful biomarker for identifying HD patients at increased risk of developing second cancers. These results need to be verified in a larger cohort with a longer follow-up time.

Baseline and mitomycin C(MMC)-induced sister-chromatid exchanges in XX and XY cells of Callithrix jacchus chimeric twins

Lymphocytes from chimetric individuals of the species Callithrix jacchus (Primates) were examined to evaluate differences in the frequency of sister chromatid exchanges (SCE) between XX and XY cells. The aim was to discover whether SCE differ according to genetic sex and whether XX and XY cells show a different sensitivity to SCE inducing agents. This experimental model has enabled us to eliminate the possible differences caused by environmental factors. The results obtained do not reveal significant differences between male and female cells, in either the baseline SCE frequency or that induced by mitomycin C at concentrations of 0.01 and 0.03 μg/ml. No signficant differences were observed in the distribution of high SCE frequency cells (HFC), even if it is possible to observe a higher level of exchanges in XX cells in each trial. With regard to the phenotypic sex, there appears to be a trend towards slightly higher SCE rates in females, even if results are not statistically significant.

Increased frequencies of sister chromatid exchange in soldiers deployed to Kuwait.

Frequencies of sister chromatid exchange (SCE), a measure of genotoxic exposure, were assessed in military troops deployed to Kuwait in 1991. Soldiers completed health questionnaires and had blood collected prior to, during and following deployment to Kuwait. Frequency of spontaneous SCE was determined on blood samples as a measure of mutagenic exposure. Compared to pre-deployment baseline SCE frequency means, levels obtained 2 months into the Kuwaiti deployment were significantly increased (P < 0.001) and persisted for at least 1 month after return to Germany. Outcome was unaffected by known personal SCE effect modifiers including smoking, age and diet. Potential sources of the apparent mutagenic exposure are discussed.

Differences in the response to mutagens between two V79 sublines

Two V79 Chinese hamster sublines (V79-UL and V79-MZ) which differed markedly with respect to their spontaneous pattern of mutations at the HPRT locus were comparatively investigated in genotoxicity tests with ethyl methanesulphonate (EMS). EMS-induced frequencies of HPRT mutations and sister chromatid exchanges (SCEs) were much higher in V79-MZ than in V79-UL. V79-MZ were not hypersensitive against EMS and had normal frequencies of spontaneous gene mutations and chromosome aberrations. Baseline SCE frequencies at various BrdUrd concentrations were slightly increased compared to V79-UL. EMS induced a similar amount of chromosome aberrations in both cell lines but exchange figures occurred with lower frequency in V79-MZ. The results indicate that specific and significant differences in the response to mutagens may exist between ‘normal’ Chinese hamster cell lines which might be relevant for genotoxicity testing.

Genetic variations in baseline and ultraviolet light-induced sister chromatid exchanges in peritoneal lymphocytes among different mouse strains

The variation in the induction of sister chromatid exchanges (SCEs) in the peritoneal lymphocytes of different mouse strains was investigated. For the baseline SCEs, BALB/c and outbred ICR showed the lowest frequency and DBA/2 and C57BL/6 the highest. BDF 1 (C57BL/6 × DBA/2) was ranked among the highest, while CDF 1 (BALB/c × DBA/2) was intermediate between the parental strains. Regarding UV-induced SCEs, BALB/c was less susceptible as compared to DBA/2 and C57BL/6. Both BDF 1 and CDF 1 showed values significantly higher than BALB/c, but not significantly different from DBA/2 or C57BL/6. ICR was ranked in the susceptible group. For the baseline SCEs of bone marrow cells, the overall ranking among strains was essentially the same as that for the baseline, but different from that for the UV-induced, SCEs in peritoneal lymphocytes. The present results can be explained by assuming that the major genetic factor contributing to the strain-dependent difference in the baseline SCEs is due to a codominant trait of a single, allele, but that the UV-induced SCEs are complicated by other genetic factor(s).

Baseline and phosphoramide mustard-induced sister-chromatid exchanges in pharmacists handling anti-cancer drugs

Determinations of baseline and mutagen-induced sister-chromatid exchanges (SCE) have been used as indicators of previous mutagen exposure in several human populations. Mutagen-induced SCE is based on the premise that a genetic outcome may depend not only on a present exposure, but also on a cell's “memory” of previous exposure. The genotoxicity of some anti-cancer drugs including cyclophosphamide (CP) has been studied by determining baseline and mutagen-induced SCE in peripheral blood lymphocytes in treated cancer patients. This study examined the in vivo genotoxic effects of occupational exposure to anti-cancer drug handling by relating baseline and phosphoramide mustard (PM) -induced SCE levels with duration of anti-cancer drug handling as a surrogate for anti-cancer drug exposure dose. The mean baseline SCE for the population was 5.19 ± 0.17 and was not correlated with duration of drug handling. However, a strong correlation was demonstrated between inducible SCE values and life-time duration of drug handling with r = 0.63 ( p < 0.0001 for low-dose PM challenge (0.1 mg/ml PM) and r = 0.67 ( p < 0.0001) for high-dose PM challenge (0.25 mg/ml PM). A similar relationship was seen for PM-induced SCE and duration of anti-cancer drug handling for the workers' present job with correlations obtained being r = 0.63 ( p < 0.0001) for low-dose PM and r = 0.59 ( p < 0.0001) for high dose PM. The short-lived nature of the baseline SCE lesion is discussed as a limitation in population surveillance studies, as it reflects primarily recent mutagen exposure and persists only for days to weeks after exposure. The induced SCE measure is postulated to provide an integrating dosimeter of remote previous exposure, improving upon the current limitation of the baseline SCE measure and allowing the “unmasking” of previous exposure in a provocative framework.

Baseline Sister Chromatid Exchange in Human Cell Lines with Different Levels of Poly(ADP-Ribose) Polymerase

Poly(ADP-ribose) polymerase is a chromatin enzyme which adds long chains of ADP-ribose to various acceptor proteins in response to DNA strand breaks. Its primary function is unknown; however, a role in DNA repair and radiation resistance has been postulated based largely on experiments with enzyme inhibitors. Recent reports of mutant cell lines, deficient in poly(ADP-ribose) polymerase activity, have supported previous studies with inhibitors, which suggests the involvement of poly(ADP-ribose) polymerase in maintaining baseline levels of sister chromatid exchanges. Mutant cells with even slightly depressed enzyme levels show large elevation of baseline sister chromatid exchanges. Since intracellular poly(ADP-ribose) polymerase levels can vary greatly between different nonmutant cell lines, we surveyed levels of baseline sister chromatid exchange in normal and tumor human cell lines and compared them with endogenous levels of poly(ADP-ribose) polymerase. Despite 10-fold differences in poly(ADP-ribose) polymerase, the baseline level of sister chromatid exchanges remained relatively constant in the different cell lines (0.13 +/- 0.03 SCE/chromosome), with no indication of a protective effect for cells with high levels of the enzyme.

Bimodal distribution of sensitivity of SCE induction by diepoxybutane in human lymphocytes. II. Relationship to baseline SCE frequency

Environmental and genetic factors have been implicated as important sources of individual variation in baseline sister-chromatid exchange (SCE) frequency in humans. The current study was designed to test whether the frequency of baseline SCEs in 58 normal blood donors is associated with previously observed variations in SCE frequencies induced by diepoxybutane (DEB). Because 12 subjects were current cigarette smokers and smoking is known to be an in vivo inducer of baseline SCE frequencies, we specifically tested whether higher baseline SCE frequencies in smokers would be associated with in vitro sensitivity to SCE induction by DEB. Analysis of variance showed that DEB-induced SCE frequencies were significantly associated with baseline SCE frequencies; those who were sensitive to SCE induction by DEB were more likely to have higher baseline SCE frequencies. This effect, however, was independent of in vivo induction of SCE by smoking. Chromosomal sensitivity to the induction of SCE by DEB explained approx. 15–20% of the variation in baseline SCE. This was similar in magnitude to the effect of cigarette smoking. Because increased sensitivity to DEB-induced SCEs is common in normal blood donors (approx. 24%) and is associated with an increase in baseline SCE s, it should be investigated as a source of bias and/or a potential marker of sensitivity to environmental mutagens in future cytogenetic studies.

Erythrocytes modulate the baseline frequency of sister-chromatid exchanges and the kinetics of lymphocyte division in culture

The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.

Baseline and phosphoramide mustard-induced sister-chromatid exchanges in cancer patients treated with cyclophosphamide

Determinations of baseline sister-chromatid exchanges (SCE) have been used extensively as indicators of previous mutagen exposure in both animals and humans. Hypersensitivity to mutagen-induced SCE levels has also been studied in a variation on the basic technique as an indication of previous mutagen exposure in a stressed or provocative test system. The genotoxicity of the alkylating anti-cancer drugs including cyclophosphamide (CP) has been examined previously by determining baseline SCEs in peripheral blood lymphocytes from treated cancer patients. This study examined the in-vivo genotoxic effects of CP therapy by comparing baseline and phosphoramide mustard (PM)-induced SCEs in therapeutically (in-vivo) treated cancer patients with SCE levels in newly diagnosed, but not treated patients. Therapeutically treated patients showed statistically higher baseline SCE frequencies than untreated control patients with a mean SCE/cell of 6.95 vs. 5.25, p < 0.016. When net SCE values (induced minus baseline) were determined in PM-exposed cells in-vitro both at low dose (0.1 μg/ml PM) and high dose (0.25 μg/ml PM) however, the difference was not significant between therapeutically treated and untreated control patients. The return to control SCE levels as a function of time since last therapeutic treatment was also evaluated and no difference was found between the rate of decline of PM-induced SCEs and baseline SCE levels over time.

Comparison of baseline Sister-Chromatid Exchanges (SCE), cyclophosphamide-, ethylnitrosourea (ENU)-induced SCE, ENU-induced cell-cycle delay and chromosome aberrations between Peru and laboratory mice

The baseline sister-chromatide exchange (SCE) frequency and sensitivity to the effects of the mutagens cyclophosphamide (CPP) and ethylnitrosourea (ENU) in bone-marrow cells descedants of wild mice trapped from Rimac valley in Peru (Peru mice) were studied and compared to the same effects in laboratory mice. Baseline SCE of the Peru mice were significantlyhigher than those of the C57BL/6J and DBA/2 mice. The average SCE/cell of 4 Peru mice was 5.4 (range 3.8–7.6), while the average of SCE/cell of either 4 C57BL or 5 DBA mice was 3.2 (range 3.0–3.4). The variation of SCE/cell among Peru mice studied was statistically significant whereas among C57BL or DBAmice it was not. SCE frequencies of primary cultures derived from the ear tissue of 10 peru (mean SCE/cell = 8.5) were also significantly higher than those of 6 C57BL mice (mean SCE/cell = 7.4). CPP treatment resulted in a dose-dependent increase of SCE frequencies in bone-marrow cells of all the mice. However, some Peru mice treated with CPP had significantly higher SCE than the other Peru mice and than all of the C57BL and DBA mice treated with equivalent dose. ENU induced increased SCE frequencies in EPru and C57BL mice. Again some of peru mice either had significantly higher SCE, greater induced cell-cycle delay or chromosome aberrations (CA) than other Peru mice and than of all the C57BL mice treated with equivalent dose. These data suggest that there are two classes of Peru mice, one with high baseline SCE, which is possibly sensitive to the mutagenic effects, as measured by induction of SCE, cell cycle delay and CA; and one class with low baseline SCE which behaves the same as the laboratory mice.

Sister chromatid exchange and proliferation pattern in lymphocytes from newborns, elderly subjects and in premature aging syndromes

Sister chromatid exchange (SCE) frequency and cell proliferation were examined in lymphocyte cultures from a group of newborns, a group of elderly subjects and from patients with syndromes who exhibit progeriform characteristics (progeria, Cockayne syndrome, Rothmund-Thomson syndrome and Christ-Siemens-Touraine syndrome) by using the bromodeoxyuridine incorporation differential staining technique. We observed a significantly increase in basal SCE frequency and a less intensive cell proliferation in cultures from elderly subjects than from newborns, as shown by the significant increase in percentage of cells in first generation simultaneous with a reduction of cells in more advanced generations. Lymphocyte cultures from each one of the patients studied also showed a decreased cell proliferation in relation to their respective control and to newborn cultures. Each of these syndromes showed higher baseline SCE levels than the control and than the newborn and elderly groups. Only the patient with progeria showed values similar to those for the elderly group. Thus, in addition to showing clinical characteristics similar to those observed during the normal aging process, these progeriform syndromes also show cytogenetic characteristics similar to those of older individuals.

Effects of antiepileptic drugs and organic brain damage on SCE frequency in cerebral palsies.

In order to assess the genotoxic effects of antiepileptic drugs (AEDs), analysis of sister chromatid exchanges (SCEs) was performed in lymphocytes of three groups of males; epileptics with AED therapy, non-epileptics without AED therapy and healthy controls. The epileptics and non-epileptics were cases of severe cerebral palsies due to perinatal asphyxia. Possible confounding factors of SCE frequencies, such as age, smoking habit, drug usage other than AED, recent history of viral infection, etc. were controlled. The frequency of SCE per cell was 4.63 +/- 0.71 (mean +/- S.D.) in epileptics, 4.70 +/- 0.89 in non-epileptics and 3.84 +/- 0.56 in healthy controls. SCEs in both epileptics and non-epileptics were significantly higher (p less than 0.05) than those in controls. There was no significant difference of SCE frequency between epileptics and non-epileptics. These results suggested that no mutagenic effect of AED could be demonstrated as revealed by SCEs, and organic brain damage per se might influence the baseline SCE frequency. The possible explanations for such observations are discussed.