M. Meijers

Molecular mechanisms involved in the production of cromosomal aberrations II. Utilization of neurospora endonuclease for the study of aberration production by X-rays in G1 and G2 stages of the cell cycle

Chinese hamster ovary cells (CHO) were X-irradiated in G1 and G2 stages of the cell cycle and subsequently Neurospora endonuclease (NE) (E.C.3.1.4), an enzyme which is specific in cleaving single-stranded DNA, was introduced into the cells, after making the cells permeable by treatment with inactivated Sendai virus. With this treatment all classes of X-ray-induced chromatid aberrations increased in G2 cells, whereas in G1 cells an increase in chromosome type of aberrations was found, associated with a profound induction of chromatid type of aberrations as well. Duration of the availability of single-strand gaps for the action of NE has been studied in G2 cells following X-irradiation and the influence of different parts of the G2 stage on the type and frequencies of chromatid aberrations was discerned. While the increase in chromosome type of aberrations by NE in X-irradiated G1 cells has been interpreted as due to the conversion of DNA single-strand breaks or gaps to double-strand breaks by NE, the induction of chromatid aberrations in G1 has been assumed to be due to conversion of some of the damaged bases into strand breaks by NE. Biochemical evidence is presented for the conversion by NE of DNA single-strand breaks induced by X-rays into double-strand breaks using neutral sucrose gradient centrifugation.

Ctrogenetic effects of mutagens/carcinogens after activation in a microsomal system in vitro I. Induction of chromosome aberrations and sister chromatid exchanges by diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) in CHO cells in the presence of rat-liver microsomes

A rat-liver microsomal system in vitro has been used to activate two indirectly acting carcinogens, DMN and DEN. On activation, both compounds were extremely potent in inducing chromosomal aberrations as well as sister chromatid exchanges in Chinese hamster cells. The implications of these findings and the potential utility of this technique to detect mutagens/carcinogens are discussed.

The nature and repair of DNA lesions that lead to chromosomal aberrations induced by ionizing radiations

Short treatment (up to 1 h) of cytosine arabinoside (araC) increases the frequencies of aberrations induced by X-rays in human lymphocytes, evaluated at the first mitosis following stimulation, or as prematurely condense chromosomes of G0 nuclei. Parallel biochemical experiments using nucleoid sedimentation technique, demonstrate that araC inhibits rejoining of DNA-strand breaks effectively. These results point out that X-ray-induced short-lived DNA strand breaks lead to chromosomal aberrations in human lymphocytes.

Mechanisms of induction of chromosomal aberrations and their detection by fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) technique using chromosome specific probes has revolutionized the field of radiation cytogenetics in the last few years. Some of the new insights on the origins of radiation induced chromosome aberrations in human, mouse and Chinese hamster, using FISH are reviewed in this paper.

Kinetics of the formation of chromosome aberrations in X-irradiated human lymphocytes, using PCC and FISH.

In order to study the initial frequencies and define kinetics of the formation of chromosomal exchanges in X-irradiated human lymphocytes, the premature chromosome condensation (PCC) technique was employed in combination with fluorescence in situ hybridization (FISH) with a composite probe for human chromosome 8 and a pan-centromeric probe for the whole genome. Human lymphocytes were X-irradiated (0.5, 1, 2, 3, 4 and 6 Gy), fused with mitotic Chinese hamster ovary (CHO) cells immediately or 1, 3, 6, 12 and 18 h after irradiation. Immediately after irradiation chromosomal breaks, dicentrics and translocations showed a linear dose-response. Unrejoined chromosome breaks were the most frequent types of aberrations (about 85%) observed. About 15% of total aberrations were chromosome exchanges of 65% of these were translocations and 35% were dicentrics. The chromosomal exchanges initially observed were mostly incomplete, with no complex exchanges at doses of 1 and 2 Gy, at higher doses (3-6 Gy) complex exchanges were observed and their frequencies increased with increasing post incubation time. Following different recovery times, repair kinetics of breaks for different doses of irradiation was studied. The shapes of the curves obtained for breaks as well as chromosome exchanges were linear-quadratic. The linear yield component, alpha, is formed entirely in the fast process that can be manifested in the early plateau, while component beta developed slowly in the subsequent hours. The kinetics of breaks rejoining was exponential, almost 50% of breaks rejoined after 1 h and at 18 h about 20% of breaks remained. At low doses of 1 and 2 Gy most of the exchanges were formed immediately and at higher doses, the frequency of exchanges increased with kinetics similar to that observed for the rejoining of breaks. However, the kinetics was different for different doses of irradiation. The frequency of dicentrics increased at doses above 2 Gy following 3 h recovery time, but for the translocations effect was pronounced even at 1 h recovery time. The frequency of incomplete exchanges (i.e., terminal translocations) decreased with post irradiation time and at 18 h was 30-40% less than the frequency obtained immediately after irradiation. The increase in the total translocations as a function of time between irradiation and fusion was due to a rapid increase in complete exchanges (i.e., reciprocal translocations). The frequency of ring chromosomes immediately after irradiation, also increased linearly, however, it was 3-5 times lower than dicentrics and remained almost constant in number for different doses and at different post-irradiation times.

Induction of sister-chromatid exchanges by restriction endonucleases

Restriction endonucleases Cfo 1, Pvu II, Sma I, Hpa II, Taq I and Hae III were tested for their ability to induce SCEs in CHO cells. The results indicate that the DNA double-strand breaks induced during S-phase by these enzymes lead to an increase in the frequencies of SCEs.

Ionizing radiation induced DNA lesions which lead to chromosomal aberrations

Several radiosensitive mutant cell lines of CHO and V79 cells have been studied to explore a possible correlation between radiation induced DNA lesions and chromosomal aberrations. In the xrs mutants which are deficient in DNA double strand break (DSB) repair, there is a correlation between the extent of the defect in repair and the frequencies of radiation induced chromosomal aberrations. In another type of radiosensitive mutant (V-C4), which has no detectable defect in DNA DSB repair, the frequencies of X-ray induced aberrations are high in comparison to wild type V79 cells. However, following treatment with restriction endonucleases or fission neutrons, the frequencies of aberrations are similar to those in V79, indicating that V-C4 cells are defective in repair of X-ray induced lesions other than DSBs. Though DSBs are the most important lesions leading to chromosomal aberrations, in repair deficient mutants, radiation induced lesions other than DSBs can lead to chromosomal aberrations.

Attempts to detect ataxia telangiectasia (AT) heterozygotes by cytogenetical techniques

AT cells are known to be more sensitive than normal human cells to ionizing radiation as well as other agents, as judged by cell survival, induced chromosomal aberrations, and micronuclei. The inhibition of DNA replication following X-irradiation is less in AT cells than in normal cells. Four different cell lines heterozygous for AT were investigated for their response to various treatments in order to see whether they behaved in an intermediate way. In most of the tests, the heterozygous AT cells behaved like normal cells. In the frequencies of induced chromosomal aberrations following X-rays, there was heterogeneity in response, with one cell line showing a definite intermediate response.

Induction of mitotic aneuploidy using Chinese hamster primary embryonic cells. Test results of 10 chemicals.

Using primary Chinese hamster embryonic cells, 10 known or suspected aneugens supplied as a part of the EC 4th Environmental Research and Development Programme were evaluated by the technique described by Dulout and Natarajan (1987). The chemicals included cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vincristine. All chemicals except pyrimethamine gave clearly positive effect at most of the doses tested. The ease with which the assay is performed and reproducible results that are obtained with the suspected compounds indicate that this in vitro test using primary embryonic fibroblasts is a promising one for routine screening.

A correlative study on the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice.: III. 1,3-Bis (2-chloroethyl)-3-nitrosourea (BCNU)

Cytogenetic damage induced by various concentrations of BCNU was evaluated by determining the frequencies of (a) micronuclei in polychromatic erythrocytes of bone marrow, (b) chromatid aberrations in bone marrow, (c) chromatid aberrations in spermatogonia, and (d) reciprocal translocations induced in spermatogonia and scored in spermatocytes. The order of sensitivity for the four parameters tested was: micronuclei greater than chromatid aberrations in bone marrow greater than aberrations in spermatogonia greater than translocations in spermatocytes, a situation similar to that found in an earlier study with CNU-ethanol. When the effect of concentration of the chemical was taken into consideration there was no correlation among the four parameters tested, so that information on induced frequencies of one parameter does not have predictive value for the frequencies of the others. A comparison of the results obtained with the bifunctional BCNU and the mono-functional CNU-ethanol at equal concentrations indicated that BCNU had a similar or a lesser clastogenic effect than did CNU-ethanol. In an experiment in vitro the situation was different in that BCNU was more effective than CNU-ethanol.