B.A. Kihlman

Use of the 5-bromodeoxyuridine-labelling technique for exploring mechanisms involved in the formation of chromosomal aberrations I. G2 experiments with root-tips of Vicia faba

Abstract Chromosomes in root-tip cells of Vicia faba were exposed in the G2 phase to long-wave (320–380 nm) ultraviolet radiation and to X-rays. Before exposure, 5-bromouracil had been substituted for thymine in various numbers of DNA strands in these chromosomes. The experiments involved cells with chromosomes of the following constitutions: TT—TT (both chromatids of the chromosome containing unsubstituted DNA), TT—TB (one chromatid with unsubstituted DNA and one with unifilarly substituted DNA), TB—TB (both chromatids containing unifilarly substituted DNA) and TB—BB (one chromatid with unifilarly and one with bifilarly substituted DNA). The sister chromatids in chromosomes of the TT—TB and TB—BB constitution could be distinguished by differential staining with the FPG technique. Long-wave UV induced no aberrations in chromosomes with unsubstituted DNA. In chromosomes with 5-bromouracil-substituted DNA, long-wave UV, like X-rays, induced sub-chromatid and chromatid aberrations. Because these aberrations were found in the first mitosis after exposure of G2 cells, the effect of long-wave UV was of the S-independent, ionizing type. The sensitivity to the production of aberrations by X-rays increased with increasing number of 5-bromouracil-substituted DNA strands, i.e. TT—TT The results of both the UV and the X-ray experiments clearly indicate that two lesions are required to produce one exchange, thus providing direct evidence in support of an indirect conclusion arrived at long ago on the basis of the dose—effect relationship for radiation-induced exchanges.

Use of the 5-bromodeoxyuridine-labelling technique for exploring mechanisms involved in the formation of chromosomal aberrations.

Synchronized G1 CHO cells with chromosomes of TB or TT constitution were irradiated with X-rays, short-wave UV and long-wave UV. The types and frequencies of chromosomal aberrations observed in the ensuing mitosis were studied. X-Rays induced predominantly chromosome types of aberration in chromosomes of TT constitution, whereas both chromosome- and chromatid-types of aberration were induced in cells with chromosomes of TB constitution. Short-wave UV induced only chromatid types of aberration in cells containing chromosomes of TT constitution, but both chromosome and chromatid types of aberration in cells with chromosomes of TB constitution. Long-wave UV induced chromosome and chromatid types of aberration in cells with chromosomes of TB constitution and no aberrations in cells containing chromosomes of TT constitution. Long-wave UV-irradiation of cells containing chromosomes of TB constitution increases the frequencies of SCEs. The relationship between chromosome constitution (TT or TB), the type of lesions induced by the 3 different agents employed, and the types of chromosomal aberration induced are discussed.

Effects of chromosome-breaking purine derivatives on nucleic acid synthesis and on the levels of adenosine 5′-triphosphate and deoxyadenosine 5′-triphosphate in bean root tips

Abstract The effects of 8-ethoxycaffeine (EOC), adenine, and deoxyadenosine (AdR) on nucleic acid synthesis and the levels of adenosine 5′-triphosphate (ATP) and deoxyadenosine 5′-triphosphate (dATP) in excised and intact bean root tips have been studied. As shown by biochemical and autoradiographic experiments all three purine derivatives produced a strong inhibition of the synthesis of DNA. RNA synthesis was strongly inhibited by EOC and adenine, but only very slightly by AdR. In the biochemical experiments, nucleic acid synthesis was determined by measuring the incorporation of 32 P-labelled phosphate or 14 C-labelled thymidine into the DNA and 32 P-labelled phosphate into the RNA fractions of the roots; in the autoradiographic experiments the incorporation of tritium-labelled thymidine into nuclear DNA was studied. Treatments with AdR resulted in an accumulation of dATP in the root tips. The level of dATP was not affected by adenine, but appeared to be slightly increased by EOC. About a 40% increase of the ATP level was obtained with EOC in the absence of inorganic phosphate. In the presence of a 6.67 mM phosphate buffer, the ATP level was reduced by EOC to one-third of its normal value. Treatment with adenine resulted in a doubling of the ATP level in the roots both in the presence and in the absence of phosphate. The results and their possible significance for the chromosome-breaking effect of the three purine derivatives are discussed.

Deoxyribonucleic acid synthesis and the production of chromosomal aberrations by streptonigrin, 8-ethoxycaffeine and 1,3,7,9-tetramethyluric acid

Abstract Autoradiographic experiments, in which Vicia faba roots were treated with [3H]thymidine in combination with streptonigrin (SN), 1,3,7,9-tetramethyluric acid (TMU) or 8-ethoxycaffeine (EOC), were performed in order to investigate the sensitivity of the various phases of the cell cycle to the production of chromosomal aberrations. It appeared in these studies that SN, TMU and EOC were all able to induce chromatid aberrations during the post-DNA-synthesis or G2 phase. During the period of DNA synthesis (the S phase), chromatid aberrations were produced by SN and EOC. Streptonigrin appeared to be at least as effective during S as during G2. The effect produced by EOC during the S phase was considerably weaker than that produced in G2 cells and consisted mainly of isolocus breaks localized in the nucleolar constriction (Fig. 2a). The results further indicate that SN and EOC are able to induce chromosome type aberrations in the G1 phase, i.e., the interphase period between telophase and S. TMU did not produce any aberrations in S and G1 cells. In experiments with excised Vicia roots, approx. 50% inhibition of the incorporation of 32P-labelled phosphate into DNA was obtained with 1.5·10−5 M SN and with 1.5·10−2 M TMU, the period of treatment being 3 h in both cases. The incorporation of 32P-labelled phosphate into RNA was also affected but to a lesser extent, the figures obtained being 27 and 37% for SN and TMU, respectively. In a previous study12, EOC was found to be even more effective as an inhibitor of DNA and RNA synthesis. The results and the mechanisms of action of SN, TMU and EOC are discussed. It is concluded that the chromosomal aberrations and the inhibition of nucleic acid synthesis are two independent affects of changes produced by the substances in chromosomal DNA.

Interphase sensitivity to the chromosome-breaking effect of 2′-deoxyadenosine: An autoradiographic study

Abstract Root tips of Vicia faba were treated with 7.5–8.10 −3 M 2′-deoxyadenosine (AdR) and exposed to 2 μC/ml tritiated thymidine before, after or simultaneously with the AdR treatments. Various times after the treatments, root tips were fixed and slides prepared as Feulgen squashes. Stripping film autoradiographs were then made and after a 14-day exposure the slides were developed and analyzed for aberration yield and the presence of silver grains over-laying the chromosomes. The results obtained suggest that there are two types of interphase cells which are sensitive to the chromosome-breaking effect of AdR: (1) cells which are preparing for DNA synthesis and chromosome duplication (cells in late G 1 or very early S ) and (2) cells which have completed chromosome duplication and are preparing to divide (cells in G 2 ). The aberrations induced in late G 1 or early S were mainly chromatid exchanges and isolocus breaks, but a few chromosome-type exchanges were also found. The majority of the chromatid interchanges produced in these cells were localized in the nucleolar constriction. Most of the cells affected by AdR in G 2 proceeded without much delay towards mitosis. The aberrations found in these cells were of the fragment type, i.e. , they consisted mostly of gaps and chromatid breaks. However, the data suggest that a fraction of the cells affected while in G 2 were considerably delayed and did not reach mitosis until about the same time as the cells which were affected by AdR while in the G 1 . These cells contained aberrations of the exchange type.

Induction of chromosomal aberrations by camptothecin in root-tip cells of Vicia faba.

When root-tip cells of Vicia faba were exposed during early and middle interphase to camptothecin (Cpt), the aberrations obtained were exclusively of the chromatid type and tended to be localized in late replicating heterochromatic regions of the chromosomes. In these respects the clastogenic effect of Cpt resembles that of agents that act by an S-phase-dependent mechanism. In contrast to typical S-phase-dependent agents, Cpt produced lesions capable of giving rise to aberrations only in S-phase cells that were synthesizing DNA at the time of treatment. The dependence on ongoing DNA synthesis was suggested in autoradiographic experiments and by the fact that the clastogenic effect of Cpt was strongly suppressed by hydroxyurea, an inhibitor of DNA synthesis. After Cpt treatments, there were many more cells with 3-12 aberrations and far fewer cells with 0, 1 or 2 aberrations than expected on the basis of a Poisson distribution. Cpt further differed from typical S-phase-dependent agents by being capable of inducing lesions in the G2 phase that give rise to chromosomal aberrations in the first mitosis after treatment. This effect was obtained at Cpt concentrations around 10 microM, whereas only 0.03 microM Cpt was required to produce chromatid aberrations in the S phase. Results of G2-phase experiments with Cpt and 5-fluorodeoxyuridine, an inhibitor of DNA synthesis, suggest that DNA synthesis is required for the clastogenic effect of Cpt not only during the S phase, but also during the G2 phase, although the DNA syntheses involved are both quantitatively and qualitatively different.

Effects of G2 treatments with inhibitors of DNA synthesis and repair on chromosome damage induced by X-rays and chemical clastogens in root tips of Vicia faba. Comparison with corresponding effects in cultured human lymphocytes.

The frequencies of chromatid aberrations produced in roots of Vicia faba by clastogenic (chromosome-damaging) agents were strongly enhanced by exposing the root-tip cells to inhibitors of DNA synthesis during the G2 phase. Chromosome damage produced by both S-dependent (maleic hydrazide, methyl methanesulfonate, thio-TEPA) and S-independent (X-rays, streptonigrin) mechanisms was enhanced by the inhibitor treatments. The types of aberrations affected by the inhibitors were mainly chromatid gaps and breaks and isochromatid breaks of the non-union type. Most effective among the inhibitors tested were hydroxyurea (HU) and 5-fluorodeoxyuridine (FdUrd). Post-treatments with caffeine were effective in enhancing clastogen-induced chromosome damage when given during the S phase. All types of aberrations, exchanges as well as breaks, were enhanced by the post-treatments. When given during the G2 phase, caffeine enhanced only the frequency of chromatid aberrations produced by X-rays. The enhancement was slight and obtained only when the cells were irradiated in the G2 phase and immediately post-treated with caffeine. Clastogen-treated cultures of human lymphocytes responded to post-treatments with inhibitors of DNA synthesis in very much the same way as clastogen-treated root-tip cells of Vicia faba. Thus, the frequencies of chromatid gaps and breaks and isochromatid breaks of the non-union type were strongly enhanced by exposing clastogen-treated lymphocytes to inhibitors of DNA synthesis during the G2 phase. The efficiency of the inhibitors, however, varied considerably in the two materials. On the whole, the number of inhibitors capable of enhancing induced chromosome damage was much larger in lymphocytes than in bean root tips. Only HU was equally effective in both materials. The most striking difference between the two materials was found when caffeine was given as a post-treatment. Thus, in human lymphocytes the frequencies of chromatid aberrations induced by most clastogenic agents were strongly enhanced when caffeine was given during the G2 phase, but little affected by post-treatments with caffeine during the S phase.

Enhancement and reduction by methylated oxypurines of the frequencies of chromatid aberrations induced by camptothecin in root-tip cells of Vicia faba

In root-tip cells of Vicia faba the frequencies of chromatid aberrations induced by 3-h treatments with 0.05 microM camptothecin were strongly modified when the treatments were carried out in the presence of caffeine at concentrations above 1 mM. Depending on the concentration of caffeine, the clastogenic effect of camptothecin was either enhanced or reduced. At concentrations between 1 and 6 mM, caffeine increased the camptothecin-induced chromosome damage, the strongest enhancement being obtained at 5 mM. A reduction of the chromosome damage was apparent at caffeine concentrations above 10 mM, and in the presence of 20 mM caffeine the clastogenic effect of camptothecin was almost completely suppressed. When present during the camptothecin treatment, theophylline, 8-chlorocaffeine and 1,3,7,9-tetramethyluric acid influenced the induced chromosome damage in a similar way as caffeine, although with varying efficiency. If the concentrations required to produce the two types of modifying effect are used as a criterion, 8-chlorocaffeine was the most effective and 1,3,7,9-tetramethyluric acid the least, whereas caffeine and theophylline were about equally effective.