DRUG SENSITIZATION: PHOTOBINDING OF SULFANILAMIDE TO BOVINE SERUM ALBUMIN

Abstract— Photobinding of sulfanilamide to bovine serum albumin (BSA) was investigated by irradiating BSA and buffered BSA/drug solutions with UV light (Λ= 300 nm) under anaerobic conditions. The protein solutions were then denatured and the unbound sulfanilamide removed. Marked differences in the UV and fluorescence spectra of the solutions before and after irradiation were observed, suggesting covalent binding of the drug to BSA. This was confirmed using [¹⁴C]labelled sulfanilamide. The extent of photobinding of sulfanilamide determined using the radiolabeled drug, was concentration dependent. The binding ratio varied from 3 mol drug per mol BSA for a 10^-4 M drug concentration, to 10 mol drug per mol BSA for 10^-2 M drug concentration.
The protein solutions were hydrolysed under acid conditions and the amino acids obtained were analysed by ion exchange chromatography. The hydrolysate of irradiated BSA (10^-4 M ) -sulfanilamide (10^-2 M ) mixture lost about 10 mol of cystine per mol of BSA. This loss was not observed after hydrolysis of irradiated alone or non-irradiated BSA. Irradiation of cystine with [¹⁴C]sulfanilamide in HC1 solutions produced the same compound as was found after hydrolysis of irradiated BSA/sulfanilamide mixtures. This was demonstrated by autoradiography of paper chromatograms. The same compound was also detected in an irradiated [³⁵S]cystine non-labelled sulfanilamide mixture. It was not detected, however, after irradiation of a mixture of all amino acids of BSA excluding cystine. These data suggest that cystine residues are involved in the photobinding of sulfanilamide (or its photoproducts) to BSA.     

4',5'-SUBSTITUTED METHYLANGELICINS: PHOTOCYCLOADDUCTS WITH PYRIMIDINE BASES OF DNA

The isolation and characterization of photocycloadducts with pyrimidine bases from DNA samples irradiated (365 nm) in the presence of four 4',5'-substituted methylangelicins was performed. All these furocoumarins yielded mainly the cis-syn furan-side cycloadduct with thymine. For 4',5'-dimethyl-, 5,4',5'-trimethyl- and 6,4',5'-trimethylangelicin this adduct was accompanied by two pyrone-side adducts ( cis-syn and cis-anti ), whereas the 4,4',5'-trimethyl derivative gave the furan-side adduct with cytosine.
The characterization of the regio- and stereochemistry of the adducts was accomplished by ¹H NOE (nuclear Overhauser effect) and ¹H-¹³C HMBC (heteronuclear multiple-bond connectivity) spectroscopies.
The formation of different cycloadducts in DNA by the various derivatives highlights the role of the methyl groups in determining the regio- and stereochemistry of the cycloaddition.     

INTRODUCTION OF PYRIMIDINE DIMERS INTO DIFFERENT INTRACELLULAR FORMS OF SIMIAN VIRUS 40

We examined the production of pyrimidine dimers by UV radiation in different intracellular forms of simian virus 40 DNA. Virus and chromatin or previrions were selectively labeled with [^l4C]-thymidine and [³H]-thymidine, respectively, in the same monolayer of infected cells. Viral DNA was extracted immediately after irradiation, and pyrimidine dimers were detected as sites sensitive to the UV-endonuclease encoded by bacteriophage T4. No difference in the number of dimers introduced into chromatin, previrions. or virions was detected.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract
I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (< 290 nm) and UV-B irradiation (290–320 nm) in the 2'-deoxy-[³H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m^-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol . 52 , 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10–20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

Induction of 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Ultraviolet Radiation in Calf Thymus DNA and HeLa Cells

Abstract— The levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in purified calf thymus DNA and HeLa cells were measured following exposure to either UVC, UVB or UVA wavelengths. This DNA damage was quantitated using HPLC coupled with an electrochemical detector. The 8-oxodGuo was induced in purified DNA in a linear dose-dependent fashion by each portion of the UV spectrum at yields of 100, 0.46 and 0.16 8-oxodGuo per 10⁵ 2'-deoxyguanosine (dGuo) per kJ/m² for UVC, UVB and UVA, respectively. However, the amount of 8-oxodGuo in HeLa cells irradiated with these UV sources decreased to approximately 2.0, 0.013 and 0.0034 8-oxodGuo per 10⁵ dGuo per kJ/m², respectively. In contrast, the levels of cyclobutyl pyrimidine dimers were similar in both irradiated DNA and cells. Therefore, 8-oxodGuo is induced in cells exposed to wavelengths throughout the UV spectrum although it appears that protective precesses exist within cells that reduce the UV-induced formation of this oxidative DNA damage. Cell survival was also measured and the number of dimers or 8-oxodGuo per genome per lethal event determined. These calculations are consistent with the conclusion that dimers play a major role in cell lethality for UVC- or UVB-irradiated cells but only a minor role in cells exposed to UVA wavelengths. In addition, it was found that the relative yield of 8-oxodGuo to dimers increased nearly 1000-fold in both UVA-irra-diated cells and DNA compared with cells subjected to either UVC or UVB. These results are supportive of the hypothesis that 8-oxodGuo, and possible other forms of oxidative damage, play an important role in the induction of biological effects caused by wavelengths in the UVA portion of the solar spectrum.     

LEAKAGE OF 86Rb+ AFTER ULTRAVIOLET IRRADIATION OF Escherichia coli K-12

Abstract— Stationary phase cultures of a DNA repair proficient Escherichia coli K-12 strain showed a release of intracellular material as assessed by three different methods (260 nm absorption; [methyl-³H]thymidine leakage and ⁸⁶Rb⁺ leakage) after broad-band (Black-Light Blue) near-UV radiation but not after far-UV (254 nm) radiation. As a control response for membrane damage to cells, this leakage of intracellular material was also determined by each method after mild-heat (52°C) treatment of E. coli K-12. An action spectrum for the release of ⁸⁶Rb⁺ from E. coli K-12 after irradiation with monochromatic wavelengths, from 254 to 405 nm, is also presented. The action spectrum for lethality (F₃₇ values) obtained for this strain, shows that leakage of ⁸⁶Rb⁺ occurs at fluences equivalent to or slightly less than fluences causing inactivation at wavelengths above 305 nm. In contrast, at wavelengths below 305 nm, leakage of ⁸⁶Rb⁺ from irradiated cells can be induced but only at fluences significantly greater than was required to cause cell inactivation. These results indicate, therefore, that near-UV radiation can induce a damaging effect on the cell's permeability barrier which may be significant in causing the death of the cell, whereas the effect is not significant in causing the death of cells by far-UV radiation where DNA damage is known to be the main cause of lethality.     

THE EFFECT OF THE ANTIPSORIATIC DRUG METABOLITE ETRETIN (Ro 10-1670) ON UVB IRRADIATION INDUCED CHANGES IN THE METABOLISM OF ARACHIDONIC ACID IN HUMAN KERATINOCYTES IN CULTURE

[¹⁴C]Arachidonic acid was avidly incorporated into human keratinocytes in culture and following exposure to UVB irradiation of 9 mJ/cm² (erythemally effective, EE) substantial amounts of ¹⁴C-radiolabel were released from the cells. The release of radiolabel was accompanied by a decrease in the labelling of phosphatidylethanolamine whereas the labelling of triacylglycerols and cholesteryl esters was increased. Keratinocytes produced significant amounts of prostaglandin E₂ (PGE₂) and following UVB irradiation of 9 mJ/cm² (EE) the formation of prostaglandin E₂ was increased.
Etretin (Ro 10-1670), the active metabolite of the antipsoriatic drug etretinate (Ro 10-9359), affected significantly neither the total release of radiolabel induced by UVB nor the formation of prostaglandin E₂. However, in the presence of etretin the UVB irradiation induced transfer of [^l4C]arachidonic acid into triacylglycerols and cholesteryl esters was not increased as much as in the corresponding experiments without etretin. On the basis of the present study it appears that etretin does not interfere with the release of arachidonic acid in amounts which could be related to the therapeutic effects of the combination of retinoids with UVB irradiation (Re-UVB) in the treatment of psoriasis.     

ALTERATIONS IN DNA IRRADIATED WITH ULTRAVIOLET RADIATION—I. THE FORMATION PROCESS OF CYCLOBUTYLPYRIMIDINE DIMERS: CROSS SECTIONS, ACTION SPECTRA and QUANTUM YIELDS

Abstract— We have determined the dimerization and monomerization cross sections of the Thy < > Thy (cyclobutyl dimer of thymine and thymine) and the Cyt < > Thy (cyclobutyl dimer of cytosine and thymine) dimers in Escherichia coti [³H]-DNA ([³H]-thymine labeled DNA) at five wavelengths in the range 240–300 nm. It may be concluded from the dimerization action spectra for the two dimers that the excitation of Thy (thymine) is mainly responsible for the photochemical dimerization reaction in both cases. The calculated quantum yields of dimerization and monomerization are also presented in this paper and several questions, raised by the results obtained at 300 nm, are discussed.     

PHOTOADDITION OF CHLORPROMAZINE TO GUANOSINE-5'-MONOPHOSPHATE

Abstrart—The photochemistry of chlorpromazine (CPZ) with guanosine-5'-monophosphate (GMP) was studied as a model for the photoaddition of CPZ to DNA. Irradiation of CPZ with calf thymus DNA produced a product emitting at 520 nm, whereas with GMP emission was at 495 nm. HPLC separation of photolysis mixtures of [³H]CPZ with GMP and [¹⁴C]GMP with CPZ indicated that three photoadducts were formed. One of the adducts fluoresced at 500 nm and appeared to be the product detected but not separated by Fujita et al. (Photochem. Photobiol . 1981, 34 , 101–105). A second adduct emitted at 460 nm, and the third was nonfluorescent. The photoadduct emitting at 500 nm was characterized by UV, fluorescence, and NMR to be an adduct from coupling of the C-8 position of guanine to the C-2 position of the phenothiazine ring of CPZ. The cation radical of CPZ (CPZ ⁺) does not appear to be an intermediate since enzymatically generated CPZ ⁺ formed a product that eluted with a retention time close to that of the photoadducts, but did not emit at 520 nm.     

SYNTHESIS OF DOUBLY AND MULTIPLY ISOTOPICALLY LABELED RETINALS

Abstract— In this paper a consecutive synthetic scheme is described that allows the synthesis of multiply ¹³C labeled retinals with labels in the conjugated chain. This scheme can be extended to incorporation of ²H labels in the conjugated chain. Also carotenoids can be multiply ¹³C labeled with this strategy. The same approach has been used to label amino acids.     

OXYGEN-DEPENDENCE OF NEAR UV (365 NM) LETHALITY AND THE INTERACTION OF NEAR UV AND X-RAYS IN TWO MAMMALIAN CELL LINES

Abstract— The colony-forming ability of Chinese hamster cells (V-79) and HeLa cells has been measured after near-ultraviolet (UV) irradiation, predominantly at 365 nm. To avoid the production of toxic photoproducts, cells were irradiated in an inorganic buffer rather than in tissue culture medium. Under these circumstances near-UV lethality was strongly oxygen-dependent. Both cell lines were approximately 10⁴ times more sensitive to 254 nm irradiation than to 365 nm radiation when irradiated aerobically. Pretreatment with 6 times 10⁵ Jm^-2 365 nm radiation sensitised the HeLa, but not the V-79 cell line to subsequent X-irradiation. Pretreatment of cells with 17 Jm^-2 254 nm radiation, a dose calculated to produce twenty times more pyrimidine dimers than the 365 nm dose, produced only slight sensitisa-tion to X-rays. It is suggested that the sensitisation to X-rays seen in the HeLa cells after 365 nm treatment is not the result of lesions induced in DNA by the near-UV radiation, but may reflect the disruption of DNA-repair systems.     

IMPAIRED REPAIR OF UVC-INDUCED DNA DAMAGE IN L5178Y-R CELLS: DNA UNWINDING STUDIES WITH THE USE OF 1-β-D-ARABINOFURANOSYL CYTOSINE

Abstract— L5178Y-R (LY-R) and L5178Y-S (LY-S) cells differ in sensitivity to UVC radiation (D₀: 2.8 and 9.0 J/m²respectively, for cells exposed in Fischer's medium). We used these cells and a DNA unwinding technique in conjunction with 1-β-D-arabinosyl cytosine to determine DNA strand breaks accumulating as a result of enzymatic incision during DNA repair. Following UVC exposure DNA strand break accumulation was observed in LY-S cells but not in LY-R cells. The repair defect in LY-R cells is accompanied by a delayed recovery of [³H]thymidine incorporation.     

BIOLOGICAL CHEMILUMINESCENCE

Abstract
The nucleobase 5-methylcytosine ( I ) is a minor component of eukaryotic DNA thought to be important in regulation of gene expression. The photochemical reactions of this nucleobase and its 2'-deoxyribonucleoside, 5-methyl-2'-deoxycytidine ( II ), in water have been studied. These reactions lead, respectively, to 3-amino-2-methylacrylamidine ( Ib ) and 3-(2- erythro - d -pentopyranos-1-yl) amino-2-methylacrylamidine ( IIb ) as the main photoproducts. The structure of the photoproducts was established by spectroscopic methods (¹H and ¹³C NMR, UV spectroscopy, electron impact and liquid secondary ion mass spectrometry); in the case of Ib , confirmatory evidence was obtained by chemical methods (photolysis of 5-methyl[2–¹³C]cytosine, hydrolysis of N -carbomethoxy-3-amino-2-methylacryl-amidine and reaction of Ib with 1,1'-carbonyldiimidazole to give I ). The quantum yield for formation of Ib was determined to be 1.8 × 10^-3at pH 7.5 while the quantum yield for formation of IIb has a lower value of 0.2 × 10^-3 at pH 7.5. These quantum yields depend strongly on pH and reach maximum values of 2.0 × 10^-3 at pH 7.0 ( Ib ) and 0.6 × 10^-3 at pH 5.0 ( IIb ). The mechanism of formation of Ib (or IIb ) is proposed to involve nucleophilic attack of water on the C-2 position of photoexcited I (or II ), followed by ring opening and decarboxylation of an intermediate carbamic acid.     

MITOCHONDRIAL DNA SYNTHESIS IN NON-GROWTH CONDITION (LIQUID HOLDING) IN Saccharomyces cerevisiae. RELATION WITH GROWTH STAGES and MITOCHONDRIAL DNA REPAIR AFTER UV-IRRADIATION

Abstract— The synthesis of DNA as measured by incorporation of [¹⁴C]adenine in Saccharomyces cerevisiae liquid held in non-growth conditions was followed in controls and UV-irradiated cells. The incorporation into mitochondrial DNA relative to total DNA was higher in these conditions than that observed in growth medium, especially in liquid held stationary phase cells. The absolute amount of mitochondrial DNA synthesized during liquid holding was larger in exponential than in stationary phase cells and increased after UV-irradiation.
The data reported here are discussed in relation to the effects of liquid holding on the UV-induction of rho ⁻ mutants, such effects depending upon the growth stage of the cells at the time of irradiation. A correlation has been found between the initial ability of the cells to synthesize mitochondrial DNA in liquid holding conditions and their capacity to repair UV-induced lesions in this DNA. We propose the hypothesis that the opposite effects of liquid holding on the UV-induction of rho ⁻ mutants observed in exponential versus stationary phase cells are not due to the action of different repair pathways, but result essentially from quantitative differences in mtDNA synthesis.     

Irradiation of DNA with 193 nm Light Yields Formamidopyrimidine-DNA Glycosylase (Fpg) Protein-Sensitive Lesions

Abstract— Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample φ_ssb= [1.5 ± 0.1] ± 10⁻⁴, argon-saturated sample φ_ssb= [0.9 ± 0.1] ± 10⁻⁴). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimi-dine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand breakage (air-saturated sample φ_fpg= [33.1 ± 3.1] ± 10⁻⁴, argon-saturated sample φ_fpg= [23.8 ± 2.6] × 10 ⁴). This result indicates that 193 nm light induces other modification) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.     

The Structure of the Aggregate Form of Bacteriochlorophyll c Showing the Qy Absorption above 740 nm as Determined by the Ring-current Effects on 1H and 13C Nuclei and by 1H–1H Intermolecular NOE Correlations

¹³C-enriched bacteriochlorophyll c (S[I, E] BChl c _F) was suspended in a 1:3 mixture of methylene chloride and carbon tetrachloride to form an aggregate showing the Q_y absorption above 740 nm; changes in the ¹³C chemical shifts were traced when methanol was titrated to dissolve the aggregate, and then, the changes were correlated to the ring-current effects due to the neighboring macrocycles in the aggregate. A pair of aggregate structures has been proposed based on the ring-current effects on both ¹H and ¹³C nuclei; the monomeric units are stacked together to form an inclined column with different sliding directions, in which the y-axis of the molecule is parallel to the long axis of the column. In order to confirm this pair of models, the ring-current effects on the ¹H and ¹³C nuclei were calculated based on both the magnetic-dipole and the loop-current approximations. Further, an application of three-dimensional F1 ¹³C-edited F3 ¹³C-filtered heteronuclear single-quantum correlation-nuclear Overhauser effect spectroscopy to the above aggregate consisting of a 1:1 mixture of ¹³C-labeled and unlabeled BChl c succeeded in detecting selectively the ¹H–¹H intermolecular nuclear Overhauser effect correlations, which established the coexistence of the above pair of stacked structures in the aggregate.     

THE EFFECTS OF HIGH-INTENSITY UV-RADIATION ON NUCLEIC ACIDS AND THEIR COMPONENTS—I. THYMINE

Abstract— The set of final products of thymine conversion induced by high-intensity UV irradiation (λ= 266nm, intensity 10²⁴-5 × 10²⁹ photons·s⁻¹·m⁻², pulse duration 10ns) of the dilute aqueous solution to the first approximation is similar to that formed with ionizing irradiation (γ-irradiation of aqueous solution or autoradiolysis of a solid 2-[¹⁴C]-thymine). The data obtained suggest that high-intensity UV-induced photochemical conversion of thymine involves photoionization and/or photodissociation. These processes pass through the higher excited state(s) populated as a result of the second photon absorption by excited (most probably in the T₁ triplet state) thymine molecules.     

SUNLIGHT-INDUCED PYRIMIDINE DIMERS IN HUMAN SKIN FIBROBLASTS IN COMPARISON WITH DIMERIZATION AFTER ARTIFICIAL UV-IRRADIATION

Abstract— We compared artificial UV-sources such as germicidal- or sun-lamps with summer noon sunlight in Switzerland for selective efficiency in the induction of pyrimidine dimers in the DNA of human cells. In our studies we determined cytosine-thymine (C-T) as well as thymine-thymine dimer densities (T-T) by high pressure liquid chromatography in cultures of xeroderma pigmentosum cells of group A. Using far-UV light from a germicidal lamp, we found a rate of formation per Jirr² for C-T and T-T of 0.0019% and 0.0024%, respectively, of the total thymine radioactivity in hydrolysates of [³H]thymidine labeled cells. After irradiation with an unfiltered sunlamp we measured a rate of formation of 0.0005% per Jm-² both for C-T and T-T, based on the sunlamp emission of 297 ±4 nm wavelength. Utilization of Kodacel- or Mylar-filters lowered the rate of dimerization by a factor of 2 and 60, respectively. One hour of irradiation with noon summer sunlight induced 0.038 ±0.012% C-T and 0.036 ±0.011% T-T. This extent of dimer production is equivalent to 15 Jm^-2 of far-UV exposure at 254 nm.     

DIRECT PHOTO-AFFINITY LABELING WITH CYCLIC NUCLEOTIDES: ACTION SPECTRA

Abstract— When [³H]-cyclic adenosine 3',5'-monophosphate and [³H]-cyclic guanosine 3',5'-monophos-phate are irradiated at varying UV wavelengths in the presence of cyclic nucleotide receptor proteins, photo-incorporation is maximal in the region of 280 nm. This suggests that covalent coupling occurs by means of photo-activation of an entity other than the ligand cyclic nucleotide. The action spectra more closely resemble the absorbtion spectrum of the reacting protein, making it likely that absorhtion of radiant energy by components of the protein receptor leads to photo-affinity labeling.