USE OF THE DYE HOECHST 33258 IN A MODIFICATION OF THE BROMODEOXYURIDINE PHOTOLYSIS TECHNIQUE FOR THE ANALYSIS OF DNA REPAIR

Abstract— A modification of the bromodeoxyuridine photolysis technique is described in which the cells are treated with the fluorescent dye Hoechst 33258 to enhance the bromodeoxyuridine photolysis produced by 365 nm or by black light. Use of the modified assay overcomes several difficulties encountered in the original 313 nm photolysis technique and demonstrates that excision repair of damage produced in the DNA by 7,12-dimethylbenz(fl)anthracene 5,6-oxide takes place by a long patch type of repair pathway.     

INDUCTION OF DNA-PROTEIN CROSS-LINKS IN CHINESE HAMSTER CELLS BY THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT

Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivation of Chinese hamster V79 cells. In order to investigate possible molecular mechanisms in the photo-dynamic action of CAPC and visible light, the induction and repair rate of two classes of DNA lesions have been determined, i.e. DNA single-strand breaks and DNA-protein cross-links. In cells pretreated with 1 μ.M CAPC, a fluence of 12 kJ/m² of red light (>600 nm) kills approximately 50% of the cells and induces 3 to 3.5 Gy-equivalents of single-strand breaks. The repair of these breaks was slower than the repair of single-strand breaks induced by -irradiation. The photodynamic action of CAPC also induces a large number of DNA-protein cross-links which, in contrast to -radiation-induced DNA-protein cross-links, do not appear to be repaired during 4 h of post-treatment incubation in fresh medium. These studies suggest that DNA may be an important target for the cytotoxicity of CAPC + red light.     

Induction of Chromosome Aberrations and Delayed Genomic Instability by Photochemical Processes

Exponentially growing cells cultured in medium containing bromodeoxyuridine, then exposed to UVA light in the presence of the dye Hoechst 33258, show significant levels of DNA strand breaks and base damage. This dye–bromodeoxyuridine–UVA photolysis treatment is markedly cytotoxic. We now demonstrate that exposure of cells to the agents used in photolysis leads directly to the formation of chromosome aberrations. Furthermore, we demonstrate that this photochemical treatment induces delayed chromosomal instability in clonal populations derived from single progenitor cells surviving photolysis. These results suggest that photolysis-induced DNA damage leads to chromosome rearrangements that could account for the observed cytotoxicity. Furthermore, in those cells surviving photolysis, the delayed effects of this treatment can be observed several generations after exposure and are manifested as compromised genomic integrity.     

SITES SENSITIVE TO S1 NUCLEASE and DISCONTINUITIES IN DNA NASCENT STRANDS OF ULTRAVIOLET IRRADIATED MOUSE CELLS

Abstract Mouse 3T3 cells irradiated with ultraviolet light synthesize DNA containing sites sensitive to the single-strand specific SI nuclease. The appearance of these sites correlates well with the presence of discontinuities in nascent strands, detected by the methodology of sedimentation in alkaline sucrose gradient. Thus, both the sites sensitive to SI nuclease and the discontinuities in nascent strands (i) are stabilized by caffeine; (ii) are no longer formed late after irradiation and (iii) disappear faster when a certain UV fluence is split into two fluences whose sum equals the single fluence. Moreover, the recovery in synthesizing DNA without SI sensitive sites is not dependent on excision repair of pyrimidine dimers or on continuous DNA synthesis. These SI sensitive sites are exclusive of replicative structures of irradiated cells and should correspond to stretches of single-strand DNA (gaps) formed during replication.     

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.     

PHOTOCHEMISTRY OF THE BISBENZIMIDAZOLE DYE 33258 HOECHST WITH BROMODEOXYURIDINE AND ITS BIOLOGICAL EFFECTS ON BrdUrd-SUBSTITUTED ESCHERICHIA COLI

Abstract— Exposure of BrdUrd-substituted E. coli cells to 360 nm light in the presence of the bisbenzimi-dazole dye 33258 Hoechst increases their sensitivity dramatically. Mutant cells deficient in excision repair of DNA damage ( uur B) are more sensitive than wild type cells, indicating that the cells are able to repair this type of damage. However, they perform only a limited amount of liquid holding recovery (LHR). Exposure of the dye with BrdUrd to near UV light in solution results in the appearance of two BrdUrd derived photoproducts. One appears to be deoxyuridine, and the other — an adduct of BrdUrd-dye. The adduct is acid labile and as a result only uracil is observed in acid-hydrolyzates of DNA after exposure of BrdUrd-substituted cells to 360 nm light in the presence of 33258 Hoechst. The production of uracil is linearly dependent on light exposure. Cells in which 85% of thymidine was replaced by BrdUrd are unable to remove more than 5–10% of uracil from their DNA during postirradiation incubation. However, when only 4% of thymidine is replaced, about 50% of the uracil is removed during 30min incubation after exposure. The results are consistent with our previous work, indicating that BrdUrd interferes with repair via excision-resynthesis. A working hypothesis is suggested to explain this interference.     

ACETONE-SENSITIZED PHOTOINACTIVATION OF TRANSFORMING DNA

Abstract— Irradiation of Haemophilus influenzae transforming DNA with 313 nm radiation in the presence of acetone leads to inactivation of transforming activity. Some of the lesions produced are substrate for photoreactivating enzyme and the dark-repair mechanism. In addition, other lesions are produced which, if their production is not prevented by the presence of EDTA during irradiation, render the DNA less photoreactivable. The possibility is discussed that, among others, single-strand breaks are responsible for this loss in photoreactivability.     

8-METHOXYPSORALEN-PHOTOINDUCED DNA CROSSLINKS AS DETERMINED IN YEAST BY ALKALINE STEP ELUTION UNDER DIFFERENT REIRRADIATION CONDITIONS. RELATION WITH GENETIC EFFECTS

Abstract— DNA damage induced by 8-methoxypsoralen (8-MOP) plus near UV light (UVA) was analyzed in diploid yeast using the alkaline step elution technique. The presence of 8-MOP and UVA induced DNA interstrand cross-links was revealed by the increase of DNA retained on elution filters as compared to untreated controls. The fraction of DNA retained on filters increased linearly with UVA dose. The amount of cross-links was estimated from the fraction of DNA retained on filters using a dose of -radiation leading to a number of DNA strand breaks at least equivalent to the number of 8-MOP induced photoadducts.
When 8-MOP treated cells were exposed to monochromatic light, 365 nm light induced monoadducts and cross-links whereas 405 nm light induced only monoadducts. When submitting 8-MOP plus 405 nm light treated cells to 365 nm irradiation, after removal of unbound 8-MOP by washing, a portion of 8-MOP plus 405 nm light induced monoadducts was converted into cross-links. The amount of monoadducts transformed into cross-links was dependent on the dose of 365 nm irradiation up to a maximum likely to correspond to the number of suitably positioned furan-side monoadducts that could be converted into biadducts. When 8-MOP plus 365 nm light treated cells were reirradiated with 365 nm light, following the same protocol, the maximum level of cross-linking obtainable in yeast was lower than that obtained with 8-MOP in a 405 nm plus 365 nm reirradiation protocol.
In the presence of 8-MOP single exposures to 405 nm light were found to be only slightly genotoxic. However, when followed by second exposures to 365 nm light, a dose-dependent increase in genetic effects, i.e. mutation and gene conversion, was observed in parallel to the induction of DNA crosslinks. These results stress again the prominent role of DNA cross-links in the genotoxicity of 8-MOP.     

Single-strand breaks in the DNA of human cells exposed to visible light from phototherapy lamps in the presence and absence of bilirubin

Clinical evidence indicates that phototherapy of hyperbilirubinaemia in newborn infants is a safe and efficient form of therapy. The short-term side effects are not serious and seem to be well controlled. There are few long-term follow-up studies of phototherapy-treated infants. Therefore one cannot completely exclude the possibility that side effects can be found in future studies. With this background we undertook the present study of possible genotoxic effects of phototherapy. Human cells of the established glioblastoma cell line TMG-1 were used. The cells were exposed to visible light in the presence of different concentrations of bilirubin or in the absence of bilirubin. DNA was unwound in alkaline solution and the induction of strand breaks was assayed by a method taking advantage of the fluorescence from the dye Hoechst 33258. Blue light induced single-strand breaks in the DNA of cells in culture in the absence of bilirubin. During irradiation of bilirubin solutions with blue and green phototherapy light, long-lived toxic photoproducts were formed under in vitro conditions. At high and clinically relevant bilirubin concentrations, the effects of blue and green light were relatively similar. At low concentrations, there was a smaller effect of green light as expected from the absorption spectrum of bilirubin. It remains to be seen whether the genotoxic effect observed in the present studies can occur in vivo.     

SINGLE-STRAND BREAKS IN THE DNA OF THE uvrA AND uvrB STRAINS OF ESCHERICHIA COLI K-12 AFTER ULTRAVIOLET IRRADIATION

Abstract— DNA single-strand breaks were produced in uvrA and uvrB strains of E. coli K-12 after UV (254 nm) irradiation. These breaks appear to be produced both directly by photochemical events, and by a temperature-dependent process. Cyclobutane-type pyrimidine dimers are probably not the photoproducts that lead to the temperature-dependent breaks, since photoreactivation had no detectable effect on the final yield of breaks. The DNA strand breaks appear to be repairable by a process that requires DNA polymerase I and polynucleotide ligase, but not the recA, recB, recF, lexA 101 or uvrD gene products. We hypothesize that these temperature-dependent breaks occur either as a result of breakdown of a thermolabile photoproduct, or as the initial endonucleolytic event of a uvrA , uvrB -independent excision repair process that acts on a UV photoproduct other than the cyclobutane-type pyrimidine dimer.     

DNA polymerase I-mediated repair of 365 nm-induced single-strand breaks in the DNA of Escherichia coli.

Abstract. Irradiation of closed circular phage Λ DNA in vivo at 365 nm results in the induction of single-strand breaks and alkali-labile lesions at rates of 1.1 × 10^-14, and 0.2 × 10^-14/dalton/J/m², respectively. The sum of the induction rates is similar to the rate of induction of single-strand breaks plus alkali-labile lesions (1 × 10^-14/dalton/J/m²) observed in the E. coli genome. Postirradiation incubation of wild-type cells in buffer results in rapid repair of the breaks (up to 80% repaired in 10 min). No repair was observed in a DNA polymerase I-deficient mutant of E. coli.     

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)     

Fluorometric analysis of DNA unwinding (FADU) as a method for detecting repair-induced DNA strand breaks in UV-irradiated mammalian cells

Fluorometric analysis of DNA unwinding (FADU assay) was originally designed to detect X-ray-induced DNA damage in repair-proficient and repair-deficient mammalian cell lines. The method was modified and applied to detect DNA strand breaks in Chinese hamster ovary (CHO) cells exposed to ionizing radiation as well as to UV light. Exposed cells were allowed to repair damaged DNA by incubation for up to 1 h after exposure under standard growth conditions in the presence and in the absence of the DNA synthesis inhibitor aphidicolin. Thereafter, cell lysates were mixed with 0.15 M sodium hydroxide, and DNA unwinding took place at pH 12.1 for 30 min at 20 degrees C. The amount of DNA remaining double-stranded after alkaline reaction was detected by binding to the Hoechst 33258 dye (bisbenzimide) and measuring the fluorescence. After exposure to X-rays DNA strand breaks were observed in all cell lines immediately after exposure with subsequent restitution of high molecular weight DNA during postexposure incubation. In contrast, after UV exposure delayed production of DNA strand break was observed only in cell lines proficient for nucleotide excision repair of DNA photoproducts. Here strand break production was enhanced when the polymerization step was inhibited by adding the repair inhibitor aphidicolin during repair incubation. These results demonstrate that the FADU approach is suitable to distinguish between different DNA lesions (strand breaks versus base alterations) preferentially induced by different environmental radiations (X-rays versus UV) and to distinguish between the different biochemical processes during damage repair (incision versus polymerization and ligation).     

PHOTOREACTIVATION and EXCISION REPAIR OF UV INDUCED PYRIMIDINE DIMERS IN THE UNICELLULAR CYANOBACTERIUM GLOEOCAPSA ALPICOLA (SYNECHOCYSTIS PCC 6308)

Abstract— The survival curve obtained after UV irradiation of the unicellular cyanobacterium Synecho-cystis is typical of a DNA repair competent organism. Inhibition of DNA replication, by incubating cells in the dark, increased resistance to the lethal effects of UV at higher fluences. Exposure of irradiated cells to near ultraviolet light(350–500 nm) restored viability to pre-irradiation levels. In order to measure DNA repair activity, techniques have been developed for the chromatographic analysis of pyrimidine dimers in Synechocystis. The specificity of this method was established using a haploid strain of Sacchar-omyces cerevisiae. In accordance with the physiological responses of irradiated cells to photoreactivating light, pyrimidine dimers were not detected after photoreactivation treatment. Incubation of irradiated cells under non-photoreactivating growth conditions for 15 h resulted in complete removal of pyrimidine dimers. It is concluded that Synechocystis contains photoreactivation and excision repair systems for the removal of pyrimidine dimers.     

Exacerbating effect of vitamin E supplementation on DNA damage induced in cultured human normal fibroblasts by UVA radiation

The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320-380 nm) (UVA). Cells were incubated in medium containing alpha-tocopherol, alpha-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2.-) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2.- reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.     

THE EFFECT OF TEMPERATURE AND WAVELENGTH ON PRODUCTION AND PHOTOLYSIS OF A UV-INDUCED PHOTOSENSITIVE DNA LESION WHICH IS NOT REPAIRED IN XERODERMA PIGMENTOSUM VARIANT CELLS

Abstract— Ultraviolet light causes a type of damage to the DNA of human cells that results in a DNA strand break upon subsequent irradiation with wavelengths around 300 nm. This DNA damage disappears from normal human fibroblasts within 5 h, but not from pyrimidine dimer excision repair deficient xeroderma pigmentosum group A cells or from excision proficient xeroderma pigmentosum variant cells. The apparent lack of repair of the ultraviolet light DNA damage described here may contribute to the cancer prone nature of xeroderma pigmentosum variant individuals. These experiments show that the same amount of damage was produced at 0^°C and 37^°C indicating a photodynamic effect and not an enzymatic reaction. The disappearance of the photosensitive lesions from the DNA is probably enzymatic since none of the damage was removed at 0^°C. Both the formation of the lesion and its photolysis by near ultraviolet light were wavelength dependent. An action spectrum for the formation of photosensitive lesions was similar to that for the formation of pyrimidine dimers and(6–4) photoproducts and included wavelengths found in sunlight. The DNA containing the lesions was sensitive to wavelengths from 304 to 340 nm with a maximum at 313 to 317 nm. This wavelength dependence of photolysis is similar to the absorption and photolysis spectra of the pyrimidine(6–4) photoproducts     

PHOTODYNAMIC DAMAGE AND ITS REPAIR IN Vibrio cholerae

Abstract— Repair of photodynamic damage induced by acriflavine and visible light has been examined in three strains of Vibrio cholerae differing in their capabilities to repair ultraviolet (UV) light induced DN A damage. Excision repair deficient wild type cells of strain 154 are more sensitive to photodynamic treatment compared to repair proficient cells of strain 569B. However, no difference in their capabilities to repair of damage following photodynamic treatment can be detected. No single-strand breaks in the irradiated cell DNA are observed when the cell survival is more than 10%. Single-strand breaks observed at cell survival less than 5% are not dark repairable even in excision repair proficient wild type cells. Repair of membrane damage can partially account for the recovery observed at low doses. In contrast, radiation-sensitive mutant 569B_s cells which lack both excision and medium-dependent dark repair for UV-lesions are most efficient in repairing damage induced by photodynamic treatment.     

DNA SINGLE-STRAND LESIONS DUE TO ''SUNLIGHT'' AND UV LIGHT: A COMPARISON OF THEIR INDUCTION IN CHINESE HAMSTER AND HUMAN CELLS, AND THEIR FATE IN CHINESE HAMSTER CELLS

Abstract— It has been shown that the lethal properties of germicidal UV light (254 nm) and sunlight-simulating near UV light are qualitatively different (Elkind et al ., 1978). Further to compare these two radiations, the induction of single-strand DNA breaks (i.e. frank breaks plus alkali-labile lesions) was measured in two cell lines. Equal numbers of breaks in Chinese hamster cells require a dose of UV 5.5% of a near UV dose but in HeLa cells a UV dose of 7.6% of a near-UV dose is required. The rate of break production by these radiations is about 1/10-th of that due to X-rays when a comparison is made on an equal killing dose basis. The inventory of breaks in Chinese hamster cells was also followed and was found to be characteristically different for UV compared to near UV light. These data indicate that significant differences exist, at a molecular level, in the effects produced by ultraviolet and sunlight-simulating light, and further emphasize the need for caution in attempting to extrapolate from observed molecular or biological effects due to the former to those to be expected from the latter.     

CU(II) SENSITIZES pBR322 PLASMID DNA TO INACTIVATION BY UV-B(280–315 nm)

Abstract— Copper(II), in the presence of UV-B radiation(280–315 nm), can generate single-strand breaks in the sugar-phosphate backbone of pBR322 plasmid DNA. A low level of single-strand backbone breaks occurs in the presence of Cu(II) alone, but UV-B irradiation increases the rate by the more than 100-fold. Concomitant with the damage to the DNA backbone is a loss of transforming activity. Oxygen is required for generation of the single-strand breaks but not for the loss of transforming activity. A DNA glycosylase (Fpg), which participates in the repair of certain DNA nitrogenous base damage, does not repair plasmid DNA damaged by Cu(II). The hydroxyl radical scavenging compound DMSO is only somewhat effective at protecting the physical and biological properties of the DNA. These results with Cu(II) are compared to those obtained previously with pBR322 plasmid DNA in the presence of Fe(III) and UV-A.     

POSTREPLICATION REPAIR IN AN EXCISION-DEFECTIVE MUTANT OF ESCHERICHIA COLI: ULTRAVIOLET LIGHT-INDUCED INCORPORATION OF BROMODEOXYURIDINE INTO PARENTAL DNA*

Abstract— Ultraviolet (UV) light-induced incorporation of bromodeoxyuridine (BrdUrd) into parental DNA of an excision-defective mutant of Escherichia coli has been observed by selective photolysis of bromouracil (BrUra)-containing regions in the parental DNA. It appears that the BrUra-containing regions occur only in that DNA which has served as a template for normal semiconservative replication. After an exposure at 254 nm which results in one pyrimidine dimer per 45times 10⁶ daltons, incubation in BrdUrd resulted in BrUra–containing regions ˜ 1.5 times 10⁴ nucleotides in length at intervals of ˜ 55 times 10⁶ daltons in the parental DNA. Thus approximately one BrUra-containing region has occurred for every 1.2 pyrimidine dimers in the parental DNA. The observed incorporation of BrdUrd is interpreted in terms of a proposed model for postreplication repair in which genetic exchanges produce single-strand gaps in the parental DNA.