Showing papers on "Sister chromatid exchange published in 1993"

Sister chromatid fusion initiates amplification of the dihydrofolate reductase gene in Chinese hamster cells.

Abstract: We have utilized a dihydrofolate reductase (DHFR) probe in combination with selected probes from other positions along the 2q chromosome arm in a two-color fluorescence in situ hybridization analysis of early DHFR gene amplification events in CHO cells. These studies show clearly that the most frequent initiating event is the formation of a giant inverted duplication, resulting either from chromosome breakage and terminal fusion or a reverse unequal sister chromatid exchange. The dicentric chromosomes thus formed initiate bridge/breakage/fusion cycles that appear to mediate subsequent amplification steps to higher copy number.

Regulation and mechanisms of mammalian gene amplification.

Abstract: Publisher Summary This chapter discusses the possible genetic abnormalities that lie in the basis of regulation of amplification and in the nature of the primary events. The regulation of amplification has two different aspects: permissivity and probability. An important primary event in amplification begins with recombination at or near telomeres to generate dicentric sister chromatids. The expression of proteins involved in the amplification process can be induced transiently or stably and provide the basis of efforts to identify the proteins. Normal human or rodent cell strains failed to give resistant colonies containing amplified DNA when selected with cytotoxic drugs that readily select such colonies from immortal cell lines. Mammalian cells respond to DNA damage or arrest of DNA synthesis in several ways, including transient inhibition of cell growth and induction of gene amplification. Different mechanisms may be responsible for amplification of different genes in the same cell or of the same gene in different cells. The primary mechanisms of amplifications includes unequal sister chromatid exchange, telomeric fusions and bridge-breakage-fusion cycles, centromere recombination, and the chromosome breakage-acentric element model.

Polymorphism of glutathione conjugation of methyl bromide, ethylene oxide and dichloromethane in human blood: Influence on the induction of sister chromatid exchanges (SCE) in lymphocytes

Abstract: A hitherto unknown glutathione-S-transferase in human erythrocytes displays polymorphism: three quarters of the population (“conjugators”) possess, whereas one quarter (“non-conjugators”) lack this specific activity. A standard method for the identification of conjugators and non-conjugators with the use of methyl bromide and gas chromatography (head space technique) is described. Three substrates of the polymorphic enzyme, methyl bromide, ethylene oxide and dichloromethane (methylene chloride), were incubated in vitro with individual whole blood samples of conjugators and non-conjugators. All three substances led to a marked increase of sister chromatid exchanges (SCE) in the lymphocytes of the non-conjugators but not in those of conjugators. A protective effect of the glutathione-S-transferase activity in human erythrocytes for the cytogenetic toxicity of these chemicals in vitro is thus confirmed. Since the enzyme activity is not found in erythrocytes of laboratory animals, species extrapolations for risk assessment of methyl bromide, ethylene oxide and dichloromethane should be reconsidered.

ReviewSister-chromatid exchange: second report of the Gene-Tox program

Abstract: This paper reviews the ability of a number of chemicals to induce sister-chromatid exchanges (SCEs). The SCE data for animal cells in vivo and in vitro, and human cells in vitro are presented in 6 tables according to their relative effectiveness. A seventh table summarizes what is known about the effects of specific chemicals on SCEs for humans exposed in vivo. The data support the concept that SCEs provide a useful indication of exposure, although the mechanism and biological significance of SCE formation still remain to be elucidated.

In vitro cytogenetic effects of 2450 MHz waves on human peripheral blood lymphocytes

Abstract: Cytogenetic analyses were performed on human peripheral blood lymphocytes exposed to 2450 MHz microwaves during 30 and 120 min at a constant temperature of 36.1 degrees C (body temperature). The temperature was kept constant by means of a temperature probe put in the blood sample which gives feedback to a microcomputer that controls the microwave supply. We found a marked increase in the frequency of chromosome aberrations (including dicentric chromosomes and acentric fragments) and micronuclei. On the other hand the microwave exposure did not influence the cell kinetics nor the sister chromatid exchange (SCE) frequency.

Origin of the de novo duplication in Charcot — Marie — Tooth disease type 1A: unequal nonsister chromatid exchange during spermatogenesis

Abstract: A 1.5 Mb duplication within 17p11.2 is the major mutation causing both autosomal dominant and sporadic Charcot-Marie-Tooth disease type 1A (CMT1A). An independent origin for the mutation in each family has been postulated. The proposed genetic mechanism causing the CMT1A duplication is unequal nonsister chromatid exchange at meiosis (unequal crossing-over). We studied the parental origin of the duplication in nine genetically sporadic CMT1A patients and demonstrated that in all cases the mutation was the product of an unequal nonsister chromatid exchange during spermatogenesis. The fact that only paternal de novo duplications were observed in the sporadic CMT1A patients suggests that male specific factors may be operating during spermatogenesis that either help forming the duplication and/or stabilize the duplicated chromosome.

Contribution of O6‐alkylguanine and N‐alkylpurines to the formation of sister chromatid exchanges, chromosomal aberrations, and gene mutations: New insights gained from studies of genetically engineered mammalian cell lines

Abstract: O6-methyl- and O6-ethylguanine are the major premutagenic and precarcinogenic lesions induced in DNA by monofunctional alkylating agents, albeit formed in minor amounts. The involvement of these lesions in SCE and aberration formation is less clear. We have analyzed the contribution of O6-alkylguanine to SCE and aberration formation, as well as its toxic and point mutation inducing effect in transgenic Chinese hamster ovary (CHO) cell lines that express variable amounts of human O6-methylguanine-DNA methyltransferase (MGMT). Cells that overexpress MGMT (or the bacterial Ada protein) gained resistance to the formation of alkylation-induced SCEs and aberrations, as compared to MGMT deficient cells. A correlation was apparent between the level of protection for SCEs and cell killing, indicating that both phenomena are interrelated. The protective effects were dependent on the level of MGMT expression, the agent used for alkylation, and cell cycle progression. Our data suggest that at least 2 kinds of lesions are responsible for SCE and aberration formation, namely, O6-alkylguanine and one or various N-alkylation products. The probability that O6-methylguanine is converted into cytogenetic effects has been estimated to be about 1:30 for SCEs, and 1:147,000 and 1:22,000 for chromosomal aberrations in the first and second post-treatment mitosis, respectively. The induction of SCEs and likely also of aberrations by O6-methylguanine requires two replication cycles and is supposed to involve the formation of secondary DNA lesions. Increased repair of 3-methyladenine and 7-methylguanine in CHO cells that overexpress the N-methylpurine-DNA glycosylase (MPG) after transfection with the human MPG-cDNA did not give rise to protection against methylation-induced SCEs and aberrations, probably because of incomplete excision repair. MPG overexpressing cells reacted even more sensitively to methylating agents, suggesting apurinic sites formed as a result of MPG action to be SCE and aberration-inducing lesions.

Replication-dependent sister chromatid recombination in rad1 mutants of Saccharomyces cerevisiae.

Abstract: Homolog recombination and unequal sister chromatid recombination were monitored in rad1-1/rad1-1 diploid yeast cells deficient for excision repair, and in control cells, RAD1/rad1-1, after exposure to UV irradiation. In a rad1-1/rad1-1 diploid, UV irradiation stimulated much more sister chromatid recombination relative to homolog recombination when cells were irradiated in the G1 or the G2 phases of the cell cycle than was observed in RAD1/rad1-1 cells. Since sister chromatids are not present during G1, this result suggested that unexcised lesions can stimulate sister chromatid recombination events during or subsequent to DNA replication. The results of mating rescue experiments suggest that unexcised UV dimers do not stimulate sister chromatid recombination during the G2 phase, but only when they are present during DNA replication. We propose that there are two types of sister chromatid recombination in yeast. In the first type, unexcised UV dimers and other bulky lesions induce sister chromatid recombination during DNA replication as a mechanism to bypass lesions obstructing the passage of DNA polymerase, and this type is analogous to the type of sister chromatid exchange commonly observed cytologically in mammalian cells. In the second type, strand scissions created by X-irradiation or the excision of damaged bases create recombinogenic sites that result in sister chromatid recombination directly in G2. Further support for the existence of two types of sister chromatid recombination is the fact that events induced in rad1-1/rad1-1 were due almost entirely to gene conversion, whereas those in RAD1/rad1-1 cells were due to a mixture of gene conversion and reciprocal recombination.

Acrylamide; induction of DNA damage, chromosomal aberrations and cell transformation without gene mutations

Abstract: The genotoxic potential of acrylamide monomer (AA), a compound familiar as a raw material of polyacrylamide electrophoresis gel, was extensively investigated in vitro. The results were clear cut: AA did not induce any gene mutations in Salmonella/microsome test systems (TA98, TA100, TA1535, TA1537), Escherichia coli/microsome assay (WP2 uvrA-) up to a dose of 50 mg AA/plate, or in HPRT-locus in Chinese hamster V79H3 cells (AA, 1-7 mM, 24 h treatment). On the other hand, AA showed a strong positive response: (a) in a Bacillus subtilis spore-rec assay (DNA damage) at 10-50 mg/disc, (b) to a chromosomal structural change test (AA, 2-5 mM, 24 h treatment), (c) to a polyploidy test (AA, 1-5 mM, 24 h treatment) in Chinese hamster V79H3 cells, (d) to a cell transformation assay in mouse BALB/c3T3 cells (AA, 1-2 mM, 72 h treatment). Sister chromatid exchange was also weakly but significantly induced by AA (AA, 1-2.5 mM, 24 h treatment) in Chinese hamster V79H3 cells. Carcinogenic potential of AA was reported in mice and rats several years ago. AA thus seems to be a typical clastogenic rodent carcinogen without any gene mutation potential. Furthermore, this experiment showed for the first time positive response of AA to a microbial test system (B. subtilis spore-rec assay).

Cellular damage by ferric nitrilotriacetate and ferric citrate in V79 cells: interrelationship between lipid peroxidation, DNA strand breaks and sister chromatid exchanges.

Abstract: Ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-citrate) were used to study the cellular damage mediated by iron overload with respect to cytotoxicity, lipid peroxidation, DNA strand breaks and sister chromatid exchanges (SCE) At non-toxic concentrations, Fe-NTA induced lipid peroxidation, DNA strand breaks and SCE in a dose- and time-dependent manner Comparing the time courses of the different events, the increase in lipid peroxidation seems to be associated with the generation of DNA strand breaks, since both types of cellular damage were observed after 1-3 h of incubation In contrast, the induction of SCE was low after 24 h and increased after 48 and 72 h treatment, indicating the requirement of other types of DNA damage Fe-citrate was inactive in the induction of lipid peroxidation and SCE, and no significant number of DNA strand breaks were generated, as determined by the alkaline unwinding method Our results suggest that the induction of lipid peroxidation and DNA strand breaks by iron overload depend on special features of the iron complex applied, while the chromosomal and genetic effects require site specific DNA damage dependent on intracellular iron metabolism It is concluded that iron overload is able to induce genetic damage in intact cells provided that iron is present in a bioavailable form

Induction of chromosomal aberrations and sister chromatid exchange in vivo and in vitro by the insecticide cypermethrin

Abstract: The induction of chromosomal aberrations and sister chromatid exchange in vivo in mouse spleen and bone marrow as well as in vitro in cultured mouse spleen cells by the insecticide 'Cypermethrin' (cis-trans 1:1) was investigated. The percentage of chromosomal aberrations in the spleen and in the bone marrow as almost the same and reached its maximum 6 h following i.p. injection. The aberrations induced were chromatid and chromosome gaps, fragments and tetraploidy. The insecticide caused a significant and dose-dependent increase in the frequency of sister chromatid exchanges (SCEs) in mouse bone-marrow cells: it reached 11.12 +/- 0.05 per cell after treatment with Cypermethrin at 300 mg kg-1 body wt. compared with 3.7 +/- 0.14 per cell and 4.4 +/- 0.26 per cell in the solvent and control, respectively. The percentage of viable cells in mouse spleen cell cultures reached 87.4% and 99.9% relative to the control after treatment of the cell cultures with 10(-3) and 10(-7) Cypermethrin, respectively. All the tested concentrations of Cypermethrin (0.25-400 micrograms ml-1) induced a high percentage of metaphases with chromosomal aberrations after 4 h of treatment. The mean frequency of SCEs per cell reached 15.1 +/- 0.05 after treatment with Cypermethrin at 4.00 micrograms ml-1 compared with 8.6 +/- 0.23 and 5.9 +/- 0.39 in the solvent and control, respectively. The results indicate that Cypermethrin is genotoxic in mouse spleen and bone marrow as well as in cultured mouse spleen cells.

Mechanisms for induction of mutations and chromosome alterations.

Abstract: Genotoxic agents induce chromosomal alterations, such as aberrations, micronuclei, and sister chromatid exchanges as well as mutations both in vivo and in vitro. Ionizing radiation and typical radiomimmetic agents such as bleomycin are very efficient inducers of chromosomal aberrations. The type of aberrations induced by these agents are cell-cycle dependent, i.e., chromosome type in pre-replication stages and chromatid type in post-replication stages of the cell cycle. Under optimal DNA repair conditions, DNA double-strand breaks (DSBs) appear to be the most important lesion responsible for the production of aberrations. In human lymphocytes, fast-repairing DSBs lead to exchange-type aberrations. The fact that the dose-response curves for induction of exchange aberrations induced by ionizing radiation are similar in vitro and in vivo allows one to use the yield of induced aberrations to estimate absorbed radiation dose in the case of accidents. In this respect, frequencies of translocations detected by the chromosome painting technique appear to be more sensitive. Mutations do not express immediately after exposure and require an expression time before they can be detected. In humans, it is estimated that for the mutations induced in bone marrow, it takes about 2 months for them to express and to be detected in peripheral blood lymphocytes. Hence, frequency of mutations is of limited value for estimating radiation doses immediately after an accident. This holds true for chemical exposure as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome aberrations in lymphocytes of high-voltage laboratory cable splicers exposed to electromagnetic fields.

Abstract: Thirteen high-voltage laboratory employees and 20 referents participated in a cross-sectional, matched-pairs study of cytogenetic damage. During cable testing the workers were exposed to static, alternating, or pulsed electric and magnetic fields. The alternating magnetic field levels of 50 Hz were 5-10 microT, occasionally much higher. Chromosome aberrations, sister chromatid exchanges, and aneuploidy were studied in peripheral blood lymphocytes. In addition, chromosome aberrations were investigated in lymphocyte cultures treated with hydroxyurea and caffeine, to inhibit deoxyribonucleic acid synthesis and repair. Among seven smoking laboratory employees the mean number of chromosome breaks/200 cells was 2.3, as compared with 0.7 for the job-matched referents. The comparable figures for inhibited cultures were 12.0 versus 6.0. No increase was detected in nonsmokers with either method. The other genetic parameters showed no differences between the exposed workers and the referents. The results support, to some extent, the hypothesis of an increased risk of genotoxic effects among high-voltage laboratory workers.

Metabolic activation of chemicals to mutagenic carcinogens by human hepatoma microsomal extracts in Chinese hamster ovary cells (in vitro)

Abstract: The efficiency of human hepatoma (Hep G2) S9 microsomal fractions to activate indirectly acting genotoxic carcinogens was evaluated. The extract was prepared from Hep G2 epithelial cells, following sonication and centrifugation. The mutagenic activity of cyclophosphamide, benzo[a]pyrene, pyrene, hexamethylphosphoramide and safrole was assessed by the ability of their activated metabolites to induce sister chromatid exchange (SCE) and micronuclei (MN) in Chinese hamster ovary cells (CHO) (treated in vitro). All promutagenic carcinogens tested were found to be effective only following metabolic activation by Hep G2 cell extracts. Non-carcinogen pyrene was not able to induce an increase in the frequencies of SCE or MN in CHO cells even in the presence of Hep G2 S9 microsomal fractions. Parallel experiments were carried out using rat liver homogenate (S9 fraction) as an exogenous activation system, and comparisons were made between these two in vitro systems and in vivo assays using the rodent.

Genetic toxicity of fluoride

Abstract: F- is not mutagenic in standard bacterial systems, but produces chromosome aberrations and gene mutations in cultured mammalian cells. Although there is disagreement in the literature concerning the ability of F- to induce chromosome aberrations in cultured human and rodent cells, the weight of the evidence leads to the conclusion that F- exposure results in increased chromosome aberrations in these test systems. NaF induced primarily chromatid gaps and chromatid breaks, indicating that the rodent cells are responsive in the G2 stage of the cell cycle. In contrast, studies with synchronized human cells indicated that the S phase was the most sensitive. If F- does have a cell cycle-specific effect, it could be expected that differences in the cell treatment and harvest protocols could lead to conflicting results for the induction of chromosome aberrations. Gene mutations were produced in cultured rodent and human cells in the majority of the studies. Unfortunately, a number of the in vitro and in vivo cytogenetic studies are of questionable utility because of the protocols used, the quality of the responses reported, or the interpretations of the data. The conflicting results in the in vivo cytogenetic studies are difficult to reconcile. There are reports of increased chromosome aberrations in rat bone marrow and testes, but other studies, using similar protocols and dose ranges, have reported no induced chromosome damage. Although some of the studies were performed at toxic levels of F-, other studies, including those that showed positive results, were at F- concentrations (1-5 ppm) equivalent to human exposure levels. In the majority of studies that were reported to be positive, there were high background frequencies, or the investigators reported categories of nuclear or chromosome damage that are difficult to interpret. Interestingly, many of the positive results were obtained when anaphase cells were scored, whereas similar treatment protocols in other laboratories yielded negative results when metaphase cells were the only cell type examined. It is difficult, without additional data, to determine the reasons for finding chromosome breaks in anaphase, but not metaphase, cells. Other reports have presented insufficient information to allow adequate evaluations. Therefore, at this time, the question of whether F- produces chromosome damage in vivo should be considered unresolved.

Relative efficiency of Phyllanthus emblica fruit extract and ascorbic acid in modifying lead and aluminium-induced sister-chromatid exchanges in mouse bone marrow

Abstract: The identification of desmutagens and bioantimutagens in plants has prompted the search for additional plant extracts capable of modifying adverse cellular effects of environmental toxicants. The protective action of crude extracts of Phyllanthus emblica fruits (PFE) against lead (Pb) and aluminium (Al)-induced sister chromatid exchanges (SCEs) was studied in bone marrow cells of Mus musculus. The modifying effect of the crude extract was compared with that of comparable amounts of synthetic ascorbic acid (AA), a major component of the fruits. Oral administration of PFE or AA for 7 consecutive days before exposure of mice to the metals by intraperitoneal injections reduced the frequencies of SCEs induced by both metals. PFE afforded a more pronounced protective effect than AA in counteracting the genotoxicity induced by both Al and Pb: This difference was significant with Pb. The higher protection afforded by PFE may be attributed to the interaction of AA with other natural ingredients present in the crude fruit extract.

Cryopreservation of mouse oocytes: mutagenic effects in the embryo?

Abstract: We have shown in previous studies that the complete cycle of cryopreservation and prefreezing manipulations increases the degeneration and decreases the fecundability of mouse oocytes. The present study confirms these results. Moreover, we show that the increase of polyploidy previously observed in one-cell zygotes derived from frozen-thawed oocytes persists during the early stages of embryonic development. Furthermore, embryos obtained from frozen oocytes or oocytes exposed to prefreezing manipulations show an increase in the frequency of sister chromatid exchanges. Since the estimation of sister chromatid exchange is a sensitive test of mutagenicity, this suggests that the complete cycle of cryopreservation might alter the oocyte and, more particularly, induce DNA damage.

Induction of chromatid breaks and tetraploidy in Chinese hamster ovary cells by treatment with sodium arsenite during the G2 phase.

Abstract: Treatment of Chinese hamster ovary (CHO) cells with sodium arsenite during the G2 phase induced poorly condensed chromosomes and chromatid breaks. The induction of chromatid breaks was confirmed by the appearance of micronucleated cells after arsenite-treated G2 cells were allowed to re-enter interphase. When the duration of the G2 phase was artificially divided into 4 periods, more chromatid breaks were induced by treatment with arsenite during the very early G2 phase (or G2/S boundary). In addition to the induction of chromatid breaks, arsenite treatment also remarkably retarded the re-entry of mitotic cells into interphase. By replating and incubating arsenite-treated G2 cells in drug-free medium, we subsequently observed the appearance of a population of cells whose DNA content was between 4C and 8C, and metaphase cells with near-tetraploid chromosome numbers in the next mitotic division.

Measurement of chromosomal aberrations, sister chromatid exchange, hprt mutations, and DNA adducts in peripheral lymphocytes of human populations at increased risk for cancer.

Abstract: Using a multidisciplinary approach, we have measured various indicators of DNA damage in peripheral lymphocytes of human populations potentially at increased risk for cancer. Sister chromatid excha...

Genetic toxicity of 1,3-butadiene and styrene.

Abstract: 1,3-Butadiene and styrene (vinyl benzene) are indirect genotoxins, which require metabolic activation to an epoxide form in order to bind covalently to DNA. Styrene 7,8-oxide, the active metabolite of styrene, is a carcinogen in rodents and has been shown to be genotoxic in most in-vitro test systems and at various genetic endpoints. The few studies available on the genotoxicity of styrene 7,8-oxide in vivo have yielded negative or (in mice) weakly positive results. Styrene is not usually genotoxic in vitro in assays employing a microsomal preparation from rat liver for metabolic activation, but positive effects have been obtained when other sources of metabolic activation, such as human erythrocytes, were provided. In vivo, styrene has been found repeatedly to be weakly genotoxic in the assay for sister chromatid exchange, especially in mice. Cytogenetic damage (usually chromosomal aberrations) has been reported in many studies of workers, mainly from the reinforced plastics industry where ambient concentrations of styrene may be high (50-100 ppm), while most negative findings are associated with exposure to lower levels. Butadiene is metabolized to two reactive forms, 1,2-epoxy-3-butene and further to 1,2:3,4-diepoxybutane, both of which are genotoxic in various test systems in vitro. The lowest effective dose of the latter is 1-2 orders of magnitude higher than that of the respective monoepoxide. Butadiene itself has not been tested extensively for genotoxicity in vitro. A species-specific difference in the responses of mice and rats at various cytogenetic end-points is seen in vivo, the lowest effective concentrations in rats being clearly higher than those in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of sister chromatid exchange and micronuclei in primary cultures of rat and human hepatocytes by the peroxisome proliferator, Wy-14,643

Abstract: The ability of peroxisome proliferators to induce hepatocellular carcinomas in rodents has been known since the mid 1970's, but the mechanism of tumor formation is still poorly understood. In this study, we have used primary cultures of both rat and human hepatocytes to address the question of whether the peroxisome proliferator, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643), causes genotoxic damage in hepatocytes as measured by sister chromatid exchange (SCE), micronuclei formation, and chromosomal aberrations. We have found that in rat hepatocytes the number of SCEs per chromosome increased in a dose-dependent manner from a background level of 0.7 to a maximum of 1.1 in cells exposed for 48 h to 100 μM of Wy-14,643. In contrast, no increase in SCE frequency was observed in rat hepatocytes exposed to Wy-14,643 for 3 h. A dose-dependent increase in micronuclei formation was also seen in the 48 h but not in the 3 h cultures. The maximum frequency of micronuclei formation after a 48 h exposure occurred at 20 μM Wy-14,643 and was 2.3 times that for control cells. At this concentration of Wy-14,643, the frequency of chromosomal aberrations was increased by more than 10-fold. A 48 h exposure to Wy-14,643 also significantly increased micronuclei formation in human hepatocytes, but it was less effective than in rat hepatocytes. To investigate the potential role of peroxisome proliferation in these genotoxic responses, we measured the activities of palmitoyl-CoA β-oxidase in hepatocytes exposed for 48 h to Wy-14,643. A dose-dependent increase in palmitoyl-CoA β-oxidase activity was observed in rat hepatocytes, but notin human hepatocytes. The SCE frequency in rat hepatocytes correlated well with the degree of peroxisome proliferation, however, the increased formation of micronuclei in both rat and human hepatocytes occurred by a mechanism that appeared to be independent of peroxisome induction. In summary, these results demonstrate that the peroxisome proliferator, Wy-14,643, causes genotoxic damage in primary cultures of both rat and human hepatocytes.

Cytogenetic effects of CPT-11 and its active metabolite, SN-38 on human lymphocytes.

Abstract: CPT-11, a new camptothecin analogue, has been demonstrated to be a promising antineoplastic agent. Late side effects of carcinogenicity and teratogenicity have been unclear from clinical phase I and II trials. In order to elucidate the carcinogenicity and teratogenicity of CPT-11, we have examined the cytogenetic changes in human peripheral blood lymphocytes induced by CPT-11 and its active metabolite, SM-38. We have also analyzed the correlation between chromosomal damage and acute clinical side effects. When peripheral blood lymphocytes obtained from a healthy donor were exposed to CPT-11, SN-38, cisplatin and mitomycin C, a significant dose-dependent increase of sister chromatid exchange (SCE) was obtained. The SCE frequency per cell cultured with 0.244 nM SN-38 was similar to that cultured with 100 nM CPT-11, 300-500 times the concentration of SN-38. A transient increase in SCE frequency was also observed in the peripheral blood lymphocytes of 11 cancer patients receiving 100 mg/m2 of CPT-11 intravenously, compared with pretreatment values (P = 0.0001). In addition, a significant correlation was observed between the frequency of SCE on day 3 and the degree of decrease in platelet count (P = 0.012). In conclusion, SN-38 might possibly have a high risk of mutagenicity and carcinogenicity; and measurement of SCE values in peripheral blood lymphocytes appears to have a potential application in the clinical prediction of chemotherapy-induced side effects.

Okadaic acid, a ptotein phosphatase inhibitor, induces sister-chromatid exchanges depending on the presence of bromodeoxyuridine

Abstract: Okadaic acid (OA), a potent tumor promoter and an inhibitor of protein phosphatase 1 and 2A, induced sister-chromatid exchanges (SCEs) in human lymphoblastoid cells and Chinese hamster ovary cells at low concentrations of 2-10 nM, when the cells were grown for two cell cycles in the presence of OA and bromodeoxyuridine (BrdUrd). Prolonged treatment with OA prior to addition of BrdUrd did not induce SCEs, indicating an essential role of BrdUrd. A similar important role of BrdUrd in SCE induction has been reported in the cases of benzamide (BA) (Natarajan et al., 1981) and camptothecin (CPT) (Zhao et al., 1992), which are inhibitors of poly(ADP-ribose)polymerase and DNA topoisomerase I (topo I), respectively. Unlike many DNA-damaging agents, they are required to be present during S phase along with BrdUrd in the medium and/or in the parental DNA as BrdUMP. Thus OA, like BA and CPT, is a new type of SCE inducer. Exposing cells to a combined treatment with OA, BA and CPT, a significantly higher level of SCEs was induced than that expected if the numbers of SCE caused by these three inhibitors were additive, while no such synergistic increase was seen in every combination of two agents. Since both phosphorylation and poly(ADP-ribosyl)ation have been known to modify topo I activity, the results suggest a common involvement of topo I for SCE formation by OA, BA and CPT. In addition to SCE induction, OA resulted in an increase of mitotic cells which were characterized by a marked chromosome condensation. OA also induced chromosome fragmentation/pulverization in human lymphoblastoid cells and fragmented nuclei in Chinese hamster cells.

Selective aflatoxin B1-induced sister chromatid exchanges and cytotoxicity in differentiating B and T lymphocytes in vivo.

Abstract: The purpose of this study was to assess the genotoxic and cytotoxic effects of the fungal metabolite aflatoxin B1 (AfB1) on the developing immune system of the chick embryo, a model in vivo system. Of particular interest was the assessment of AfB1 -mediated selective toxicity toward developing B lymphocytes as compared to T lymphocytes. In vivo bromodeoxyuridine (BrdU) labelling of DNA was used to detect the induction of sister chromatid exchanges (SCE) in lymphocytes and to assess the progression of these cells through successive cell cycles. Cytotoxicity was also assessed by studying the entrance and maintenance of cells in mitosis (mitotic index). Graded doses of AfB1 (1.09–17.4 μ/g embryo) were applied to chick embryos of 18 days of incubation (Dl). Embryos also received two doses of BrdU at 3 mg/200 μ (3 hr apart) to provide continuous labelling of B and T lymphocyte replicating DNA. B and T lymphocytes were harvested 20 hr post-AfB1/BrdU exposure from the bursa and thymus, respectively, and were processed for cytogenetic analyses. AfB1 induced dose-related increases in SCE in B lymphocytes; this induction was 6- to 8-fold that of controls at the higher doses tested, AfB1 -mediated induction of SCE in T cells was just 2-fold that of controls at the highest dose tested. AfB1 reduced the progression of B cells and to a lesser extent T ceels through successive rounds of replication. Furthermore, AfB1 dramatically reduced the mitotic index of B cells but not of T cells. These data indicate both selective genotoxicity and cytotoxicity of AfB1 toward B cells in the late stage embryo. © 1993 Wiley-Liss, Inc.

The spontaneous frequency of chromosomal aberrations and sister-chromatid exchanges in cultured peripheral lymphocytes of a single blood donor sampled more than 200 times.

Abstract: Blood samples were taken from a single individual over 200 times during a 12-year period and the lymphocytes cultured with the intention of examining the frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE). The data presented here are a compilation of the aberration frequencies in untreated control cultures from all experiments in which at least 100 metaphases were analysed for chromosomal aberrations (135 experiments), and at least 25 for the frequency of SCE (24 experiments). Since 52-h cultures displayed a high mitotic index and a low percentage of second-division metaphases (7%), this culture time was chosen for 70 experiments investigating the frequency of chromosomal aberrations. The inter-experimental variation in aberration frequency was found to be small, 0–4 aberrations per 100 cells, with a mean of 0.79±0.94. The fact that the spontaneous frequency of chromosomal aberrations was distributed according to the Poisson expectation indicates that the mean aberration frequency for a single subject is constant over time. On average, 80% of the aberrations were chromatid breaks (0.59 aberrations per 100 cells). Isochromatid breaks (or chromosome deletions) were not uncommon (0.11). Chromatid exchanges (0.03), as well as dicentric (0.014) and ring (0.014) chromosomes were rare. The incidence of SCE was examined in lymphocytes grown for 72 h in the presence of 20 μM 5-bromodeoxyuridine. The mean number of spontaneous SCE/cell was 6.41±0.69. The variation in SCE frequency between experiments was small, the coefficient of variation being only 0.11.