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.

Linear induction of DNA double-strand breakage with X-ray dose, as determined from DNA fragment size distribution.

Pulsed-field gel electrophoresis has been applied to separate DNA from mouse L1210 cells exposed to X-ray doses of 1 to 50 Gy. Simultaneous separation of marker chromosomes in the range 0.1 to 12.6 Mbp allowed calculation of the size distribution of the radiation-induced fragments. The distribution was consistent with a random induction of double-strand breaks (DSBs). A theoretical relationship between the size distribution of such fragments and the average number of induced breaks was used to calculate the yield and dose response. The DNA distribution was determined by both radiolabeling and fluorescence staining. Two independent methods were used to evaluate the radiation-induced yield of DSBs, both assuming that all DNA is broken at random. In the first method we compared the theoretical and experimental fraction of DNA that is below a given size limit. By this method we estimated the yield to be 0.006-0.007 DSB/Gy per million base pairs using the radiolabel and 0.004-0.008 DSB/Gy per million base pairs by fluorescence staining. The dose response was linear in both cases. In the second method we looked only at the size distribution in the resolving part of the gel and compared it to the theoretical distribution. By this method a value of approximately 0.012 DSB/Gy/Mbp was found, using fluorescence as a measure of DNA distribution. In a normal diploid mammalian genome of size 6000 Mbp, this is equivalent to a yield of 25-50 DSBs/Gy or 70 DSBs/Gy, respectively. The second approach, which looks only at the smaller fragments, may overestimate the yield, while the first approach suffers from uncertainties about the fraction of DNA irreversibly trapped in the well. The assay has the capacity to detect a dose of less than 1 Gy.

Clonogenic cell survival and rejoining of DNA double-strand breaks: comparisons between three cell lines after photon or He ion irradiation.

Three cell lines, human glioma U-343MG, Chinese hamster V79-379A and human colon carcinoma LS-174T, were analysed for clonogenic survival and with pulsed-field gel electrophoresis regarding induction and rejoining of double-strand breaks (dsb) in DNA. The cells were irradiated with either intermediate linear energy transfer (LET approximately 55 keV/microns) He ions or with 60Co photons and showed cell type-specific variations in their survival curves. The induction of dsb, per DNA unit, increased linearly with dose for both radiation qualities, and there were no cell type-dependent differences. However, the dsb induction frequency increased for all three cell lines with an RBE of 1.31 +/- 0.08 (SD) after He ion irradiation. All three cell lines showed biphasic dsb rejoining patterns with cell type-specific differences. The differences between the cell lines were somewhat smaller after irradiation with He ions, although the patterns were very similar to those seen after photon irradiation. The shoulders in the survival curves disappeared after He ion irradiations, however the cells retained the capacity to rejoin dsb. After 8-h rejoining the increase of LET to intermediate values did not give a higher fraction of residual dsb.

On the mechanism of the hepatocarcinogenicity of peroxisome proliferators.

The absence of a genotoxic action in the rat of several peroxisome proliferators (PP) has been confirmed by measuring gross degradation, unscheduled DNA-synthesis (UDS), as well as by measurement of single strand breaks using alkali unwinding in absence and presence of inhibitors of DNA-repair. Similar results were obtained even after drastically lowering the glutathione content of liver. Further, after oral administration of ciprofibrate, no potentiating effect was found in vivo on the generation of micronuclei in hepatocytes by ionizing radiation. The metabolically inert PP, perfluorooctanoic acid, was found to act as a promoter of liver tumors in the rat induced by diethylnitrosamine in an initiation-selection-promotion protocol. The results are discussed in light of available information concerning the mechanism of action of PPs.

Single-strand breaks in DNA of various organs of mice induced by methyl methanesulfonate and dimethylsulfoxide determined by the alkaline unwinding technique.

The method for determination of single-strand breaks (SSB) in DNA by the technique of alkaline unwinding and hydroxylapatite chromatography has been applied for cell nuclei from organs of mice. Male mice were given methyl methane-sulfonate (MMS) and dimethylsulfoxide (DMSO) by i.p. administration. Cell nuclei were prepared from various organs and then lysed in alkali. The amount of DNA was determined by fluorometry using 4',6-diamidino-2-phenylindole.2HCl. The relative level of SSB in DNA was determined in various organs (liver, kidney, lung, spleen, testis and brain) 1-24 h after administration of the agent. After MMS-treatment the number of SSB in DNA increased to about the same extent in all organs 1 h post-treatment but then decreased by time. The SSB persisted for the longest time in brain- and lung-DNA. DMSO induced SSB only in DNA of kidney.

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-beta-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.

Studies on the nature of the inhibitory effect of trypsin on the photosynthetic electron transport of system II in spinach chloroplasts.

The effect of trypsin on the photosynthetic electron transport of spinach chloroplasts has been investigated by measurements of the flash-induced absorption changes, indicating chlorophyll a1 at 703 nm, chlorophyll aII at 690 nm and at 515 nm via electrochromism the electrical potential gradient across the thylakoid membrane, respectively, and of the fluorescence induction caused by moderate actinic light. It was found: (1) In the presence of benzyl viologen as electron acceptor and with water as natural electron donor trypsin, incubation leads to a complete suppression of the absorption changes of the electrochromic effect and of chlorophyll aI and chlorophyll aII. (2) Addition of System I electron donors (N-methylphenazonium sulfate plus ascorbate or 2,6-dichlorophenolindphenol plus ascorbate) fully restores the chlorophyll aI photoreaction, whereas the initial amplitude of the electrochromic absorption change at 515 nm amounts about 50% of the control value without trypsin. The chlorophyll aII inhibition remains uneffected by System I electron donors. (3) System II electron donors (benzohydroquinone plus ascorbate or TPB) are unable to overcome the inhibition of electron transport by trypsin. (4) The fluorescence induction curve in 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea-blocked chloroplasts is modified by trypsin. The level of maximal fluorescence is remarkably decreased, whereas the initial fluorescence remains constant. The rise in kinetics is slightly decelerated. From these results, it is concluded that in the linear electron transport from water to benzyl viologen, mild trypsin treatment specifically attacks System II at a site very close to the reaction center, either on the oxidizing or on the reducing side. The reaction center of System II itself is relatively stable against trypsin. Arguments are presented which argue in favor of the trypsin attack being primarily directed at the reducing side of System II.

The action of tetraphenylboron as a system II electron donor and its effect on the decay of the electrical field across the thylakoid membrane.

The effect of tetraphenylboron on the O2 evolution, the electron transport in normal and in Tris-washed chloroplasts, and on the decay of the electrical field across the thylakoid membrane has been investigated in spinach chloroplasts. It was found that: (1) The average O2 yield per flash as a function of the tetraphenylboron concentration is dependent on the number of excitation flashes. With increasing flash number, the tetraphenylboron concentration which is required for 50% suppression of the average O2 yield per flash shifts toward higher values. (2) After the irreversible consumption of tetraphenylboron by System II oxidizing equivalents, the O2 evolution reappears. (3) The electron flow from System II to System I, as indicated by the amplitude of the reduction kinetics of the 703 nm absorption change, remains unaffected in the tetraphenylboron concentration range and at flash numbers where the O2 evolution is totally suppressed. (4) Tetraphenylboron restores electron transport in Tris-washed chloroplasts. This tetraphenylboron-mediated electron transport is completely blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). (5) Preliminary titration experiments favor the assumption that tetraphenylboron acts in chloroplasts rather as a 1-electron donor than as a 2-electron donor. (6) Tetraphenylboron accelerates the decay of the electrical field across the thylakoid membrane. Because of its ability to act as a System II electron donor, the accelerating effect on the electrical field is transitory if System II is active. By contrast, in DCMU-blocked chloroplasts with active System I electron transport [mediated by the 2,6-dichlorophenolindophenol (DCIP) cycle], a permanent accelerating effect on the electrical field is observed. From these results it has been concluded, that tetraphenylboron acts as an efficient 1-electron donor for System II. The functional integrity of the water-splitting enzyme system Y is not required for this donation. Tetraphenylboron itself does not destroy the O2 evolution capability of chloroplasts, it rather acts as a competitive electron donor. However, an inhibitory effect arises probably from the oxidation products of tetraphenylboron. The possible modes of action of tetraphenylboron are discussed.