[Show abstract][Hide abstract] ABSTRACT: Previously we found that Rad54/Rad54B cells are more sensitive towards mitomycin C (MMC) as compared to wild-type (WT) cells. This difference in sensitivity was absent upon exposure to other clastogens like bleomycin (BLM) and gamma-radiation. In order to get further insight into possible underlying mechanisms, gene expression changes in WT and Rad54/Rad54B MEFs (mouse embryonic fibroblasts) after exposure to the clastogens MMC and BLM were investigated. Exposures of these cells to mutagens (N-ac-AAF and ENU) and vehicle were taken as controls.
Most exposures resulted in an induction of DNA damage signaling and apoptosis genes and a reduced expression of cell division genes in cells of both genotypes. As expected, responses to N-ac-AAF were very similar in both genotypes. ENU exposure did not lead to significant gene expression changes in cells of both genotypes, presumably due to its short half-life. Gene expression responses to clastogens, however, showed a genotype-dependent effect for BLM and MMC. MMC treated Rad54/Rad54B MEFs showed no induction of p53-signaling, DNA damage response and apoptosis as seen for all the other treatments.
These data support our finding that different types of clastogens exist and that responses to these types depend on the DNA repair status of the cells.
[Show abstract][Hide abstract] ABSTRACT: The clastogenic effects of MMC and BLM and the mutagenic effects of B[a]P, N-ac-AAF and ENU were studied in mouse embryonic fibroblasts derived from wild-type (WT) and Rad54/Rad54B-deficient mice. Clastogens as well as mutagens showed a statistically significant induction of mutations in the lacZ reporter gene both in a WT and Rad54/Rad54B-deficient genetic background. Rad54/Rad54B MEFs appeared equally sensitive to the clastogens compared to WT MEFs, except for MMC. The type of mutations induced by the different compounds was investigated further by hybridizing the mutant colonies with total mouse DNA. An obvious increased number of mouse DNA positive clones was observed after BLM and MMC exposure, indicating that after these treatments genome rearrangements/translocations had occurred. In this hybridization assay, Rad54/Rad54B MEFs did not show more rearrangements/translocations than WT MEFs. As expected, the mutagens used showed no increase in chromosomal rearrangements or transloctions in MEFs derived from both genotypes. These results show that WT MEFs carrying the lacZ reporter gene on a plasmid are capable to detect both clastogenic as well as mutagenic effects of compounds in vitro. Deletion of the Rad54 and Rad54B genes did not further enhance the sensitivity of MEFs towards clastogens.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 05/2009; 666(1-2):50-6. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The sensitivity of DNA-repair-deficient Rad54/Rad54B mice for clastogens was studied and compared to that of wild-type mice. LacZ mutant frequencies (MF) in Rad54/Rad54B mice, after treatment with mitomycin C (MMC), bleomycin (BLM) and gamma-irradiation, were compared to those of the wild-type mice following the same treatments. While none of the clastogens showed an induction of the lacZ MF in the wild-type mice, there was a significant increase of the lacZ MF in the bone marrow of the Rad54/Rad54B mice after treatment with BLM and gamma-irradiation and in the spleen after MMC treatment. As expected, the positive control ENU showed a significant increase in the lacZ MF in all tested organs in wild-type mice. Mutant colonies were hybridized with total mouse DNA in order to discriminate between small gene mutations and large DNA rearrangements and translocations (size-change mutations). The hybridization studies showed a significant increase in mouse DNA positive clones 4 days after treatment with MMC and BLM in the bone marrow of the wild-type mice, which is indicative for chromosomal rearrangements and translocations to occur. An even more pronounced increase was seen 28 days after treatment with the same compounds in the Rad54/Rad54B mice.
[Show abstract][Hide abstract] ABSTRACT: In the present paper the capacity of the pUR288 plasmid mouse model and the MutaMouse model to detect the clastogens bleomycin, m-AMSA, o-AMSA and camptothecin, was investigated. Ethylnitrosourea (ENU) served as a positive control, methylcellulose as a negative control. Only bleomycin induced a slight but significant increase in lacZ mutant frequency (MF) in bone marrow of pUR288 plasmid mice. Exposure to the other compounds did not result in an increase in the MF in bone marrow and liver in both mouse models. For the MutaMouse this result was expected, for the plasmid mouse an increase in MF after clastogen exposure was expected. The positive control ENU induced statistically significant increases in MF compared with the negative control in both models and in both tissues analyzed. Hybridisation of DNA of mutant colonies derived from plasmid mice with labelled total mouse DNA (Hybridisation Assay) demonstrated an increase in the percentage of colonies hybridised with total mouse DNA as compared with the negative control, which suggests that there was indeed a biological response associated with treatment. The latter results indicate that the plasmid mouse assay may still be a promising model for the detection of clastogens.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 05/2008; 652(2):151-7. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several studies suggest that MutaMouse is insensitive to clastogens, including the accompanying paper by Mahabir et al., which describes a study with bleomycin, camptothecin, m-AMSA (4'-(9-acridinylamino)-methanesulfon-m-anisidide) and its ortho-analogue, o-AMSA (4'-(9-acridinylamino)-methanesulfon-o-anisidide). Only camptothecin was clastogenic in MutaMouse and none of these four compounds induced mutations at the lacZ locus. However, to improve exposure, dose range-finding studies were performed in CD2F1 mice, the parental strain of MutaMouse. Male CD2F1 mice (n=3) were treated with bleomycin (25-100 mg/kg bw, p.o. and i.p.), camptothecin (1-10 mg/kg bw p.o.) and m-AMSA (10-50mg/kg bw p.o. and 1-5 mg/kg bw i.p.) for 5 days and blood was sampled on day 3 and/or day 6 for analysis by flow cytometry to determine % MN-RETs. Camptothecin (1 mg/kg bw, day 6) induced a 3.6-fold increase in % MN-RET (P<0.05) but was toxic at higher doses. All day-3 camptothecin samples were positive (P<0.05). Bleomycin was negative when administered p.o. but positive at all doses on both days when given i.p. (P<0.05) whereas m-AMSA was negative when given i.p. or orally. Based on these results, male MutaMouse mice (5 per group) were dosed daily with bleomycin (50 mg/kg bw) for 5 days or with camptothecin (5 mg/kg bw) for 2 days. Peripheral blood was sampled 24 h after the final dose in each group and tissues were sampled 37 days later. Both compounds induced significant increases in % MN-RET, but only bleomycin induced a significant increase in MF (6-fold in liver, 4.5-fold in kidney and 2-fold in lung) compared with the untreated control. These studies support the view that MutaMouse is insensitive to compounds where the genotoxic mechanism of action is predominantly clastogenesis, but demonstrates that the peripheral blood micronucleus test is a useful adjunct to the transgenic gene-mutation assay.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/2008; 652(2):145-50. · 3.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Genotoxic agents are a major threat to the integritiy of chromosomes and viability of cells, specially if the damage is not repaired, because it can lead to chromosome instability, cell cycle arrest, cell dysfunction, induction of apoptosis or carcinogenesis. For genotoxicity, two main endpoints are gene mutations and chromosome aberrations; the latter can either be structural (clastogenic) or numerical (aneugenic). There are several in vitro and in vivo genotoxicity tests available. However, none of these tests are capable to detect both endpoints of genotoxicity simultaneously. The pUR288 mouse model, carrying the lacZ transgene on a plasmid vector, used for this thesis, is in theory capable of detecting large deletions (>500 base pairs) in addition to small deletions and point mutations. Since it was not previously demonstrated that the pUR288 mouse model is indeed sensitive enough to detect clastogens, a second mouse model was used, the Rad54/Rad54B deficient mice. This model has a defect in the repair of chromosomal aberrations, caused by clastogens. Determination of the lacZ mutant frequency (lacZ MF) as well as hybridization studies showed that the Rad54/Rad54B deficient mice are only moderately more sensitive towards clastogens as compared to pUR288 mice. It was concluded that, although still a promising test, both in vivo models were not sensitive enough to detect the effect of exposure to clastogens. In contrast to the in vivo studies, the in vitro studies using mouse embryonic fibroblasts (MEFs) derived from pUR288 and Rad54/Rad54B mice, have shown a dose-dependent induction in the lacZ MF and the micronuclei frequency (for the MNT) after exposure to both mutagens as well as clastogens in both MEFs models. Moreover, MMC-treatment resulted in a higher lacZ MF in Rad54/Rad54B MEFs compared to pUR288 MEFs. This genotype effect after MMC-exposure, also shown in microarray studies, was due to an impaired DNA damage response in MMC-treated Rad54/Rad54B MEFs. This will lead to a weaker DNA repair response and therefore a larger percentage of the cells will carry a lacZ mutation, hence a higher lacZ MF in the Rad54/Rad54B MEFs compared to pUR288 MEFs. Since MMC induces predominantly DNA crosslinks, the obtained results suggests that the Rad54 and/or Rad54B genes are involved in DNA crosslink damage recognition and repair. As a conclusion, the lacZ MF assay using pUR288 MEFs is able to detect both mutagens as well as clastogens simultaneously. Using this in vitro test can be of great advantage, since this can lead to a reduction in the number of tests needed. The reduction in time and costs and the increase in the sensitivity make this in vitro lacZ assay a good alternative for existing tests for screening of genotoxic compounds. However, before this test can be used as a high-throughput screenings test, it needs to be validated with a wider range of genotoxic compounds, non-genotoxic compounds, non-carcinogens and most importantly also low potency genotoxic compounds. Then it can be assessed whether this assay is reproducible and whether the results obtained can replace existing genotoxicity assays