Mitomycin C-induced damage and repair in human and pig lymphocytes

Human and pig lymphocytes were used to compare the chromosomal sensitivity to MMC and the efficiency of repair of MMC-induced DNA adducts. No significant interspecies differences were found. The results obtained show that SCE frequencies are linearly correlated with MMC doses. During the G0 period there are indications that lymphocytes may half-repair the DNA-interstrand crosslinks transforming bi- into mono-adducts. SCEs induced by MMC decrease to near control levels in the second cell cycle. Therefore, most MMC lesions responsible for SCEs should be repaired between the moment in the first S phase in which they induce the exchanges and the onset of the second S period.     

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.     

Endogenous sex hormones affect the mutagen-induced chromosome damage by altering a caffeine-sensitive checkpoint

In the present study we analysed the effect of endogenous sex hormones on the SCE frequencies induced in vitro by mitomycin C (MMC), a bifunctional alkylating agent producing high chromosome damage and mitotic arrest. The analysis has been performed on lymphocytes obtained at three different phases of menstrual cycle, from women with regular cycle and hormones dosage. At all phases we further analysed the effect of a post-treatment with caffeine, an agent that it is known to overrride the DNA damage checkpoints.

After MMC, the cultures obtained at ovulation and luteal phases have SCE frequencies statistically higher than the cultures obtained at the progestogenic phase, showing increases of 15 and 25%, respectively. After caffeine, the MMC treated cultures which were set up at the progestogenic phase show a high potentiation of SCE frequencies (28%) whereas the treated cultures set up at ovulatory and luteal phases show little or no potentiation.

These findings demonstrate that the endogenous hormones greatly modulate the SCE frequencies induced by the mutagen; they also indicate that hormones action competes with the caffeine effect. Caffeine acts by abrogating the mitotic arrest produced by DNA damage and induced cells with a higher chromosome damage into a premature mitosis. Our findings suggest that endogenous hormones could overcome the checkpoint controls activated in cells after mutagenic exposure. This action may be an epigenetic mechanism relevant in hormone carcinogenesis.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Proliferation-dependent reduction of sister-chromatid exchange frequency induced by mitomycin C in human lymphoblastoid cells and its suppression by inhibitors of DNA replication

A high frequency of sister-chromatid exchange (SCE) induced in cells of a human lymphoblastoid cell line, NL3, by 2-h treatment with 1 microM mitomycin C (MMC) was maintained after holding the treated cells in a nonproliferating state for 48 h before cells were transferred into the BrdUrd-containing medium for SCE assay. The same was observed in cells treated with 4-nitroquinoline 1-oxide (4NQO) or ethyl methanesulfonate (EMS). In contrast, when MMC-treated cells were transferred into a growth medium and allowed to proliferate for various periods of time before SCE assay, MMC-induced SCE frequency decreased with time and reached near control level after 48 h. The reduction in SCE was also observed in 4NQO-treated cells, though to a lesser extent, but not in EMS-treated cells. When hydroxyurea or 1-beta-D-arabinofuranosylcytosine was given as a post-MMC treatment during this recovery process, such a reduction of SCE frequency was suppressed and the extent of the suppression appears to be roughly parallel to their ability to inhibit DNA replication. Cycloheximide and 5-azacytidine also exerted a similar inhibitory effect on the reduction of SCE. Benzamide and caffeine had no appreciable effect. Our results indicate that the SCE-forming lesions induced by MMC can be eliminated only in proliferating cells, probably during DNA replication.     

Mutagenicity, cytotoxicity and DNA crosslinking in V79 Chinese hamster cells treated with cis- and trans-Pt(II) diamminedichloride.

The mutagenicity and cytotoxicity of cis- and trans-Pt(II) diamminedichloride (PDD) were examined in V79 Chinese hamster lung cells and compared with effects on DNA measured by alkaline elution. DNA--protein crosslinks and DNA interstrand crosslinks were detected following doses of cis-PDD which reduced cell survival 80--90% and which produced a mutant frequency of 3 X 10(-4) at the HGPRT locus. Equitoxic doses of trans-PDD were much less mutagenic than cis-PDD. At equitoxic doses, trans-PDD produced more DNA-protein crosslinking than did cis-PDD, but interstrand crosslinking for the two isomers was comparable. Hence, the interstrand crosslink could be the cytotoxic lesion produced by these Pt compounds whereas neither of these DNA lesions are necessarily mutagenic. The mutagenesis produced by cis-PDD could be due to crosslinks of a different type than those produced by trans-PDD or it may be due to monofunctional damage.     

SCE evaluations in human lymphocytes after G0 exposure to mitomycin C Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions

We conducted a series of experiments designed to determine whether DNA damage induced in G₀ lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3–4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G₀ lymphocytes are probably expressed as SCEs during the first period of mitogeninduced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.     

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.     

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O6-guanine and to form interstrand cross-links (ISC) has been studied using Mer+ and Mer- human embryonic cells. Mer+ (IMR-90) cells have been reported to contain an O6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer- (VA-13) cell line, which is deficient in the repair of O6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.     

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.     

Differential response to mitomycin-C- and cis-diamminedichloroplatinum(II)-induced damage in normal human fibroblasts during confluent holding

Confluent cultures of normal human fibroblasts were treated with the chemotherapeutic agents, mitomycin C (MMC) and cis-diamminedichloroplatinum(II) (cis-Pt(II]. This treatment induced a decrease in clonogenic cell survival. Recovery of the cytotoxic effect was observed in the case of cis-Pt(II)-treated cultures maintained at confluence for 1-5 days. No such recovery was observed after treatment with MMC. These data suggest that contrary to potential lethal damage induced by cis-Pt(II) which is repaired in confluent cells, DNA damage induced by MMC is not repaired in confluent cells.     

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.     

Reduced frequencies of Mitomycin-C induced sister chromatid exchanges in AKR Mice

Summary The frequencies of base-line and Mitomycin-C (MMC) induced sister chromatid exchanges (SCE) were surveyed in four inbred strains of mice. In contrast to the C57B1/6J, CBA/J, and A/J strains where frequencies of SCE increased linearly with increasing dose of MMC, levels of SCE were significantly lower in AKR/J mice at high MMC concentrations. At a dose of 5 mg/kg MMC, chromosomal aberrations were more frequent in bone marrow cells of AKR/J mice than in C57B1/6J mice. These observations suggest an altered response to DNA damage in the AKR mouse strain.     

黄鳝染色体体内SCE检测系统的建立

应用IdU-毛玉米油体内SCE技术,以不同剂量的典型诱变剂MMC和CP对70尾黄鳝的脾、肾、血淋巴细胞进行了体内诱发SCE敏感性测试。结果:三种细胞的染色体SCE自发频率均较低,不同剂量MMC和CP诱发黄鳝三种细胞SCE频率均较对照组显著增加。诱变剂剂量与诱发SCE频率呈线性关系。三种细胞染色体SCE对MMC和CP的敏感性次序为肾>脾>血淋巴细胞。与几种鱼和其它动物比较,黄鳝三种细胞的SCE自发频率均较低,对MMC和CP诱发SCE的敏感性均较高,因此认为黄鳝可作为较理想的体内SCE检测系统。     

Effects of repair inhibition in the G1 phase of clastogen-treated human lymphocytes on the frequencies of chromosome-type and chromatid-type aberrations and sister-chromatid exchanges

It has been shown that certain types of DNA lesions induced by an S-dependent clastogen are converted to chromosome-type aberrations when their repair is inhibited in the G1 phase of the cell cycle. The purpose of the present study was to investigate which kinds of repair inhibitors have the ability to induce chromosome-type aberrations in cells having DNA lesions and which kinds of DNA lesions will be converted to chromosome-type aberrations when their repair is inhibited. For this purpose, human peripheral blood lymphocytes, which were treated with a clastogen in their G0 phase, were post-treated with one of several kinds of repair inhibitors in the G1 phase, and resulting frequencies of both chromosome-type and chromatid-type aberrations as well as of sister-chromatid exchanges (SCEs) were compared with those of the control cultures: chromatid-type aberrations and SCEs were adopted as cytogenetic indicators of lesions remaining in S and G2 phases. Chemicals used for the induction of DNA lesions were 4-nitroquinoline 1-oxide (4NQO), methyl methanesulfonate (MMS) and mitomycin C (MMC); inhibitors used were excess thymidine (dThd), caffeine, hydroxyurea (HU), 5-fluoro-2'-deoxyuridine (FdUrd), 1-beta-D-arabinofuranosylcytosine (ara C), 9-beta-D-arabinofuranosyladenine (ara A), 1-beta-D-arabinofuranosylthymine (ara T) and aphidicolin (APC). Induction of chromosome-type aberrations was observed in cells pretreated with 4NQO or MMS followed by ara C, ara A, ara T or APC, whereas other combinations of a clastogen and an inhibitor did not induce them. Among the inhibitors, ara C alone induced chromosome-type aberrations in cells without pretreatment. Chromatid-type aberrations were increased only in cells pretreated with MMC and their frequency was enhanced further by post-treatment with ara C. All of the clastogens used in the present experiments induced SCEs. Most inhibitors did not modify the SCE frequencies except for ara C which synergistically increased the frequency in MMC-treated cells. The present study offers further evidence that the lesions responsible for chromosome-type aberrations are those which are repaired quickly, and that they are converted to chromosome-type aberrations when repair by polymerase alpha is inhibited. The effects of ara C on MMC-induced lesions are considered residual effects of ara C treatment in the S or G2 phases rather than repair inhibition in the G1 phase.     

Recombination repair pathway in the maintenance of chromosomal integrity against DNA interstrand crosslinks

DNA interstrand crosslinks (ICL) present a major threat to cell viability and genome integrity. In eukaryotic cells, the ICLs have been suggested to be repaired by a complex process involving Xpf/Ercc1-mediated endonucleolytic incision and homologous recombination (HR). However, the entire feature of the ICL tolerating mechanism is still poorly understood. Here we studied chromosome aberrations (CA) and sister chromatid exchanges (SCE) by the use of the crosslinking agent mitomycin C (MMC), in chicken DT40 cells with the HR genes disrupted by targeted replacement. The disruption of the Rad54, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3 genes resulted in a dramatic reduction of spontaneous and MMC-induced SCEs. Interestingly, while HR-deficient cells were hypersensitive to cell killing by MMC, MMC-induced CAs were also suppressed in the HR-deficient cells except for Rad51D-, Xrcc2- and Xrcc3-deficient cells. These observations indicate that DNA double strand breaks (DSB) at stalled replication forks and those arising as repair intermediates present strong signals to cell death but can be tolerated by the HR repair pathway, where Rad54, Rad51B and Rad51C have an initiative role and repair can be completed by their paralogs Rad51D, Xrcc2 and Xrcc3. The impairment of the HR pathway, which otherwise leads to cell death, may be somewhat substituted by an alternative mechanism such as the Mre11/Rad50/Nbs1 pathway, resulting in reduced frequencies of SCEs and CAs.     

Low efficiency of repair of critical DNA damage induced by low doses of radiation

This study provides an analysis of the development of cellular response to the critical DNA damage and the mechanisms for limiting the efficiency of repairing such damages induced by low doses of ionizing radiation exposure. Based on the data of many studies, one can conclude that the majority of damages occurring in the DNA of the cells after exposure to ionizing radiation significantly differ in their chemical nature from the endogenous ones. The most important characteristic of radiation-induced DNA damages is their complexity and clustering. Double strand breaks, interstrand crosslinks or destruction of the replication fork and formation of long single-stranded gaps in DNA are considered to be critical damages for the fate of cells. The occurrence of such lesions in DNA may be a key event in the etiology and the therapy of cancer. The appearance in the cells of the critical DNA damage induces a rapid development of a complex and ramified network of molecular and biochemical reactions which are called the cellular response to DNA damage. Induction of the cellular response to DNA damage involves the activation of the systems of cell cycle checkpoints, DNA repair, changes in the expression of many genes, reconstruction of the chromatin or apoptosis. However, the efficiency of repair of the complex DNA damage in cells after exposure to low doses of radiation remains at low levels. The development of the cell response to DNA damages after exposure to low doses of radiation does not reach the desired result due to a small amount of damage, with the progression of the phase cell cycle being ahead of the processes of DNA repair. This is primarily due to the failure of signalization to activate the checkpoint of the cell cycle for its arrest in the case of a small number of critical DNA lesions. In the absence of the arrest of the phase cell cycle progression, especially during the G2/M transition, the reparation mechanisms fail to completely restore DNA, and cells pass into mitosis with a damaged DNA. It is assumed that another reason for the low efficiency of DNA repair in the cells after exposure to low doses of radiation is the existence of a restricted access for the repair system components to the complex damages at the DNA sites of highly compacted chromatin.     

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.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on induction of sister-chromatid exchanges in Chinese hamster V79 cells treated with mutagens

The effect of a tumor promoter, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) alone and in combination with mitomycin C (MMC) or cyclophosphamide (CPP) on the induction of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells was investigated. TPA alone at various doses and durations caused no increase of SCE frequency. MMC either at the dose of 0.03 or 0.003 μg/ml alone or in combination with TPA (2 μg/ml) all caused a significant increase of SCE frequencies. There was no difference in SCE frequencies between the cultures treated with MMC alone at 0.03 μg/ml and those treated with MMC plus TPA. However, cultures treated with MMC at 0.003 μg/ml plus TPA had significantly and consistently higher SCE frequencies than those treated with MMC alone at all durations. Treatment of CPP at 1 μg/ml activated by S9 mix caused significant increase of SCE frequencies. Surprisingly, the cultures treated with CPP, S9 mix plus TPA (2 μg/ml) caused a drastic reduction of SCE frequencies as compared to those treated with CPP and S9 mix only at all durations. These results indicate that TPA alone had no effect on SCE in V79 cells. TPA enhanced the SCE induction in V79 cells treated with MMC at a low dose, i.e. 0.003 μg/ml, but it inhibited SCE induction in cultures treated with the indirect mutagen CPP. Thus, TPA has no direct effect on genetic materials but it may indirectly alter the effects of a mutagen.