化学诱变物诱发小鼠脾脏细胞和大麦根端分生组织细胞姐妹染色单体交换的比较研究

诱变剂诱发SCE的增加在大麦细胞中比在小鼠脾脏细胞中所需浓度较低,说明植物SCE比动物体内SCE敏感。所检测的诱变剂大多均能在小鼠脾脏细胞和大麦细胞中诱发SCE的增加,两者之间的SCE/细胞、SCE/pgDNA、SCE比率、SCE/pgDNA增加等项数值均显示极显著相关性,表明所检测的几种诱变剂在小鼠脾脏细胞和大麦细胞中最终诱发SCE的效应是一致的。     

非诱变剂对姐妹染色单体交换的诱导效应

检测了16种非诱变剂对大麦根端分生细胞姐妹染色单体交换(SCE)的影响及其中9种成份对人外周血淋巴细胞SCE的影响。在大麦细胞中,用高浓度的葡萄糖、氯化钠、维生素、氨基酸及脱氧核糖核苷三磷酸(dNTP)处理均能显著提高SCE频率。维生素、氨基酸和dNTP对人外周血淋巴细胞中的SCE频率也有显著影响。实验结果表明,不只是诱变剂能提高SCE频率,非诱变剂在一定高剂量时也能提高SCE频率。非诱变剂影响SCE具有特殊的S CE增长曲线,SCE频率达一定值后不再随处理浓度的增大而提高。     

毒性弥漫性甲状腺肿患者外周血淋巴细胞SCE须率的研究

姊妹染色单体交换（SCE）系一条染色体中 两条姊妹染色单体之间同源节段的互换。SCE 可以自发产生,各物种细胞中自发SCE频率 相对比较稳定。但若用诱变剂或致癌剂处理细 胞,则SCE频率可显著提高u1。已经证实,一 些物质的诱变、致癌力与诱发SCE能力之间 存在很高的相关性[=,330目前,国内SCE技术 已用于多种遗传病、肿瘤和病毒性传染病的病 因学研究,以及化学“三致即物质作用机理的研 究［[3,A)。对毒性弥漫性甲状腺肿患者细胞SCE 频率尚未见报道。为了从细胞及亚细胞水平探 索本症的病因．,作者对20例毒性弥漫性甲状腺 肿患者外周血淋巴细胞SCE频率和20例正 常人细胞SCE频率进行了分析研究,现报告 如下。     

吸烟与姐妹染色单体交换

用荧光加Giemsa(FPG)术区分姐妹染色单体,分析了13名中年男子外周血淋巴细胞的SCE,比较吸烟者和不吸烟者“自发”的和用丝裂霉素(MMC)诱发的SCE频率变化。发现吸烟者的SCE频率(8.33±1.08)显著地高于不吸烟者(4.41±0.72),p<0.001。用MMC诱发后吸烟者各剂量的SCE值也明显地高于不吸烟者(P<0.01),并随着MMC剂量的增加,两者之间的差值增大。由于SCE的形成同DNA损伤后的修复机制有关,SCE频率的不同反映交换产生过程中机体重组修复系统的差异。因此,吸烟者与不吸烟者SCE频率的差异,表明两者在DNA修复机制上存在着差异。吸烟很可能改变了机体DNA损伤后的修复能力。     

DNA修复酶系统的研究 Ⅱ.紫外线和丝裂霉素C诱发着色性干皮病外周血淋巴细胞的姐妹染色单体互换的观察

本文报告4例着色性干皮病病人和3例正常人外周血淋巴细胞经紫外线照射和/或丝裂霉素C处理诱发SCE的观察结果。经紫外线照射的3例着色性干皮病病人淋巴细胞的诱发SCE频率比未照射的自发SCE频率均有显著增高(P<0.01),而正常人的淋巴细胞经紫外线照射诱发的SCE频率与未照射的自发SCE频率之间无显著差异(P>0.05)。经丝裂霉素C处理的2例着色性干皮病病人和1例正常人淋巴细胞的诱发SCE频率比未处理的自发SCE频率均有明显增加,但病人的增加幅度显著大于正常人(P<0.01)。结果表明,这4例着色性干皮病病人都存在着DNA切除修复功能缺陷。此外,还发现分离淋巴细胞的自发SCE频率比微量全血培养的显著地高(P<0.01)。     

草鱼体内肾细胞姐妹染色单体分化及交换的初步研究

本文确立了一个以草鱼体内肾细胞姐妹染色单体交换频率为指标的检测环境诱变或致癌物质的短期试验系统。采用硫堇-UV-Giemsa染色法,分析了草鱼体内肾细胞的SCD-2(注射BrdU后第二个细胞周期的中期分裂相的SCD)频率和SCE频率。用500微克/克体重BrdU体内标记5天,草鱼肾细胞SCD-2频率为8.58±0.22%;SCE频率为3.05±2.523 SCE_5/细胞。以丝裂霉素C(Mitomycin C,MMC)作为阳性对照,分析了化合物亚硝基胍(N-methyl-N~1-nitro-N-nitrosoguanidine,MNNG)和农药叶蝉散(Mipc)诱发SCE的能力。这项工作在评价水质污染方面将起一定的作用。     

mmc诱导绒毛细胞和淋巴细胞SCE的对比研究

用不同剂量的mmc分别诱导孕早期孕妇绒毛细炮和淋巴细胞SCE,发现除16ng对比组外,二 类细胞相同处理组间的mmc诱发的SCE无显著性差异。同时发现mmc剂量与二类细胞SCE频 率所形成的二条回归直线的斜率无显著性差异。这些结果表明在一定毒性剂量范围内,二类细胞SCE 对mmc的反应敏感性可能是相似的。对油胞PRI的分析结果提示,细胞PRI的改变会对其SCE 频率产生影响。     

丝裂霉素C诱发中国仓鼠体外培养细胞姐妹染色单体互换的研究

本文对不同年龄中国仓鼠各种组织的体外培养细胞姐妹染色单体互换(SCE)频率进行了比较研究。实验结果表明,体外培养细胞的自发SCE频率与动物年龄无关,相同组织细胞经MMC处理后,老龄仓鼠SCE频率比幼龄仓鼠SCE频率明显低。心脏和皮肤细胸的SCE频率高于肺和尾的SCE频率。     

微核形成与细胞周期关系的初步研究 Ⅲ.丝裂霉素c诱发人淋巴细胞染色体畸变与不同周期阶段的微核形成

为了研究染色体畸变与微核形成的关系,本实验用不同浓度的丝裂霉素C(MMC,0.025—0.4μg/ml),处理人外周血淋巴细胞,观察中期染色体畸变与不同细胞周期形成的微核间的关系。获得如下主要结果:(1)MMC诱发的染色体畸变细胞率(ACF),未经培养的G_0期淋巴细胞的微核细胞率(NC-MNCF)以及培养的淋巴细胞的微核细胞率(C-MNCF),在一定剂量范围内均呈剂量依赖性增加,并可用幂回归方程描述;(2)微核形成与染色体畸变全然无关的NC-MNCF,和C-MNCF一样,与ACF呈良好的正相关;(3)用胞质分裂阻滞(CB)法,检测MMC诱发的CB-MNCF,较C-MNCF无显著提高,MNCF/ACF的比值较小,并随着MMC剂量增加从0.15左右降到0.03。所有上述结果表明,不能简单理解微核形成与染色体畸变间的关系,在分裂的细胞群体中,中期染色体畸变可能仅是微核形成的一种来源。     

人白血病细胞系（J6-1,J6-2和J6-3）的SCE及其对MMC敏感性的研究

染色体或染色单体断裂可作为研究DNA 损伤的一个细胞遗传学指标,但是在正常情况 下染色体和染色单体断裂的出现率并不高,而 姐妹染色单体互换（简称SCE）频率则较高,因 而后者是研究染色体稳定性的一个较好指标。 丝裂霉素（MMC）是一种强的致突变和 致癌剂^[2],同时又是强的SCE诱发剂。     

Rad51C-deficient CL-V4B cells exhibit normal levels of mitomycin C-induced SCEs but reduced levels of UVC-induced SCEs

The mechanisms of sister chromatid exchanges (SCEs) are not known. One hypothesis is that SCE is a manifestation of Rad51-dependent homologous recombination repair. In order to test this hypothesis, we have compared the frequencies of SCEs induced by mitomycin C (MMC) and 254nm ultraviolet radiation (UVC) in wt V79B and the Rad51C-deficient CL-V4B cells. SCEs were analysed in the first (M1) and second (M2) post-treatment mitoses. In M1 MMC induced the same frequencies of SCEs in CL-V4B and V79B cells, while the UVC-induced SCE frequencies were lower in CL-V4B than V79B cells. In CL-V4B cells, MMC-induced SCEs were higher in M2 than in M1, suggesting that interstrand cross-links (ICL) are either not removed completely or are transformed into another form of DNA damage that persists until the next cell cycle. We suggest that SCEs may represent a mechanism to bypass MMC-induced ICL without their removal.     

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.     

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.     

Mitomycin-C-induced sister-chromatid exchanges and cell-cycle kinetics in lymphocytes from patients with Klinefelter syndrome

The chromosomal sensitivity to mitomycin-C (MMC) and cell-cycle kinetics in cells from patients with Klinefelter syndrome, a sex chromosomal disorder giving a high risk of malignant tumor, were studied by techniques of sister-chromatid exchanges (SCEs). The frequencies of MMC-induced SCEs increased in proportion to the increase in MMC concentration in both patient and normal control cells. At low levels of MMC there were no significant differences in SCE frequencies between the patient and normal control cells, but at MMC concentrations of 3 X 10(-8) M (p less than 0.05) and 1 X 10(-7) M (p less than 0.01), significant increases in the frequency of MMC-induced SCEs were observed in cells from patients compared to cells from normal controls. Although the analysis of cell-cycle kinetics both after various culture times and after treatment with MMC revealed that there were no significant differences between the patient and normal control cells, patients with Klinefelter syndrome showed a tendency to cell-cycle delays after treatment with MMC in comparison with normal controls.     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

Effects of temperature on chemically induced sister-chromatid exchange in human lymphocytes

Lymphocytes from healthy adults were studied for sister-chromatid exchanges (SCEs) when pulse-treated in G0 with mitomycin C (MMC), ethyl methanesulfonate (EMS), or 4-nitroquinoline N-oxide (4NQO) at various temperatures ranging from 0 degrees C to 41 degrees C and then cultured in medium containing 5-bromodeoxyuridine at 37 degrees C. The results showed that the frequencies of SCEs induced by MMC or EMS varied according to the treatment temperature. In MMC- or EMS-exposed cultures, the SCE frequency increased continuously with increasing treatment temperature; treatment at 37 degrees C resulted in a 3-4 times greater induction of SCEs than did that at room temperature (25 degrees C). On the other hand, SCE frequencies in cells exposed to 4NQO remained within normal deviation, showing no temperature-dependent changes. Baseline SCE frequencies remained almost constant within the temperature range tested. These data indicate that treatment temperature is a very critical factor in determining the sensitivity of cells to the chemical induction of SCEs.     

Sister-chromatid exchanges induced by triethylenemelamine: in vivo and in vivo/in vitro studies in mouse and Chinese hamster bone marrow and spleen cells

This study was designed to obtain sister-chromatid exchange (SCE) frequencies in bone marrow and spleen cells of mice and Chinese hamsters under in vivo and in vivo/in vitro systems following treatment of animals with varying doses (15-405 micrograms/kg) of triethylenemelamine (TEM). A dose-related SCE response was found in both species, tissues, and systems analyzed following TEM treatment. In vivo, similar responses were noted for both tissues in both species. However, in vivo/in vitro, the response was lower than in vivo and it varied with the tissue. The spleen cells were more sensitive and gave higher numbers of SCEs than bone marrow of both species at the two highest doses tested (135 and 405 micrograms/kg). These differences may be attributed to cell-culturing effects, type of cells analyzed, species and tissue specificities, and pharmacokinetic properties of the chemical. This study lends support to recently established in vivo/in vitro cell culture methodologies employing mice and Chinese hamsters for comparative cytogenetic analysis.     

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. I. Five different genes participate in the formation of baseline sister-chromatid exchanges and spontaneous chromosomal aberrations

In a search for cell mutants that show an increase or a decrease in the frequency of baseline sister-chromatid exchanges (SCEs) or spontaneous chromosomal aberrations (CAs), large numbers of mutagen-sensitive clones previously isolated from mouse lymphoma L5178Y cells were analyzed. In addition to two SCE mutants (ES 4 and AC 12) previously reported, three other mutants were identified as an SCE mutant. An ethyl methanesulfonate-sensitive mutant ES 2 and an alkylating agent-sensitive mutant MS 1 exhibited, respectively, 1.4-fold and 1.8-fold higher baseline SCE frequencies than did the parental L5178Y. In contrast, M10, which is sensitive to X-ray and 4-nitroquinoline 1-oxide, showed a reduced frequency of baseline SCEs (0.65-fold). These 5 mutants including ES 4 and AC 12 had 3--9-fold increases in spontaneous CA frequencies. Measurement of baseline SCE formation in inter-mutant hybrids revealed that M10 mutation is dominant, MS 1 and ES 4 mutations are semidominant, and ES 2 and AC 12 mutations are recessive. Because SCE frequencies in hybrids formed between pairs of 4 mutants (ES 2, MS 1, ES 4 and AC 12) were significantly lower than those in the tetraploid mutant cells, these 4 mutants probably belong to different complementation groups. Since M10 behaved dominantly with respect to SCE phenotype, it was not possible to determine by complementation test whether it belongs to a different group from the other mutants. However, the finding that M10 is complemented by other mutants for EMS sensitivity indicates that the M10 mutation is different from the other mutations. From these results, it is concluded that at least 4 different genes participate in the formation of high levels of baseline SCEs. The defects in ES 2, MS 1, ES 4, and AC 12 produce common lesions responsible for the formation of both SCEs and CAs. In contrast, the defect in M10 is associated with a high increase in spontaneous CA frequency, but conversely associated with a decrease in baseline SCE frequency. This suggests that M10 is defective in the process involved in the formation of baseline SCEs.     

Influence of low doses of BrdU and estimation of spontaneous SCE in CHO chromosomes: three-way differential staining and an immunoperoxidase method

The influence of low doses of 5-bromodeoxyuridine (BrdU) on the occurrence of sister chromatid exchanges (SCEs) during the first cell cycle, when unsubstituted DNA templates replicate in the presence of the halogenated nucleoside (SCE1) has been assessed in third mitosis (M3) Chinese hamster ovary (CHO) cells showing three-way differential (TWD) staining. In addition, lower concentrations of BrdU, not detectable by Giemsa staining, have been tested by a high resolution immunoperoxidase method (anti-BrdU monoclonal antibody) and SCEs were scored in second mitosis (M2) cells. Our findings was a dose-response curve for SCE1 that allows an estimated mean spontaneous yield of 1.32/cell per cell cycle by extrapolation to zero concentration of BrdU. On the other hand, when the total SCE frequency corresponding to the first and second rounds of replication (SCE1+SCE2) found in M3 chromosomes was compared with the yield of SCEs scored in M2 cells grown in BrdU at doses lower than 1 M no further reduction was achieved. This seems to indicate that SCEs can occur spontaneously in this cell line, though the estimated frequency is higher than that reported in vivo.by S. Wolff