Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells. II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro.

To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.     

小鼠体内脾脏,骨髓及精原细胞姐妹染色单体交换的比较研究

本文对几种化学诱变剂诱发小鼠体内脾脏、骨髓和精原细胞的SCE进行了比较研究,同时分析了几类常见化合物在小鼠脾脏细胞中诱发SCE的活力。结果显示诱变剂在脾脏细胞中诱发SCE比骨髓和精原细胞敏感。几类化合物都能显著地诱发小鼠脾脏SCE的增加,与对照相比差异显著(P<0.05)或极显著(P<0.01),说明利用小鼠脾脏细胞检测环境诱变物是相当灵敏的。     

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

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

Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro

To increase the sensitivity of cytogenetic surveillance of exposure to mutagens in the peripheral lymphocyte assay, structural chromosome aberrations (CA) were studied after inhibition of DNA synthesis and DNA repair with hydroxyurea and caffeine in culture 3 h prior to harvesting. CA and sister-chromatid exchanges (SCE) from conventional cultures from the same subjects were used for comparison. Smoking was used as exposure parameter. Thirty-two smokers and 35 nonsmokers were studied. In the inhibited cultures a significantly higher number of aberrations was found in lymphocytes from smokers than nonsmokers: chromatid breaks (20.4 vs. 11.8, p = 0.0002), chromosome breaks (4.5 vs. 1.7, p = 0.0003), and the number of cells with aberrations (18.9 vs. 12.4, p = 0.0001), when 50 cells per subject were analyzed. In conventional cultures no increase in gaps, chromatid and chromosome breaks or number of cells with aberrations was found in smokers when 100 cells from each subject were studied. Smokers showed an increased number of SCE (6.8 vs. nonsmokers 5.9, p = 0.02). A significant positive linear correlation (r = 0.39, p = 0.01) was seen between SCE and the number of cells with chromatid breaks from inhibited cultures. The present results indicate that adding hydroxyurea and caffeine to lymphocyte cultures for the last 3 h prior to harvesting may enhance the detection of cytogenetic damage from previous in vivo exposure to mutagens.     

Sister-chromatid exchanges in a special form of xeroderma pigmentosum (form II)

The frequency of sister-chromatid exchanges (SCE) was studied in peripheral blood lymphocytes from a xeroderma pigmentosum (form II, XPII) patient. The cells were irradiated with UV or X-rays. In some experiments novobiocin (NB), inhibitor of topoisomerase II, or caffeine (CA), inhibitor of DNA repair were added to the cultures. The level of spontaneous SCE in the patient's lymphocytes was found to be significantly increased in comparison to that in the cells from normal donors. The inhibitors and UV-light caused a rise in the frequency of SCE in the cells taken from normal donors and except for NB, in the lymphocytes from the patient XPII. X-Rays did not increase SCE frequency in normal lymphocytes and lowered it in the patient's cells. SCE frequency rose when inhibitors of DNA replication and repair were used in combination with mutagens.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.     

Correlation of the sister chromatid exchange level with chromosome aberrations induced by chemical mutagens in vivo

The data on the dose dependencies of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations during exposure of mouse bone marrow cells in vivo to 5 alkylating substances are provided. The efficacy of SCE induction was found to be higher than that of chromosomal aberrations. It was established that SCE induced by chemical mutagens in vivo and in vitro are more sensitive and stable tests than chromosomal aberrations.     

Quantitative evaluation of the effectiveness of the formation of sister chromatid exchanges and chromosome aberrations as affected by chemical mutagens

Dose curves of five chemicals were studied to compare the efficiency of SCE and chromosomal aberration induction by different chemical mutagens. SCEs were found to increase linearly with the dose, whereas chromosomal aberrations--nonlinearly. Using regression coefficients obtained from the dose curves it was found that the efficiency of the studied chemical mutagens in induction of SCEs is 100-300 times as high as that in the induction of chromosomal aberrations.     

The use of sister-chromatid exchange in Chinese hamster primary lung cell cultures to measure genotoxicity

Primary cell cultures derived from Chinese hamster lung (CHL) were established, and their response for the induction of sister-chromatid exchange (SCE) by direct- and indirect-acting mutagens was characterized. An increase in SCE frequency was induced in CHL cells by 3-methylcholanthrene (MCA), benzo[a]pyrene (BaP), and 2-aminoanthracene (2AA). The SCE frequency increased slightly after exposure to cyclophosphamide, but did not respond to the hepatocarcinogen dimethylnitrosamine (DMN). A slight increase in SCE frequency by DMN was observed in the CHL system with use of Aroclor-1254-induced rat liver homogenate fraction (S9). This response to DMN in CHL cells was lower than that seen when CHO cells were the target in the presence of S9. At low (1) and high (20) passages, the CHL cells responded with a similar dose-related increase in SCE frequency to direct- (ethyl methanesulfonate, EMS) and indirect- (MCA) acting mutagens. This response indicates that even after prolonged culturing in vitro, the cells retained the ability to metabolically activate xenobiotic promutagens. The induction of SCE by MCA occurred at concentrations that also induced macromolecular binding. SCE induction was also examined in primary lung cell cultures from animals exposed by nose-only inhalation to MCA aerosol. A significant increase in SCE frequency above controls was observed in cells from animals after a single exposure to MCA. No detectable increase in SCE frequency was observed after repeated inhalation exposures. Because CHL cells are of lung origin and showed metabolic activity, the CHL system appears to be appropriate for study of the genotoxic potential of inhaled compounds.     

Lack of association between induction of dominant-lethal mutations and induction of heritable translocations with benzo[a]pyrene in postmeiotic germ cells of male mice

Benzo[a]pyrene was tested for induction of dominant-lethal mutations in germ cells of male mice. Clear-cut dominant-lethal effects were induced in middle and early spermatoza. In contrast to the dominant-lethal effects observed the study showed no detectable increase in hertiable translocations for these stages over the spontaneous level. Thus, the results provide another example of a chemical mutagen that is effective in inducing dominant-lethal mutations but relatively ineffective in inducing heritable translocations in male postmeiotic germ cells.     

Developmental and cytogenetic effects of caffeine on mouse blastocysts, alone or in combination with benzo(a)pyrene

Mouse blastocysts were treated with caffeine and/or benzo(a)pyrene (BP), and the effects on development and on induction of sister chromatid exchanges (SCEs) were examined. Caffeine interfered with blastocyst development in a dose-related manner. At 4 mM, the highest concentration tested, caffeine interfered with development of blastocysts to all four endpoints: hatching, trophoblast outgrowth, inner cell mass (ICM) growth, and two-layer (primary endoderm and ectoderm) differentiation of ICMs. At 2 mM, caffeine reduced the incidence of both ICM growth and differentiation but did not affect hatching or formation of trophoblast outgrowths. At 1 mM, caffeine interfered only with ICM differentiation. Cell proliferation was least sensitive to caffeine and was reduced at concentrations of greater than or equal to 2 mM. Induction of SCEs was most sensitive to caffeine exposure; an increase in SCE frequency was observed at 0.1 and 0.5 mM. When caffeine was added to cultures with BP (1 microM, a concentration that was not embryotoxic and did not induce SCEs), both embryotoxic effects and SCE frequency were increased. The enhancing effect on SCE induction was particularly marked; as little as 0.1 mM caffeine was sufficient to cause doubling of induced SCE frequencies when added to cultures with BP.     

In vivo sister-chromatid exchange: A sensitive measure of DNA damage

A variety of chemical agents and X-irradiation were examined for their abilities to induced sister-chromatid exchanges (SCE) in vivo. In addition to demonstrating the several known mutagens and carcinogens are capable of inducing SCE in vivo, our studies indicate that the suspected carcinogen, tris-bromophosphate, can significantly elevate SCE levels. Comparison of the effects of these agents on SCE levels, chromosomal-aberration frequencies and cell-replication kinetics reveals that no consistent relationship exists between SCE levels and other indicators of cellular DNA damage.

It is proposed that analysis of SCE induction in vivo may provide a useful technique for the screening of mutagenic and carcinogenic compounds.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Effect of caffeine on intrachromosomal distribution of chromatid aberrations induced by chemical mutagens in Crepis capillaris L

The intrachromosomal distribution patterns of chromatid aberrations induced by N-methyl-N-nitrosourethane (MNU), N-ethyl-N-nitrosourethane (ENU) and ethyleneimine (EI) were compared with those induced by combined treatment with the same mutagens and caffeine, the latter being considered as an inhibitor of post-replication repair of DNA.Chromatid aberrations induced by mutagens alone were distributed non-randomly along the chromosomes. In certain regions few aberrations were located; in others pronounced clustering of aberrations was observed and these regions were considered to be hot spots. This refers especially to MNU- and EI-induced aberrations, whereas ENU-induced chromatid aberrations showed a more length-proportional distribution. In ENU experiments, certain chromosomal segments also represented hot spots, but these were less pronounced. The distribution patterns of chromatid aberrations induced by combined treatment with mutagens and caffeine differed significantly from those observed in experiments with the mutagens only. There seemed to be a tendency to approach random distribution here. This was a result both of the decrease in the quantity of the aberrations in the regions, which in the experiments with mutagens only were hot spots, and of its increase in other chromosomal regions. Some of these regions were considered as hot spots but they were less pronounced. These tendencies refer to MNU and EI. Certain differences between the two variants, with the without caffeine, in ENU experiments were observed but these were of lower expressivity.The causes od differential sensivity of chromosomal regions are discussed. The conclusion is drawn that clustering of chromatid aberrations in certain chromosomal regions is due to differences in the repair systems acting in heterochromatic and euchromatic regions.     

Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells

Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. In the present study, we conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1) Continuous post-UV treatment with 1 mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating or the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. (4) Neither BrdUrd in the medium nor the incorporated 5-bromodeoxyuridine monophosphate (BrdUMP) in DNA plays an appreciable role in the expression of the enhancing effect of caffeine. The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists until the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency.     

Caffeine induces sister-chromatid exchanges during the whole S-phase of the cell cycle

The capacity of caffeine to induce sister chromatid exchanges (SCEs) in different cell cycle stages and the proliferation kinetics were studied. Continuous treatment with this xanthine during the whole second cycle significantly increased the baseline SCE frequency. Pulse-treatment experiments showed that the induction of SCEs by caffeine, which was dose-dependent, was restricted to the S-phase of the cell cycle without effect on G1 or G2 cells. Moreover, unlike other SCE-inducing agents, such as DNA-synthesis inhibitors and DNA-damaging agents, caffeine produced similar SCE increases in cells treated at different times throughout the S-phase. In the light of Painter's model for SCE formation and the known effects of caffeine on the DNA replication pattern, the most likely mechanism of SCE induction by caffeine is an increase in the number of DNA-replication sites.     

Reduced N-methyl-N′-nitro-N-nitrosoguanidine sister chromatid exchange induction in chinese hamster V79 cells pre-exposed to 5-bromodeoxyuridine

The sequence in which N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and 5-bromodeoxyuridine (BrdU) are added to cell cultures affects the number of sister chromatid exchanges (SCE) induced by MNNG. When V79 Chinese hamster cell monolayer cultures were treated with MNNG for 2 h prior to addition of BrdUrd, approximately a 4–5-fold increase in SCE was observed at the second division metaphases compared to controls exposed to BrdU alone. This effect was independent of whether one or three DNA strands had been substituted as a result of incubating the cells through one or two DNA synthesis periods in the presence of BrdU. This increase in SCE also occurred after MNNG exposure and BrdU incubation was extended for three division cycles. In contrast, when BrdU incorporation preceded MNNG treatment, the average number of SCE/metaphase was reduced 70–80% at the second division cycle and 60% relative to the total number found in three division cycles. SCE induction by MNNG does not involve a caffeine sensitive step since caffeine had no effect on the SCE frequency regardless of the treatment protocol. The conditions in which BrdU preceded MNNG exposure may be responsible for either reducing the number of DNA sites available for interaction with MNNG or preventing the expression of SCE.     

Differences in sensitivity of murine spermatogonia and somatic cells in vivo to sister-chromatid exchange induction by nitrosoureas

Previously published data indicate that spermatogonia (SPG) are less sensitive to a sister-chromatid exchange (SCE) induction for different mutagens. In an earlier study, we have observed that bromodeoxyuridine (BrdU) substituted murine SPG are less sensitive to SCE induction by gamma ray in cells, than bone marrow (BM) and salivary gland (SG) cells in vivo. This was interpreted to mean that SPG are more efficient in DNA repair or are less prone to SCE induction. That the lower induction of SCE could be due to a reduced accessibility of mutagens to the SPG by virtue of a physiological barrier, was discarded by using gamma radiation. The aim of the present study was to establish whether or not there are differences in SCE induction by nitrosoureas among SPG, SG and BM cells with BrdU substituted or unsubstituted DNA. It was observed that SCE induction by methylnitrosourea (MNU) or by ethylnitrosourea (ENU) in SPG was, respectively, five and two times lower than in SG, and ten and three times lower than in BM. In SPG after BrdU incorporation, there was no increase in efficiency of SCE induction; in fact, there was even a slight decrease by exposure to MNU or ENU. BM and SG cells showed an increased efficiency in SCE induction after BrdU incorporation. This implies that SPG are also less sensitive to SCE induction by nitrosoureas, which cause a different kind of damage from previously assayed mutagens.     

Suppressive effects of coffee on the SOS responses induced by UV and chemical mutagens

SOS-inducing activity of UV or chemical mutagens (AF-2, 4NQO and MNNG) was strongly suppressed by instant coffee in Salmonella typhimurium TA1535/pSK1002. As decaffeinated instant coffee showed a similarly strong suppressive effect, it would seem that caffeine, a known inhibitor of SOS responses, is not responsible for the effect observed. The suppression was also shown by freshly brewed coffee extracts. However, the suppression was absent in green coffee-bean extracts. These results suggest that coffee contains some substance(s) which, apart from caffeine, suppresses SOS-inducing activity of UV or chemical mutagens and that the suppressive substance(s) are produced by roasting coffee beans.