MMC-induced Chromosome Aberrations Research Articles

In Vitro Anticlastogenic and Antioxidant properties of extracted and Pure form of Curcumin against Mitomycin C in Human Peripheral Leukocytes

Objectives: Turmeric is native to India and is widely used in Indian cuisine. Curcumin, the active principle of turmeric used primarily as a coloring agent in food, drug and cosmetics. The medicinal properties of curcumin are well known in ancient Indian Ayurvedic Medicine. Mitomycin C (MMC) is an antineoplastic agent used to fight a number of different cancers including cancer of the stomach, colon, rectum, pancreas, breast, lung, uterus, cervix, bladder, head, neck, eye and esophagus. It is a potent DNA cross-linker. But it has severe side effect, the prolonged use of the drug may result in permanent bone marrow damage and other various types of secondary tumors in normal cells. The present study was conducted to examine the anticlastogenic action of extracted curcumin from dry turmeric and pure curcumin against the MMC-induced chromosomal aberrations. For this purpose, in the present study, clastogenic parameter like chromosomal aberration test was conducted in peripheral human leukocytes. Methods: The antioxidant property of both extracted and pure curcumin was also evaluated by phosphomolybdenum method. Results: Our results demonstrated that, extracted as well as pure Curcumin, a strong antioxidant phyto-molecule is effective in counteracting the clastogenicity in human leukocyte in vitro. Conclusion: These results suggest that the use of turmeric in diet may be an effective protection against the health crisis generated by harmful agents.

Genotoxic and antigenotoxic effects of luteolin against MMC-induced chromosomal aberrations in human lymphocytes in vitro

Genotoxic and antigenotoxic effects of luteolin against MMC-induced chromosomal aberrations in human lymphocytes in vitro

MODULATION OF THE GENOTOXIC AND CYTOTOXIC EFFECTS OF THE ANTICANCER DRUGS CYCLOPHOSPHAMIDE AND MITOMYCIN C BY N-ACETYLCYSTEINE

This study aimed to evaluate the possible protective role of the antioxidant N-acetylcysteine (NAC) against the genotoxic and cytotoxic effects of two widely-used anticancer drugs namely, cyclophosphamide (CP) and mitomycin C (MMC). Such evaluation has been monitored by using chromosomal aberrations analysis and micronucleus assay of bone marrow cells of male CD-1 mice. The results indicated that NAC treatment at three tested doses (100,150 and 200 mg/kg body weight) one hour prior to CP, significantly inhibited the incidence of CP-induced chromosomal aberrations and micronuclei in bone marrow cells of mice in a dose-dependent manner. This protective effect of NAC could be attributed to its nucleophilicity and antioxidant activity as well as its role as a precursor of reduced glutathione (GSH). NAC pretreatment also markedly restored the mitotic activity of bone marrow cells, which had been suppressed by CP. On the other hand, NAC pretreatment at the three tested doses potentiated MMC-induced chromosomal aberrations and micronuclei in a dose-dependent manner and resulted in suppression of mitotic activity of bone marrow cells. This can be explained by the further reduction of the initially-formed MMC semiquinone free radical by glutathione. In conclusion, supplementation with antioxidants as chemo-preventatives may not always provoke the desired protective effect; such effect may depend on the specificity of interaction between the used antioxidant and the anticancer agents.

Structural insights into the function of FANCM‐mediated complexes

FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FA‐BRCA pathway of DNA damage response and repair. FANCM is a highly conserved protein that belongs to the SF2 superfamily of helicases/translocases. Previously, we have reported the isolation and characterization of two novel histone‐fold‐containing FANCM‐associated proteins, MHF1 and MHF2. These proteins form a complex that is important for DNA damage‐induced monoubiquitination and foci formation of FANCD2, as well as chromatin localization of FA nuclear core complex proteins. Depletion of MHF complex resulted in elevated MMC‐induced chromosome aberrations, and sensitivity of cells to MMC and camptothecin. Biochemical characterization of the MHF complex showed that it assembles into a heterodimer that binds double stranded DNA and most importantly enhances the DNA branch migration activity of FANCM. We have carried out structural work to characterize the MHF complex by X‐ray crystallography. Based on the high resolution crystal structure of the MHF complex we have designed and tested mutants of MHF complex that are defective in DNA binding and FANCM interaction and showed that both protein‐protein interactions and DNA binding of MHF complex are needed for stimulation of FANCM activity.

Rad51C is essential for embryonic development and haploinsufficiency causes increased DNA damage sensitivity and genomic instability

Homologous recombination is essential for repair of DNA interstrand cross-links and double-strand breaks. The Rad51C protein is one of the five Rad51 paralogs in vertebrates implicated in homologous recombination. A previously described hamster cell mutant defective in Rad51C (CL-V4B) showed increased sensitivity to DNA damaging agents and displayed genomic instability. Here, we identified a splice donor mutation at position +5 of intron 5 of the Rad51C gene in this mutant, and generated mice harboring an analogous base pair alteration. Rad51C splice heterozygous animals are viable and do not display any phenotypic abnormalities, however homozygous Rad51C splice embryos die during early development (E8.5). Detailed analysis of two CL-V4B revertants, V4B-MR1 and V4B-MR2, that have reduced levels of full-length Rad51C transcript when compared to wild type hamster cells, showed increased sensitivity to mitomycin C (MMC) in clonogenic survival, suggesting haploinsufficiency of Rad51C. Similarly, mouse Rad51C splice/neo heterozygous ES cells also displayed increased MMC sensitivity. Moreover, in both hamster revertants, Rad51C haploinsufficiency gives rise to increased frequencies of spontaneous and MMC-induced chromosomal aberrations, impaired sister chromatid cohesion and reduced cloning efficiency. These results imply that adequate expression of Rad51C in mammalian cells is essential for maintaining genomic stability and sister chromatid cohesion to prevent malignant transformation.

MHF1-MHF2, a Histone-Fold-Containing Protein Complex, Participates in the Fanconi Anemia Pathway via FANCM

FANCM is a Fanconi anemia nuclear core complex protein required for the functional integrity of the FANC-BRCA pathway of DNA damage response and repair. Here we report the isolation and characterization of two histone-fold-containing FANCM-associated proteins, MHF1 and MHF2. We show that suppression of MHF1 expression results in (1) destabilization of FANCM and MHF2, (2) impairment of DNA damage-induced monoubiquitination and foci formation of FANCD2, (3) defective chromatin localization of FA nuclear core complex proteins, (4) elevated MMC-induced chromosome aberrations, and (5) sensitivity to MMC and camptothecin. We also provide biochemical evidence that MHF1 and MHF2 assemble into a heterodimer that binds DNA and enhances the DNA branch migration activity of FANCM. These findings reveal critical roles of the MHF1-MHF2 dimer in DNA damage repair and genome maintenance through FANCM.

Inhibition of mitomycin C-induced chromosomal aberrations by micrometer powder of selenium-enriched green tea in mice spermatocytes

The anticlastogenic effect of micrometer powder of selenium-enriched green tea (MSTP) was evaluated by using a chromosomal aberration assay in mouse testicular cells. Animals fed with a Se-deficient diet were treated with MSTP, micrometer powder of regular green tea (MRTP), selenite, and MRTP + selenite for 30 days by an intragastric route, followed by treatment of mitomycin C (MMC) on day 19 through intraperitoneal injection (ip). Selenium status and antioxidant enzymes were measured. Results indicated that MSTP showed a significant capability to reduce the incidence of MMC-induced chromosomal aberrations in spermatocytes from 22.7% to 6.7%. This inhibitory was highest, for MSTP, at 73.1%, while it was only 38.4% for MRTP. After 30 days of a Se-deficient diet, mice, either with or without the MMC treatment, showed a lower selenium concentration in blood and liver as well as lower enzyme activity of the antioxidants, GPx and SOD. Supplementation with MSTP, selenite, or selenite + MRTP enhanced the activities of these antioxidant enzymes. This enhancement was accompanied with a concomitant elevation of selenium levels, which favored the synthesis of the seleno-enzyme GPx and protected the cells from the MMC-induced oxidative stress. Our results indicate that MSTP is both able to prevent the chromosomal aberrations induced by MMC in mouse spermatocytes and to enhance GPx and SOD activity in blood serum and liver.

Cellular characterization of cells from the Fanconi anemia complementation group, FA-D1/BRCA2

Fanconi anemia (FA) is an inherited cancer-susceptibility disorder, characterized by genomic instability and hypersensitivity to DNA cross-linking agents. The discovery of biallelic BRCA2 mutations in the FA-D1 complementation group allows for the first time to study the characteristics of primary BRCA2-deficient human cells. FANCD1/ BRCA2-deficient fibroblasts appeared hypersensitive to mitomycin C (MMC), slightly sensitive to methyl methane sulfonate (MMS), and like cells derived from other FA complementation groups, not sensitive to X-ray irradiation. However, unlike other FA cells, FA-D1 cells were slightly sensitive to UV irradiation. Despite the observed lack of X-ray sensitivity in cell survival, significant radioresistant DNA synthesis (RDS) was observed in the BRCA2-deficient fibroblasts but also in the FANCA-deficient fibroblasts, suggesting an impaired S-phase checkpoint. FA-D1/BRCA2 cells displayed greatly enhanced levels of spontaneous as well as MMC-induced chromosomal aberrations (CA), similar to cells deficient in homologous recombination (HR) and non-D1 FA cells. In contrast to Brca2-deficient rodent cells, FA-D1/BRCA2 cells showed normal sister chromatid exchange (SCE) levels, both spontaneous as well as after MMC treatment. Hence, these data indicate that human cells with biallelic BRCA2 mutations display typical features of both FA- and HR-deficient cells, which suggests that FANCD1/BRCA2 is part of the integrated FA/BRCA DNA damage response pathway but also controls other functions outside the FA pathway.

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles. In two of these revertants, V-C8-Rev 1 and V-C8-Rev 6, the reversions lead to the wild-type Brca2 sequence. The V-C8 revertants did not gain the entire wild-type phenotype and still show a 2.5-fold increased sensitivity to mitomycin C (MMC), higher levels of spontaneous and MMC-induced chromosomal aberrations, as well as abnormal centrosomes when compared to wild-type cells. Our results suggest that Brca2 heterozygosity in hamster cells primarily gives rise to sensitivity to DNA cross-linking agents, especially chromosomal instability, a feature that might also be displayed in BRCA2 heterozygous mutation carriers.

Mammalian Rad51C contributes to DNA cross-link resistance, sister chromatid cohesion and genomic stability.

The eukaryotic Rad51 protein is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Five paralogs of Rad51 have been identified in vertebrates, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3, which are also implicated in recombination and genome stability. Here, we identify a mammalian cell mutant in Rad51C. We show that the Chinese hamster cell mutant, CL-V4B, has a defect in Rad51C. Sequencing of the hamster Rad51C cDNA revealed a 132 bp deletion corresponding to an alternatively spliced transcript with lack of exon 5. CL-V4B was hypersensitive to the interstrand cross-linking agents mitomycin C (MMC) and cisplatinum, the alkylating agent methyl methanesulfonate and the topoisomerase I inhibitor campthotecin and showed impaired Rad51 foci formation in response to DNA damage. The defect in Rad51C also resulted in an increase of spontaneous and MMC-induced chromosomal aberrations as well as a lack of induction of sister chromatid exchanges. However, centrosome formation was not affected. Intriguingly, a reduced level of sister chromatid cohesion was found in CL-V4B cells. These results reveal a role for Rad51C that is unique among the Rad51 paralogs.

Antimutagenic effects of centchroman--a contraceptive and a candidate drug for breast cancer in multiple mutational assays.

Centchroman (CC), a non-steroidal oral contraceptive and a candidate drug for breast cancer, has been reported to exhibit partial to complete remission of lesions in 40.5% of breast cancer patients. The potent anti-oestrogenic activity, negligible side-effects and anti-breast cancer activity of CC prompted us to evaluate the antimutagenic effects of this compound in a bacterial mutagenicity assay and CHO/HPRT and AS52/GPT mutation assays in vitro and in vivo in female Swiss albino mice as measured by both sister chromatid exchange (SCE) and chromosome aberrations (CA) against three known positive mutagen compounds, dimethylbenz[a]anthracene (DMBA), cyclophosphamide (CP) and mitomycin C (MMC). Antimutagenicity assays in Salmonella strains TA97a, TA100, TA98 and TA102 were carried out against commonly used known positive mutagens, sodium azide, 4-nitro-o-phenylenediamine, cumine hydroperoxide, 2-aminofluorene and danthron. A significantly reduced number of bacterial histidine revertant colonies was observed in the plates treated with 0.1, 1, 5 and 10 microg/plate CC and a positive compound when compared with bacterial plates treated with the respective positive compound alone. Ethyl methanesulfonate (EMS), a commonly used positive mutagen for CHO/HPRT and AS52/GPT gene mutation assays, was used for antimutagenicity assay in these cells. CC exhibited protective effects against the mutagenicity of EMS in these two mammalian cell mutation assays, CHO/HPRT and AS52/GPT. In the in vivo studies, pretreatment with CC reduced DMBA-induced SCE and CA and CP- and MMC-induced CA when compared with the group treated only with the positive compounds. These results indicate that CC can reduce the mutagenic effects of known genotoxic compounds.

Comparative studies on genotoxicity and antigenotoxicity of natural and synthetic β-carotene stereoisomers

To evaluate the practical value of natural β-carotene (NβC) and to elucidate the apparent discrepancy between epidemiological observations and intervention trials on the role of β-carotene (βC) in tumor prevention, the genotoxicity and the antigenotoxicity of NβC and synthetic βC crystal (SβCC) stereoisomers were studied comparatively using chromosome aberration analysis and the micronucleus test in human lymphocytes in vitro. NβC was extracted from the halotolerant algae Dunaliella salina. The NβC crystal (NβCC) preparation is about 70% all- trans (TβC) and 8% 9- cis (CβC). The NβC oil (NβCO) preparation is about 40% all- trans and 38% 9- cis. SβCC is more than 97% all- trans, and the 9- cis can not be detected. The mixture of βC (βCM) preparation is 74% SβCC and 26% NβC. Our results show no genotoxicity of 1–30 μg/ml NβCC, but this concentration of NβCC inhibited significantly γ-ray-induced micronucleus formation in human lymphocytes in vitro. One to thirty μg/ml NβCO was most effective against both γ-ray-induced and spontaneous micronucleus formation. However, no influence of NβCO on spontaneous chromosome aberrations in human lymphocytes in vitro was observed. NβCO suppressed significantly mitomycin C (MMC)-induced chromosome aberrations. One to thirty μg/ml SβCC induced a dose-dependent increase in micronucleus frequency, and also inhibited γ-ray-induced micronucleus formation. No effect of βCM on spontaneous chromosome aberrations was found. One to thirty μg/ml βCM is more effective against MMC-induced chromosome aberrations than NβCO. These results suggest that CβC might play a critical role in the genotoxicity and antigenotoxicity of SβCC and NβC. The genotoxic activity of SβCC might be involved in carcinogenesis. NβC or βCM could be of practical value in tumor prevention and supplementary treatment.

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were posttreated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (≦ 6.7 μg/ml) with S9 mix. At a high concentration of tea tannin components (20 μg/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G 1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.

Cocultivation of Fanconi anemia cells and of mouse lymphoma mutants leads to interspecies complementation of chromosomal hypersensitivity to DNA cross-linking agents

We have studied the effects of cocultivation on the frequency of mitomycin C (MMC)-induced chromosomal aberrations. This was carried out by cocultivating Fanconi anemia (FA) cells from the genetic complementation groups A and B with both normal mouse lymphoma L5178Y cells and the derived "FA-like" mutant cells, MCN-151 and MCE-50, assigned to complementation groups I and II, respectively. The results show a partial complementation of the defect (i.e. a reduction in the frequency of chromosomal aberration) in FA group A cells cocultured with normal or group II mouse cells, and a partial correction of mouse group I cells cocultived with normal or FA group B human cells. No reciprocal effects were observed between FA group A cells and mouse group I mutant cells; the frequencies of MMC-induced chromosomal aberrations in these cells remained unchanged by cocultivation. Moreover, no complementation was observed for both FA group B cells and mouse group II cells, after cocultivation with normal cells of either mouse or human origin. This implies that a diffusible factor released by normal human and mouse cells, and by FA group B and mouse group II mutant cells, can correct at least in part the chromosomal defect of FA group A and mouse group I mutant cells. With normal human or mouse cells, the frequency of chromosomal breakage after cocultivation remains the same as that observed in non-cocultived cells. This suggests that no detectable clastogenic factor is released by human FA or "FA-like" mouse cells.

Differential sensitivity of peripheral blood lymphocytes of untreated leprosy patients to mitomycin C

The effects of a bifunctional alkylating agent mitomycin C (MMC), an effective inducer of chromosome aberrations and sister-chromatid exchanges (SCEs), have been studied in untreated leprosy patients. This was done to study the mutagen sensitivity of the leprosy patients. The frequency of chromosomal aberrations induced by MMC (conc. 0.01 μg/ml) was 2.5% in controls, 3.6% in paucibacillary (PB), and 6.8% in multibacillary (MB) patients. The difference in the frequency of MMC-induced chromosome aberrations between the 3 groups studied was highly significant ( p < 0.01). Cultures grown with MMC showed the frequency of SCEs/cell to be 12.70 ± 1.19 in controls, 19.97 ± 3.51 in PB, and 29.66 ± 5.92 in MB patients. The differences in the frequency of MMC-induced SCEs between the 3 groups were found to be highly significant ( p < 0.01). The enhanced frequencies of spontaneous and MMC-induced chromosome aberrations and SCEs observed in PB and MB patients indicate a clear differential mutagen sensitivity between PB and MB patients who are known to have different immunological status and thereby differ in the severity of the disease.

Studies of gene transfer and reversion to mitomycin C resistance in Fanconi anemia cells

As a first step to the cloning of the Fanconi anemia (FA) gene, we have attempted to correct the sensitivity of FA cells to DNA crosslinking agents by the introduction of wild-type DNA. The protocol involved the introduction of both genomic and pRSV neo DNA, selection for G418-resistant colonies and the subsequent selection of mitomycin C-resistant cells from the latter. Preliminary experiments indicated that untransformed FA cells were not suitable recipients for the introduction of foreign DNA, so all experiments were performed with an SV40-transformed FA cell line. Approximately 40 000 G418-resistant colonies were obtained in 5 separate experiments at an overall frequency of about 5 × 10 −4. These were then selected in mitomycin C and 15 colonies were recovered. Colonies were obtained with wild-type DNA (both human and rodent) and with FA DNA at about the same frequency of 2× 10 −7. Colonies were isolated and shown to have a stable, partial (from 25 to 90% of wild-type) resistance to mitomycin C. One colony was also shown to be partially resistant to two other DNA crosslinking agents, diepoxybutane and nitrogen mustard. This clone also had an intermediate level of spontaneous and MMC-induced chromosome aberrations. pRSVneo, but not rodent, DNA could be demonstrated in the high molecular weight fraction of several colonies. Thus, it is likely that these colonies represent partial revertants rather than transfectants. These mitomycin C-resistant FA cells should be useful for the biochemical analysis of the FA mutation.