AN INSTRUMENT FOR ACTION SPECTRUM STUDIES IN DERMATOLOGY

Abstract— An instrument designed for convenient determination of action spectra for cutaneous photo-responses in man and experimental animals is described. Light from 450 W Xe lamp is dispersed by a concave holographic grating. The spectrum from 244 to 616 nm is projected as a planar strip (2 times 17 cm) intercepted by a grid with 31 ports. The bandwidth at each port is 12 nm and the size of the port increases from about 4 × 4 mm to 6 × 8 mm from the low to high wavelength limits, respectively. Typical fluence rates in quanta m^-2 s^-1 are 4.0 times 10¹⁹ at 298 nm, 16 times 10¹⁹ at 394nm and 22 times 10¹⁹ at 538 nm. Responses due to delayed erythema in normal skin and to musk ambrette photoallergy and solar urticaria in patients skin have been elicited with this instrument.     

PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

PRIOR EXPOSURE OF HUMAN CELLS TO NEAR UV-RADIATION GIVES A DECREASE IN THE AMOUNT OF THE UNSCHEDULED DNA-SYNTHESIS INDUCED BY FAR UV-RADIATION

Pretreatment of human cells with near UV radiation (UVA) in fluences exceeding 5 × 10⁴ Jm⁻² caused a decrease in the amount of the unscheduled DNA synthesis induced by far UV radiation (UVC). The DNA repair synthesis, as measured by the incorporation of [³H] -thymidine, is reduced by nearly a factor of 2 for a UVA radiation exposure of 1.5 × 10⁵ Jm⁻². Since solar UVA fluence rate is rather independent of latitude, this figure corresponds to a UVA exposure time of 50-60 min from noon sunlight in the summer time.     

SKIN DAMAGE IN ADULT AMPHIBIANS AFTER CHRONIC EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The effects of repeated UV exposure on the skin of the European crested newt, Triturus cristatus carnifex , have been investigated. The animals were irradiated 3 times per week with a Westing-house FS40T12 fluorescent sun lamp (wavelength spectrum 275–350 nm). Two groups of animals received the same total fluence of 1.3 × 10⁵ J/m² in single fluences of either 1570 J/m² (group A) or 9430 J/m² (group C), and one group received a total fluence of 2.6 × 10⁵ J/m² in single fluences of 4710 J/m² (group B). All the animals were killed 7 months after the first UV exposure, but at different intervals after the last exposure. Striking epidermal hyperplasia was found in the newts irradiated at the lower fluence rate (group A). In the animals given the higher total fluence (group B), the most prominent skin changes were dermal fibrosis and irregular thinning and thickening of the epidermis. No significant skin changes were found in group C., in which if there had been UV lesions, they had been repaired during the 5 month interval between the last irradiation and the killing of the animals. No skin tumors developed in any experimental group.     

REC A +-DEPENDENT SYNERGISM BETWEEN 365 NM AND IONIZING RADIATION IN LOG-PHASE ESCHERICHIA COLI: A MODEL FOR OXYGEN-DEPENDENT NEAR-UV INACTIVATION BY DISRUPTION OF DNA REPAIR

Abstract —The oxygen dependence of 365 nm inactivation of colony-forming ability of Escherichia coli has been investigated in two series of DNA repair-deficient K12 mutants grown to mid-exponential phase. All strains except a uvr A rec A double mutant are more sensitive to inactivation under O₂ and show a lower threshold dose. The inactivation of photoreactivating enzyme in a crude cell extract and DNA repair disruption are both reduced when irradiation is carried out under nitrogen. The rec A gene-dependent synergism between 365 nm and ionising radiation is reversible if cells are incubated in full growth medium before ionising radiation treatment. In a wildtype strain, incubation for 2.5 h in full growth medium after 10⁶ J m^-2 365 nm radiation changes a sensitised response to a protection from ionising radiation. Protection is not seen at 1.5 times 10⁶ J m^-2. A tentative model for near UV lethality in logarithmic phase cells is suggested which proposes two classes of lesions. One requires oxygen for it's induction, is rapidly fixed as a lethal event as a result of repair disruption, and is primarily responsible for cell death after aerobic 365 nm irradiation. The other lesion, possibly pyrimidine dimers, may lead to cell death under anaerobic conditions.     

Luminol Oxidation by Hydrogen Peroxide Catalyzed by Tobacco Anionic Peroxidase: Steady-State Luminescent and Transient Kinetic Studies

The properties of a newly isolated anionic tobacco peroxidase from transgenic tobacco plants overexpressing the enzyme have been studied with respect to the chemiluminescent reaction of luminol oxidation. These were compared to the properties of horseradish peroxidase in the cooxidation of luminol and p -iodophenol, the enhanced chemiluminescence reaction. The pH, luminol and hydrogen peroxide concentrations were optimized for maximum sensitivity using the tobacco enzyme. The detection limit for the latter under the optimal conditions (2.5 m M luminol, 2 m M hydrogen peroxide, 100 m M Naborate buffer, pH 9.3) was about 0.1 p M , which is at least five times lower than that for horseradish peroxidase in enhanced chemiluminescence with p -iodophenol. The rate constants for the elementary steps of the enzyme-catalyzed reaction have been determined: k ₁= 4.9 × 10⁶ M ⁻¹ s¹, k ₂= 7.3 × 10⁶ M ⁻¹ s⁻¹, k ³= 2.1 × 10⁶ M ⁻¹ s⁻¹ (pH 9.3). The similarity of these rate constants is unusual for plant peroxidases. The high catalytic activity of tobacco peroxidase in the luminescent reaction is explained by the high reactivity of its Compound II toward luminol and the high stability of the holoenzyme with respect to heme dissociation. This seems to be a unique property of this particular enzyme among other plant peroxidases.     

THE CYTOTOXIC AND MUTAGENIC EFFECTS OF UVA RADIATION ON L5178Y MOUSE LYMPHOMA CELLS

Abstract— A broad-band UVA source that emits primarily350–400 nm radiation and no measurable radiation below 340 nm was used to test toxicity and mutagenicity at the thymidine kinase locus in L5178Y, subclone 3.7.2C (TK⁺/^-) mouse lymphoma cells. Cells were exposed to a fluence of 0 to 80 × 10⁴ J/m². The relationship between UVA fluence and survival was found to have a shoulder region followed by an exponential decrease in survival at higher fluence levels. An exposure-dependent increase in mutation was observed with increasing fluences from 0 to about 60 × 10⁴ J/m². An approximately 3- to 4-fold increase in mutations (trifluorothymidine resistance) over unexposed, control cells was seen at a fluence that resulted in 90% cell killing. We conclude that UVA radiation is a mutagen in the L5178Y mouse lymphoma cells used in this study.     

NEAR-UV INDUCTION OF SISTER CHROMATID EXCHANGES UNDER AEROBIC AND ANAEROBIC CONDITIONS

Abstract— The induction of sister chromatid exchanges (SCE) and cell sensitivity in mouse myeloma cells (66.2 subclone of MPC11) by irradiation with monochromatic near-UV (365 nm) light were studied under aerobic and anaerobic conditions. Sister chromatid exchanges were studied using the fluorescence-plus-Giemsa technique, and sensitivity was determined by the ability of irradiated and nonirradiated control cells to form colonies in soft agar. Cells were found to be 16 times more sensitive to near-UV light under aerobic exposure, producing an F₃₇ value of 7 × 10⁴ J/m² compared to the F₃₇ value of 11.5 × 10⁵ J/m² under anaerobic conditions. The induction of SCE was also 12 times more efficient for aerobic irradiation than for anaerobic irradiation. The data suggest that the SCE-inducing potential of DNA lesions differs when near-UV irradiation is performed in the presence or absence of air. In addition, the DNA lesions responsible for lethality and also those lesions leading to SCE induction may differ under the two irradiation conditions.     

CAROTENOIDS AFFECT DEVELOPMENT OF UV-B INDUCED SKIN CANCER

One large dose of UV-B (8 × 10¹⁴ J m^-2: 290-320 nm) has been found to cause the development of skin tumors in hairless mice, and carotenoid pigments prevent or delay tumor development in this system. This model was used to determine if the protective effect of carotenoid pigments against UV-B induced skin tumors occurred during the induction phase or progression phase of UV-B carcinogenesis. The results indicate that the carotenoids canthaxanthin and (J-carotene interfered with the progression phase in this system; whether they also had an effect during induction could not be definitely determined from our data.     

ANALYSIS OF FLUORESCENCE KINETICS AND ENERGY TRANSFER IN ISOLATED α SUBUNITS OF PHYCOERYTHRIN FROM Nostoc sp.

Abstract— The fluorescence decay profiles, relative quantum yield, and transmission of the phycoerythrin a subunit, isolated from the photosynthetic antenna system of Nostoc sp., were measured using single picosecond laser excitation. The fluorescence decay profiles were found to be intensity independent for the intensity range investigated (4 × 10¹³ and 4 × 10¹⁵ photons-cm-² per pulse). The decay profiles were fitted to a model assuming both chromophores absorb and fluoresce. The inferred total deactivation rates for the two chromophores, in the absence of energy transfer and when the effects of the response time of the streak camera and the finite pulse width are properly included, are 1.0 × 10¹⁰s' and 1.0 × 10⁹ s ¹ for the s and f chromophores. respectively, whereas the transfer rate between the two fluorophorcs is estimated to be 1.0 × 10¹⁰ s⁻¹ giving a s→ f transfer rate on the order of (100 ps)⁻¹. Steady-atate polarization measurements were found to be equal to those calculated using the rate parameters inferred from the kinetic model fit to the fluorescence decays. The apparent decrease in the relative fluorescence quantum yield and increase of the relative transmission with increasing excitation intensity is suggestive of ground state depletion and upper excited state absorption. Evidence suggests that exciton annihilation is absent within isolated α subunits for the intensity range investigated (4 × 10¹³ to 4 × 10¹⁵ photons-cm ² per pulse).     

REACTIONS OF PHOTOCHEMICALLY FORMED TRANSIENTS FROM 2-NITROTOLUENE

Abstract— Kinetic data are reported for the thermal decay of colored transients formed by U.V. irradiation of aqueous solutions of 2-nitrotoluene. The transients display an acid-base equilibrium with a pK value of 3.7. The decay is catalyzed by acids and the following rate constants in liter sec^-l mole^-1 were evaluated for the base form of the transient reacting with an acid at 30.0°C: 3.5 × 10^-3 (H₂O), 2.6×10³ (CH₃COOH), 4.7×10⁴ (⁺NH₃CH₂COOH) and 4.2×10⁵(H+).     

QUENCHING OF EXCITED TRIPLET LUMIFLAVIN BY LUMIFLAVIN RADICALS FORMED IN ITS T-T REACTION

Abstract— Reactions of the triplet state of lumiflavin (³LF) in water adjusted at pH 7.2 were reexamined by means of a Xe-flash photolysis and a laser photolysis. Measurements of the decay of ³LF were made on solutions of LF ranging in the concentration from 4 to 61 times 10^-6 mol/dm³. A one-electron reduced and a one-electron oxidized species of lumiflavin (LF^- and LF⁺) were produced in the first decay stage of ³LF with a high efficiency (0.6 ± 0.1) in a bimolecular triplet-triplet reaction. The product radicals (LFH^- and LF⁺) quench ³LF very efficiently (3 ± 0.8 × 10⁹ mol^-1dm³ s^-1) compared with LF in the ground state (> 2 × 10⁷ dm³ mol^-1).     

PHOTOLYSIS OF PHOSPHODIESTER BONDS IN PLASMID DNA BY HIGH INTENSITY UV LASER IRRADIATION

Abstract— The cleavage of phosphodiester bonds in DNA exposed to high intensity UV laser pulses in aerated aqueous solution has been investigated using a krypton fluoride excimer laser (248 nm) and bacterial plasmid DNA. The dependence of strand breakage on fluence and intensity has been studied in detail and shows that the process is non-linear with respect to intensity. The relationship between the quantum yield for strand breakage and intensity shows that the strand breakage reaction involves two-photon excitation of DNA bases. The quantum yield rises with intensity from a lower value of 7 times 10^-5 until a maximum value of 4.5 times 10^-4 is attained at intensities of 10¹¹ W m^-2 and above. This value is approximately fifty-fold higher than the quantum yield for strand breakage induced by exposure to low density UV irradiation (254 nm, 12 W m^-2). DNA sequencing experiments have shown that strand breakage occurs by the specific cleavage of the phosphodiester bond which lies immediately 3' to guanine residues in the DNA, leaving some alkali-labile remnant attached to the terminal phosphate. A mechanism for DNA strand breakage which involves the generation of guanine radical cations is proposed.     

FLASH PHOTOLYSIS STUDIES OF OROTIC ACID

Abstract— The triplet state of orotic acid has been studied by flash photolysis. The rate for dimerization has been observed to vary from 2 × 10⁹ M ^-1 sec^-1 at pH 1 where both the triplet and ground state molecules are neutral, to under 10⁸ M^-1 sec^-1 above pH 9 where both the triplet and ground state molecules are doubly ionized. The p K of the triplet state has been measured as 4.6. The rate of oxygen quenching for the triplet is 2–3 × 10⁹ M^-1 sec^-1 while the rate of radiationless decay in solution is 0.73 × 10⁴ sec^-1. The triplet absorption spectra have been measured for the two ionic forms of the triplet.     

ON THE PRODUCTION OF NITROXIDE RADICALS BY SINGLET OXYGEN REACTION: AN EPR STUDY

Abstract— The production of free radicals by reaction of 2,2,6,6-tetramethyl-4-piperidinol with singlet oxygen was studied by EPR spectroscopy. The rate constant of the amine was found to be equal to 8 ×10⁵ M ^-1s^-1 in ethanol and to 4 × 10⁷M^-1s^-1 in phosphate buffer (pH 8). Competition experiments were performed with singlet oxygen quenchers such as NaN₃, DABCO and the quenching rate constants were found to be consistent with the literature values. The EPR method proved to be a valuable technique to study the reaction of singlet O₂ with the sterically hindered amine without any interfering effect.     

DESIGN AND PERFORMANCE OF THE OKAZAKI LARGE SPECTROGRAPH FOR PHOTOBIOLOGICAL RESEARCH

A computer-operated spectrograph was recently built at Okazaki, Japan. Different specimens can be placed on a horseshoe-shaped focal curve (10 m long) covering a wavelength range of 250 to 1000 nm so they can be irradiated simultaneously. The linear dispersion is about 0.8 nm/cm. The photon fluence rate on the focal curve is 5 x 10¹⁵. photons x cm^-2 x s^-1 at 300nm and 1 x 10¹⁶ photons x cm^-2 x s^-1 at 600 and at 900 nm. The spectral half width is 5.5 nm or less on the focal curve. The stray light content is about 10^-5 of the main peak at the peak wavelength ± 100 nm. Specimens are set in microcomputer-controlled threshold boxes so that wavelengths, photon fluence rates, photon fluences and timing of irradiations are controlled automatically according to a pre-programmed schedule. An optical fiber system is also provided for remote irradiations.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED SISTER CHROMATID EXCHANGES IN POTOROUS CELLS

Abstract— Exposure to visible light after UV-irradiation showed a remarkable effect on UV-induced sister chromatid exchanges (SCEs). After 6-h exposure to visible light (3 × 10⁵ J/m²), two-thirds of the UV-induced SCEs were prevented, confirming Kato's findings. Exposure to visible light before UV irradiation had no effect. This effect of visible light on UV-induced SCEs was temperature dependent, suggesting the presence of enzymatic photoreactivation.     

THE SINGLET OXYGEN REACTIVITY OF BILIVERDIN

Abstract— In 1, 1, 2-trichlorotrifluoroethane solution biliverdin physically quenches singlet oxygen at a rate of 8 × l0^sM-¹s^-1 and reacts chemically at 6 × 10 ⁵M-¹s^-1 to give a red product. In D, O solution the rate constants are PD dependent and range from 1.5–6 times 10¹⁰M-¹s^-1 for quenching and the chemical rate varies from 3–5 × 10⁸ M^-1 s^-1 to give colorless products.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.