Sister-chromatid exchanges in lymphocytes from patients with Schistosoma hematobium

Sister-chromatid exchange (SCE) frequencies were studied in peripheral blood lymphocytes from 19 patients (13 males and 6 females) with Schistosoma hematobium, prior to the initiation of chemotherapy. The mean frequency of SCE per metaphase for the patients (both sexes) was 10.4 +/- 4.2 which was significantly higher (P less than 0.01) than the mean SCE (6.4 +/- 1.1) score for 35 healthy controls. A highly significant reduction in lymphocyte division and delay in cell-cycle progression as a result of infection were also noticed. These data indicate that infection with S. hematobium could increase SCEs in the host somatic cells.     

Induction of sister chromatid exchanges in the central mudminnow following in vivo exposure to mutagenic agents.

Sister-chromatid exchange (SCE) studies using the central mudminnow, Umbra limi, demonstrated its usefulness as a model in vivo system for the detection of genetically active substances (mutagens and/or carcinogens). 5 days following the injection of 500 micrograms/g of 5-bromodeoxyuridine (BrdU), SCE rates were found to be quite low in the gills (2.0--3.3 SCEs/metaphase), kidneys (2.6--3.4 SCEs/metaphase), and intestines (3.7--4.5 SCEs/metaphase). However, after i.p. exposure to microgram quantities of methyl methanesulfonate (MMS) or cyclophosphamide (CP), large linear dose-dependent increases in SCE rates were observed in all tissues examined. amined. On a microgram/g basis, CP was found to be a 2--4 times more potent inducer of SCE than was MMS. Studies involving the addition of neutral red dye (NR) to the fish's aquarium water revealed that the mudminnow could concentrate the dye in its gill and kidney tissues. This dye was found to cause significant increases in SCE rates at water levels of less than 0.1 ppm. However, the concentration of dye in the tissues did not show a correlation with the SCE rate. Possible explanations for this observation are presented in the text. These findings demonstrate the feasibility of using a fish such as the mudminnow to investigate cytogenetic consequences of aquatic pollution.     

Propofol anesthesia in children does not induce sister chromatid exchanges in lymphocytes

BACKGROUND: Propofol is frequently used for general anesthesia in children although little is known about possible genotoxic effects in humans. We investigated the formation of sister chromatid exchanges (SCE) in metaphase chromosomes of T-lymphocytes of children as a marker for possible genotoxocity following total intravenous anesthesia with propofol for minor surgical procedures. METHODS: 40 children ASA classification I-III were included (ASA I n=34, ASA II n=5, ASA III n=1) in the study. Anesthesia was induced by propofol (3mg/kg) and alfentanil. Succinylcholine or rocuronium were administered for muscle relaxation. After tracheal intubation anesthesia was maintained by continuous propofol infusion at 12 mg/(kgh). Blood samples were drawn before induction and after termination of anesthesia. Following a 72 h cell culture period, 25 T-lymphocyte metaphases per blood sample for all children were analyzed for SCE frequencies. RESULTS: Total intravenous anesthesia with propofol on children did not influence SCE rates in metaphase chromosomes of T-lymphocytes. No SCE differences could be detected between blood samples before initiation and after termination of anesthesia (Wilcoxon signed rank test). Slightly elevated SCE rates were obtained in T-lymphocytes of girls compared to boys, but these differences did not reach statistical significance. CONCLUSIONS: Propofol anesthesia under the chosen conditions did not induce the formation of SCE in children in vivo. No genotoxic effect of a short term exposure to propofol during pediatric anesthesia had been observed.     

Propofol anesthesia in children does not induce sister chromatid exchanges in lymphocytes

Background: Propofol is frequently used for general anesthesia in children although little is known about possible genotoxic effects in humans. We investigated the formation of sister chromatid exchanges (SCE) in metaphase chromosomes of T-lymphocytes of children as a marker for possible genotoxocity following total intravenous anesthesia with propofol for minor surgical procedures.Methods: 40 children ASA classification I–III were included (ASA I n=34, ASA II n=5, ASA III n=1) in the study. Anesthesia was induced by propofol (3 mg/kg) and alfentanil. Succinylcholine or rocuronium were administered for muscle relaxation. After tracheal intubation anesthesia was maintained by continuos propofol infusion at 12 mg/(kg h). Blood samples were drawn before induction and after termination of anesthesia. Following a 72 h cell culture period, 25 T-lymphocyte metaphases per blood sample for all children were analyzed for SCE frequencies.Results: Total intravenous anesthesia with propofol on children did not influence SCE rates in metaphase chromosomes of T-lymphocytes. No SCE differences could be detected between blood samples before initiation and after termination of anesthesia (Wilcoxon signed rank test). Slightly elevated SCE rates were obtained in T-lymphocytes of girls compared to boys, but these differences did not reach statistical significance.Conclusions: Propofol anesthesia under the chosen conditions did not induce the formation of SCE in children in vivo. No genotoxic effect of a short term exposure to propofol during pediatric anesthesia had been observed.     

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.     

Rodent species and strain specificities for sister-chromatid exchange induction and gene mutagenesis effects from ethyl carbamated,ethyl N-hydroxycarbamate,and vinyl carbanate

Ethyl carbamate (EC) and two related carcinogens, ethyl N-hydroxycarbamate (ENHC) and vinyl carbamated (VC), caused species-specific increase in sister-chromatid exchange (SCE) formation in the bone marrow cells of rodents. Mice exposed to 400 mg/kg of EC had SCE increases of 6-times-baseline, while rats, Chines hamsters, and golden hamsters showed 3- to 4-times-baseline increases in response to this dose. Lesser, but still significant, differences were found for ENHC and VC; the severest effects consistently occured in mice. Control bone marrow cell-cycle kinetics among the rodent species were similar. Mouse strains A and C57BL/6, which have high and low susceptibilities to EC induction of lung adenomas, respectively, showed nearly identical levels of SCE induction after in vivo exposure to these carbamate. However, testing of VC, a possible metabolite of EC, in vitro revelaed strain-dependent liver enzyme (Aroclor-induced S-9 fraction) capabilities to convert VC to genotoxic products. SCE induction, gene mutation for 6-thioguanine and ouabain resistance, and cytotoxicity in Chinese hamster V79 cells were significantly greater when A strain S-9 enzymes were used as compared with C57BL/6 strain S-9 enzyme preparations. No effect of SCE of reseeding, compared with no reseeding, of VC-treated V79 cells was observed. At a concentration of 25 μg/ml, VC cause 6-times-baselin induction of SCE in the presence of A strain S-9 mix and 4-times-baseline induction in the presence of C57BL/6 strain S-9 mix. These in vitro strain-dependent patterns of response are relevant to the current theory that VC amy be a proximate carcinogenic metabolite of EC.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by the biotic ketoaldehyde methylglyoxal

The number of sister-chromatid exchanges (SCEs) per metaphase was determined in Chinese hamster ovary cells after 16 h exposure to methylglyoxal (MG) concentrations ranging from 0.1 to 0.75 mM. MG produced an increase of SCE frequency that proved to be dose-dependent, and to reach a maximum of 2 X baseline at the highest nontoxic concentration (0.5 mM).     

Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine

An in vivo system for the detection of sister chromatid exchange (SCE) in the central mudminnow, Umbra limi, is presented. Sister chromatid differential (SCD) and SCE were demonstrated by fluorescent and Giemsa procedures 5 to 6 days after the fish were injected with 500 g/g of BrdU. The exchange rate was found to be 2.64 SCEs metaphase in the intestines and 2.42 SCEs/metaphase in the gills. SCE analysis in U. limi should be a useful tool for measuring the mutagenicity of water-borne chemicals.     

Genotype- and age-associated in vivo cytogenetic alterations following mutagenic exposures in mice

We studied mice from eight genetic strains at two ages (young, 10 weeks; and old, more than 80 weeks) for cytogenetic alterations (sister chromatid exchange (SCE), micronuclei, and metaphase indices) following challenges by two known mutagens: N-nitrosoethyl urea (ENU, 17 mg/kg) and cyclophosphamide (CP, 4.5 mg/kg) on bone marrow cells in vivo. The data were used to evaluate the effect of age, genotype, and differential aging patterns of genotypes in relative susceptibility to chromosomal breakage and instability in otherwise normal individuals. The older animals had a higher frequency of micronuclei, reduced metaphase indices, and lower SCE/cell as compared with their younger counterparts. Treatment with both mutagens significantly increased micronuclei and SCEs/cell in almost all strains at both ages but had little effect on the frequency of cells in metaphase. Among individual differences for SCEs/cell at most treatment combinations were not significant. In general, the induced SCEs (treatment-control) are significantly higher in older animals, variable among strains, and relatively higher as a result of CP than the ENU treatment. When the age effect was evaluated as the difference of SCE/cell in old and SCE/cell in young animals of each genotype-treatment combination, an age-dependent pattern was evident. In the presence of a mutagen the pattern in aging response was highly variable and strain (genotype) dependent. This variability may be viewed as subtle inherent genetic predisposition of sensitivity to mutagens that could be evaluated only using sensitive measures (e.g., SCE and not micronuclei) following more than one mutagenic challenges. These subtle differences could become pronounced when these parameters are evaluated at different ages on the same genotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of bromodeoxyuridine on the proliferation and growth of ethyl methanesulfonate-exposed P3 cells: Relationship to the induction of sister-chromatid exchanges

Although sister-chromatid exchange (SCE) analysis is recognized as an indicator of exposure to DNA-damaging agents, the results of these analyses have been confounded by the use of bromodeoxyuridine (BrdUrd) to differentially label the sister chromatids. Not only does BrdUrd itself induce SCE, it also modulates the frequency of SCE induced by certain DNA-damaging agents. In order to examine this effect of BrdUrd on SCE frequency, an indirect method which lends itself to measurements both with and without BrdUrd was employed. Human teratocarcinoma-derived (P3) cells were exposed to ethyl methanesulfonate (EMS) and cultured with increasing concentrations of BrdUrd for lengths of time corresponding to one, two, and three generations of cell growth. At each time point, the distribution of nuclei among the phases of the cell-cycle and cell growth were evaluated for each concentration and chemical. A statistical model was employed which tested both for the main effects of chemicals and culture times and for interactions between these factors. Both EMS and BrdUrd significantly affected the percentages of nuclei within the cell-cycle. Exposure to EMS resulted in decreases in the percentages of nuclei in G0 + G1 and increases in the G2 + M compartment. Exposure to BrdUrd affected the size of the G0 + G1 compartment as well as the percentage of S-phase nuclei. Cell growth was reduced as a consequence of increasing EMS concentration and as a function of BrdUrd concentration; the effects of these chemicals were more readily apparent at the later time points. Most importantly, for both the cell-cycle kinetics data and the cell growth data, no evidence of an interaction between the effects of EMS and the effects of BrdUrd was detected statistically. These results may be interpreted to mean that while both EMS and BrdUrd affect the induction of SCE, under the conditions of this experiment, the effects are additive rather than interactive.Abbreviations: EMS, ethyl methanesulfonate - BrdUrd, bromodeoxyuridine - BrdUTP, bromodeoxyuridine triphosphate - dCTP, deoxycytidine triphosphate - SCE, sister-chromatid exchange - P3, human teratocarcinoma derived - HBSS, Hank's Balanced Salt Solution - HOUR, culture time - REP, replicate     

Sister-chromatid exchanges in lymphocytes from infants with Down's syndrome

Sister-chromatid exchange (SCE) frequencies were studied in blood lymphocytes from 12 patients (3 females and 9 males) with Down's syndrome (DS). The mean frequency of SCE per metaphase for the patients (both sexes) was 9.2 +/- 0.8 which was significantly higher (P less than 0.01) than the mean SCE value (5.1 +/- 0.2) scored for 16 healthy infants (8 females and 8 males). A significant increase in the mean frequency of SCE in 12 parents of infants with DS (8.7 +/- 0.9 SCE/cell) was noticeable when compared with 20 parents of normal infants (6.3 +/- 0.1 SCE/cell). Increases in cellular division with reduction in their replication were also observed in patients with DS. Treatment with mitomycin C (0.05 micrograms/ml), hycanthone (0.1 micrograms/ml) and gamma-radiation (0.1 Gy) revealed a significant (P less than 0.01) increase in frequencies of SCE in DS lymphocytes and in those of their parents as compared to controls. These data may reveal a familial hypersensitivity reaction to these agents. The results indicate a genomic instability and deranged DNA-repair mechanisms which are accentuated by exposure to mutagenic agents, the underlying causal factor for which might be genetic.     

Chemical induction of sister-chromatid exchanges in human lymphocytes treated in G0 prior to stimulation by different mitogens and revealed 72 h later in second division cells

Frequencies of sister-chromatid exchanges (SCE) were determined in second-division metaphases of human lymphocytes, exposed for 1 h during the G0 phase to mitomycin C (MMC) alone or to cyclophosphamide (CP) in the presence of S9 mix. The cells were then cultured for 72 h in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), Wistaria floribunda (WFA) or Lens culinaris (LcH-A) extracts. Large differences in mitotic indices (MI) and cell-cycle kinetics were observed among cells subjected to the various treatments. However, in the controls as well as in the cultures submitted to a G0 mutagenic exposure, the yield of SCE was not influenced by the mitogenic agent and was, therefore, independent of the proliferation properties of the cultured lymphocyte population.     

SCE induction and cell-cycle delay by toxaphene

Toxaphene is genotoxic in mammalian cell systems and also inhibits cell replication. It was therefore used to investigate possible masking of SCE induction due to cell-cycle delay. In this study, toxaphene-treated Chinese hamster lung (Don) cells exhibited a dose-dependent decrease in cell-cycle progression compared with untreated cells. At high, nontoxic toxaphene levels (15 micrograms/ml), cell cycling also slowed as the toxaphene treatment time was increased. Toxaphene induced significantly higher numbers of SCEs in treated cells, demonstrating a dose- and treatment time-relationship. Slopes of dose-response curves were 0.29, 0.43 and 0.77 SCE/micrograms toxaphene for 20.5 h, 24.5 h and 28.5 h incubation, respectively. There were no changes in SCE values in control cultures even when slower dividing cells were sampled e.g. at longer incubation times. Thus, higher SCE values in Chinese hamster cells were not associated per se with slower or more delayed cells. The results demonstrate that longer toxaphene treatment times were not necessary for obtaining sufficient harlequin-stained cells for SCE analysis, but that higher numbers of SCEs occurred in slower dividing cells, following prolonged incubation of cultures treated with toxaphene.     

Sister-chromatid exchanges and cell-cycle kinetics in human lymphocyte cultures exposed to alkylating mutagens: apparent deformity in dose-response relationships

Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and of the duration of 5-bromodeoxyuridine (BrdUrd) treatment before fixation on sister-chromatid exchange (SCE) frequencies following exposure to mitomycin C (MMC). Cells were pulse treated for 1 h with 3 X 10(-6) M MMC at G1, and then sampled at 4-h intervals up to 88 h after stimulation of cultures with phytohemagglutinin (PHA). Results showed that this MMC treatment induced a 5-6 h proliferation delay per cell cycle, and that SCE frequencies first increased with time of fixation, peaking at 68 h, and then decreased. When cells were similarly treated with MMC, but subsequently exposed to BrdUrd for various times before fixation of cultures at 72 h, the SCE frequencies markedly increased with increasing durations of BrdUrd incubation times. These data indicate that, in mutagen-treated cultures, lymphocytes having relatively longer cell-cycle times show a higher mean frequency of SCEs. In a subsequent experiment, cells were treated for 1 h with increasing doses of MMC or 4-nitroquinoline 1-oxide (4NQO) at 0, 24, or 48 h, and then fixed at 72 h after PHA stimulation. Results showed that the optimal treatment times at which the agents could most efficiently produce SCEs were different for MMC and 4NQO, and that the dose-response curves tended to 'bend down' at very high doses; that is, treatments with very high doses induced smaller than expected numbers of SCEs. However, cells similarly treated with very high doses showed a higher, expected frequency of SCEs when sampled at 84 h, but again had a lower than expected SCE frequency when fixed at 96 h. The results indicate that there is an optimal time for sampling at which one can observe the maximum increase in SCE frequencies following mutagen exposure, and strongly suggest that the higher the dose, the later the optimal sampling time. Because of the apparent deformity of dose-response curves obtained after various treatments and sampling times, it seems necessary that extra fixation-time points be included in test protocols so as to avoid false negatives or confirm possible positives.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

Induction of sister chromatid exchanges by styrene and its presumed metabolite styrene oxide in the presence of rat liver homogenate

Styrene and its metabolite styrene oxide were tested for their ability to induce sister chromatid exchanges (SCE) in CHO cells. Styrene oxide appeared to be a potent inducer of SCE. Styrene itself did not increase the number of SCE per metaphase, even in the presence of a metabolic activation system. The metabolic activation system decreased the SCE induction caused by styrene oxide. Induction of SCE by styrene in the presence of metabolic activation occurred when cyclohexene oxide was used as an inhibitor of the enzyme epoxide hydrase.     

Frequencies of sister-chromatid exchanges in relation to cell kinetics in lymphocyte cultures

The frequency of sister-chromatid exchanges (SCE) was determined on second-division metaphase of lymphocytes stimulated by phytohaemagglutinin (PHA) during 9 days of culture.

By using either a continuous or a pulsed bromodeoxyuridine (BUdR) treatment, cells were selected that had divided only twice, or at least twice, after different culture periods. No significant differences were observed in the SCE frequencies among the various samples. The incidence of SCE appears to be independent of the proliferation properties of cultured lymphocytes, such as length of cell cycle, fast or delayed response to PHA and number of divisions performed in vitro.     

Induction of sister-chromatid exchanges and chromosomal aberrations in hematopoietic tissue of a marine fish following in vivo exposure to genotoxic carcinogens

The development of procedures to assess genetic damage in fish exposed in situ to point sources of aquatic pollution can be expected to contribute to the evaluation of the role of genotoxic contaminants in epizootic neoplasia in fish populations. To this end methods have been developed for assessing the in vivo induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in tissues of a marine teleost, the oyster toadfish, which may be applicable to other species. An alternative to the solid tissue and squash techniques for metaphase preparation permits the resolution of more than 100 SCEs/metaphase in toadfish kidney cells, which have moderately large chromosomes (0.122 pg DNA/chromosome). The bleeding of toadfish which have been injected with 5-bromodeoxyuridine (BrdUrd) and the subsequent use of hematopoietic tissue (kidney) for cytogenetic analysis was shown to increase the metaphase yield and provide a more predictable production of second-division metaphases required for SCE analysis. With these methods linear dose-dependent increases in chromatid-type exchange CAs and SCEs were obtained with i.p. exposure to ethyl methanesulfonate (EMS) and cyclophosphamide (CP). The doses required to double the observed control SCE frequencies (least effective doses) were 170 mg/kg for EMS and 7.4 mg/kg for CP. which are comparable to those reported for rodent bone marrow assays. A BrdUrd-sensitive site for chromatid breakage was observed on a pair of apparently homologous acrocentric chromosomes for the toadfish.     

Long dwell-time exposure of human chorionic villi to transvaginal ultrasound in the first trimester of pregnancy induces activation of caspase-3 and cytochrome C release

Bioeffects after exposure to ultrasound are correlated to its duration. Although diagnostic ultrasound has been suggested to induce apoptosis, the underlying signal transduction pathway remains elusive. In this study, women in the first trimester of pregnancy were exposed to transvaginal diagnostic ultrasound with 5.0-MHz frequency for 0, 10, 20, or 30 min. The chorionic villi were obtained 4 h after exposure and activation of caspase-3 and cytochrome c release were analyzed by Western blotting. In contrast with the 0- and 10-min groups, cleavage products of active caspase-3 and cytochrome c release significantly increased in 20- and 30-min groups in a time-dependent manner. We show that long-duration exposure to transvaginal ultrasound activates effector caspase-3-mediated apoptotic cascade of chorionic villi in the first trimester of pregnancy. This occurs through the intrinsic death pathway involved in cytochrome c release. Our findings provide a molecular rationale for discriminant use of transvaginal ultrasound at the early stage of pregnancy.