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M J Peak

Publications and source records attributed to M J Peak.

At least 19 recordsLinked to original sources
PubMed1991

Comparison of initial yields of DNA-to-protein crosslinks and single-strand breaks induced in cultured human cells by far- and near-ultraviolet light, blue light and X-rays.

The initial yields of DNA-to-protein crosslinks (dpc) caused by ionizing and nonionizing radiations were compared, with emphasis upon values within the biological dose ranges (D0). Induction of dpc in cold (0-0.5 degrees C) human P3 teratocarcinoma cells was measured by using alkaline elution techniques after exposure to monochromatic UVC (254 nm), UVB (313 nm), UVA (365 and 405 nm), and blue light (434 nm). UVC and UVB light induced detectable numbers (about 100 dpc per cell per D0). Monochromatic UVA radiations produced yields about 8 times higher than UVC or UVB (for 365 nm, about 1500 dpc per cell per D0) Similar results at low doses were obtained for measurements of single-strand breaks induced by the different radiations. The action spectra for dpc were closely similar. The biological significance of these relatively high numbers of DNA lesions caused by environmental nonionizing radiation that readily penetrates into human skin is not understood.

Cells, Cultured↗
PubMed1991

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.

Catalase↗
PubMed1991

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)

Animals↗
PubMed1991

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.

Cell Line↗
PubMed1991

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)

Animals↗
PubMed1991

Comparison of repair of DNA double-strand breaks caused by neutron or gamma radiation in cultured human cells.

The dose-response for the induction of initial double-strand breaks (dsb) in DNA of human epithelioid cells by JANUS 0.85 MeV fission-spectrum neutrons was parabolic as assayed by a calibrated neutral filter elution technique. The relative biological effectiveness (RBE) of these neutrons relative to 60Co gamma-rays was unity. The kinetics of repair after a 60 Gy gamma-ray exposure were biphasic. About 65% of these dsb were rapidly repaired (T 1/2 of approximately 2 min), and the remainder were almost completely removed after 150 min at a slower rate (T 1/2 = 30 min). After the same dose of JANUS neutrons, the rapid repair component was markedly reduced (possibly not a significant repair component), and the bulk of the dsb were sealed more slowly (T 1/2 = 90 min). After 150 min, 25% remained unsealed. Even after a lower neutron dose (20 Gy), a proportion of the dsb were refractory to repair. Thus, unrepaired (or irreparable) dsb induced by high energy neutrons might explain the high RBE of neutrons for cell killing.

Cell Line↗
PubMed1990

Photosensitized damage to supercoiled plasmid DNA induced by 334-nm radiation in the presence of 2-thiouracil consists of alkali- and piperidine-labile sites as well as frank strand breaks.

A covalently closed, supercoiled plasmid was irradiated with 334-nm ultraviolet radiation in the presence of the naturally occurring photosensitizer 2-thiouracil (s2Ura). After irradiation, some DNA samples were treated to reveal labile sites. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed forms, and the DNA bands were quantitated by fluorescence scanning. Irradiation of the plasmid in the absence of s2Ura induced small numbers of frank DNA strand breaks (FSB), alkali-labile sites (ALS), and piperidine-labile sites (PLS). The induction of each of these lesions was enhanced 30 times when s2Ura was present during aerobic irradiation. Anoxia, as well as the hydroxyl radical scavengers acetate and formate, inhibited the formation of all three lesion types. The relative proportions of the three lesion types produced by several DNA damaging treatments were measured. Hydrogen peroxide, gamma-irradiation, and s2Ura photosensitization produced nearly identical damage proportions, with PLS: FSB ratios of 1.25:1, 0.78:1, and 0.84:1, respectively. Treatment with singlet oxygen [data from Blazek et al. (1989) Photochem. Photobiol. 48, 607-613] produced much different proportions, with a PLS:FSB ratio of 4.1:1. These results may indicate a role for hydroxyl radical in s2Ura-photosensitized DNA damage.

DNA Damage↗
PubMed1990

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.

Cell Line↗
PubMed1990

Induction and repair of DNA strand breaks in Bacteroides fragilis.

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.

Anaerobiosis↗
PubMed1990

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.

Acetates↗
PubMed1990

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.

Animals↗
PubMed1989

Effects of UV radiation of cells.

UV radiation interacts with mammalian cells in a very complex manner, although DNA appears to be the main chromophore. Recent literature within this field is reviewed. The review is concentrated on the following main topics: Chromophores for UV action, photoproducts in DNA, repair of UV-induced DNA damage, wavelength interactions, inactivation, mutagenesis, transformation and protection mechanisms against UV damage.

Animals↗
PubMed1989

Chlorpromazine reduces UV-induced squamous cell carcinogenesis in hairless mice and enhances UV-induced DNA damage in cultured cells.

Administration of the photoactivable compound chlorpromazine (CPZ) to SKH-1 hairless mice via their drinking water (CPZ, 0.01%) significantly reduced the rates of accumulation and yields of squamous cell carcinomas induced by long-term repeated exposures of these animals to solar UV radiation. This protective effect of CPZ was partially reversed in mice given a single injection of ethyl nitrosourea at birth. In in vitro studies, the presence of CPZ (0.2 mM) in mammalian cell cultures enhanced the yield of DNA single-strand breaks induced in the cells by exposure to monochromatic UVA radiation at 334 nm. Collectively, the results suggest that CPZ may exert antineoplastic effects against UV-induced skin tumours by the induction of DNA damage.

Animals↗
PubMed1989

DNA damage and repair in rodent and human cells after exposure to JANUS fission spectrum neutrons: a minor fraction of single-strand breaks as revealed by alkaline elution is refractory to repair.

We have examined the induction and repair of breaks induced in the DNA of Chinese hamster V79 and human P3 epithelial teratocarcinoma cells by JANUS fission-spectrum neutrons (mean energy 0.85 MeV) and 60Co gamma radiation in the biological dose range, using alkaline filter elution methods. Fission-spectrum neutrons induce fewer immediate single-strand breaks (ssb) per gray of absorbed dose than do gamma rays, as measured by alkaline elution methods. Previous survival measurements have indicated incomplete recovery after neutron exposures. The present data demonstrate that whereas most ssb caused by exposure to fission-spectrum neutrons can be rapidly repaired by both cell lines, a small but statistically significant fraction of the ssb induced by exposure to 6 Gy of neutrons is refractory to repair. In contrast, all measurable ssb induced by 3 Gy gamma rays are rapidly repaired.

Animals↗
PubMed1989

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.

DNA Damage↗
PubMed1989

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.

Alkalies↗