HOECHST 33258 DYE GENERATES DNA-PROTEIN CROSS-LINKS DURING ULTRAVIOLET LIGHT-INDUCED PHOTOLYSIS OF BROMODEOXYURIDINE IN REPLICATED AND REPAIRED DNA

Abstract— Substitution of bromodeoxyuridine for thymidine in the DNA of mammalian cells sensitizes them to a range of wavelengths of ultraviolet light. Cells are also sensitized to photochemical reactions involving dyes such as Hoechst 33258, which is used to produce differential staining of chromatids according to their bromodeoxyuridine content. Irradiation with 313 nm light of human and hamster cells containing bromodeoxyuridine in their DNA produced single-strand breaks, detectable by alkaline elution, but no DNA-protein cross-links. Irradiation with 360 nm light in the presence of Hoechst 33258 produced extensive DNA-protein cross-linkage as well as single-strand breaks. These cross-links were observed in DNA containing bromodeoxyuridine incorporated by either semiconservative or repair replication, and may provide a method for identification of proteins in close proximity to replication forks or repair sites. When the protein was removed with proteinase K, bromodeoxyuridine in repair patches after irradiation by doses of ultraviolet (254 nm) light as low as 0.26 J/m² could readily be detected. Hoechst 33258-mediated photolysis, therefore, provides a sensitive new technique for measuring repair replication after ultraviolet light irradiation.     

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     

Survival, excision repair and inhibition of DNA synthesis in normal human skin fibroblasts exposed to simulated sunlight.

The responses of normal human skin fibroblasts exposed to simulated sunlight produced by a solar simulator were examined. The parameters investigated were cellular survival, excision repair and the inhibition and recovery of DNA synthesis. The latter two effects were examined using the bromodeoxyuridine photolysis assay and the alkaline step elution assay respectively. The results of these experiments are consistent with the conclusion that the lesions induced by simulated sunlight represent a mixture of damage which elicits cellular responses and repair mechanisms similar to those manifested by cells irradiated with UVC and UVA radiation.     

SPIN-TRAPPING and ESR STUDIES OF THE DIRECT PHOTOLYSIS OF AROMATIC AMINO ACIDS, DIPEPTIDES, TRIPEPTIDES and POLYPEPTIDES IN AQUEOUS SOLUTIONS—II. TYROSINE and RELATED COMPOUNDS

Abstract— The UV-photolysis of peptides containing tyrosine (Tyr) was investigated in aqueous solutions at room temperature at 220 and 265 nm. The short-lived free radicals formed during photolysis were spin-trapped by t-nitrosobutane and identified by electron spin resonance. For N-acetyl- and N-formyl-L-Tyr and for peptides containing L-Tyr as the middle residue, photolysis at 265 nm under neutral conditions produced mainly spin-adducts due to the scission between the alpha carbon and the methylene group attached to the aromatic ring, while at 220 nm decarboxylation radicals were spin-trapped. Photolysis of di- and tripeptides at 275 nm in alkaline solutions predominantly generated deamination radicals. The radicals produced in the photolysis of the oxidized A chain of insulin were tentatively characterized by comparison with the results for di- and tripeptides.     

THE PHOTOLYSIS OF TRYPTOPHAN WITH 337.1 nm LASER RADIATION

Abstract— Aqueous solutions of L-tryptophan were photolyzed by exposure to 337.1 nm radiation from a pulsed nitrogen laser. These data were compared with results for the 290 nm conventional-source photolysis of tryptophan. The progress of photolysis was monitored by fluorescence analysis of tryptophan. UV absorption spectroscopy, HPLC, TLC, and proton NMR spectroscopy. The loss of Trp was observed to be first order for 290 nm photolysis but of mixed order for 337.1 nm photolysis. Five photolysis products were detected by TLC analysis, including: N-formylkynurenine. kynurenine, tryptamine (detected after 290 nm photolysis but not 337.1 nm photolysis) and two unknown products. The tryptophan-containing peptides N-acetyl-tryptophanamide (NATA) and tryptophylglycine (Trp-Gly) were also observed to photolyze upon 337.1 nm laser radiation demonstrating that this phenomenon is not restricted to free tryptophan monomer.
Since Trp is not ordinarily thought to absorb U V radiation at wavelengths as long as 337.1 nm. a number of experiments were performed in an effort to determine the mechanism of photolysis at this wavelength. Evidence is presented which indicates that the 337.1 nm laser photolysis of Trp does not result from two photon absorption, dielectric breakdown, or other laser-specific processes. Instead. it is concluded that this photolysis results either from a very weak absorption tail extending to 337.1 nm in tryptophan itself or from a reaction involving an impurity sensitizer which absorbs the 337.1 nm radiation. The sensitizing impurity. if present, could not. however, be removed by preparative HPLC and could not be detected by TLC or fluorescence analysis.     

THE USE OF SPECIFIC RADIOIMMUNOASSAYS TO DETERMINE ACTION SPECTRA FOR THE PHOTOLYSIS OF (6-4) PHOTOPRODUCTS

Abstract. A radioimmunoassay (RIA) was developed which specifically detects a photoproduct produced by the near-UV photolysis of pyrimidine(6-4)pyrimidone photoproducts. This assay was used in conjunction with a previously characterized RIA which specifically detects (6-4) photoproducts to determine the relative efficiency of wavelengths between 265 and 435 nm for photolysing these lesions. The rate of loss of antibody-binding sites associated with (6-4) photoproducts correlates with the production of those associated with its photolysis product. Action spectra for both the loss of (6-4) photoproducts and the induction of the photolysis product parallel the absorption spectrum of the (6-4) photoproduct.     

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.     

INTERMEDIATES IN THE ROOM TEMPERATURE FLASH PHOTOLYSIS OF ADENINE AND SOME OF ITS DERIVATIVES

The transient absorption spectra of aqueous solutions of adenine, 2′-deoxyadenosine, 2′-deoxyadenosine-5′-phosphate and 2′-deoxyadenylyl-(3′-5′)-2′-deoxyadenosine have been determinated at different pH values using conventional flash photolysis. Reactives intermediates produced in the flash photolysis of these adenine derivatives present similar absorption regions: two higher intensity bands in the UV and 560–720 nm wavelength region and a third weaker band at 420–560 nm. On the basis of the effects produced by triplet quenchers and/or electron scavengers the bands have been assigned to hydrated electrons, radical cations, radical anions and/or neutral radicals resulting from neutralization reactions of the charged radicals. The results indicate that the bases photoionize via a triplet state under these conditions.     

RADICAL CATION PRODUCTION IN THE PHOTOLYSIS OF 5-M-ETHOXY-1-M-ETHYLINDOLE

Abstract— The flash photolysis of 5-m-ethoxy-1-m-ethylindole in aqueous media was studied for the purpose of assigning the absorption spectrum of the radical cation. Transients produced in this study were analogous to those formed in the photolysis of 5-m-ethoxyindole. The major transient observed with an absorption maximum of 460 nm was O₂-s_-ensitive and had a lifetime of 20 μs in nitrogen saturated solutions. One radical species is produced with absorption maxima at 445 and 530 nm. Ionic strength effects on the reaction of this species with I⁻ confirms that it is the radical cation of 5-m-ethoxy-l-methylindole. The effect of H⁺ and Br⁻ on the fluorescence, radical cation and triplet yields is discussed in relation to the mechanism of transient formation.     

PHOTOCHEMISTRY OF BIOLOGICAL MOLECULES-V. A MECHANISTIC STUDY OF THE PHOTOLYSIS OF CYCLOALANYLALANINE IN THE SOLID STATE

Abstract— Earlier studies of the photodamage induced by 254 nm irradiation of linear alanine peptides in the solid state have been supplemented by an investigation into the gaseous photoproducts from the cyclic dipeptide, 3,6-dimethyl-2,5-diketopiperazine. The trans and cis isomers have been prepared and the photoproducts compared with those from the DL-mixture. The conformation of the molecule does influence the yield of gaseous products. CO was produced by peptide bond rupture with concomitant release of hydrogen. CO₂ was also produced. The use of N- and C-deuterated analogues together with relevant crystallographic and EPR data has enabled a detailed study of the mechanism of photode-gradation to be made, from which it is concluded that the methyl protons are not inert but rather are the major source of the hydrogen observed on photolysis.     

ON THE PHOTOSENSITIZING PROPERTIES OF N-FORMYLKYNURENINE AND RELATED COMPOUNDS

Abstract— The photochemical and photosensitizing properties of N -formylkynurenine (FK) and related compounds have been investigated using the laser flash photolysis technique by exciting water solutions with 265 nm or 353 nm radiation. The FK molecules in their first excited singlet state readily react with water leading probably to OH formation. FK triplet state reacts with many biological compounds including vitamins, amino acids and nucleic acid bases. The semi-reduced FK thus formed can, in turn, reduce substrates such as cytochrome c or O₂.     

DNA REPAIR IN V-79 CELLS TREATED WITH COMBINATIONS OF PHYSICAL AND CHEMICAL CARCINOGENS*

Excision repair of DNA damage was measured by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair in Chinese hamster V-79 cells treated with 254 nm of ultraviolet radiation (UV), 7,12-dimethylbenz[a]anthracene 5,6-oxide (DMBA-epoxide), N-acetoxy-2-acetylaminofluorene (AAAF), 4-nitroquinoline 1-oxide (4NQO), 2-methoxy-6-chloro-9-[3(ethyl-2-chloroethyl)-aminopropylamino]acridine dihydrochloride (ICR-170), X-rays, ethylmethanesulfonate (EMS), methyl methanesulfonate (MMS) and combinations of these agents. Compared to normal human cells V-79 were defective in repair of UV lesions and the lesions induced by the UV-mimetic chemicals. The extent of the defects varied from 10 to 50% and was similar to those in Xeroderma pigmentosum group C cells (XP C). V-79 cells repaired X-ray damage and damage from the alkylating agents EMS and MMS to the same extent as human cells. Repair was additive after a combination of UV plus MMS indicating, as expected, that there are different rate-limiting steps for removal of the damages from these agents. Repair was less than additive in cells treated with UV plus ICR-170, AAAF plus ICR-170, AAAF plus 4NQO, and 4NQO plus ICR-170 and approximately equal to that observed for the higher of the two agents separately, indicating that there may be similar rate-limiting steps for removal of lesions. Although the results on repair after combinations of UV plus 4NQO, UV plus DMBA-epoxide or X-rays plus MMS were difficult to interpret, there was not any inhibition of repair in these combinations.     

ON THE MECHANISMS OF PHOTOLYSIS OF COMPOUNDS I AND II OF HORSERADISH PEROXIDASE AT 77 K*

Abstract—When Compounds I and II of horseradish peroxidase in glycerol/water glasses at 77 K were irradiated with the light of a mercury lamp, some changes in optical spectra as well as the development of strong EPR signals of a free-radical type were detected. In case of photolysis of Compound I the light of wavelengths around 400 nm was the most effective, while only short wavelength (≥ 280 nm) light affected Compound II. The results of experiments with thawing and freezing the product of photolysis of Compound I (called Intermediate Y) provide further evidence for the suggestion that the ferric state of the heme iron is generated in the course of the photolysis. The quantum yields of the photolysis of Compound I at different wavelengths of irradiating light were obtained and mechanisms of the photolysis of Compounds I and II are proposed.     

Formation of hydroxyl radical from the photolysis of salicylic acid

Photodissociation dynamics of salicylic acid (SA) in the gas phase at different photolysis wavelengths (266, 315-317 nm) is investigated by probing the nascent OH photoproduct employing the single-photon laser-induced fluorescence (LIF) technique. At all the photolysis wavelengths it is found that the nascent OH radicals are produced mostly in a vibrationally ground state (υ' = 0) and have similar rotational state distributions. The two spin-orbit and Λ-doublet states of the OH fragment formed in the dissociation are measured to have a nonstatistical distribution at each photolysis wavelength. The LIF signal of the OH could be observed upon photolysis at 317 nm but not at 317.5 nm. The threshold of OH formation from SA photodissociation is estimated to be 98.2 ± 0.9 kcal/mol. The effect of the phenolic OH group on the dissociation of SA is discussed.     

有机锡化合物光解活泼自由基的ESR研究

本文用自旋捕捉技术与ESR相结合的方法,研究了六种有机锡化合物的光解反应历程。结果表明:有机锡化合物紫外光照时,碳-锡键发生均裂,产生碳中心和锡中心自由基。其中碳中心自由基易被捕捉剂α-苯基-N-特丁基氮氧化物(PBN)或2,3,5,6-四甲基亚硝基苯(ND)所捕获;而锡中心自由基可与菲醌形成稳定的环状加合物。由于环状加合物中配体的不同,产生的空间效应也不同,从而导致加合物中的未偶电子云密度发生变化,给出相异的ESR信号。     

THE EFFECT OF pH ON THE AEROBIC and ANAEROBIC PHOTOLYSIS OF TRYPTOPHAN AND SOME TRYPTOPHAN-CONTAINING DIPEPTIDES

Abstract— Rates of photolysis, quantum yields of fluorescence, and fluorescence emission maxima for the dipeptides glycyltryptophan (Gly-Trp) and tryptophylglycine (Trp-Gly) and for free tryptophan (Trp) were determined under both degassed and aerated conditions in the pH range 4.5-10.0. The photolyses were performed at 25°C using 290 nm radiation from a 1000 W xenon lamp. Photolysis rates were determined by monitoring tryptophan fluorescence loss with time. It was found that Trp-Gly and free Trp showed similar behavior in that their fluorescence quantum yields and photolysis rates increased significantly above neutral pH. In contrast, the Gly-Trp fluorescence yield was smaller than that of Trp or Trp-Gly, showing no significant increase at high pH and the photolysis rate for Gly-Trp decreased with increasing pH. In comparing aerated to degassed samples, it was found that degassing had a far greater effect on the photolysis rates of Trp and Trp-Gly than on the photolysis rate of Gly-Trp especially at higher pH. But, degassing did not change the relative fluorescence quantum yields or fluorescence emission maxima of any of the three compounds. Possible mechanisms for photolysis under various experimental conditions were examined in light of the data.     

芳基重氮盐及其冠醚络合物光解活泼自由基的ESR研究

本文用自由基捕捉剂2,3,5,6-四甲基亚硝基苯(ND)及苯亚甲基叔丁基氮氧化物(PBN)与ESR相结合的方法研究了芳基重氮盐RC_6H_4N~+_2BF~-_4(R=F, Cl, Br, I, NO_2, COOH,OCH_3及H)及其与18-冠-6、二苯并-24-冠-8的络合物在苯中的光解过程。结果表明它们光解产生了活泼自由基RC_6H_4并可被ND及PBN捕捉....     

THE VACUUM ULTRAVIOLET PHOTOCHEMISTRY OF NUCLEOTIDES

Abstract— Photodestruction of nucleotides by vacuum UV (λ < 200 nm) and near UV radiation in thin films has been studied; the products were analyzed by thin-layer chromatography. Vacuum UV photolysis leads to the rupture of the N-glycosidic bond with liberation of bases, the quantum yield of the process being about 2 × 10^-4 for guanosine-5'-monophosphate. No bases have been found among the products of near UV photolysis.
Vacuum UV irradiation of aqueous solutions of adenosine-5'-monophosphate and guanosine-5'-monophosphate produces, along with bases, some other fluorescent substances. The identity of photoproducts of vacuum UV photolysis with those of γ-radiolysis is established. The mechanism of photochemical conversion is discussed.     

THE PHOTOLYSIS RATES OF SOME DI- AND TRIPEPTIDES OF TRYPTOPHAN

We have measured the relative rates of photolysis of free tryptophan (trp), the dipeptides Gly-Trp, Trp-Gly, Leu-Trp, and Trp-Leu, and the tripeptides Gly-Trp-Gly and Leu-Trp-Leu. The photolyses were performed in neutral 0.1 mM aqueous solutions at 25°C using monochromatic 290 nm Xe arc radiation. Tryptophan loss was monitored by absorption, fluorescence and phosphorescence spectroscopy. The rate of tryptophan fluorescence loss was found to be different in the di-and tripeptides than in tryptophan monomer. These rate differences depended on both the identity of the neighboring amino acid (gly or leu) and on the nature of the linkage, e.g., the rate of Gly-Trp photolysis was more than 10 times greater than the rate of Trp-Gly photolysis. Degassing was found to markedly reduce (factor of 8) the photolysis rates of Trp, Trp-Gly, and Trp-Leu, but degassing only slightly reduced (less than a factor of 2) the photolysis rates of the other di-and tri-peptides. Photochemical product structures were not determined, but absorption and fluorescence spectra were obtained and products could be inferred in some cases by comparison with data of previous workers. The products appeared to differ greatly among the various peptides studied; Trp, Trp-Gly, and Trp-Leu gave oxidation products, while Gly-Trp and Leu-Trp apparently gave ring closure products, not requiring oxygen.     

PRIMARY PRODUCTS IN THE FLASH PHOTOLYSIS OF TRYPTOPHAN*

There has been considerable interest in the photochemistry of tryptophan in connection with ultraviolet inactivation of enzymes. Earlier flash photolysis work has demonstrated that the hydrated electron (e^-_aq) is an initial product in the irradiation of indole derivatives, accompanied by a longer-lived transient absorption near 500 nm attributed to an aromatic radical species[1–5]. Similar transients were observed in a recent flash photolysis study of lysozyme[6] in which it was proposed that inactivation is a consequence of electron ejection from 1 to 2 essential tryptophan residues in the active center. However, there has been uncertainty concerning the tryptophan radical structure and its relationship to the triplet state and radical spectra reported for tryptophan photolysis in low-temperature rigid media. This note reports a flash photolysis investigation of L-tryptophan (Trp) and 1-Methyl-L-tryptophan (1-MeTrp) undertaken to clarify these points. The flash photolysis apparatus and methods employed are described in Ref. [6].