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.     

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.     

Physiological Doses of Ultraviolet Irradiation Induce DNA Strand Breaks in Cultured Human Melanocytes, as Detected by Means of an Immunochemical Assay

Abstract— An immunochemical assay, i.e. sandwich enzyme-linked immunosorbent assay, has been modified to detect UV-induced damage in cellular DNA of monolayer-grown human melanocytes. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The melanocytes derived from human foreskin of skin type II individuals were suspended and exposed to UVA, UVB, solar-simulated light or γ-rays. Following physiological doses of UVA, UVB or solar-simulated light, a dose-related DNA unwinding comprising a considerable number of single-strand breaks (ssb) was observed. No correlation was found between different seeded cell densities or different culturing periods and the UVA sensitivity of the cells. After UVA irradiation, 0.07 ssb/10¹⁰ Da/kJ/m² were detected and after UVB irradiation 1.9 ssb/10¹⁰ Da/kJ/m² were seen. One minimal erythema dose of solar-simulated light induced 2.25 ssb/10¹⁰ Da. Our results from melanocytes expressed in ssb/Da DNA are comparable and have the same sensitivity toward UVA as well as toward UVB as nonpigmented skin cells. As low doses of UVA have already been shown to induce detectable numbers of ssb, this assay is of great interest for further investigations about the photoprotecting and/or photosensitizing effects of melanins in human melanocytes derived from different skin types.     

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.     

PHOTOINDUCTION OF PROTOPERITHECIA IN NEUROSPORA CRASSA BY BLUE LIGHT

Blue light induces the formation of Neurospora crassa protoperithecia.This photoinduction is completed in less than 24 h. Its threshold is about 4.2 J/m². Red light is ineffective. The Bunsen-Roscoe law is obeyed at the fluence of 12.6 J/m² for fluence rates from 5.25 × 10 ² to 1.05 W/m².     

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.     

PHOTOCHEMICAL ALTERATIONS IN DNA REVEALED BY DNA-BASED LIQUID CRYSTALS

Abstract The preparations of chicken erythrocyte linear double-stranded DNA and superhelical plasmid pBR322 DNA were irradiated by continuous low-intensity UV radiation (I = 25-50 W/m², λ= 254 nm) as well as by highintensity picosecond laser UV radiation (I = 10¹¹-10¹³ W/m², λ= 266 nm). The effect of DNA secondary structure alterations on the formation of liquid-crystalline dispersions from UV-irradiated DNA preparations was studied. It was shown that in the case of linear DNA, watching the disappearance of abnormal optical activity characteristic for cholesteric liquid crystal we managed to detect the presence of photochemical alterations in DNA irradiated by low-intensity UV radiation at an absorbed energy of more than 20 quanta per nucleotide. In the case of superhelical DNA using enzyme treatment of liquid-crystalline dispersions and monitoring the appearance of abnormal optical activity, we detected the presence of photochemical alterations in DNA molecules after low-intensity UV irradiation at an absorbed energy of less than 4 quanta per nucleotide. Under the latter approach using picosecond UV laser irradiation at three different light intensities we were able to distinguish the different mechanisms of fine alterations in DNA secondary structure at an absorbed energy value of about 3 quanta per nucleotide.     

CAROTENOID PIGMENT ADMINISTRATION and DELAY IN DEVELOPMENT OF UV-B-INDUCED TUMORS

—The results of two studies are reported. In the first study, the carotenoid pigments, β-carotene. canthaxanthin and phytoene were administered to mice after one UV-B-induced skin tumor had developed, to see if carotenoid administration would delay the development of subsequent tumors. Canthaxanthin significantly delayed the development of subsequent tumors. In the second study, the same pigments were administered starting 10 weeks before, and for 24 weeks after, exposure to a large single fluence (8 × 10⁴J/m²) of UV-B radiation, to determine the effect of the pigments on tumor development. Phytoene significantly prevented the development of tumors to this fluence of radiation.     

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.     

IMPAIRED REPAIR OF UVC-INDUCED DNA DAMAGE IN L5178Y-R CELLS: SEDIMENTATION STUDIES WITH THE USE OF 5''-BROMODEOXYURIDINE PHOTOLYSIS

Abstract Relative to their L5178Y-S counterparts, L5178Y-R cells have an impaired capacity to form patches in DNA after exposure to UVC radiation. The photolysis of 5'-bromodeoxyuridine (BrdUrd) incorporated into DNA was used to estimate the number of 'repair patches'formed in response to a 254 nm UV (UVC) exposure. L5178Y-S cells, typical of rodent cell lines, formed a small number of patches in exposed DNA (1-2 patches per 1 times 10⁸ dalton during a 6 h recovery after an exposure of 20 J/m²). In contrast, DNA extracted from L5178Y-R cells exposed to UVC and subsequently incubated with BrdUrd for 6 h showed no evidence of BrdUrd incorporation indicating no capacity to form sites of repair (fewer than 0.5 sites of BrdUrd incorporation per 1 times 10⁸ dalton). Moreover, in L5178Y-R cells high fluences of UVC caused an extensive DNA degradation. Such degradation was not observed in L5178Y-S cells during the 24-h post-exposure period. These results are consistent with the notion that L5178Y-R cells have a reduced capacity to repair DNA damage induced by UVC radiation.     

Intensity-Dependent Enzyme Photosensitization Using 532 nm Nanosecond Laser Pulses

Abstract— The intensity dependence of the rose bengal (RB)-photosensitized inhibition of red blood cell acetylcholinesterase has been studied experimentally and the results compared to a quantitative excitation/deactivation model of RB photochemistry. Red blood cell membrane suspensions containing 5 μ M RB were irradiated with 532 nm, 8 ns laser pulses with energies between 1 and 98.5 mJ. A constant dose (7 J) was delivered to all samples by varying the total number of pulses. At incident energies greater than ∼ 4.5 mJ/pulse, the efficiency for photosensitized enzyme inhibition decreased as the energy/pulse increased. The generation of RB triplet state was monitored as a function of laser energy and the triplet-triplet absorption coefficient was determined to be 1.9 × 10⁴ M ⁻¹ cm⁻¹ at 530 nm. The number of singlet oxygen molecules produced at each intensity was calculated from both the physico-mathematical model and from laser flash photolysis results. The results indicated that the photosensitized inhibition of acetylcholinesterase was exclusively mediated by singlet oxygen, even at the highest laser intensities employed.     

FREQUENCY OF ULTRAVIOLET RADIATION-INDUCED MUTATION AT THE hprt LOCUS IN REPAIR-PROFICIENT MURINE FIBROBLASTS TRANSFECTED WITH THE denV GENE OF BACTERIOPHAGE T4

Abstract— Thc frequency of spontaneous and ultraviolet radiation (UVR)-induced mutation at the hprt locus was determined in control and denV-transfected, repair-proficient murine fibroblasts. Control cells removed an average of 25% of pyrimidine dimers induced by exposure to 150 J/m²UVR from an FS40 sunlamp within 24 h; under the same conditions of induction and repair, denV-transfected cells removed an average of 71% of pyrimidine dimers. Control cells were somewhat more resistant than denV-transfected cells to killing by UVR. The average frequency of spontancous mutation at the hprt locus for control and denV-transfected cells was 3 and 15 6-thioguanine (6-TG)-resistant colonies per 10⁶ surviving cells, respectively; there was no statistically significant difference between control and dcnV-transfected cells. However, after exposure to 75 or 150 J/m² UVR, denV-transfected cells had a significantly lower frequency of mutation to 6-TG resistance. After exposure to a fluence of 75 J/m², the average frequency of UVR-induced mutation at the hprt locus was 166 mutant colonies per lo^h surviving cells for control cells and 92 mutant colonies for denV-transfectcd cells; after 150 J/m², control cells had 205 6-TG-resistant colonies per 10⁶ cells, while dmV-transfected cclls had 61 mutant colonies. These results demonstrate that UVR-induced pyrimidine dimers are mutagenic photoproducts in mammalian cells.     

THE EFFECTS OF FAR ULTRAVIOLET RADIATION ON RESPIRATION AND CATION ACCUMULATION BY ISOLATED MITOCHONDRIA OF PHASEOLUS VULGARIS

Abstract. The respiration rates and respiratory control ratios of isolated bean mitochondria have been measured following exposure to 0, 150, 300 and 900 J/m² of far UV radiation (190–300 nm) from a mercury vapour light source with 90% total radiant intensity at 254 nm. Loss of respiratory control occurred at 150 J/m² and inhibition of respiration was significant at the highest exposure dosage. The uptake of both ⁴⁵Ca and ⁸⁵Sr have been measured following a 10min incubation of isolated mitochondria with 2 m M cation. Significant decreases in cation accumulation were observed following exposure to 900 J/m². The effect seemed to be associated with loss of active transport of the ions as a result of respiratory uncoupling or reduced electron transport. There was no significant effect of storage on respiration or ion transport nor was there any indirect effect of irradiated suspending medium on mitochondria.     

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.     

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).     

IMPROVED QUANTITATION OF DNA-PROTEIN CROSSLINKING CAUSED BY 405-nm MONOCHROMATIC NEAR-UV RADIATION IN HUMAN CELLS

An improved alkaline elution analysis has been used to estimate the yield of DNA-protein crosslinks induced in human P3 cells by monochromatic 405-nm near-ultraviolet radiation. Crosslinks are induced linearly over the fluence range studied, at a rate of 1.22 crosslinks per 10¹⁰ daltons per MJ m^-2± 0.066 (one standard error of the slope, N = 37). This rate is equivalent to 1360 crosslinks per cell genome per lethal event.     

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.     

EFFECT OF PHOTOREACTIVATING LIGHT ON LETHAL AND PRE-MUTATIONAL UV LESIONS IN ESCHERICHIA COLI WP2s CARRYING THE R46 MUTATOR PLASMID

Abstract—An excision-deficient E. coli strain carrying the R46 mutator plasmid showed a different response towards photo-reactivation after UV irradiation than the same strain without plasmid. While the photoreactivation of lethal lesions was comparable in both strains, the number of UV-induced mutants per 10⁶ survivors was slightly reduced for the plasmid bearing strain by photoreactivating light at UV fluences below 60 mJ/m² but increased at higher fluences. To explain this it is proposed that some UV photoproduct(s) of DNA other than cyclobutane dipyrimidine dimers are pre-mutational lesions for error-prone DNA repair by the plasmid, P-repair, but not for SOS-repair.     

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.     

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.