BASE-SPECIFIC DAMAGE INDUCED BY 4-THIOURIDINE PHOTOSENSITIZATION WITH 334-nm RADIATION IN M13 PHAGE DNA

Abstract— Site-specific DNA damage caused by 334-nm radiation in the presence of the rare Escherichia coli base 4-thiouridine was investigated in vitro by detecting the sites of the termination of DNA synthesis with irradiated M13 phage DNA used as a template. Single-strand breakage was also examined. The results indicate that 334-nm radiation at very low fluences in the presence of 4-thiouridine induces termination of strand synthesis at thymine base sites and at the base immediately prior to thymine. Termination at these sites was diminished by treatment with hot piperidine. Strand cleavage by piperidine treatments was observed preferentially at the guanine site, but only after irradiation at much larger fluences. It is hypothesized that at low fluences 4-thiouridine forms photoadducts with thymine that block DNA synthesis, while at high fluences the guanine site is damaged via oxygen species.     

OBSERVATIONS ON THE PHOTOSENSITIZED BREAKAGE OF DNA BY 2-THIOURACIL AND 334-nm ULTRAVIOLET RADIATION

Abstract— Breaks induced in purified DNA by 334-nm ultraviolet (UV) radiation are enhanced 30 times when 2-thiouracil (s²Ura) is present during aerobic irradiation. This enhancement by s²Ura is maximally effective at a concentration of about 1 m M. Anoxic irradiation reduces the s²Ura-enhanced breakage by 90%, indicating a Type II photosensitization. Benzoate, glycerol, diazabicyclo[2.2.2.]octane (DABCO) and histidine all inhibit formation of s²Ura photosensitized breaks, unlike diethylenetriaminepenta-acetic acid (DETAPAC) and catalase, which do not. The relationships between the concentration of DABCO. benzoate and histidine and their protection against induction of single strand breaks (SSBs) were similar, with little inhibition below 10 m M and maximal inhibition near 0.1 M for all compounds. Irradiation of the DNA-s²Ura mixture dissolved in D₂O instead of H₂O enhanced the rate of induction of SSBs in DNA by 334-nm light almost five times. Addition of superoxide dismutase (40, 80 and 200 μg/ml) decreased the rate of induction of breaks in DNA by 334-nm radiation plus s²Ura (in H₂O) by about 40%. Boiled superoxide dismutase had no effect.     

THE ROLE OF 4-THIOURIDINE IN LETHAL EFFECTS AND IN DNA BACKBONE BREAKAGE CAUSED BY 334 nm ULTRAVIOLET LIGHT IN Escherichia coli

Strains of Escherichia coli that lack 4-thiouridine (⁴Srd) are killed by monochromatic 334 nm UV light (UV) less efficiently than their wild-type parents, which contain ⁴Srd. Oxygen enhancement ratios (OER) at 10% survival are 3.3 for a strain that possesses ⁴Srd, and 2.6 for one that lacks ⁴Srd. Single-strand breaks in DNA caused by 334 nm UV accumulate more than twice as fast in the wild-type strains than in the strains lacking ⁴Srd. The results suggest that ⁴Srd is an important chromophore in some near-UV lethal effects. The results also suggest that the excitation energy from 334 nm UV light may be passed from RNA to DNA, resulting in single-strand breaks.     

INACTIVATION BY MONOCHROMATIC NEAR-UV RADIATION OF AN Escherichia coli hemA8 MUTANT GROWN WITH AND WITHOUT δ-AMINOLEVULINIC ACID: THE ROLE OF DNA vs MEMBRANE DAMAGE

Abstract— Escherichia coli strain RT8 hemA8 [blocked in biosynthesis of δ-aminolevulinic acid (δ-ALA), and unable to manufacture porphyrins unless exogenously supplied with δ-ALA] is inactivated more efficiently by monochromatic 334- and 405-nm radiations if the cells are grown with δ-ALA supplementation. The fiuence enhancement factor for δ-ALA sensitization is larger for light at 405 nm than at 334 nm. Both irradiation conditions (plus or minus δ-ALA) showed prominent oxygen enhancement ratios, which were also larger at 405 nm than at 334 nm. At 334 nm, δ-ALA supplementation did not affect the accumulation of DNA breaks, while at 405 nm, the induction of DNA breaks doubled for cells supplemented with δ-ALA. Rubidium leakage caused by 405-nm radiation occurred at a smaller fiuence in cells supplemented with higher concentrations of δ-ALA than in cells supplemented with a lower concentration. The results suggest that (1) porphyrin derivatives may have a role in cell killing by near-UV radiations, and (2) damage to cytomembranes may be a critical lesion produced by these events, whereas DNA breakage may not.     

PHOTOSENSITIZED INACTIVATION OF DNA BY MONOCHROMATIC 334-nm RADIATION IN THE PRESENCE OF 2-THIOURACIL: GENETIC ACTIVITY AND BACKBONE BREAKS

Abstract Monochromatic 334-nm radiation delivered under aerobic conditions inactivates the genetic activity (ability to transform auxotrophic recipient cells to nutritional prototrophy) of isolated transforming Bacillus subtilis DNA. The presence of superoxide dismutase (SOD), catalase, and mannitol reduces the 334-nm inactivation. The rate of inactivation of the genetic activity by 334-nm radiation is enhanced fivefold by the sensitizer 2-thiouracil (s²Ura). This enhancement is substantially reversed when the irradiations are performed in the presence of mannitol, and, to a lesser extent, SOD. Catalase slightly reduces the s²Ura enhancement of 334-nm inactivation of transforming activity. Backbone breaks induced in the same DNA by aerobic 334-nm radiation were also enhanced markedly by the presence of s²Ura; this enhancement was reversed by the presence of mannitol and, to a lesser extent, SOD during irradiation. Catalase had no effect upon s²Ura-enhanced, 334-nm-induced SSBs. Whereas DNA breakage may be responsible for a portion of the inactivation of the DNA by the photosensitized reaction between s²-Ura and 334-nm radiation, it is not the only inactivating lesion, because the yield of SSBs per lethal hit per unit length of DNA is not constant for all the irradiation conditions studied. The results support a complex role for active oxygen species in inactivation of transforming activity and DNA breakage by s²Ura-enhanced 334-nm radiation. They are also consistent with the formation of superoxide anion, hydroxyl radical, and possibly also singlet molecular oxygen, generated from ground-state molecular oxygen by reactive s²Ura in both Type I and II reactions.     

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.     

INDUCTION OF DIRECT AND INDIRECT SINGLE-STRAND BREAKS IN HUMAN CELL DNA BY FAR- AND NEAR-ULTRAVIOLET RADIATIONS: ACTION SPECTRUM AND MECHANISMS

Abstract— An action spectrum for the immediate induction in DNA of single-strand breaks (SSBs, frank breaks plus alkali-labile sites) in human P3 teratoma cells in culture by monochromatic 254-, 270-, 290-, 313-, 334-, 365-, and 405-nm radiation is described. The cells were held at +0.5C during irradiation and were Iysed immediately for alkaline sedimentation analysis following the irradiation treatments. Linear fluence responses were observed over the fluence ranges studied for all energies. Irradiation of the cells in a D₂O environment (compared with the normal H₂O environment) did not alter the rate of induction of SSBs by 290-nm radiation, whereas the D₂O environment enhanced the induction of SSBs by 365- and 405-nm irradiation. Analysis of the relative efficiencies for the induction of SSBs, corrected for quantum efficiency and cellular shielding, revealed a spectrum that coincided closely with nucleic acid absorption below 313 nm. At longer wavelengths, the plot of relative efficiency vs . wavelength contained a minor shoulder in the same wavelength region as that observed in a previously obtained action spectrum for stationary phase Bacillus subtilis cells. Far-UV radiation induced few breaks relative to pyrimidine dimers, whereas in the near-UV region of radiation, SSBs account for a significant proportion of the lesions relative to dimers, with a maximum number of SSBs per lethal event occurring at 365-nm radiation.     

SINGLE-STRAND BREAKS INDUCED IN BACILLUS SUBTILIS DNA BY ULTRAVIOLET LIGHT: ACTION SPECTRUM and PROPERTIES

Abstract— The induction of single-strand breaks (alkali-labile bonds plus frank breaks) in the DNA of Bacillus subtilis irradiated in vivo by monochromatic UV light at wavelengths from 254 to 434 nm was measured. The spectrum consists of a major far-UV (below 320 nm) component and a minor near-UV shoulder. A mutant deficient in DNA polymerase I accumulates breaks caused by near-UV (above 320 nm) wavelengths faster than the wild-type strain proficient in polymerase I. Measurable breaks in extracted DNA are induced at a higher frequency than those induced in vivo. Anoxia, glycerol, and diazobicyclo (2.2.2.) octane inhibit break formation in extracted DNA. Alkali-labile bonds induced by 365-nm UV radiation are largely (78%) covalent bond chain breaks, the remainder consists of true alkali-labile bonds, probably apurinic and apyrimidinic sites.     

A5–4 PYRIMIDINE-PYRIMIDONE PHOTOPRODUCT PRODUCED FROM MIXTURES OF THYMINE AND 4-THIOURIDINE IRRADIATED WITH 334 nm LIGHT

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and ¹H- and ¹³C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(β-D-erythro-pentofuranosyl)-4′-pyrimidin-2′-one]dihydrothymine (o⁶hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90d?C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.     

INHIBITION OF UV-INDUCED DNA STRAND BREAKAGE IN DICTYOSTELIUM DISCOIDEUM BY 2,4–DINITROPHENOL

Abstract— In UV-irradiated vegetative cultures of the cellular slime mold Dictyostelium discoideum NC-4, single strand breaks appeared in the DNA very rapidly and at low temperatures (0–4°C). However, when these cells were incubated, prior to UV irradiation, in the presence of 2 m M 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, breaks did not appear in the DNA. Extracts prepared from cells that had been incubated either in the presence or absence of DNP were tested for endonucleolytic activities on a UV-irradiated exogenous DNA template (φX-174 RF I). Results suggested that DNP might mediate its effect by interfering with the action of a UV-specific endonuclease.     

PHOTOCHEMISTRY OF 4-THIOURIDINE AND THYMINE

Abstract— When thymine is irradiated in aqueous solution with monochromatic 334-nm UV radiation in the presence of 4-thiouridine a photoproduct of thymine is formed, as shown by thin-layer chromatography and autoradiography. The quantum yield for the formation of thymine photoproduct (θ=0.017) is greater than that for cytosine photoproduct formation (θ= 0.0015). The identity of the photoproduct is not known: one possibility is the formation of an adduct between the sensitizer and the base yielding a pyrimidine-pyrimidone type of photoproduct.     

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.     

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.     

Generation of thiyl radicals by the photolysis of 5-iodo-4-thiouridine

The photochemistry of 2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine (3) in deoxygenated 1:1 CH(3)CN-H(2)O pH 5.8 (phosphate buffer) solution has been studied by means of steady-state and nanosecond laser flash photolysis methods. Under steady-state irradiation (lambda > or = 334 nm), the stable photoproducts were iodide ion, 2',3',5'-tri-O-acetyl-4-thiouridine (4), and two disulfides. The disulfides were the symmetrical bis-(2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine) (5) and unsymmetrical 6, which contains both 4-thiouridine and 5-iodo-4-thiouridine residues. The formation of the dehalogenated photoproduct suggests that C(5)-I bond cleavage is a primary photochemical step. Attempts to scavenge the resulting C(5)-centered radical by suitable addends, bis-(N-alpha-acetyl)cystine-bis-N-ethylamide or benzene, were unsuccessful. Analysis of the photoproducts formed under these conditions showed that the S-atom is the reactive center. The photoproduct 4, obtained by irradiation of 3 in CD(3)CN-H(2)O, followed by reversed-phase HPLC isolation using nonlabeled eluents, did not contain deuterium. An analogous experiment performed in CH(3)CN-D(2)O gave deuterated product 4-d with 88% of the deuterium incorporated at C(5). Transient absorption observed upon laser excitation (lambda= 308 nm) of 3 was assigned to the 4-uridinylthiyl radical on the basis of the similarity of this spectrum with that obtained upon laser photolysis of the disulfide: bis-(2',3',5'-tri-O-acetyl-4-thiouridine) 14. On the basis of the results of steady-state and laser photolysis studies, a mechanism of the photochemical reaction of 3 is proposed. The key mechanistic step is a transformation of the C(5)-centered radical formed initially by C(5)-I bond cleavage into a long-lived S-centered radical via a 1,3-hydrogen shift. Theoretical calculations confirmed that the long-lived S-centered radical is the most stable radical derived from the 4-thiouracil residue.     

REDUCTION BY NEAR-ULTRAVIOLET (334 nm) LIGHT OF E. COLI CAPACITY FOR PHAGE GROWTH DEPENDS UPON THE rel GENE AND 4-THIOURIDINE

Abstract— Near-ultraviolet radiation (near UV; 300–380 nm) has long been known to produce a transient reduction of the capacity of bacteria to support phage growth. The present work shows that, at high fluenœs (40–100 kJ/m²), 85% of 334-nm-induced reduction of capacity in Escherichia coli B/r requires the rel gene; that is, it results from rel -gene activity caused by the near-UV treatment. This rel -gene activity leads to (1) a bacterial growth delay and concomitantly lowered bacterial metabolism, and (2) a parallel delay in phage development, with a considerable depression of burst size. We propose that the observed effects on phage development are a consequence primarily of the lowered bacterial metabolism, but they may also result partly from a direct inhibition of phage DNA synthesis by the rel gene product, these effects together leading to the observed reduction of capacity in a rel ⁺ strain. The remaining 15% of capacity reduction, observed in a rel strain, has an unknown mechanism, but does appear to involve a delay in phage development.
At least 95% of the total capacity reduction observed in the rel ⁺ strain in the range 40–100 kJ/m² requires the presence of 4-thiouridine, an unusual base in E. coli transfer RNA, which is presumably both the chromophore and the target for near-UV-induced capacity reduction.     

RNA PROTEIN CROSSLINKS INTRODUCED INTO E. coli RIBOSOMES BY USE OF THE INTRINSIC PROBE 4-THIOURIDINE

Abstract— 70S Ribosome substituted by the uridine photoactivable analogue 4-thiouridine has been prepared by an in vivo method (substitution level 4.5%). The r-proteins crosslinked to 16S and 23S rRNA before and after 366-nm photoactivation were identified. Proteins S2-S7-S9/11-S18 are found linked to 16S RNA in dark-prepared 30S subunits. Illumination increases uniformly their binding by a factor of 2.5. Similarly, proteins L5-L15-L18-L23-L28-L32 are found crosslinked to 23S RNA in dark-prepared 50S subunits. Photoactivation increases their binding but in addition promotes the covalent linking of proteins L1-L3-L4.     

EFFECTS OF GLYCEROL UPON THE BIOLOGICAL ACTIONS OF NEAR-ULTRAVIOLET LIGHT: SPECTRA AND CONCENTRATION DEPENDENCE FOR TRANSFORMING DNA AND FOR ESCHERICHIA COLI B/r

The concentration dependence for the protection of isolated transforming DNA and Escherichia coli by glycerol against 365-nm monochromatic near-ultraviolet light (UV) was measured. Glycerol protection saturates at a concentration of about 0.1 M for DNA and 1.0 M for E. coli. Action spectra for glycerol protection of transforming DNA (tryptophan and histidine markers) are similar to those obtained previously for diazobicyclo[2.2.2.˜octane (DABCO) protection, with protection reaching a maximum near 350-nm UV and decreasing rapidly at wavelengths above and below 350 nm. However, glycerol protects against near-UV about twice as efficiently as DABCO. The action spectrum for protection of E. coli by glycerol against the lethal effects of near-UV was not the same as the spectrum for DNA since glycerol sensitized the cells, but not the DNA, at wavelengths longer than about 380 nm. A possible role of hydroxyl or other radicals was supported by the observation that benzoate also protected DNA against inactivation by 334-nm UV.     

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)     

NONRECIPROCAL SYNERGISTIC LETHAL INTERACTION BETWEEN 365-nm and 405-nm RADIATION IN WILD TYPE and uvrA STRAINS OF ESCHERICHIA COLI*

Abstract— Lethality by 405-nm radiation in three repair-proficient and two uvrA strains of Escherichia coli that belong to two isogenic series was greatly enhanced by prior exposures to 365-nm radiation at fluences greater than 1 times 10₆Jm_-2. Fluences at 365 nm that yielded a surviving fraction of 0.10 (>1 times 10₆ Jm_-2) in the 5 strains tested resulted in the following 405-nm fluence enhancement factors (FEF, ratio of the 405-nm F₃₇ in the absence of a prior 365-nm irradiation to that in the presence): strain K.12 AB1157 (wild type), 8.7; strain B/r (wild type), 52; strain WP2 (wild type), 25; strain WP2s (uvrA), 13; strain K.12 AB1886 (uvrA), 15. The maximal 405-nm FEF value obtained after a prior 365-nm irradiation at greater fluences was 83 in the wild-type strain B/r. Enhancement of anoxic 405-nm radiation after a prior aerobic 365-nm exposure was not detectable, suggesting that prior aerobic irradiation at 365-nm increased the effects of damage produced at 405 nm by means of an oxygen-dependent process. Single-strand breaks (or alkali-labile bonds) were produced by 405-nm radiation at 3.0 times 10_-5 breaks per 2.5 times 10₉ daltons per Jm_-2 in the polA strain P3478; pyrimidine dimers were not detected by biological assay (photoreactivation) at 405 nm. Although the introduction of different DNA lesions produced by 365- and 405-nm radiations cannot be ruled out, we propose that the strong synergistic effect of 365-nm irradiation on 405-nm lethality is the consequence of pronounced inhibition by 365-nm radiation of components of the DNA-repair systems that can mend or bypass damage produced by 405-nm radiation.     

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.