Urocanic acid photobiology. Photochemical binding to calf thymus DNA

Abstract— Urocanic acid (UA) has previously been shown to photochemically react with N, N-dimethylthymine and with phage G4 single-stranded DNA. In this study, E-[ring-2-¹⁴C]-U A and calf thymus DNA have been irradiated with UV light (Λ > 270 nm) in buffered, aqueous solutions. Rc-isolation of the DNA indicates covalent binding of UA at levels of up to 80 nmol UA/mg DNA. Binding is observed for both native and heat denatured DNA. Equilibrium dialysis studies give no evidence for complexation of UA to either form of DNA in the dark. Enzymatic cleavage of the UA bound DNA and analysis by HPLC shows peaks for ^l4C-labelled products with retention volumes identical to those of a marker mixture prepared by irradiating UA with [³H-methyl]thymidine. Photolysis of the DNA before irradiation with U A leads to the formation of a second product with a retention volume corresponding to that for a deoxyadenosine/UA marker.     

ULTRAVIOLET LIGHT IRRADIATION OF DEFINED-SEQUENCE DNA UNDER CONDITIONS OF CHEMICAL PHOTOSENSITIZATION

Abstract— The formation of cyclobutane pyrimidine dimers and UV light-induced (6-4) products was examined under conditions of triplet state photosensitization. DNA fragments of defined sequence were irradiated with 313 nm light in the presence of either acetone qr silver ion. UV irradiation in the presence of both silver ion and acetone enhanced the formation of TT cyclobutane dimers, yet no (6-4) photoproducts were formed at appreciable levels. When photoproduct formation was also measured in pyrimidine dinucleotides, only cyclobutane dimers were formed when the dinucleotides were exposed to 313 nm light in the presence of photosensitizer. The relative distribution of each type of cyclobutane dimer formed was compared for DNA fragments that were irradiated with 254, 313, or 313 nm UV light in the presence of acetone. The dimer distribution for DNA irradiated with 254 and 313 nm UV light were very similar, whereas the distribution for DNA irradiated with 313 nm light in the presence of acetone favored TT dimers. Alkaline labile lesions at guanine sites were also seen when DNA was irradiated with 313 nm light in the presence of acetone.     

Theoretical investigation of the photosensitization mechanisms of urocanic acid

The photosensitization mechanisms of urocanic acid (UA), the main skin chromophores of ultraviolet (UV) light, are investigated by means of theoretical calculations. The results indicate that the direct photooxidative damage to DNA bases by triplet state UA through electron transfer reaction is not favorable on thermodynamic grounds. However, UA can photogenerate various reactive oxygen species (ROS, e.g., (1)O(2), O(2)(-)) theoretically and the ROS-generating mechanisms are illustrated as follows. Firstly, the (1)O(2)-generating pathway involves direct energy transfer between triplet state UA and (3)O(2). Secondly, UA gives birth to O(2)(-) through two pathways: (i) direct electron transfer between triplet state UA and (3)O(2); (ii) electron transfer between anion radical of UA (generated through autoionization reactions) and (3)O(2).     

RELAXATION OF SUPERCOILED DNA BY AMINOMETHYL TRIMETHYLPSORALEN AND UV PHOTONS: ACTION SPECTRUM

Abstract— An action spectrum of the relaxation of supercoied plasmid DNA(induction fo the firt single-strand break) by photoactivated 4'-aminomethyl-4, 5',8-trimethylpsoralen(AMT) has been determined using monochormatic UV photons from 254 to 405 nm. The spectrum of AMT-induced plasmid DNA relaxation fits closely with the absorbance spectrum of AMT in the spectral region between 313 nm and 405nm but deviates at wavelengths shorter than 313nm. This asay also reveals that the psoralen photosensitization reaction with DNA also produces piperidinelabile sites. Addition of mannitol and azide partially quenches the supercoil realaxation reaction, evidence for a role of Type II photosensitization pathway.     

MECHANISM OF HIGH POWER PICOSECOND LASER UV INACTIVATION OF VIRUSES AND BACTERIAL PLASMIDS

Abstract— The mechanism of inactivating action of high-power picosecond laser UV radiation (λ= 266 nm) on the λ and φX174 bacteriophages and the pBR 322 plasmid has been studied. It has been shown that at UV radiation intensities from 10¹¹ to 10¹³ W/m², inactivation of viruses and bacterial plasmids occurs mainly on account of single-strand breaks in the DNA chain unlike the case of less powerful UV radiation where the inactivation is associated with the formation of pyrimidine dimers.     

Photochemistry and Photobiology of lmidazole-4(5)-Methylidene Malonic Acid: An Analog of Both E- and Z-Urocanic Acid*

Imidazole-4(5)-methylidene malonic acid (IMMA) may be thought of as having its imidazole and carboxyl functionalities permanently fixed in a configuration that is simultaneously analogous to both E- (trans) and Z- (cis) urocanic acid (UA). Because the UA isomers affect the photoinactivation of bacteriophage single-stranded DNA differently (E-UA increases and Z-UA decreases viral DNA inactivation), IMMA was similarly tested and was found to change the inactivation rate by a factor of 0.43, which is intermediate between the values for E- and Z-UA (1.6 and 0.014, respectively). The IMMA likewise sensitizes double-stranded DNA by a factor of 10.3 compared to a value of 13 for UA. In several ways the effects of IMMA parallel the distinctive effects of UA on the UV inactivation of single- and double-stranded DNA, including the ability to prevent the formation of cyclobutane pyrimidine dimers in irradiated single-stranded DNA and to sensitize a large increase in the formation of those dimers in irradiated double-stranded DNA. The IMMA photodecarboxylates to UA with a low quantum efficiency (ca 1 × 10^?3) and photochemically binds to calf-thymus DNA.     

PROFLAVINE MEDIATED PHOTOINACTIVATION OFBACTERIOPHAGE φX174 AND ITS ISOLATED DNA: EFFECTS OF AGENTS MODIFYING VARIOUS PHOTOCHEMICAL PATHWAYS

Abstract. Thiols and disulfides protect both φX174 phage and its isolated DNA from the lethal action of proflavine plus light. The protective ability of these compounds appears to be attributed to the -SH or the -S-S- group and the property to interact with the proflavine-phage DNA complex. The phage inactivation efficiency per proflavine bound to DNA is reduced by 50 to 30% upon addition of cysteine or cystamine. Substances that affect the lifetime of singlet oxygen modify the rate of phage photoinactivation in the presence of proflavine; the inactivation rate is decreased by N^-₃ and increased by D₂O. Irradiation under N₂ atmosphere markedly decreases the phage photosensitization by proflavine. Irradiation with monochromatic light of 440 nm is less efficient than irradiation with light of 440 nm plus 360 nm, and the difference is more pronounced in N₂ than in air. These results are discussed in relation to various possible photochemical pathways.     

ACTION SPECTRUM FOR THE OXYGEN-INDEPENDENT INACTIVATION OF HAEMOPHILUS INFLUENZAE TRANSFORMING DNA WITH NEAR ULTRA-VIOLET LIGHT*

Abstract— The oxygen-independent inactivation of Haemophilis influenzae transforming DNA by near UV light (300–380 nm) has an action spectrum in which the efficiency of inactivation drops rapidly between 313 and 334 nm and more slowly between 334 and 405 nm, with a shoulder between 334 and 365 nm.     

PROTECTION BY DABCO AGAINST INACTIVATION OF TRANSFORMING DNA BY NEAR-ULTRAVIOLET LIGHT: ACTION SPECTRA AND IMPLICATIONS FOR INVOLVEMENT OF SINGLET OXYGEN

Abstract— Diazobicyclo (2.2.2) octane (DABCO) protects the genetic activity of purified transforming Bacillus subtilis DNA against inactivation by near-, but not far-, UV light. The maximum dose-modifying factor is 0.4, at 0.1 M DABCO. Maximal protection is at about 350 nm and no protection occurs below 313 nm. The spectrum for protection is similar to that described for 2-aminoethylisothiouronium bromide hydrobromide. The relevance of these observations with regard to the role of singlet oxygen in near-UV effects is discussed.     

Photogenotoxicity of mammalian cells: a review of the different assays for in vitro testing

During the past several years, phototoxicity has been studied at the molecular level, and these studies have provided new insights in the field of DNA lesion characterization, DNA repair and cell response to ultraviolet (UV)-induced stress. The development of new antibiotics and antiinflammatory drugs has highlighted the necessity to develop the assessment of phototoxicity in the safety evaluation of new chemical compounds. This paper aims at reviewing the known molecular mechanisms of the cellular response to UV-induced stress, the in vitro methods that can be proposed and used to screen for toxicity of sunlight and the photosensitization process resulting from the activation of drugs by light. UV sources, biological systems and endpoints of interest in that particular objective are listed. Phototoxic effects span from the cytotoxic-apoptotic effect to the induction of primary DNA damage, DNA repair and a variety of stress genes acting on the cell cycle and the fate of the cell. Ultimately, it can lead to the induction of hereditary DNA modification. A variety of assays are proposed to specifically address all these particular consequences of UV-induced toxicity.     

PHOTOSENSITIZED INACTTVATION OF INFECTIOUS DNA BY UROCANIC ACID, INDOLEACRYLIC ACID AND RHODIUM COMPLEXES

Naked, infectious single-stranded (ss) and double-stranded (ds) DNA from phages SI3 and G4 were irradiated with 308 nm UV radiation in the absence and presence of several photobiologically active compounds: E - and Z -urocanic acid ( E - and Z -UA), their methyl esters ( E - and Z -MU), E - and Z-indoleacrylic acid ( E - and Z -IA), cis -dichlorobis(1,10-phenanthroline)rhodium(III) chloride (cDCBPR) and tris(1,10-phenanthroline)rhodium (III) perchlorate (TPR). E -urocanic acid protects against cyclobutane pyrimidine dimer formation in ssDNA but concomitantly photosensitizes the formation of other lesions that inactivate ssDNA. Z -urocanic acid also protects ssDNA against such dimerization but without the associated sensitized damage. The methyl ester isomers behave similarly. There is no such differential activity observed for the IA isomers, both of which sensitize the inactivation of ssDNA. Photostationary state mixtures of both UA and IA efficiently sensitize the inactivation of dsDNA, and cDCBPR strongly protects ssDNA from UV damage, while TPR is a significant sensitizer. Both of these metal complexes sensitize the inactivation of dsDNA slightly. For all compounds, cyclobutane pyrimidine dimers were the predominant lethal lesions produced by sensitization of the dsDNA, but they were not the major lethal lesions created by sensitization of the ssDNA. In the case of dsDNA, both UA and IA created pyrimidine dimers with a high degree of potential for mutagenesis, as determined by an assay that monitors the frequency of mutations following the spontaneous deamination of cytosine in photodimers.     

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.     

ULTRAVIOLET AND N-FORMYL-KYNURENINE-SENSITIZED PHOTOINACTIVATION OF BOVINE CARBONIC ANHYDRASE: AN INTERNAL PHOTODYNAMIC EFFECT

Abstract —Direct photoinactivation by UV light of bovine carbonic anhydrase, as well as its photosensitization by N -formyl-kynurenine, a tryptophan photooxidation product, have been investigated. In the presence of oxygen both methods lead to similar results: the enzyme loses its activity, the tryptophanyl, histidyl and, to a lesser extent, tyrosyl residues being destroyed. In nitrogen-saturated solutions, a dramatic drop is observed in the photoinacitivation yield under the direct action of ultraviolet light, whereas histidyl residues remain intact. Evidence indicates an internal photodynamic action of N -formyl-kynurenine in the protein core produced by the UV photooxidation of tryptophanyl residues. Photoinactivation of oxygenated enzyme solutions by external and internal photodynamic action correlates with histidyl residue destruction via singlet oxygen. The possible importance of the photodynamic ability of N -formyl-kynurenine in the photochemistry of proteins, DNA, and cells is discussed.     

NEAR ULTRAVIOLET INACTIVATION STUDIES ONESCHERICHIA COLI TRYPTOPHANASE AND TRYPTOPHAN SYNTHETASE

Abstract— –The response of two pyridoxal-phosphate-requiring enzymes of E. coli, tryptophanase and tryptophan synthetase, to near UV light (320–400 nm) has been studied. Tryptophanase is inactivated both in vivo and in vitro, but tryptophan synthetase is resistant to near UV under both conditions. This shows that near UV inactivation is not general for pyridoxal-phosphate-requiring enzymes. Substrate protection against light inactivation is demonstrated for tryptophanase. It is furthermore shown that pyridoxal phosphate is required for inactivation of this enzyme. However, the action spectrum for this inactivation does not coincide with the absorption spectrum of tryptophanase or of pyridoxal phosphate.     

ACTION SPECTRA FOR KILLING NON-DIVIDING NORMAL HUMAN AND XERODERMA PIGMENTOSUM CELLS

Abstract— Non-dividing human cells degenerate and eventually detach from a culture vessel surface when exposed to UV light. Action spectra for this kind of cell inactivation were determined using eight monochromatic wavelengths from 240 to 313 nm and both a normal DNA excision-repair-proficient strain and a repair-deficient Xeroderma pigmentosum (XP12BE) strain. The action spectra for both strains have similar shapes with a broad peak between 254 and 280 nm followed by a steep decline at wavelengths greater than 280 nm. The relative action spectra are similar to those for inactivation of reproductive capacity and pyrimidine dimer formation in rodent cells suggesting that the critical target and critical damage for inactivation of non-dividing human cells is DNA and damage to DNA, respectively. Normal repair-proficient cells are 5–7 times more resistant at all wavelengths, based on a comparison of D_o values, than repair-deficient XP12BE cells, supporting the conclusion that the inactivating damage at all wavelengths is to DNA.     

DIFFERENT PHOTOINACTIVATION MECHANISMS IN Propionibacterium acnes FOR NEAR-ULTRAVIOLET and VISIBLE LIGHT

The light sensitivity of Propionibacterium acnes was investigated when the cells were exposed to anoxia, sodium azide, D₂O or superoxide dismutase in combination with visible light (broad band red light and 415 nm) and near-ultraviolet irradiation (360 and 320 nm). During anoxia the cells became less sensitive when the irradiation wavelength increased. Oxygen increased the photodamage to a greater extent in the case of visible light than of near-UV light. The photosensitization effects were, however, more or less oxygen dependent at all wavelengths used. An effect of azide and D₂O on the light sensitivity was observed for visible light, while superoxide dismutase was effective only at 320 nm.
The results support the hypothesis that inactivation of P. acnes with near-UV and visible light is based on different mechanisms. Porphyrin photosensitization, accomplished by singlet oxygen, is the most important mechanism when visible light is used. At shorter wavelengths (320 and 360 nm) singlet oxygen is not involved and for 320 nm the destruction might occur via superoxide anion formation.     

PHOTOSENSITIZED CYCLODIMERIZATION OF 5-FLUOROURACIL

Abstract—The photosensitization by acetone or N-methyllutidone yields a C₄-cyclodimer from 5-fluorouracil in aqueous solution. with a quantum yield of 2 × 10^--³. The dimer isolated has been characterized by UV, IR. NMR, and mass spectra. The stereochemistry of the dimer has been determined to be anti head-to-tail configuration from the NMR spectral analysis.     

PHOTOPROTECTION OF E. COLI B/r: RESPIRATION,GROWTH, MACROMOLECULAR SYNTHESIS AND REPAIR OF DNA*

Abstract— Photoprotecting effects of near UV radiations (300–400 nm, maximum at 360 nm) against far UV radiations (primarily 254 nm) have been studied in Escherichia coli B/r cells in minimal medium with glycerol as a carbon source. Near UV light (10⁵ Jm^-2) has a negligible effect on survival, but causes transitory inhibition of respiration, growth, DNA, RNA, and protein syntheses and cell division. Far UV (52 J m^-2) reduces survival to about 0.5 per cent; respiration, growth and RNA and protein syntheses proceed for about 60 min, after which they nearly cease for several hours. Near UV given before this fluence of far UV increases survival 10-fold and the above processes resume at times and with kinetics characteristic of those produced by lower fluences of far UV. Single-strand breaks appear in the DNA of both unprotected and photoprotected cells; repair of the breaks is essentially complete in protected but not unprotected cells. The viability kinetics for far-UV-irradiated cells with and without photoprotecting treatment are identical except that the curve for the latter is displaced upward about 1 log; exponential increases (cell division) begin at 120 min in each case. The data suggest that, in B/r cells grown under our particular conditions, namely in minimal medium with glycerol, photoprotection is not the result of growth or division delays, but reflects an increased repair capability due to continued respiration.     

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.     

INDUCTION OF FREE RADICALS IN DNA BY PROFLAVINE AND VISIBLE LIGHT: INFLUENCE OF OXYGEN AND IONIC STRENGTH

Abstract —The photosensitization of native DNA is observed as an induction of free radicals in the DNA moiety of proflavine-DNA complexes. The intensity of the electron paramagnetic resonance spectra (at 77 K) is a measure of the number of free radicals present in frozen solutions of DNA-proflavine complexes after irradiation with visible light (Λ > 320 nm). In the absence of O₂, the photosensitization is significant but very low; it increases slightly with increasing NaCl ionic strength; it appears to be due to intercalated dye molecules and the qualitative analysis of the spectra obtained shows that mainly thymidine is involved. The reaction measured after saturation with O₂ is the same as the reaction in air but is quantitatively higher; the free radicals observed are peroxides. This induction of free radicals appears to be due to the intercalated dye molecules, each molecule acting independently. The important observation is a very sharp and large (around a hundred-fold) increase in the photosensitizing efficiency of the bound dye molecules occurring in NaCl between μ, # 0–25 and μ= 0–5 and in MgCl₂ between μ# 0–01 and μ=0–1.