Induction of pyrimidine dimers in epidermal DNA of hairless mice by UVB: an action spectrum.

An action spectrum for the induction of pyrimidine dimers in the epidermis of hairless mice was determined between 288 and 307 nm. The presence of pyrimidine dimers in tritium-labeled DNA extracted from exposed SKH:hairless-1 mouse skin was determined using dimer-specific nucleases from Micrococcus luteus in conjunction with sedimentation of the irradiated DNA in alkaline sucrose gradients. The rate of induction of pyrimidine dimers was maximal at 293 nm. These values were used to propose a UVB transmission curve for mouse epidermis.

Effects of hydroxyl radical scavengers on relaxation of supercoiled DNA by aminomethyl-trimethyl-psoralen and monochromatic UVA photons.

The ability of scavengers of hydroxyl radical (OH radical) to modulate the photosensitized relaxation (induction of the first single-strand break) of supercoiled plasmid DNA with UVA photoactivated 4'-aminomethyl-4,5',8-trimethylpsoralen was examined by comparing the dose reduction factor (DRF: the ratio of fluence required to induce the same degree of relaxation in the absence to the presence of OH radical scavengers). The addition of mannitol, azide, acetate, or formate at concentrations inversely proportional to the value of the rate constants for the scavenging of OH radicals partially attenuated the supercoiled DNA relaxation. The degrees of protection afforded by the four scavengers in the presence of AMT photoactivated by either 334 nm or 365 nm monochromatic photons were similar, giving an average DRF of about 0.25 in all cases. Given the diverse chemical nature of the scavengers and their wide range of concentrations utilized, these findings are evidence for the involvement of a Type I photosensitization in the induction of DNA single-strand breaks by photoactivated AMT.

Comparison of the inactivation of Bacillus subtilis transforming DNA by the potassium superoxide and xanthine-xanthine oxidase systems for generating superoxide.

Potassium superoxide (KO2) and xanthine-xanthine oxidase (X-XO), which are known generating systems for the superoxide anion, have different inactivating actions on Bacillus subtilis transforming DNA in vitro. Superoxide dismutase and CuSO4 enhanced the inactivation for KO2, but not for X-XO. Mannitol, a hydroxyl radical scavenger, protected against the inactivation by X-XO, but not by KO2. The results obtained with X-XO were consistent with the involvement of Fenton reactions, in which hydroxyl radical is the reactive species that ultimately causes damage. On the other hand, KO2-induced inactivation was partly due to the effect of H2O2. Differences in inactivation between the KO2 and X-XO systems may result from the different rates of production of the superoxide anion.

Similar ultraviolet action spectra for the protection by glycerol of transforming DNA against single-strand breaks and inactivation of biological activity.

An action spectrum for the protection of purified DNA by glycerol against the induction of single-strand breaks in the DNA by ultraviolet (uv) light is described. Protection was not observed below 300 nm, was maximal between 334 and 365 nm, and decreased at 405 nm. This spectrum closely matched the spectrum for the protection by glycerol against the inactivation of biological transforming activity by near uv, described previously. Also, deviations from the reciprocity rule are similar for inactivation of transforming activity and for induction of DNA breaks by 365-nm radiation. That is, the deviations for the two end points are quantitatively the same, such that high fluence rates are less effective than low fluence rates.

Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra.

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells.

Evidence from nondenaturing filter elution that induction of double-strand breaks in the DNA of Chinese hamster V79 cells by gamma radiation is proportional to the square of dose.

We have used nondenaturing filter elution performed at both pH 7.2 and pH 9.6 to measure the induction of double-strand breaks (DSBs) in the DNA of Chinese hamster V79 cells by 60Co gamma-radiation doses between 10 and 120 Gy. The absolute DSB yields as measured by this assay were determined by using our recent calibration of the assay based upon disintegrations of 125I incorporated into the DNA. An analysis of the dose-response relationship for the induction of DSBs by 60Co gamma rays showed that the number of DSBs induced per dalton of DNA was proportional to the square of the applied dose throughout the dose range used. The contribution made by the dose to the first power was small at pH 9.6 and negligible at pH 7.2. These results suggest that DSB induction in cells by gamma rays may be entirely a two-hit event.

Measurement of double-strand breaks in Chinese hamster cell DNA by neutral filter elution: calibration by 125I decay.

We labeled the DNA of Chinese hamster lung V79 cells with 125I in the form of iododeoxyuridine and subsequently measured the elution of the DNA through polycarbonate filters at pH 9.6 and pH 7.2. Since decay of incorporated 125I produces predominantly double-strand breaks (DSB) in DNA at a rate close to one DSB per 125I decay, this measurement provides an absolute calibration for the assay of DSBs by neutral filter elution. Neutral elution profiles are not first order with respect to elution time; thus we have examined the relationships between accumulated 125I decays and several functions of retention of DNA on the filter at various times during the elution process. At both pH 9.6 and pH 7.2 there were linear relationships between accumulated decays and certain retention functions. The retention function most closely correlated to 125I decays for both pH values was the logarithm of the ratio of the retention of control DNA to that of 125I-labeled DNA, both evaluated at the 9th fraction (13.5 h of elution). The linear relationship between this ratio and 125I decays allows DSB induction to be determined directly from retention values. The calibration was used to measure DSBs induced by X rays.

Evidence that hydrogen peroxide generated by 365-nm UVA radiation is not important in mammalian cell killing.

We compared measurements of cell survival and DNA single-strand breaks (SSBs) caused by hydrogen peroxide (H2O2) and UVA radiation (365-nm) in both a parental and a H2O2-resistant variant of the Chinese hamster ovary HA1 line derived by culturing cells in progressively higher concentrations of H2O2. Both RNA slot blot analysis and enzyme analysis confirmed that the variant possesses high levels of both catalase activity and mRNA. The variant was completely resistant to the lethal effects of H2O2 over the concentration range tested (up to 480 microM), whereas the parental strain showed less than 1% survival at this concentration. Similarly, the H2O2-resistant strain exhibited far fewer SSBs after exposure to H2O2 than the parental strain. Addition of o-phenanthroline to the parental cells during H2O2 exposure almost completely inhibited SSB induction, evidence that these SSBs are produced via the Fenton pathway of Haber-Weiss reactions. Very little difference was found between the variant and the parent after exposure to 365-nm radiation: only a minor difference in survival kinetics and no difference is SSB induction were observed between the two cell lines. These results are consistent with a hypothesis that most lethal events caused in cells by UVA occur by pathways that do not involve the H2O2 that is produced by sensitized reactions within the cells.

Human epithelial teratocarcinoma cells (P3): radiobiological characterization, DNA damage, and comparison with other rodent and human cell lines.

Survival parameters and immediate DNA damage induced by 60Co gamma rays, 50-kVp X rays, and Janus fission-spectrum neutrons in human epithelial P3 cells (derived from an embryonic teratocarcinoma) are compared with those for Chinese hamster lung V79 cells. DNA damage caused by X and gamma irradiation, measured by alkaline elution methods, is the same in both cell types, whereas the P3 cells are about two times more sensitive (as measured by Do ratios of the final survival curve slope) to the lethal effects of these radiations than are the V79 cells. Human P3 cells are also more sensitive to the lethal effects of fission-spectrum neutrons than V79 cells. Survival experiments with split radiation doses and hypertonic salt treatment indicate that both P3 cells and V79 cells can recover from radiation-induced damage efficiently.

Ultraviolet light induces double-strand breaks in DNA of cultured human P3 cells as measured by neutral filter elution.

Neutral filter elution at pH 7.2 and 9.6 was used to measure the induction of DNA lesions in human P3 teratocarcinoma cells by monochromatic 254-, 270-, 313-, 334-, 365-, and 405-nm radiation and by 60 gamma rays. In this assay DNA double-strand breaks (dsb) increase the rate of elution of DNA from cell lysates on a filter. Yields of dsb as measured by this procedure were determined by using a calibration of the assay that correlates elution parameters with number of dsb caused by disintegration of 125I incorporated into the DNA. Analysis of fluence responses obtained by using the calibrated assay indicated that the number of dsb induced per dalton of DNA as measured by this assay is proportional to the square of the fluence at all the energies of radiation studied, implying that the induction of these lesions may be a two-hit event. Analysis of the relative efficiencies for the induction of dsb by ultraviolet radiation, corrected for quantum efficiency, revealed a spectrum that coincided closely with that for the induction of single-strand breaks (ssb) in the same cells, having a close fit with the spectrum of nucleic acid in the UVC and UVB region below 313 nm, and a shoulder in the UVA region. It was calculated, however, that there may be too few ssb for dsb to result from randomly distributed closely opposed ssb.

Hydroxyl radical quenching agents protect against DNA breakage caused by both 365-nm UVA and by gamma radiation.

The ability of hydroxyl radical (.OH) scavengers to reduce DNA breakage in isolated DNA from Bacillus subtilis by either gamma radiation or monochromatic radiation in the UVA region (365 nm) was examined by comparing dose reduction factors (the ratio of dose required to induce n DNA breaks in the absence to the presence of quencher). Previous data have demonstrated that acetate, formate, azide, and mannitol protect supercoiled DNA against gamma-radiation-induced ssb (single-strand breaks-relaxation of supercoil by first nick) in close agreement with the rate at which their solutions quench .OH. Here we show that these quenchers also protect against 365-nm-induced ssb. The ratios for protection against 365-nm induced DNA ssb in isolated B. subtilis DNA by the four quenchers are also in proportion to their ability to quench .OH. In view of the diverse chemical nature of the quenchers and the wide range of concentrations involved, these findings are evidence that both these radiations may induce ssb in DNA via a common step that might involve .OH.

Convex curvatures of alkaline elution profiles of DNA from human cells irradiated with 405 nm UVA: evidence for induction of slowly developing alkali-labile sites.

The alkaline (pH 12.1) elution profiles of DNA from human P3 cells exposed to monochromatic 405 nm UVA radiation deviate from exponential: on a logarithmic plot of eluted fraction of DNA vs time of elution, the rate of elution accelerates for the first 6 h. Following this period, the profiles become exponential. In contrast, the elution profiles of DNA after 520 nm green light or ionizing radiation exposures (x- and gamma rays, and fission spectrum neutrons) are always strictly exponential, evidence that the convex profiles were not due to an artifact caused by elution technique. Holding the DNA at pH 12.1 for 6 h after 405-nm exposures before initiating elution resulted in profiles that were close to exponential, with slopes similar to the final slopes observed following the 6-h elution period in the original experiments. This is evidence that some DNA breaks develop slowly during the first 6 h of elution, as a result of exposure to alkali. Therefore, the DNA lesions induced by 405-nm light as measured by the alkaline elution technique are apparently heterogeneous and include a major class of alkali-labile sites that develop slowly during incubation at pH 12.1. Convex profiles also occur following exposure of the cells to visible light at 434 and 512 nm.

Induction of slowly developing alkali-labile sites in human P3 cell DNA by UVA and blue- and green-light photons: action spectrum.

Action spectra (365-520 nm) for the formation of DNA single-strand breaks (SSB) and slowly developing alkali-labile sites (SDALS) in human teratocarcinoma P3 cells in culture were determined. Induction of SDALS results from the absorption of blue- and green-light photons. The spectrum has a broad peak that is maximal between 400 nm to 500 nm and declines sharply above and below these wavelength regions. Negligible yields of SDALS were produced by photons at wavelengths of 365 nm or shorter and at 520 nm or longer, whereas for SSB, the action ioffeases with shorter wavelength throughout the whole spectral range studied. The configuration of the SDALS action spectrum suggests that the primary chromophore, and therefore possibly the photosensitizer, is a mixture of porphyrin and flavin residues.

Repair of near-visible- and blue-light-induced DNA single-strand breaks by the CHO cell lines AA8 and EM9.

The induction of single-strand breaks (SSB) and the kinetics of SSB repair were measured in two Chinese hamster ovary cell lines irradiated with monochromatic photons of near-visible radiation (405 nm) and blue light (434 nm). The radiosensitive and UV-A-sensitive mutant line EM9 is known to repair SSB induced by ionizing radiation or 365-nm UV-A more slowly than the parent line AA8. At the 10% survival level, EM9 cells were 1.7- and 1.6-fold more sensitive than AA8 cells to 405 and 434 nm radiation, respectively. This sensitivity was not due to differences in induction of SSB because AA8 and EM9 cells accumulated the same number of initial breaks when irradiated at 0.5 degrees C with either 405 nm (5.9 SSB per MJ/m2) or 434 nm (5.1 SSB per MJ/m2), as measured by alkaline elution. When the cells repaired these SSB at 37 degrees C in full culture medium, biphasic repair kinetics were observed for both cell lines. In both phases of repair, EM9 cells repaired breaks induced by both wavelengths more slowly than did AA8 cells. The t1/2 values for the repair phases for 405-nm-induced SSB were 3.8 and 150 min for EM9, and 1.5 and 52 min for AA8; the corresponding values for repair of 434 nm breaks were 3.7 and 39 min for EM9, and 2.0 and 30 min for AA8. Because of this slower repair, EM9 cells left more SSB unrepaired after 90 min than did AA8 cells for both wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced expression of protein kinase C gene caused by solar radiation.

Natural solar radiation (5 min of midday exposure in mid July, latitude 42 degrees N) induces protein kinase C mRNA almost two-fold in human epithelioid P3 cells in culture. This response is the same as that following tumor promotion by chemicals. The result indicates a possible role of promotion by solar UV radiation.

Singlet oxygen induces frank strand breaks as well as alkali- and piperidine-labile sites in supercoiled plasmid DNA.

A covalently closed, circular, supercoiled plasmid was exposed to singlet oxygen by a separated-surface sensitizer. For each exposure, the quantity of single oxygen entering the DNA target solution was estimated by its oxidation of histidine. After singlet oxygen exposure, some DNA samples were treated to disclose occult lesions. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed circular and linear species, and all bands were quantitated fluorometrically. Exposure of supercoiled plasmid DNA to singlet oxygen induced frank DNA strand breaks, alkali-labile sites (pH 12.5, 90 degrees C, 30 min), and piperidine-labile sites (0.4 M, 60 degrees C, 30 min), all in a dose-dependent manner. Yields of alkali-labile and piperidine-labile sites ranged from one to four times the frank strand break yield. Replacement of buffered H2O by buffered D2O as the DNA solvent for singlet oxygen exposures increased DNA lesion yields by a factor of 2.6 (averaged over lesion classes). Our data for the detection of frank strand breaks is at variance with published results from studies in which singlet oxygen was derived from a thermolabile endoperoxide dissolved in the DNA solution.

4'-Aminomethyl-4,5',8-trimethylpsoralen photochemistry: the effect of concentration and UVA fluence on photoadduct formation in poly(dA-dT) and calf thymus DNA.

The photochemistry of 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) with poly(dA-dT) and calf thymus DNA was studied. The extent of photoadduct formation and the distribution of photoadducts (3,4- and 4',5'-monoadducts and crosslinks) were determined by liquid scintillation analysis and HPLC, respectively. The adducts were characterized on the basis of their UV absorption spectra and mass spectral analysis. The high DNA binding constant for AMT (1.5 x 10(5) M-1) led to a high fraction of intercalated molecules, which contributed to the high level of AMT photoadduct formation, as many as 102 adducts per kilobase pair. In addition, there is a distinct difference in the adduct distribution compared to the previously studied 8-methoxypsoralen (8-MOP). Under the conditions employed for the photochemical studies, virtually all of the AMT molecules in solution are intercalated, occupying 25% of the base pair sites. Under similar conditions, 8-MOP molecules occupied 10 times fewer sites. Thus, for AMT, DNA base pair sites other than 5'TA, the well-characterized strong binding for psoralens in general, are an additional target for photomodification, which results in the formation of a higher percentage of monoadducts. The proportion of photoadducts formed was virtually independent of AMT concentration and UVA (320-400 nm radiation) fluence.

DNA breaks caused by monochromatic 365 nm ultraviolet-A radiation or hydrogen peroxide and their repair in human epithelioid and xeroderma pigmentosum cells.

The induction and repair of DNA single-strand breaks (SSB) assayed by alkaline filter elution was compared in human epithelioid P3 and xeroderma pigmentosum (XP) cells exposed to monochromatic 365-nm UV-A radiation and H2O2. Initial yields of SSB were measured with the cells held at 0.5 degrees C during exposure. The yield from exposure to 365-nm radiation was slightly greater in XP than in P3 cells, whereas H2O2 produced more than three times as many SSB in P3 compared with XP cells. o-Phenanthroline (50 mM) markedly inhibited the yields of SSB induced in XP cells by H2O2, but had no effect on those produced by 365-nm UV-A. These results are consistent with the fact that P3 cells, unlike XP cells, have undetectable levels of catalase. The measured production of trace amounts of H2O2 by the actual 365-nm UV-A exposures was not sufficient to account for the numbers of breaks that were observed. Single-strand breaks produced by both agents were completely repaired after 50 min in P3 cells, as were H2O2-induced SSB in XP cells. However, 25% of the 365-nm UV-A-induced SSB in XP cells remained refractory to repair after 60 min. The results show that SSB produced by these two agents are different and that 365 nm radiation produces most SSB in cells by mechanisms other than by production of H2O2.

Correlation between cell survival and DNA single-strand break repair proficiency in the Chinese hamster ovary cell lines AA8 and EM9 irradiated with 365-nm ultraviolet-A radiation.

Cell survival parameters and the induction and repair of DNA single-strand breaks were measured in two Chinese hamster ovary cell lines after irradiation with monochromatic UVA radiation of wavelength 365 nm. The radiosensitive mutant cell line EM9 is known to repair ionizing-radiation-induced single-strand breaks (SSB) more slowly than the parent line AA8. EM9 was determined to be 1.7-fold more sensitive to killing by 365-nm radiation than AA8 at the 10% survival level, and EM9 had a smaller shoulder region on the survival curve (alpha = 1.76) than AA8 (alpha = 0.62). No significant differences were found between the cell lines in the initial yields of SSB induced either by gamma-radiation (as determined by alkaline sucrose gradient sedimentation) or by 365-nm UVA (as determined by alkaline elution). For measurement of initial SSB, cells were irradiated at 0.5 degrees C to minimize DNA repair processes. Rejoining of 365-nm induced SSB was measured by irradiating cells at 0.5 degrees C, allowing them to repair at 37 degrees C in full culture medium, and then quantitating the remaining SSB by alkaline elution. The repair of these breaks followed biphasic kinetics in both cell lines. EM9 repaired the breaks more slowly (t1/2 values of 1.3 and 61.3 min) than did AA8 (t1/2 values of 0.9 and 53.3 min), and EM9 also left more breaks unrepaired 90 min after irradiation (24% vs 8% for AA8). Thus, the sensitivity of EM9 to 365-nm radiation correlated with its deficiency in repairing DNA lesions revealed as SSB in alkaline elution.(ABSTRACT TRUNCATED AT 250 WORDS)