Gunnar Ahnström

The comet assay: mechanisms and technical considerations.

The comet assay is frequently used to measure DNA damage in individual cells. In order to better understand the mechanisms behind the technique, we have studied the behaviour of DNA under different electrophoresis conditions in mammalian cells exposed to gamma radiation. The comet tails obtained after neutral electrophoresis seem to consist of DNA loops which are attached to structures in the nucleus, since the DNA cannot move in the second direction after two-dimensional electrophoresis. When the DNA is labelled by a short pulse, microautoradiography reveals that all label appear in the head of the comets when neutral electrophoresis is applied. After chase incubation, the label moves out into the tails. This gives further support to the view that the DNA loops are fixed to some structure in the nucleus where also the DNA synthesis takes place. Under alkaline electrophoresis conditions, however, the entire comet tails move in the new electrophoresis direction. Thus, it appears that the alkaline comet tails consist of free DNA fragments. Further, the effects of alkaline concentration and sodium chloride during unwinding and electrophoresis are discussed. Throughout the study, a protocol for drying and fixation of the comets has been used.

A review of dsb induction data for varying quality radiations

PURPOSE This short review summarizes the data obtained with various techniques for measuring the yields of double strand breaks (dsb) produced by particle radiations of differing linear energy transfer (LET) in order to obtain relative biological effectiveness (RBE) values. RESULTS AND CONCLUSIONS Studies aimed at understanding the interactions of different types of radiation with cellular DNA have monitored the yields of DNA dsb versus radiation quality. Several techniques have been used to measure dsb yields in mammalian cells, and these include: neutral sedimentation gradients, filter elution and more recently pulsed field gel electrophoresis techniques (PFGE). Recent developments in PFGE have allowed the measurement of both the yields and the distribution of breaks within the genome, which go part of the way to explaining the RBE values close to 1.0 previously measured using other approaches with various radiation qualities. It is clear that future studies to determine the effectiveness of radiations of differing LET must use techniques that determine both yields and distributions of dsb, and assays need to be developed to allow these measurements at biologically relevant doses.

Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: Effects of hydroxyurea, 5-fluorodeoxyuridine and 1-β-d-arabinofuranosylcytosine on the kinetics of repair

A simple and sensitive technique for detection of strand breaks in DNA has been further developed. The method has been used to follow UV-induced excision-repair in human fibroblasts. It has been possible to study the kinetics of enzymic reactions in intact cells, in which strand breaks in DNA are produced and sealed again. Hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine, potent inhibitors of DNA synthesis, drastically increased the number of breaks observed during the repair process. This was probably due to a decreased polymerase activity, which will cause the strand breaks formed by endonuclease to remain open longer. The initial rate of strand-break formation did not seem to be influenced by hydroxyurea or araC, and was about 4000 breaks per minute in a diploid genome, at a dose of 20 J/m2. After 5--30 min, depending on the dose of UV, the number of breaks reached a maximum and started to decrease again. Hydroxyurea decreased the rate of polymerization in the sites under repair. However, there was no concomitant reduction of repair-induced incorporation of [3H]thymidine and no reduction of the excision of pyrimidine dimers. It therefore seems that the action of the polymerase was not a rate-limiting event, but rather an earlier step. It is likely that the endonucleolytic activity determined the rate of repair. As a consequence, the endonuclease and polymerase cannot be bound in a permanent complex. Under certain assumptions, the time for repair of a site, i.e. the time from incision to final ligase sealing, can be estimated as between 3 and 10 min. Essentially no breaks were produced in Xeroderma pigmentosum cells belonging to complementation group A, and there was no enhancement by hydroxyurea. Cells from the variant type of Xeroderma pigmentosum behaved like normal cells in this respect.

Techniques to Measure DNA Single-strand Breaks in Cells: A Review

Alkaline sucrose sedimentation was for a number of years the standard procedure for the measurement of single-strand breaks. Some years ago a number of new techniques with improved sensitivity were introduced. The following techniques are presented and discussed: alkaline unwinding, alkaline filter elution, nucleoid sedimentation, viscoelastometry, microelectrophoresis of single cells, DNA precipitation, pulse field gel electrophoresis, fluctuation spectroscopy and nick translation.

Repair of Gamma-ray and Neutron-induced Lesions in Germinating Barley Seeds

SummaryBarley seeds were irradiated with gamma-rays or fast neutrons in the resting state and after various periods of germination. When irradiated in the resting state, about 50 krads of gamma-rays or about 1 krad of fast neutrons produced a 50 per cent inhibition of seedling growth. The radiosensitivity increased after pre-soaking and reached a maximum level after 12–15 hours of germination. At this stage the sensitivity to gamma-rays had increased by a factor of 50 and to fast neutrons by a factor of 5.The presence of caffeine (assumed to be a DNA-repair inhibitor) during the first period of germination of seeds irradiated in the resting state led to an enhancement of the gamma-induced damage by a factor of 2, but had little effect on neutron-irradiated seeds. The caffeine effect was also negligible if the gamma-irradiated seeds were allowed to germinate for at least 5 hours in water before the treatment. The effect of caffeine was also considerably lower in seeds pre-soaked for 12–15 hours.The results...

Exposure of pancreatic islets to different alkylating agents decreases mitochondrial DNA content but only streptozotocin induces long-lasting functional impairment of B-cells

Pancreatic B-cells exposed in vivo or in vitro to streptozotocin (SZ), the N-nitrosourea derivative of glucosamide, present a long-lasting impairment in the production and release of insulin while other cell functions are better preserved. This functional impairment is associated with a defective mitochondrial function. To further study the mechanisms behind SZ actions, mouse pancreatic islets were exposed in vitro to SZ (1.5 mM) or to different concentrations of methyl methanesulfonate (MMS; 2, 4 and 6 mM). The effect of the aglucone moiety of SZ, nitroso-N-methylurea (NMU; 2, 4 and 6 mM) was also tested. Islets were either studied immediately after exposure to the drugs (day 0) or after six days in culture following toxin treatment (day 6). On day 0 the islets showed a decrease in the NAD + NADH content, decreased glucose oxidation rates and an impaired insulin release in response to glucose. Six days after exposure to SZ there was still impaired glucose oxidation and insulin release, and decreased islet insulin mRNA and insulin content, but the NAD + NADH content was again similar to the control group. On the other hand, islets which survived for 6 days in culture following exposure to either MMS or NMU were able to regain normal B-cell function. The mouse islets exposed to SZ, NMU and MMS showed on day 6 a 30-40% decrease in the content of the mitochondrial DNA encoded cytochrome b mRNA and a 60-70% decrease in total mitochondrial DNA, as evaluated by dot and Southern blot analysis. Only SZ decreased the insulin mRNA content whereas both MMS and NMU decreased the glucagon mRNA content. As a whole, the data obtained indicate that SZ, NMU and MMS induce damage to the mitochondrial genome, and this may contribute to the B-cell dysfunction observed after SZ treatment. It is conceivable that the glucose moiety of SZ may direct the methylation to other intracellular sites besides the mitochondrial DNA, thus explaining the different functional responses of islets following exposure to SZ and NMU.

Energy-linked nicotinamide nucleotide transhydrogenase: Hydrodynamic properties and active form of purified and membrane-bound mitochondrial transhydrogenase from beef heart

The mitochondrial nicotinamide nucleotide transhydrogenase from beef heart was investigated with respect to minimal assembly of the purified enzyme and of the enzyme in the mitochondrial inner membrane. Studies of the hydrodynamic properties of the purified enzyme in the presence of 0.3% Triton X-100 allowed determination of the Stokes radius, sedimentation constant, partial specific volume, frictional ratio, and molecular weight. Under these conditions transhydrogenase existed as an inactive monomer, suggesting that monomerization may be accompanied by inactivation. Radiation inactivation was used to determine the functional molecular size of purified detergent-dispersed transhydrogenase and transhydrogenase in beef heart submitochondrial particles. Under these conditions the catalytic activity of both the purified and the membrane-bound enzyme was found to be catalyzed by a dimeric form of the enzyme. These results suggest for the first time that the minimal functional assembly of detergent-dispersed as well as membrane-bound transhydrogenase is a dimer, which is not functionally associated with, for example, complex I or ATPase. In addition, the results are consistent with the possibility that the two subunits of transhydrogenase are catalytically active in an alternating fashion according to a previously proposed half-of-the-sites reactivity model.

Effects of 3-aminobenzamide on the rejoining of DNA-strand breaks in mammalian cells exposed to methyl methanesulphonate; role of poly(ADP-ribose) polymerase

The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, on DNA-repair processes has been investigated after treating V79 hamster cells with methyl methanesulphonate (MMS). Repair activity was observed as changes in DNA-strand break levels. MMS induces transient strand breaks, the level of which slowly decreases with time. Addition of 3AB leads to a rapid increase in the number of breaks. The level of breaks increases linearly with time until it suddenly levels off. Increasing the concentration of 3AB does not change the slope of this curve, but the steady-state level of breaks increases. The incision-rejoining kinetics indicates that 3AB induces a delay in the strand-break rejoining process. In the absence of 3AB the breaks have a lifetime of 1-2 min and this is increased by a factor of 5 in the presence of 5 mM 3AB.

Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes

Earlier studies using the technique of premature chromosome condensation (PCC) have shown that in human lymphocytes, exchange type of aberrations are formed immediately following low doses (<2 Gy) of X-rays, whereas at higher doses these aberrations increase with the duration of recovery. This reflects the relative roles of slow and fast repair in the formation of exchange aberrations. The underlying basis for slow and fast repairing components of the DNA repair may be related to differential localization of the initial damage in the genome, i.e., between relaxed and condensed chromatin. We have tried to gain some insight into this problem by (a) X-irradiating lymphocytes in the presence of dimethyl sulfoxide (DMSO) a potent scavenger of radiation-induced .OH radicals followed by PCC and (b) probing the damage and repair in two specific chromosomes, 18 and 19, which are relatively poor and rich in transcribing genes by COMET-FISH, a combination of Comet assay and fluorescence in situ hybridization (FISH) techniques. Results obtained show (a) that both fast appearing and slowly formed exchange aberrations seem to take place in relaxed chromatin, since they are affected to a similar extent by DMSO, (b) significant differential DNA breakage of chromosome 18 compared to chromosome 19 in both G0 and G1 phases of the cell cycle as detected by Comet assay, indicating that relaxed chromatin containing high densities of transcriptionally active genes shows less fragmentation due to fast repair (chromosome 19) compared to chromosome 18, and (c) that relaxed chromatin is repaired or mis-repaired faster than more compact chromatin.

Repair of DNA damage in mammalian cells after treatment with UV and dimethyl sulphate: discrimination between nucleotide and base excision repair by their temperature dependence

Alkylating agents have been reported to give rise to both short and long patches of repair. The reason for the different patch sizes is not known. One possibility is that alkylating agents can trigger both base and nucleotide excision repair. Another possibility is that base excision repair itself can result in different patch sizes. Recognition and incision at lesions is the rate limiting step in excision repair. In order to discriminate between base and nucleotide excision repair it would be desirable to be able to distinguish between different incision activities. In order to accurately measure incision rates, the rejoining of the strand-breaks formed must be inhibited. We have used two inhibitors, aphidicolin and 3-aminobenzamide. Aphidicolin, an inhibitor of DNA polymerases alpha/delta/epsilon. caused accumulation of single-strand breaks both after UV and dimethylsulphate. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose)-polymerase caused accumulation of single-strand breaks only after alkylating agents and is thus specific for base excision repair. Enzymatic activities can be characterised by their activation energy. In order to discriminate between base and nucleotide excision repair the temperature dependence of incision activities was determined. When the temperature is decreased, the incision rate is reduced to a larger extent for UV than for DMS-induced repair. Incisions in UV-irradiated cells are practically cut off at temperatures of 15 degrees C and below, whereas DMS-exposed cells still are actively repairing at this temperature. In DMS treated cells the temperature dependence was the same whether aphidicolin or 3-aminobenzamide was used, speaking against an involvement of nucleotide excision repair. In addition, cell lines deficient in nucleotide excision repair responded in the same way to aphidicolin after DMS treatment as normal cells and were able to make incisions at 15 degrees C. This indicates that nucleotide excision repair is not to any significant amount involved in repair of DNA damage induced by a methylating agent.